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Mechanics of Oxidative Stress and Protein Turnover in C2C12 Myotubes
Ya‐tzu Chang BSc
School of Chemistry and Biochemistry School of Anatomy, Physiology and Human Biology
This thesis is presented for the degree of Master of Philosophy in Biochemistry of The University of Western Australia
2016
i
Abstract
Skeletalmuscle is amajor component of bodymass, and not only controls voluntary
movementsbutalsoservesasamajorsourceofbodyheat.Alossofmusclemass,also
described as muscle wasting or atrophy, can be detrimental to overall health. An
imbalance between protein synthesis and degradation is thought to contribute to
musclewasting.
One of the key causes of muscle wasting is oxidative stress caused by a mismatch
betweenreactiveoxygenspecies(ROS)generationanddegradation.Asthemechanisms
thatunderlyoxidativestressandmusclewastingarestillunknown,itwashypothesized
thatanincreaseinoxidativestressdecreasestherateofproteinsynthesisleadingtoa
netlossoftotalprotein.
Thefirstpartofthethesisinvestigatedtheeffectsofoxidativestressontheleveloftotal
proteincontent.UsingaC2C12myotubeculturemodel,levelsofhydrogenperoxidewere
modulated with catalase and glucose oxidase. The treated myotubes were harvested
withtrichloroaceticacid(TCA)andlevelsoftotalproteincontentweremeasuredusing
themicroBCAassay.Asexpected,theleveloftotalproteinwassignificantlyincreased
withcatalasetreatmentanddecreasedwithglucoseoxidasetreatment.
Since the level of total protein was regulated with the balance between protein
synthesis and degradation, the level of protein synthesis and the rate of protein
degradation were then measured. Unexpectedly, the level of protein synthesis was
significantly decreased with catalase treatment and not significantly affected with
glucoseoxidasetreatment.Therateofproteindegradationwassignificantlydecreased
with catalase treatment but still not significantly affected with glucose oxidase
treatment.To explainhow the levels of protein synthesiswereaffected, the signaling
pathwayswerethenobserved.
Previous studies have found the 4EBP1 signaling pathway and eIF2α pathway areaffectedbyoxidativestress.Whentherateofphosphorylationof4EBP1decreases,the
levelofproteinsynthesisisreducedbysuppressingtheactivityofeIF4E.Inthepresent
study, the rate of phosphorylation of 4EBP1 was not affected by either catalase or
glucoseoxidase.TheeIF2α pathwaywasthen investigated.While thephosphorylation
ii
ofeIF2αincreases,thelevelofproteinsynthesisisreducedbysuppressingtheactivity
ofeIF2B.Asexpected,theratesofphosphorylationofeIF2αwereincreasedafterboth
catalase and glucose oxidase treatment. This suggested that the eIF2α signaling
pathway is involved in inducing the changes in protein synthesis in myotubes in
responsetooxidativestress.
To further explore the effect of ROS on muscle proteins, the C2C12 myotubes were
treatedwithcatalaseandglucoseoxidaseandthioloxidationassessed.Thiolgroupsare
potentially powerful antioxidants which react to oxidative stress. They contain
sulfhydryl (‐SH) groups that are readily oxidized to form stable disulphide bonds.
Previousstudieshaveshownthatthestructureofthiolsinvariouscelltypesarealtered
in response to oxidative stress. In the present study, 2 tag method developed for
labelingmusclesamples(Armstrongetal.2011)wasadaptedtolabeltheC2C12culture
samples.Usingthismethod,thioloxidationinC2C12myotubeswasobservedforthefirst
time. However, unexpectedly, total thiol oxidation and thiol oxidation of specific
proteins were not found to be significantly changed by catalase or glucose oxidase
treatment.
iii
Acknowledgement
IwouldliketoextendmymostsinceregratitudetomysupervisorsA/ProfessorPeterG.
Arthur, A/Professor Tea Shavlakadze and Professor Miranda D. Grounds for their
invaluable guidance, support, advice and supervision throughout this challenging but
rewardingexperience. Iwouldalso liketoextendmymostsinceregratitudetoschool
staffDr.JoanneEdmondson,LouseWedlockandSatoJuniperfortheirinvaluableadvice
and support throughout this challenging process of composing this thesis. All their
immenseknowledge,scientificingenuity,constantenthusiasmandseeminglyunlimited
patiencearegratefullyacknowledged.
I would like to thank my dearest family and friends. It was a tough journey as an
internationalstudent.Thankstoalltheirlove,supportandencouragementthatgaveme
strengthtogetthroughthisjourney.Thankyousomuch,andloveyouall.
To themembersofGroundsandPGA labs, thankyou for all your support andadvice
throughout my entires study. I would like to especially thank Tinashe Chinzou for
gettingmefamiliarwiththe labswhileI firstarrivedthe lab, thecountry. Ialsothank
AlexArmstrong,JessicaTerrill,PearlTan,RuthChai,StevenKho,SumiiHaleemandZoe
Soffefortheirhelpandguidanceinlaboratorywork.
MysincerethankstoMrGregCozensforhisexpertadviceonmolecularbiologywork
andalwayskeepingthelaboratorywellorganized.
IalsowanttoacknowledgetheNationalHealthandMedicalResearchCouncil(NHMRC)
grantwhichsupportedtheworkofthisthesis.
Lastbutnotleast,IamreallyappreciativeoftheScholarshipsforInternationalResearch
Fees(SIRF)thatawardedbyUWAthatsupportedmystudiesinUWA.
Thankyou.
iv
Declaration of Contributions
Alltheworkpresentedinthisthesiswasperformedsolelybytheauthor.
Thisthesiswaswrittensolelybytheauthor.
I hereby declare that thework containedwithin this thesis is entirelymy ownwork,
whichhasbeencontributedisclearlystated.
Ya‐tzuChang
May2016
v
Table of Contents
Abstract .................................................................................................................................i
Acknowledgement .............................................................................................................. iii
Declaration of Contributions ............................................................................................. iv
Table of Contents ................................................................................................................ v
List of Figures ..................................................................................................................... ix
List of Tables ...................................................................................................................... xi
List of abbreviations .......................................................................................................... xii
Chapter 1 Literature review ................................................................................................ 1
1.1 Introduction ............................................................................................................................. 1
1.2 Oxidative stress ...................................................................................................................... 1
1.2.1 Overview ............................................................................................................................ 1
1.2.2 Hydrogen peroxide, catalase and glucose oxidase ........................................................... 3
1.2.3 Thiol oxidation .................................................................................................................... 4
1.2.4 Effects of oxidative stress in muscle and pathology........................................................... 5
1.3 Skeletal muscle biology ......................................................................................................... 6
1.3.1 Overview ............................................................................................................................ 6
1.3.2 Main proteins in skeletal muscle ........................................................................................ 7
1.3.3 Muscle differentiation and C2C12 myotubes model ............................................................. 8
1.3.4 Muscle wasting ................................................................................................................ 10
1.4 Protein turnover .................................................................................................................... 11
1.4.1 Overview .......................................................................................................................... 11
1.4.2 Protein degradation .......................................................................................................... 12
Ubiquitin-proteasome system (UPS) .................................................................................................... 12
Lysosomal-autophagy (LA) system ...................................................................................................... 14
1.4.3 Protein synthesis .............................................................................................................. 15
Initiation ................................................................................................................................................ 15
Elongation ............................................................................................................................................. 18
Termination ........................................................................................................................................... 19
1.5 Signalling pathway ............................................................................................................... 20
1.5.1 Overview .......................................................................................................................... 20
1.5.2 mTOR/4EBP1 pathway .................................................................................................... 21
1.5.3 PERK/eIF2α pathway ....................................................................................................... 23
Aim ...................................................................................................................................... 26
vi
Chapter 2 Material and Methods ...................................................................................... 27
2.1 Cell Culture ............................................................................................................................ 27
2.1.1 Proliferation ...................................................................................................................... 27
2.1.2 Trypsinization and seeding of myoblasts ......................................................................... 28
2.1.4 Treatment conditions ....................................................................................................... 29
2.2 Protein extraction ................................................................................................................. 29
2.2.1 TCA acetone extraction ................................................................................................... 29
2.2.2 Phospho-safe extraction .................................................................................................. 30
2.3 Protein quantification ........................................................................................................... 31
2.3.1 Bradford assay ................................................................................................................. 31
2.3.2 Micro BCA assay ............................................................................................................. 31
2.3.3 Detergent compatible (DC) protein assay ........................................................................ 32
2.4 Measurement of protein synthesis ...................................................................................... 32
2.4.1 Incorporation of radioactive leucine ................................................................................. 32
2.4.2 Harvest ............................................................................................................................. 33
2.4.3 Radiation analysis ............................................................................................................ 33
2.5 Measurements of protein degradation ................................................................................ 34
2.5.1 Incorporation of radioactive leucine ................................................................................. 34
2.5.2 Harvest ............................................................................................................................. 34
2.5.3 Radiation analysis ............................................................................................................ 35
2.6 Western Blot .......................................................................................................................... 35
2.6.1 SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE) .................................................. 36
2.6.2 Bio-Rad system ................................................................................................................ 38
2.6.3 Densitometry analysis ...................................................................................................... 39
2.7 Measurements of thiol oxidation-2 tag labeling ................................................................. 39
2.7.1 Preparation of protein samples ........................................................................................ 40
2.7.2 Dual labeling of protein thiols with fluorescent tags ......................................................... 40
2.7.3 Fluorescence measurement using a plate reader ............................................................ 41
2.7.4 SDS-PAGE ...................................................................................................................... 42
2.8 Statistics ................................................................................................................................ 43
Chapter 3: Development of methods for the study of protein content in C2C12
myotubes in response to treatment with catalase and glucose oxidase ...................... 44
3.1 Introduction ........................................................................................................................... 44
3.2 Methods ................................................................................................................................. 46
Myotube cultures .................................................................................................................................. 46
Protein extraction .................................................................................................................................. 46
Protein quantification- micro BCA assay .............................................................................................. 47
3.3 Results ................................................................................................................................... 47
vii
3.3.1 Modifying the extraction method to extract proteins from C2C12 myotubes ...................... 47
3.3.2 Method to quantify protein content in C2C12 myotubes .................................................... 49
3.3.3 Measuring the level of protein content in C2C12 myotubes in response to catalase and
glucose oxidase ........................................................................................................................ 51
3.4 Discussion ............................................................................................................................. 52
Chapter 4: Development of methods for the measurement of protein synthesis and
degradation in C2C12 myotubes in response to treatment with catalase and glucose
oxidase ............................................................................................................................... 54
4.1 Introduction ........................................................................................................................... 54
4.2 Methods ................................................................................................................................. 55
Myotube cultures .................................................................................................................................. 55
Protein synthesis .................................................................................................................................. 55
Protein degradation .............................................................................................................................. 56
4.3 Results ................................................................................................................................... 56
4.3.1 Establishment of method for measuring protein synthesis in C2C12 myotubes ................ 56
4.3.2 Establishment of method for measuring protein degradation in C2C12 myotubes ............ 57
4.3.3 Measuring protein synthesis in C2C12 myotubes .............................................................. 59
4.3.4 Measuring protein degradation in C2C12 myotubes .......................................................... 60
4.4 Discussion ............................................................................................................................. 61
Chapter 5: Development of methods for the study of signaling pathway on protein
synthesis in C2C12 myotubes in response to treatment with catalase and glucose
oxidase ............................................................................................................................... 63
5.1 Introduction ........................................................................................................................... 63
5.2 Methods ................................................................................................................................. 64
Myotube cultures .................................................................................................................................. 64
Protein extraction .................................................................................................................................. 64
Protein quantification ............................................................................................................................ 64
Western Blot- SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE) system ................................... 65
Western Blot- Bio-Rad system ............................................................................................................. 66
Antibodies ............................................................................................................................................. 66
5.3 Results ................................................................................................................................... 67
5.3.1 Optimization of method for measuring 4EBP1 phosphorylation in C2C12 myotubes ........ 67
5.3.2 Optimization of method for measuring eIF2αphopsphorylation in C2C12 myotubes ........ 68
5.3.3 Measuring the rate of phosphorylation on 4EBP1 in C2C12 myotubes ............................. 71
5.3.4 Measuring eIF2α phosphorylation in C2C12 myotubes ..................................................... 71
5.4 Discussion ............................................................................................................................. 72
viii
Chapter 6: Development of a method to measure thiol oxidation in C2C12 myotubes in
response to treatment with catalase and glucose oxidase ............................................ 74
6.1 Introduction ........................................................................................................................... 74
6.2 Methods ................................................................................................................................. 74
Myotube cultures .................................................................................................................................. 74
Protein extraction .................................................................................................................................. 75
Protein quantification- micro BCA assay .............................................................................................. 75
2 tag labeling ........................................................................................................................................ 75
Protein quantification-DC assay ........................................................................................................... 77
FLm and TRm quantification ................................................................................................................ 77
6.3 Results ................................................................................................................................... 78
6.3.1 Optimize the 2 tag method for C2C12 myotubes model .................................................... 78
6.3.2 Measuring total thiol oxidation in fluorescently labeled C2C12 myotubes using a
fluorescent plate reader ............................................................................................................ 79
6.3.3 Measuring thiol oxidation in C2C12 myotubes on actin and myosin by gel electrophoresis
.................................................................................................................................................. 80
6.4 Discussion ............................................................................................................................. 82
Chapter 7: General discussion ......................................................................................... 83
7.1 Introduction ........................................................................................................................... 83
7.2 Muscle wasting ..................................................................................................................... 83
7.3 Protein turnover .................................................................................................................... 84
7.4 Signalling pathways ............................................................................................................. 85
7.5 Thiol oxidation ...................................................................................................................... 86
7.6 Future studies ....................................................................................................................... 86
Bibliography ....................................................................................................................... 88
Appendices ...................................................................................................................... 113
ix
List of Figures
Figure 1.1 The sources and cellular responses to ROS ................................................... 2
Figure 1.2 Catalase decomposition of hydrogen peroxide .............................................. 3
Figure 1.3 Glucose oxidase composition of hydrogen peroxide..................................... 3
Figure 1.4 Structure of skeletal muscle ............................................................................. 7
Figure 1.5 Myosin structure and contractile apparatus in muscle (Richfield 2014) ....... 8
Figure 1.6 Changes in C2C12 cell morphology in response to myogenic differentiation
.............................................................................................................................................. 9
Figure 1.7 Changes in muscle mass with age ................................................................. 11
Figure 1.8 Ubiquitin-proteasome system (UPS) .............................................................. 13
Figure 1.9 Lysosomal digestion ....................................................................................... 14
Figure 1.10 Cyclical process of translation ..................................................................... 17
Figure 1.11 Translation elongation in bacteria ............................................................... 19
Figure 1.12 Translation termination ................................................................................. 20
Figure 1.13 Inhibition of translation under different types of stress ............................. 20
Figure 1.14 Regulating cap-dependent translation initiation ......................................... 21
Figure 1.15 mTORC1 pathway and ageing ...................................................................... 22
Figure 1.16 Effects of ROS on mTOR/4EBP1 pathway ................................................... 23
Figure 1.17 The relationship between ER stress and ROS ............................................ 25
Figure 2.1 Haemocytometer .............................................................................................. 28
Figure 2.2 The assemble of transfer cassette ................................................................. 37
Figure 3.1 Changes in muscle mass accompanying cancer ......................................... 44
Figure 3.2 The total protein content ................................................................................. 48
Figure 3.3 The total protein content ................................................................................. 49
Figure 3.4 The standard curve of DC assay in Tris buffer ............................................. 50
Figure 3.5 Standard curve for micro BCA assay using BSA in various buffers .......... 51
x
Figure 3.6 Total protein levels in C2C12 myotubes in response to catalase and glucose
oxidase treatment .............................................................................................................. 52
Figure 4.1 Leucine incorporation in C2C12 myotbes treated with catalase ................... 57
Figure 4.2 Radioactive leucine release from C2C12 myotubes treated with catalase and
TNF ...................................................................................................................................... 58
Figure 4.3 Radioactive leucine release from pre-labeled C2C12 myotubes with various
treatments .......................................................................................................................... 59
Figure 4.4 Protein synthesis in C2C12 Myotubes with catalase and glucose oxidase
treatment ............................................................................................................................ 60
Figure 4.5 Radioactive leucine release from C2C12 myotubes with various treatments
............................................................................................................................................ 61
Figure 5.1 Detection of phosphorylated 4EBP1 and total 4EBP1 .................................. 67
Figure 5.2 Detection of total eIF2α ................................................................................... 68
Figure 5.3 Detection of phosphorylated eIF2α ................................................................ 69
Figure 5.4 Detection of phosphorylated eIF2α ................................................................ 70
Figure 5.5 Detection of phosphorylated eIF2α ................................................................ 70
Figure 5.6 4EBP1 phosphorylation in C2C12 myotubes after catalase and glucose
oxidase treatment .............................................................................................................. 71
Figure 5.7 eIF2α phosphorylation in C2C12 myotubes after catalase and glucose
oxidase treatment .............................................................................................................. 72
Figure 6.1 Total protein levels after precipitation with ethanol and acetone ............... 79
Figure 6.2 Thiol oxidation in C2C12 myotubes in response to catalase and glucose
oxidase treatment .............................................................................................................. 80
Figure 6.3 Thiol oxidation on myosin in C2C12 myotubes in response to catalase and
glucose oxidase treatment ............................................................................................... 81
Figure 6.4 Thiol oxidation on actin in C2C12 myotubes in response to catalase and
glucose oxidase treatment ............................................................................................... 81
xi
List of Tables
Table 1.1 Formation and properties of different forms of ROS ....................................... 2
Table 1.2 Redox-regulated proteins and complexes ........................................................ 4
Table 1.3 Effectiveness of antioxidant treatment to skeletal muscle wasting ............... 5
Table 1.4 Metabolic consequences of sarcopenia and cachexia .................................. 10
Table 1.5 Eukaryotic initiation factors ............................................................................. 16
Table 2.1 Composition of resolving and stacking gel .................................................... 36
Table 2.2 Primary antibodies ............................................................................................ 38
Table 2.3 Chemiluminescence substrate solution used for protein detection ............. 38
Table 2.4 Dilution for FLm and TRm standards .............................................................. 42
Table 2.5 In-gel FLm/TRm standards ............................................................................... 42
xii
List of abbreviations
3’UTR 3’untranslatedregion
4EBP1 Eukaryotictranslationinitiationfactor4E‐bindingprotein1
5’UTR 5’untranslatedregion
ABCE1 ATP‐bindingcassettesubfamilyEmember1
AIDS AcquiredImmuneDeficiencySyndrome
Akt ProteinkinaseB
A‐site Aminoacyl‐site
ATCC AmericanTypeCultureCollection
ATF4 Activatingtranscriptionfactor4
ATP Adenosinetriphosphate
BCAassay Bicinchoninicacidassay
BSA Bovineserumalbumin
Cat. Catalase
ddi Doubledeionized
DCassay Detergentcompatibleassay
Deptor DEP‐domain‐containingmTOR‐interactingprotein
DEX Dexamethasone
DMEM Dulbecco’smodifiedEaglemedium
DMSO Dimethylsulfoxide
xiii
DTT Dithiothreitol
EDTA Ethylenediaminetetraaceticacid
eEF1A Eukaryotictranslationelongationfactor1A
eEF2 Eukaryotictranslationelongationfactor2
EF‐G ElongationfactorG
EF‐Tu Elongationfactorthermounstable
eIFs Eukaryotictranslationinitiationfactors
eIF1 Eukaryotictranslationinitiationfactor1
eIF1A Eukaryotictranslationinitiationfactor1A
eIF2 Eukaryotictranslationinitiationfactor2
eIF2α α subunitofeIF2
eIF2γ γsubunitofeIF2
eIF2B Eukaryotictranslationinitiationfactor2B
eIF3 Eukaryotictranslationinitiationfactor3
eIF3 j subunitofeIF3
eIF4B Eukaryotictranslationinitiationfactor4B
eIF4E Eukaryotictranslationinitiationfactor4E
eIF4F Eukaryotictranslationinitiationfactor4F
eIF4G Eukaryotictranslationinitiationfactor4G
eIF5B Eukaryotictranslationinitiationfactor5B
xiv
ER Endoplasmicreticulum
eRF1 Eukaryotictranslationterminationfactor1
eRF3 Eukaryotictranslationterminationfactor3
E‐site Exit‐site
FBS Fetalbovineserum
FLm BODIPYFL‐N‐(2‐aminoethyl)maleimide
GAP GTPase‐activatingprotein
GCN2 eIF2αkinase4
GDP Guanosinediphosphate
GEF Guaninenucleotideexchangefactor
GluO. Glucoseoxidase
GPx Glutathioneperoxidase
GSH Glutathione
GTP Guanosine‐5’‐triphosphate
GTPase SingularGTPase
H+ Hydrogenion
HRI Heme‐regulatedinhibitorkinase
HS Horseserum
IGF Insulin‐growthfactor‐1
Met Methionine
xv
Mg2+ Magnesiumion
mLST8 MammalianlethalwithSec13protein8
mRNA MessengerRNA
mTOR Mammaliantargetofrapamycin
NAC N‐acetylcysteine
NADPH Nicotinamideadeninedinucleotidephosphate
NPSH Intracellularnon‐proteinthiols
O2 Dioxygen
•O2‐ Superoxideanions
•OH Hydroxylradical
p4EBP1 Phosphorylated4EBP1atThr37/46
PABP Poly(A)‐bindingprotein
PBS PhosphateBufferedSaline
peIF2α PhosphorylatedeIF2αatSer51
PERK PKR‐likeER‐localizedeIF2αkinase
PGC‐1α Peroxisomeproliferator‐activated‐receptor‐gamma‐coactivator‐1α
PI3K Phosphoinositide‐3‐kinase
PIKKs PI3K‐relatedkinases
PKR ProteinkinaseR
post‐TCs Post‐terminationribosomalcomplexes
xvi
PP Proteinphosphatase
PRAS40 Proline‐richAKTsubstrate40kDa
P‐site Peptidyl‐site
PVDF Polyvinylidenedifluoride
RO• Alkaoxyl
RO2• Peroxyl
ROS Reactiveoxygenspecies
rRNA RibosomalRNA
SDS‐PAGE Sodiumdodecylsulfatepolyacrylamidegelelectrophoresis
‐SH Sulfhydryl
SOD Superoxidedismutase
TBS TrisBufferedSaline
TBS‐T TBSbufferwithTween‐20
TCA Trichloroaceticacid
TCEP Tris(2‐carboxyethyl)phosphinehydrochloride
TNF Tumournecrosisfactor
Trp Tryptophan
TRm TEXASRED‐C2‐malemide
TRx Thioredoxinreductase
tTNA TransferRNA
xvii
UCP Uncouplingproteins
UPP Ubiquitin‐proteasomepathway
UPR Unfoldedproteinresponse
UV Ultraviolet
1
Chapter 1 Literature review
1.1 Introduction
Oxidative stress occurs when there is an imbalance between oxidant generation and
degradationanditcanresultinthewastingofmusclemassindifferentconditions.This
reviewdescribestheroleofhydrogenperoxide,catalaseandglucoseoxidaseonprotein
levels,proteinturnover,signallingpathwaysandthioloxidationinskeletalmusclecells.
Thereviewbeginswithadescriptionoftheroleofoxidativestress,thioloxidation,and
effects of oxidative stress on muscle. This is followed by the description of skeletal
muscle biology, skeletal muscle models, muscular proteins and two types of muscle
wasting,sarcopeniaandcachexia.Thedescriptionsofproteindegradationandprotein
synthesisthatareinvolvedintheprocessofmusclewastingarethendiscussed.Finally,
theroleofthemTOR/4EBP1andPERK/eIF2αsignallingpathwaysthatmayinvolvein
themodulationofproteinsynthesis inmusclewastingarediscussed.Fivehypotheses
are proposed to demonstrate the possible interactions between oxidative stress and
C2C12myotubesunderdifferentlevelsofhydrogenperoxide.
1.2 Oxidative stress
1.2.1 Overview
Reactive oxygen species (ROS) is a collective term that broadly describes a group of
reactivecompoundsderivedfromoxygen(Table1.1)(Halliwelletal.1994;Circuetal.
2010). The oxidants are generated continuously as a consequence of aerobic
metabolisminorganellessuchasmitochondriaandperoxisomes(Doriaetal.2012),or
anumberofexternalagentssuchasultraviolet(UV)light(Fig.1.1)(Barbierietal.2012;
Terrilletal.2013).
2
Table1.1FormationandpropertiesofdifferentformsofROSDifferentformsofROScanbederivedfromsuperoxidebythesequentialadditionofelectrons,andeachofthemhasdifferentpropertiesinthecell.ReprintedbypermissionfromMacmillanPublishersLtd:[CurrentOpinioninClinicalNutritionandMetabolicCare](3971721443198),copyright(Arthuretal.2008).
O2+e‐ O2‐+e‐ H2O2 +e‐ •OH+e‐ H2O
Superoxide Hydrogenperoxide Hydroxylradical
Negativelychargedradicalion
Uncharged,non‐radical,relatively
stable
Highlyreactiveradical
Propertiesofinterest
Reactswithnitricoxidetoformperoxinitrite
Causeformationofdisulfidebonds
Primaryagentofprotein,DNAandlipiddamage
Duringphysiologicalhomeostasis, anoveralloxidativebalance ismaintained in tissue
bymatching theproductionofROSvia the removal actionbyavarietyof antioxidant
systems.Inthisenvironment,ROSservesasasignallingmoleculetostimulateoractasa
secondarymessengerinvarietyofsignallingtransductionpathways(Arthuretal.2008;
Barbierietal.2012;Terrilletal.2013).LowingROS levelsbelowthehomeostaticset
pointmay interrupt the physiological function in cellular proliferative responses and
host defenses. Oxidative stress also occurs when the action of antioxidants are
outweighed by the generation of ROS (Terrill et al. 2013). Oxidative stress has been
implicated in numerous conditions including ageing, inflammatory disorders, cancer,
musclewasting andmusculardystrophies (Fig. 1.1) (Finkel et al. 2000;Tidball 2005;
Barbierietal.2012;Terrilletal.2013).
Figure1.1ThesourcesandcellularresponsestoROS
3
Reprinted by permission from Macmillan Publishers Ltd: [Nature] (3971711505027), copyright(Finkeletal.2000).
1.2.2 Hydrogen peroxide, catalase and glucose oxidase
Hydrogenperoxide,anon‐radical,weakoxidantwitharelativelylonghalf‐life,isoneof
theseROS.Thissmallandstablemoleculecandiffusereadilywithincellsandacrosscell
membranesandalsoactasasignallingmoleculetoactivatealargenumberofsignalling
pathways(Bienertetal.2006;Rhee2006;Vealetal.2007;Arthuretal.2008;Paulsenet
al.2010;Barbierietal.2012).
Catalaseisanenzymethatcanremovehydrogenperoxidefromthecelltodecreasethe
levelofoxidativestress.Catalasedecomposeshydrogenperoxidetowaterandoxygen
bysuccessivereductionofthecatalaseironbyhydrogenperoxideanditsre‐oxidation
byO2(Fig1.2)(Keilinetal.1938;Jonesetal.1968).
Figure1.2Catalasedecompositionofhydrogenperoxide
Incontrast,glucoseoxidaseisanenzymethatcangeneratehydrogenperoxidethrough
the glucose/glucose oxidase pathway (Fig 1.3) (Weiss et al. 1981; Starkebaum et al.
1986; Salazar et al. 1997). This can result in the steady accumulation of hydrogen
peroxideinthecell(Starkebaumetal.1986;Dayetal.1997;Salazaretal.1997),which
canincreaseoxidativedamageandstress.
Figure1.3Glucoseoxidasecompositionofhydrogenperoxide
DifferentkindsofROSareinvolvedinavarietyofsignalingpathways.Theyalsoimpact
onawidearrayofproteinssuchaskinases,phosphatesandtranscription factors that
contain reduction‐oxidation (redox) sensitive residue (Table 1.2) (Arthur et al. 2008;
Paulsenetal.2010).
4Fe3‐+2H2O2=4Fe2‐+4H‐+2O24Fe2‐+4H‐+O2=4Fe3‐+2H2O
2H2O2=2H2O+O2
β‐D‐glucose+O2 Glucoseoxidase
D‐gluconolactone+H2O2
4
Table1.2Redox‐regulatedproteinsandcomplexesReprintedbypermissionfromMacmillanPublishersLtd:[AmericanChemicalSociety],copyright(Paulsenetal.2010).
Stimulation Organism(A) ROSsource(B) EffectofstimulationEpidermalgrowthfactor
(EGF)Hs,M,R NOX(C) Proliferation
Platelet‐derivedgrowthfactor(PDGF)
Hs,M,R NOX Proliferation/migration
Basicfibroblastgrowthfactor(bFGF)
B NOX Proliferation
Vascularendothelialgrowthfactor(VEGF)
P L Angiogenesis/proliferation
Granulocyte‐macrophagecolony‐stimulatingfactor
(GM‐CSF)
H ND Proliferation/migration
Insulin M,R NOX,Cytokines Glucoseuptake/transportLipopolysaccharide
(LPS)M NOX Inductionofimmuneresponse
Interleukin‐1β(IL‐1β) Hs,M NOX,L InductionofimmuneresponseInterleukin‐3(IL‐3) Hs ND InductionofimmuneresponseInterleukin‐4(IL‐4) Hs NOX InductionofimmuneresponseCD28stimulation Hs L Inductionofimmune
response/proliferationTumournecrosisfactor
α(TNF‐α)B,M,Hs NOX Apoptosis
Transforminggrowthfactor‐β1(TGF‐β1)
M NDAgonistofGPCRs(D)
Cellcyclearrest
AngiotensinII(AngII) R NOX HypertrophyLysophosphatidicacid
(LPA) Hs NOX,L Proliferation
Thrombin Hs NOX ProliferationSerotonin Ha NOX,
Otherstimulants
Proliferation
Wounding Z NOX LeukocyterecruitmentOxidativeStress D MT Differentiation
Reoxygenationafterhypoxia
R MT O2•‐burst
(A) B, bovine; D, Drosophila melanogaster; Ha, hamster; Hs, human; M, mouse; Z, zebrafish. (B) NOX, NADPH oxidase; M, mitochondria; L, lipoxygenase; ND, not determined. (C) For many of these cases, the specific NOX isoform activated is unknown. Each NOX isoform demonstrates disparate tissue expression, and continued studies will be required to elucidate the regulation of each NOX isoform in response to diverse external signals. (D) GPCRs= guanosine triphosphate (GTP)-binding protein (G protein)-coupled receptors.
1.2.3 Thiol oxidation
Hydrogenperoxidemodifiesproteinfunctionbyoxidizingthethiolgroupofthetarget
proteins to formdisulfide bonds (Bienert et al. 2006; Rhee 2006; Arthur et al. 2008). Thiols
areorganicsulfurderivatives,identifiedbythepresenceofsulfhydrylresidues(‐SH)at
theiractivesite.Biological thiols include low‐molecular‐weight free thiolsandprotein
thiols, the functional group of the amino acid cysteine are important in cellular
antioxidantdefencesandredoxsignalling (Batyetal.2005;Terrilletal.2013). In the
5
presence of ROS, ‐SH residues of thiol proteins (such as cysteine) may undergo
reversible structural modifications, whereby the ‐SH bonds are broken and disulfide
bondsareformed.
Themodificationonthiols isoneof themajorcellularconsequencesofROSexposure,
includinghydrogenperoxideexposure.Oxidationofthiolproteinsiscrucialtocellsasit
affectsvariouscellfunctionsincludingproteinstructure,proteintoproteininteractions,
catalysis, electron transfer, ion channel modulation, signalling pathway, post‐
translational protein modifications, and transcription activation (Baty et al. 2005;
Arthur et al. 2008; Paulsen et al. 2010; Terrill et al. 2013). Hydrogen peroxide, for
example,canaffectnumeroussignallingpathwaysbyoxidizingthethiolgroupsof the
targetproteins (Nealetal.1998;Rhee2006;Arthuretal.2008;Barbierietal.2012).
However,thismodificationcouldbereversedbacktothethiolformsviathiol/disulfide
exchangethroughtheactionofantioxidantmoleculesorenzymessuchasglutaredoxin
orperoxiredoxins(Bienert et al. 2006; Rhee 2006; Arthur et al. 2008; Terrill et al. 2013).
1.2.4 Effects of oxidative stress in muscle and pathology
Oxidative stresshasbeen implicated in thepathologyofnumerousmusculardiseases
suchasmusculardystrophies(Terrilletal.2013)thatarecharacterizedbyprogressive
skeletalmusclewastinganddegeneration.Ithasbeenshownthatmusclefunctioncan
be improved after treatment with antioxidants in different muscle wasting models
(Table1.3)(Bonettoetal.2009;Terrilletal.2013).
Table1.3EffectivenessofantioxidanttreatmenttoskeletalmusclewastingReprintedbypermissionfromMacmillanPublishersLtd:[FreeRadicalBiologyandMedicine](3971721188561),copyright(Bonettoetal.2009).
Antioxidanttreatment Effectivedisease IneffectiveoruncertainVitaminE Diabetes Ageing,ALSVitaminC Diabetes Ageing,ALSResveratrol Ageing,diabetes Cancercachexia
Dehydroepiandrosterone Diabetes, cancercachexia ALSOrnithine,cysteine,NAC Cancercachexia,DMD ALS
Carnitine Cancercachexia,ageing,ALS,diabetes
‐
Epigallocatechingallate DMD ‐Low‐intensitytraining Ageing,diabetes,cancer
cachexia,DMD‐
ALS= amyotrophic lateral sclerosisDMD= Duchenne muscular dystrophy
6
Duchenne muscular dystrophy (DMD) is one of the muscular diseases and the
relationshipbetweenDMDandoxidativestresshasbeenextensivelyinvestigated.The
severityofdystropathology invivocanbe reducedwith thiol‐reducingantioxidantN–
acetylcysteine (NAC) ,asmeasuredbydecreased levelsofplasmacreatinekinaseand
reduced myonecrosis, and the thiol oxidation can also be reduced with NAC in
dystrophicmuscle(Terrilletal.2013).
1.3 Skeletal muscle biology
1.3.1 Overview
In skeletal muscle, contractile proteins (such as troponin, tropomyosin, myosin and
actin)containthiolsidechainsthataresensitivetooxidation,andthesemodifications
may alter excitation/contraction coupling and cross‐bridge cycling, and therefore
modulatemusclecontraction(Terrilletal.2013).
About 40% of human bodymass consists of skeletalmuscle and there are over 600
individual skeletalmuscles are related in daily life such as breathing, eating, posture,
walking and reflexes (Shavlakadze et al. 2006; Saladin 2011). Muscle is highly
metabolically active,with the restingmetabolic rateof skeletalmuscle accounting for
about20‐30%ofrestingwhole‐bodyoxygenconsumptionandalsoservesasamajor
sourceofbodyheat(Zurloetal.1990).
Skeletalmuscle(Fig.1.4) iscomposedofbundlesofmuscle fibrescalled fascicles.The
cell membrane surrounding the muscle cell is called sarcolemma, and beneath the
sarcolemma is called sarcoplasm that contains the cellular proteins, organelles, and
myofibrils. The myofibrils are composed of contractile units called sarcomeres that
consist of thickmyosin filaments and thin actin filaments. The arrangement of these
filaments gives skeletal muscle its striated appearance, and the muscle contract by
slidingthethickandthinfilamentsalongeachothersotheskeletalmuscleiscapableof
remarkableadaptationsinresponsetoalteredactivity(Sakumaetal.2015).Thereare
threekindsofmuscle connective tissue: theepimysium,covers thewholemuscle; the
perimysium,coversthebundlesofmusclefibres;theendomysium,coverseachmuscle
fibre.
7
Figure1.4StructureofskeletalmuscleSkeletal muscle is made up of muscle fibres that are composed by myofibrils that consist thickmyosin filaments and thin actin filaments. There are three connective tissues: epimysium,perimysium,andendomysiumthatconnectsallmuscletissuestogether(IvyRose2003;Gebski2009;MedicaLook 2012). Reprinted by permission fromMacmillan Publishers Ltd: [Muscle and Nerve](3972350046350),copyright(Gilliesetal.2011)
1.3.2 Main proteins in skeletal muscle
Thetwomainproteinsfoundinmusclearemyosinandactinandbothareinvolvedin
muscularfunction.Myosins(Fig.1.5)areakeypartofthecontractileproteinsofmuscle
and play an important role in signal transduction and the establishment of polarity
(Bähler 2000). They also act as actin‐based motors that play a fundamental role in
differentformsofeukaryoticmotilityincludingcellcrawling,cytokinesis,phagocytosis,
growth cone extension, maintenance of cell shape, and organelle/particle trafficking
(Berg et al. 2001).
Membersofthemyosinsuperfamilyaredefinedbythepresenceofaheavychainwitha
conserved~80kDacatalyticdomain.Inmostmyosins,thecatalyticdomainisfollowed
by an α‐helical light chain‐binding region consisting of one or more IQ motifs. Most
myosinsalsohaveaC‐terminal tail and/oranN‐terminalextension thought toconfer
class‐specific properties such as membrane binding or kinase activity (Hodge et al.
2000;Bergetal.2001).
8
Figure1.5Myosinstructureandcontractileapparatusinmuscle(Richfield2014)
Actinplaysakeyroleinmusclecontraction,cellmobility,andothercellprocessesand
functions including cell division, endocytosis, secretion, signal transduction, the
regulation of enzyme activity, and themaintenance of cell shape.Actin has also been
shown to regulate the activity ofmembranes and participate in transcription,mRNA
transportandtranslationandsynaptictransmission(Khaitlina 2001).
Actin has α, β, and γ‐isoforms that have been classified according to differences in
mobilities (Storti et al. 1976; Whalen et al. 1976; Rubenstein et al. 1977; Khaitlina
2001).Theseisoformscannotsubstituteforeachother,andthehigh‐levelsynthesisof
exogenous actins lead to changes in cell organization andmorphology. This suggests
that actins are functionally specialized for the tissues in which they predominate
(Khaitlina 2001).
1.3.3 Muscle differentiation and C2C12 myotubes model
Thedevelopmentofskeletalmuscleinvivoandthedifferentiationofmyoblastsinvitro
areaccompaniedbyachangeinisoactinpatterns.Onlycytoplasmicβ‐andγ‐actinsare
participatedinearlymuscledevelopmentandinpre‐fusedculturedmyoblasts(Stortiet
al. 1976; Whalen et al. 1976). During development, the relative amount of α‐actin
increasesuntilitbecomesthepredominantactinspecies.Thisincreasehappensbyday
9
20ofembryonicdevelopment inchicken thighmuscle (Stortietal.1976). Incultured
chickenembryonicmyoblasts,thisincreasebeginsatabout44hrafterplating.At96hr,
when fusion is complete and the myotubes begin to spontaneously contract, α‐actin
becomesthemajorcomponentintheculture(Rubensteinetal.1977).
Themurineimmortalizedcellcellline,C2C12,isaninvitromodelusedforstudiesofthe
molecularbasisofskeletalmusclecelldifferentiation(Kislingeretal.2005;Montesano
etal.2013).C2C12cellswereoriginallyobtainedfromthethighmuscleofC3Hmiceafter
crush injury and are capable of differentiation (Yaffe et al. 1977). In this model,
undifferentiated myoblasts are recognized as flat, fusiform or star‐shaped cells,
scatteredonthesubstrateandrigorouslymono‐nucleated.Afterreachingconfluence,or
24hrafter serumexchange from20% fetalbovineserum(FBS) to2%horseserum
(HS),theorientation,length,andthickeningofthesemyoblastsareconsideredtobeat
anearlystageofdifferentiationat thispoint.Later, thesecellsbegin to fuseand form
multi‐nucleatedmyotubes(Fig.1.6)(Kislingeretal.2005;Koetal.2006;Montesanoet
al.2013).
Figure1.6ChangesinC2C12cellmorphologyinresponsetomyogenicdifferentiation
Light microscopy‐based images of undifferentiated proliferating C2C12 myoblasts (myoblast) anddifferentiatingcellsatvarioustimepoints(2,4,6‐day‐old)followingserumstarvation.Bar,450μmReprinted by permission from Macmillan Publishers Ltd: [Molecular & Cellular Proteomics],copyright(Kislingeretal.2005).
Myoblast 2-day-old
4-day-old 6-day-old
10
1.3.4 Muscle wasting
Loss ofmusclemass ormusclewasting, is related to a poor quality of life, increased
morbidity and mortality, and affected metabolic functions. Two common forms of
musclewastingaresarcopeniaandcachexia(Table1.4).
Table1.4MetabolicconsequencesofsarcopeniaandcachexiaReprintedbypermissionfromMacmillanPublishersLtd:[AnnualReviewofMedicine],copyright(Dodsonetal.2011)
Metaboliccondition Sarcopenia CachexiaMuscleproteinsynthesis Decreased DecreasedMuscleproteindegradation Nochange IncreasedMusclemass,strength,and
functionDecreased Decreased
Fatmass Increased NochangeordecreasedBasalmetabolicrateandtotal
energyexpenditureDecreased Increased
Inflammation Nochange IncreasedInsulinresistance Increased Increased
Severemusclewastingisknownascachexiaandoftenaccompaniesdiseasestatessuch
as cancer, immunodeficiency diseases, HIV/AIDS, rheumatoid arthritis, chronic renal
insufficiency and chronic uremia (Thomas 2007; Tazi et al. 2010;White et al. 2011;
Palusetal.2014).Cachexiaaffectsup to80%ofpatientswithadvancedcancersand
also accounts for nearly 30%of cancer‐related deaths (Glass et al. 2010; Zhou et al.
2010). Although the mechanism of cancer cachexia is poorly understood, multiple
biological pathways and factors such as tumour‐specific proteolysis‐inducing factor
(PIF) and tumournecrosis factorα (TNF‐α) are thought to be involved. In particular,
TNF‐αhasbeenshowntohaveadirectcataboliceffectonskeletalmuscleandleadsto
musclewastingthroughtheubiquitin‐proteasomesystem(UPS).Oxidativestressisalso
thoughttoplayakeyroleincachexiabystimulatingtheUPS(Lenketal.2010;Silverio
etal.2011;Sakumaetal.2012).
Sarcopeniaisassociatedwithaprogressivedeclineofmusclemass,qualityandstrength
asaresultofageingandisassociatedwithlossofmusclefibres,especiallythetypeIIa
fibres.Estimatesoftheprevalenceofsarcopeniarangefrom13%to24%inadultsover
60yearsofagetomorethan50%inadultsaged80yearsandolder(Fig.1.7).Thisloss
ofmusclemass ismostnotable in the lower limbmusclegroups,withvastus lateralis
beingmostaffected.Sarcopeniaisinvolvedinamultifactorialprocessincludesphysical
11
activity,nutritionalintake,oxidativestressandhormonalchanges(Thomas2007;Lenk
etal.2010;Sakumaetal.2012;Sakumaetal.2015).
Figure1.7ChangesinmusclemasswithageComputedtomography(CT)scanof theupper leg(midthigh level) ina25(A)and81yearold(B)male,matched forbodymass andheight.Decreasedmuscle area, increased subcutaneous fat, andincreased fat and connective tissue infiltration into themuscle canbe seen in the elderly subject.ReprintedbypermissionfromMacmillanPublishersLtd:[JournalofAppliedPhysiology],copyright(Koopmanetal.2009).
Loss ofmusclemass can result frommyofibre death or from the reduction in size of
individualmyofibresduetoanetlossofproteincontentresultingfromtheunbalanced
protein degradation and synthesis (Balagopal et al. 1997; Smith et al. 1999; Tisdale
2001;Jackmanetal.2004;Khaletal.2005;Shavlakadzeetal.2006;Moylanetal.2007;
Thomas2007;Arthuretal.2008;Bonettoetal.2009;Evans2010;Pennaetal.2010;
Sakumaetal.2015).ROSispresumedtodelaythedifferentiationprocessofmyoblastto
myotubesbyoxidativelydamagingthecell(Arthuretal.2008;Barbierietal.2012).An
imbalanceinproteindegradationandsynthesishasbeenobservedinmusclecellsafter
hydrogenperoxidetreatment(Jackmanetal.2004).
1.4 Protein turnover
1.4.1 Overview
The turnover of protein in muscle is controlled by protein degradation and protein
synthesis mechanism, and an imbalance in these two can result in muscle wasting.
A. B.
12
Proteindegradation involves therapidmodulationofcellular functionandremovalof
damaged molecules by two systems: the ubiquitin‐proteasome system (UPS) and
lysosomalautophagy(LA)system.ProteinsaretargetedfordestructionbytheUPSviaa
series of enzymatic reactions that tag themwith ubiquitin. In contrast to UPS, LA is
restricted to the cytoplasm but is capable of degrading a much wider spectrum of
substrates, which tend to be long‐lived proteins (Korolchuk et al. 2010). Protein
synthesis involves three main processes: initiation, elongation and termination.
Initiation, in particular, is involved with different kinds of initiation factors that are
regulatedbyvarioussignallingpathwaysthatmaybeaffectedbyoxidativestress.
1.4.2 Protein degradation
Ubiquitin‐proteasome system (UPS)
In eukaryotic cells, the ATP‐dependent UPS is essential for regulating protein
degradation in the cytosol and nucleus, includingmuscle proteins (Palus et al. 2014;
Sakumaetal.2015).Proteinsaretargetedforthisdegradationbyaseriesofenzymatic
reactions that label themwithubiquitin (a76aminoacid residue) inaprocesscalled
poly‐ubiquitylation (Ciechanover et al. 1980; Hershko et al. 1980; Korolchuk et al.
2010).Thismarksthetargetprotein for transportationtothe26Sproteasome,where
the protein is degraded into oligopeptides and then released into the cytoplasm or
nucleoplasmforfurtherdigestionintoaminoacidbypeptidases(Korolchuketal.2010;
Sakumaetal.2015).
The specificity and selectivity of the ubiquitylation process is controlled by three
enzymes,E1,E2andE3(Fig.1.8).E1enzymesactivateubiquitinfunctiontoattackthe
substrateaminogroup.TheactivatedubiquitinisthentransferredfromE1enzymesto
E2enzymeswhicharealsocalledubiquitin‐carriersorconjugatingproteins.Themost
remarkablefeatureofubiquitylationistheextraordinarydiversityofitstargetprotein
substrateandE3enzymes, theuniqueubiquitin ligase,are thekey in this recognition
process. The activated ubiquitin is transferred on the onto the lysine residues of the
target protein substrate by E3 enzymes after the recognition process (Pickart et al.
2004;Leckeretal.2006;Ciechanover2010;Korolchuketal.2010).Thetargetprotein
becomesmono‐ubiquitylatedinoneormoreplaceswiththisprocess,however, this is
insufficientforproteasometargetingasthistargetingrequirespoly‐ubiquitylationofat
least fourubiquitins.Thesepoly‐ubiquitinchainsare formed in subsequent roundsat
13
the lysine residues of ubiquitin (position 6, 11,27, 31, 33, 48 and 68). All these sites
couldbeanacceptorofanotherubiquitin.(Fushmanetal.2010;Korolchuketal.2010;
Sakumaetal.2015).
Figure1.8Ubiquitin‐proteasomesystem(UPS)Theareseveralsteps involved intheUPSprocess. (1)ubiquitin(Ub) isactivatedby theubiquitin‐activatingenzyme,E1enzyme; (2)activatedubiquitin is transferred toaubiquitin‐carrierprotein,E2enzyme;(3)E2enzymetransferstheactivatedubiquitintothetargetproteinsubstratewhichisbound specifically to a unique ubiquitin ligase, E3 enzyme; (4) the transfer of activated ubiquitincouldbedoneviaanadditionalthiol‐esterintermediateonE3enzyme;(5)successiveconjugationofubiquitin to one another generates a poly‐ubiquitin chain; (6) poly‐ubiquitin chain serves as thebindinganddegradationsignal for thedownstream26Sproteasome, theproteinsubstrate is thendegraded into short peptides; (7) free and reusable ubiquitin is released by de‐ubiquitinatingenzymes(DUBs)forfutureuse.ReprintedbypermissionfromMacmillanPublishersLtd:[RambamMaimonidesMedicalJournal],copyright(Ciechanover2010;Ciechanover2012).
Poly‐ubiquitylation marks the target protein for transportation to a barrel‐shaped
organelle, 26S proteasome which consists of a 20S central complex and two 19S lid
complexes.The19Scomplexescontroltheentrybyremovingthepoly‐ubiquitinchain
andunfoldingthetargetproteinbeforeenteringthe20Scomplexthroughthenarrow
catalyticpore(Nandietal.2006;Korolchuketal.2010;Sakumaetal.2015).Onceinside
the 20S complex, the proteins are exposed to trypsin‐, chymotrypsin‐ and peptidyl‐
glutamylpeptide‐hydrolyzing‐likeactivitiesoftheproteasome(Heinemeyeretal.1997;
Korolchuk et al. 2010).After thewholeUPSprocess, short peptidesderived from the
targetproteinandreusableubiquitinarethenreleased(Glickmanetal.2002).
14
Lysosomal‐autophagy (LA) system
In addition to the UPS, the degradation ofmost long‐lived proteins,macromolecules,
biological membranes, and whole organelles, including the mitochondria, ribosomes,
the endoplasmic reticulum, and peroxisomes also occurs by autophagy which is
associated with lysosomes (Sakuma et al. 2015). The various hydrolytic enzymes in
lysosome areworking optimally at an acidic environment, therefore, the lysosome is
surrounded by a membrane to protect cellular contents from enzymatic actions
(Ciechanover2010).
The digest action of lysosome is dynamic and it targets substrates specifically in
numerouswayswhichinclude:(1)receptor‐mediatedendocytosisandpinocytosis;(2)
phagocytosis also known as herterophagy; and (3) microautophagy and
macroautophagyinthelysosomallumen(Fig.1.9)(Mortimoreetal.1987;Ciechanover
2005; Ciechanover 2010). For example, mitochondria, endoplasmic reticulum (ER)
membranes, glycogenbodies andother cytoplasmic entities are degradedby lysosme
under extreme conditions bymacroautophagy (Ashford et al. 1962; Mortimore et al.
1987;Ciechanover2010).
Figure1.9LysosomaldigestionReprinted by permission fromMacmillan Publishers Ltd: [RambamMaimonidesMedical Journal],copyright(Ciechanover2010;Ciechanover2012).
15
1.4.3 Protein synthesis
Initiation
Translation is a cyclicalprocess (Fig.1.10)and ribosomal subunits thatparticipate in
initiation are recycled from post‐termination ribosomal complexes (post‐TCs) that
consistofan80SribosomeboundtomRNA,aP‐sitedeacylatedtRNAandat leastone
eukaryoticreleasefactor1(eRF1).Thesepost‐TCsaredisassembledbyreleasingthese
factors and dissociating the ribosomes into subunits before the process of initiation
(Jacksonetal.2010).
Tostartinitiation,eukaryoticinitiationfactors(eIFs)(Table1.5)suchaseIF2withthe
anticodon loop of an initiator tRNA (Met‐ tRNAMeti) and recycled 40S ribosomal unit
fromdisassembledpost‐TCsareattached together to form43Spre‐initiation complex
for the attachment tomRNA (Unbehaun et al. 2004; Fraser et al. 2007; Jackson et al.
2010).However,beforethisattachment,thesecondarystructureofmRNAneedstobe
sufficiently unwound to allow the loading of the 43S pre‐initiation complex. This
processrequires theworkofeIF4Fcomplex (consistsofeIF4E,eIF4GandeIF4A)and
eIF4Btounwindthe5’cap‐proximalregionofmRNAinanATP‐dependentmannerto
prepareit forribosomalattachment.Oncethe43Spre‐initiationcomplexbindstothis
unwoundmRNA,itstartsthescanningfortheinitiationcodonwhichisusuallythefirst
AUGtriplet(—GCC(A/G)CCAUGG—,withapurineatthe‐3andaGatthe+4position)
from5’ to3’direction(Kozak1991;Pestovaetal.2002; Jacksonetal.2010).The48S
pre‐initiationcomplexisthenformedafterthebindingofinitiatortRNA(Met‐tRNAMeti)
totheinitiationcodon.The60Sribosomalunitthebindstothis48Scomplexwiththe
release of the rest initiation factors to form 80S initiation complex to achieve next
progressoftranslation,theelongationofthepeptide(Pisarevetal.2006;Yuetal.2009;
Jacksonetal.2010).
16
Table1.5EukaryoticinitiationfactorsReprintedbypermissionfromMacmillanPublishersLtd:[NatureReviewsMolecularCellBiology](3971781259397),copyright(Jacksonetal.2010)
Name FunctioneIF2 FormsaneIF2‐GTP‐Met‐tRNAMeti ternarycomplexthatbindstothe40Ssubunit,thus
mediatingribosomalrecruitmentofMet‐tRNAMetieIF3 Binds40Ssubunits,eIF1,eIF4GandeIF5;promotesattachmentof43Scomplexesto
mRNAandsubsequentscanning;andpossessesribosomedissociationandanti‐associationactivities,preventingjoiningof40Sand60Ssubunits
eIF1 Ensuresthefidelityofinitiationcodonselection;promotesribosomalscanning;stimulatesbindingofeIF2‐GTP‐Met‐tRNAMetito40Ssubunits
eIF1A StimulatesbindingofeIF2‐GTP‐Met‐ tRNAMeti to40SsubunitsandcooperateswitheIF1inpromotingribosomalscanningandinitiationcodonselection
eIF4E Bindstothe5’cap‐proximalregionofmRNAeIF4A(1) DEAD‐boxATPaseandATP‐dependentRNAhelicaseeIF4G(2) BindseIF4E,eIF4A,eIF3,PABP,andmRNAandenhancesthehelicaseactivityofelF4AeIF4F Acap‐bindingcomplex,comprisingeIF4E,eIF4AandeIF4G;unwindsthe5′
cap‐proximalregionofmRNAandmediatestheattachmentof43Scomplexestoit;andassistsribosomalcomplexesduringscanning
eIF4B AnRNA‐bindingproteinthatenhancesthehelicaseactivityofeIF4AeIF4H AnRNA‐bindingproteinthatenhancesthehelicaseactivityofeIF4Aandishomologous
toafragmentofeIF4BeIF5 AGTPase‐activatingprotein,specificforGTP‐boundeIF2,thatinduceshydrolysisof
eIF2‐boundGTPonrecognitionoftheinitiationcodoneIF5B Aribosome‐dependentGTPasethatmediatesribosomalsubunitjoiningeIF2B AguanosinenucleotideexchangefactorthatpromotesGDP–GTPexchangeoneIF2
(1) Two paralogues (eIF4AI and eIF4AII), encoded by different genes, are functionally indistinguishable, but eIF4AIII has no activity as an eIF. (2) Two paralogues (eIF4GI and eIF4GII), encoded by different genes, are functionally similar but show some selectivity towards different mRNAs. eIF4GI is generally the more abundant.
17
Figure1.10Cyclicalprocessoftranslation(1)Recyclingofpost‐terminationcomplexes(post‐TCs)toseparate40Sand60Sribosomalsubunits;(2) formation of eIF2‐GTP‐Met‐tRNAMeti; (3) formation of 43S preinitiation complex with a 40Sribosomalsubunit,eIF1,eIF1A,eIF3,eIF2‐GTP‐Met‐tRNAMeti,andeIF5;(4)activationofmRNAbytheATP‐dependentmannerofeIF4FandeIF4B;(5)attachmentof43SpreinitiationcomplextomRNAregion;(6)scanningofthestartcodon(AUG);(7)recognitionoftheinitiationcodon;(8)joiningof60Sribosomalsubunitto48ScomplexandconcomitantdisplacementofeIF2‐GDPandotherfactors(eIF1,eIF3,eIF4B,eIF4F,andeIF5)mediatedbyeIF5B;(9)releaseofeIF1AwitheIF5Bfollowedbythe assembly of elongation‐competent 80S ribosome. Reprinted by permission from MacmillanPublishersLtd:[NatureReviewsMolecularCellBiology](3971781259397),copyright(Jacksonetal.2010).
(2)(1)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
18
Elongation
The peptide elongation process (Fig. 1.11) is highly conserved across eukaryotes,
prokaryotesandthearchaea(Spahnetal.2001;Ramakrishnan2002;Kappetal.2004).
Tostartthisprocess,apeptidyltRNAsitsintheribosomalP‐siteandanaminoacyltRNA
isbrought to the ribosomalA‐siteasa ternarycomplexwith theelongation factor1A
(eEF1A; EF‐Tu in bacteria) and GTP. tRNA, the anticodon, corresponds to the three
basesof thecodononthemRNA.Whencorrectcodon‐anticodonpairingoccurs, three
basesofrRNAinthe40Sribosomalsubunitinducetheswingoutandinteractionwith
the resulting mRNA‐tRNA duplex to stabilize the tRNA binding and prevent other
aminoacyl tRNA binding via hydrolysis of GTP by eEF1A‐GTPase (Ogle et al. 2001;
Rodninaetal.2001;Ramakrishnan2002;Kappetal.2004).TheaminoacyltRNAinthe
A‐site then swings to the peptidyl transferase site to formpeptide bond in a process
calledaccommodationafterthereleaseofeEF1A‐GDP(Ramakrishnan2002;Kappetal.
2004).
The P‐site tRNA is then deacylated and the peptide chain is transferred to the A‐site
tRNAtoformpeptidechain.TheA‐sitetRNAwithpeptidechainisthentranslocatedto
P‐siteviahydrolysisofGTPbyfactor2(eEF2;EF‐Ginbacteria).Thiscycleisrepeated
untilastopcodonisencounteredandterminationbegins(Ramakrishnan2002;Moore
etal.2003;Kappetal.2004;Peskeetal.2004).
19
Figure1.11TranslationelongationinbacteriaTheprocessofelongationishighlyconservedacrossthethreekingdomsoflife.Thisdiagramshowstheprocessofelongation inbacteriawhich issimilartoeukaryotes.ReprintedbypermissionfromMacmillanPublishersLtd:[Cell](3971790172892),copyright(Ramakrishnan2002).
Termination
Terminationoftranslation(Fig.1.12)occursinresponsetoastopcodon(5’‐UAG‐3’,5’‐
UGA‐3’,or5’‐UAA‐3’)intheribosomalA‐site(Bertrametal.2001;Ramakrishnan2002;
Kappetal.2004).UnlikeothercodonswhichisrecognizedbyaminoacyltRNA,thethree
stop codons are recognized by eukaryotic release factors (eRFs). In eukaryotes, two
releasefactors(eRF1andeRF3)functionasaterminationcomplex(Zhouravlevaetal.
1995;Keelingetal.2011).TheeRF1recognizesandbindstoallthreestopcodonsinthe
ribosomalA‐siteandmediatesthereleaseofthenascentpolypeptidefromtheribosome
(Songetal.2000;Bertrametal.2001;Kappetal.2004;Keelingetal.2011).TheeRF3
actsasaGTPasetoassisteRF1instopcodonrecognitionandreleaseofthepolypeptide
(Bertrametal.2001;Salas‐Marcoetal.2004;Keelingetal.2011).
TerminationendswithreleaseofthecompletedpolypeptidefromtheP‐sitetRNAwhich
isbelievedto involvepeptidyl transferaseat thecentreof theribosome(Caskeyetal.
1971;Arkovetal.1998;Seit‐Nebietal.2001;Zavialovetal.2002;Kappetal.2004).
20
Figure1.12TranslationterminationReprinted by permission fromMacmillan Publishers Ltd: [WILEY INTERDISCIPLINARY REVIEWS:RNA](3971790698161),copyright(Keelingetal.2011)
1.5 Signalling pathway
1.5.1 Overview
Duringtranslationandtheotherprocessesinvolvedinproteinproduction,asubstantial
amount of energy and cellular material is consumed. For these reasons, mammalian
cellshaveevolvedelaboratemechanismstoregulatetranslationinresponsetovarious
stimulithatindicatesdown‐regulationisrequitedforcellsurvival.Thesestimuliinclude
changes in nutrient availability, cellular energy, stress, hormones and growth factors
(Fig.1.13)(Maetal.2009).
Figure1.13InhibitionoftranslationunderdifferenttypesofstressReprinted by permission from Macmillan Publishers Ltd: [Molecular Cell] (3971790974348),copyright(Spriggsetal.2010)
21
Inskeletalmuscle,arangeofextracellularanabolicorcatabolicstimulusareinvolvedin
dynamicregulationofproductioninmusclefibres,andthisregulationoccursprimarily
attheinitiationoftranslation(Syntichakietal.2006;Maetal.2009;Tisdale2009).The
initiation factors eIF4E and eIF2B, in particular, from the mTOR1/4EBP1 and
PERK/eIF2αsignallingpathways,playimportantrolesinthisregulation.
1.5.2 mTOR/4EBP1 pathway
In eukaryotes, the eIF4F complex (comprised of eIF4E, eIF4G and eIF4A) plays a key
role in initiation.TheeIF4Esubunit, inparticular, isoneof themainregulatorsof the
assembly of the eIF4F complex (Duncan et al. 1987; Powers et al. 2011) and is
controlled by its reversible association with the 4E‐binding proteins such as 4EBP1
(Kimballetal.2006;Spriggsetal.2010;Powersetal.2011).The4EBP1canblockeIF4F
assemblythoughcompetitionwitheIF4GforeIF4Ebinding(Kimballetal.2006;Powers
et al. 2011). When 4EBP1 is phosphorylated via the mTOR pathway, 4EBP1 is
dissociatedfromeIF4Etoallowtranslationtoproceed(Phametal.2000;Kimballetal.
2006; Powers et al. 2011). Conversely, dephosphorylation of 4EBP1 by a protein
phosphataseresultsinincreasedassociationof4EBP1witheIF4Eandinhibitionofthe
formation of the eIF4F complex (Fig. 1.14) which leads to a decrease in translation
(Phametal.2000;Powersetal.2011).
Figure1.14Regulatingcap‐dependenttranslationinitiationTherecruitmentof the40Sribosomalsubunit to the5′endofmRNA isacrucialandrate‐limitingstep during cap‐dependent translation. A number of translation initiation factors, including the 5′cap‐bindingprotein eukaryotic translation initiation factor 4E (eIF4E), have essential roles in thisprocess. Hypophosphorylated 4E‐BPs bind tightly to eIF4E, thereby prevents its interaction witheIF4Gandthusinhibitsproteinsynthesis.ThemTORC1‐mediatedphosphorylationof4e‐BPsreleasethe 4E‐BPs from eIF4E, resulting in the recruitment of eIF4G to the 5′ cap, and thereby allowingtranslation initiation to proceed. Reprinted by permission fromMacmillan Publishers Ltd: [NatureReviewsMolecularCellBiology](3971791370596),copyright(Maetal.2009).
22
MammalianTOR(mTOR)existsintwodistinctcomplexescalledcomplex1(mTORC1)
and complex 2 (mTORC2) (Guertin et al. 2007). mTOR responds to various stresses
including genotoxic, nutrient, energy and oxidative stress (Sengupta et al. 2010) and
playsacriticalroleindiabetesandageing(Zoncuetal.2011).Studieshaveshownthe
level of protein synthesis decreases in old age due to decreased phosphorylation of
4EBP1(Fig.1.15)(Drummondetal.2008).IthasalsobeenshownthatinsulinandIGF‐1
activationofthePI3K/Akt/mTORpathwayleadstoanincreaseinproteinsynthesisand
adecrease inproteindegradation resulting inhypertrophyof themuscle (Palusetal.
2014).
Figure1.15mTORC1pathwayandageingmTOR regulated the process of ageing via different factors. With a depression in translation viaeIF4E/4EBP1 pathway, ageing was then generated. Reprinted by permission from MacmillanPublishersLtd:[Aging(AlbanyNY)],copyright(Handsetal.2009).
Many studies suggest that oxidants depress protein synthesis by decreasing
phosphorylationof4EBP1,therebyinhibitinginitiationoftranslation(Fig.1.16)(Pham
et al. 2000; Shenton et al. 2006; Zhang et al. 2009; Powers et al. 2011). Hydrogen
peroxidehasbeenfoundtostimulatedephosphorylationof4EBP1byincreasingprotein
phosphatase (PP1/PP2A) activity and resulting in an increase in the association
23
between 4EBP1 with eIF4E, and a decrease in protein synthesis (Pham et al. 2000;
Powersetal.2011).
Figure1.16EffectsofROSonmTOR/4EBP1pathwayAdaptedfrom(Powersetal.2011).
1.5.3 PERK/eIF2α pathway
eIF2α is assumed to be anothermechanism involved in the regulation of translation
inanition by phosphorylation (Spriggs et al. 2010). eIF2 consists of three subunits
(α,β,γ) and is one of the key initiation factors that carries the initiator tRNA (Met‐
tRNAMeti) with GTP to form the 43S pre‐initiation complex. During the process of
initiation,eIF2istransformedfromtheGTPformtoaGDPformbutitcanberecycled
for the next translation process by eIF2B to progress the GTP‑exchange reaction.
However,phosphorylationofeIF2α atresidueSer51preventsthisreactionbyinhibiting
the dissociation of eIF2 from eIF2B (Deng et al. 2002;DangDo et al. 2009;Ma et al.
2009;Powleyetal.2009;Spriggsetal.2010).
24
TherearefourkinasesinvolvedinthephosphorylationofeIF2αinresponsetoarange
of external stresses. These includeGCN2, PERK,HRI, andPKR (Fig. 1.13) (Deng et al.
2002;Hardingetal.2003;Cullinanetal.2006;DangDoetal.2009;Spriggsetal.2010;
Emara et al. 2012). In the mice that bear the cachexia‐inducing MAC 16 tumour,
phosphorylationofeIF2αandPKRhavebeenshowntoincreasewithoutchangesinthe
amountof eIF2α andPKR.Thesemicealso showadecrease inweightandmyosinas
phosphorylationof eIF2α increases (Eley et al. 2007).Thishas alsobeenobserved in
vitrostudiesofMCF7andMCF12Acells(Kimetal.2000).
The PERK/eIF2α pathway is also involved in the responds of endoplasmic reticulum
(ER)tostress.Tomaintainhomeostasisineukaryoticcells,ERsensesandresponsesto
cellular stresses in a range of ways including the unfolded protein response (UPR)
(Schroderetal.2005;Cullinanetal.2006;Backetal.2009;Changetal.2010).TheUPR
is reduces ER stress by clearing misfiled proteins in the ER though PERK/eIF2α
pathway(Hardingetal.2001;Ozcanetal.2004;Cullinanetal.2006;Liangetal.2006;
Shentonetal.2006;Rutkowskietal.2007;Scheuneretal.2008;Backetal.2009).This
pathway leads to a reduction in protein synthesis, which reduces protein folding
demands and allows for the clearance of misfolded proteins (Cullinan et al. 2006;
Rutkowskietal.2007;Backetal.2009).
Recentevidencesuggests that there isacloserelationshipbetweenERstress,protein
misfolding, and oxidative stress (Fig. 1.17). In this relationship, ROS leads to the
accumulationofmisfoldedproteinsintheER,creatingacycleofERstressandoxidative
stress(Teraietal.2005;Scheuneretal.2008;Backetal.2009;Changetal.2010).As
thePERK/eIF2αpathwayhasbeenshowntoplayarole in theclearanceofmisfolded
proteins, the PERK/eIF2α pathway is also likely to be involved in the response to
oxidativestress.
25
Figure1.17TherelationshipbetweenERstressandROSProtein folding within the ER lumen was ushered by a family of oxidoreductase that catalyzeddisulfidebondformationandisomerization.UnderERstress,therewasanincreaseintheformationof incorrect intermolecular and/or intramolecular disulfide bonds that leaded to the formation ofROS. Inturn,ROScouldalsocauseERstressthroughmodificationofproteinsand lipidsthatwerenecessary to maintain ER homeostasis. Reprinted by permission from Macmillan Publishers Ltd:[EndocrineReviews](3971800940503),copyright(Scheuneretal.2008).
26
Aim
An increase in oxidative stress has been seen to occur alongsidemusclewasting and
changes in protein turnover in various conditions and disease such as cancer, type 2
diabetes,chronic inflammationandageing(Sohaletal.1996;Klaunigetal.1998;Wei
1998;Finkeletal.2000;Atalayetal.2002;Evansetal.2002;Weietal.2002;Maritimet
al.2003;Robertson2004;Khaletal.2005;Phillipsetal.2005;Roloetal.2006;Valkoet
al.2006;Parketal.2007;Thomas2007;Chenetal.2008;Bonettoetal.2009;Parketal.
2009;Evans2010;Reuteretal.2010;Terrilletal.2013).Adecreaseinoxidativestress
with antioxidant has also been shown to improve muscle pathology and decrease
necrosis(Terrilletal.2013).Thissuggeststhatoxidativestressmayimpactonmuscle
wasting.Whilethesignallingpathwayinvolvedinproteinsynthesisandoxidativestress
isnotyetclear,themTOR/4EBP1andPERK/eIF2αpathwaysareconsideredthemost
likelypathwaysaffectedbyoxidativestress.
This study uses the skeletal muscle culture system of C2C12 myotubes to study the
effectsofhydrogenperoxidemediatedoxidativestress,specificallytheeffectofcatalase
andglucoseoxidaseon(i)totalproteinlevels,(ii)proteinsynthesislevelsandprotein
degradation rates, (iii) phosphorylation rates of 4EBP1 and eIF2α, and (iv) thiol
oxidation of whole protein, actin, and myosin. As direct application of hydrogen
peroxideintothemediumwouldlikelybecytotoxic(Halliwelletal.2000),catalaseand
glucoseoxidasewereapplied to theC2C12 culturemedia.Glucoseoxidase isknown to
generateendogenoushydrogenperoxidecontinuously(Boverisetal.1972;Gruneetal.
1995;Gruneetal.1997).
27
Chapter 2 Material and Methods
2.1 Cell Culture
Inthisstudy,immortalizedcultureswereusedasinvitromodelsforinvivomyofibres.
The source of the cell cultures and their preparation are described below. The C2C12
culture techniques developed in our laboratory were adapted in this study (Gebski
2009).Inthisstudy,allmyotubesweretreatedunderserum‐starvedconditiontoavoid
theanypossibleeffectofenzymesintheserum.
2.1.1 Proliferation
C2C12mousemyoblastsoriginatedfromthethighmuscleofC3Hmiceaftercrushinjury
(Yaffe et al. 1977). This cell line was purchased from the American Type Culture
Collection (ATCC, Manassas, USA). In this study, all experiments were performed on
cellsatpassage4.
TheC2C12myoblastswerestoredascryopreservedstocksinliquidnitrogen.Theywere
frozen at a concentrationof approximately0.5‐1.5× 106 cells/ml in freezingmedium
consisting of Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen, 11965‐118)
supplementedwith1%(v/v)ofpenicillin/streptomycin(Invitrogen,15070‐063),20%
Fetal Bovine Serum (FBS; Invitrogen, 16000044) and 10% (v/v) dimethyl sulfoxide
(DMSO;Sigma,D2650).
Cellswerethawedundersterileconditionsinalaminarflowhoodbyadding1mlofpre‐
warmed(37°C)proliferationmediumconsistingofDMEMsupplementedwith1%(v/v)
ofpenicillin/streptomycinand20%ofFBS.Thecellswerethawedslowlybyaspirating
with1mlofproliferationmedium.Itwasthentransferredtoafalcontubewith5mlof
proliferation medium. The cells were centrifuged at 1500 rpm for 5 min at room
temperature in a centrifuge (Eppendorf , 5702). The supernatantwas then discarded
andthecellpelletwasresuspendedin1mlofproliferationmediumandtransferredtoa
T‐75cm2flask(Falcon)with10mlofproliferationmedium.Thecellswereincubatedat
37°Cin5%CO2and95%air.
28
2.1.2 Trypsinization and seeding of myoblasts
Themyoblaststookapproximately5‐7daystoreach80‐90%confluenceinculture.For
seeding, theproliferationmediumwas firstremovedandthecellswerewashedtwice
with 5 ml phosphate‐buffered saline (PBS, Medicago, 09‐8912‐100) then 7 ml of
Trypsin/EDTAsolution(Sigma,T4049)wasaddedtotheflask.Afterincubationat37°C
for5min,celldetachmentwasconfirmedbymicroscopicexaminationoftheflask.The
trypsin reactionwas stopped by the applying 5ml of proliferationmedium. The cell
suspensionwasthentransferredtoa15mlfalcontubeandcentrifugedat1500rpmfor
5 min at room temperature. The supernatant was discarded and the cell pellet was
resuspended in1mlofproliferationmedium.10µlof cell suspensionwasdiluted20‐
timeswithTrypanBluesolution(Sigma,T8154) ina0.6mlmicrocentrifuge tubeand
transferred to a haemocytometer (Brand, 717805) to determine the total cell count
underthelightmicroscope.Fourquadrants(Fig.2.1)werecountedseparatelyandthe
totalcountwasaveraged.
Figure2.1HaemocytometerCellswerecounted inthefour largesquares(A,B,C,andD),whicharefurthersubdivided into16smallersquares.Theaveragenumberofcellswastakenandmultipliedby1×104andtheTrypanbluedilutionfactor(20).
29
Theconcentrationofthecellsuspensionwascalculatedusingthemultiplicationfactor
(104)derivedfromthevolumeofeachofthefourcountedsquares(0.1mmdeepand1
× 1mm square)which equates to a volume of 0.1mm3 or 0.0001ml (10‐4ml). The
formulausedtodeterminethecellcontentrationwas:
Thecell suspensionwas thendiluted inproliferationmediumtoa final concentration
dependingontheintendeduseofthecellsandthesizeoftheculturedishtobeseeded.
2.1.4 Treatment conditions
Catalaseandglucoseoxidasewereusedtomodulatethelevelofhydrogenperoxidein
themyotubes.Catalase is ananti‐oxidase that reduces the levelofhydrogenperoxide
andglucoseoxidaseisanoxidasethatincreasesthelevelofhydrogenperoxide.IGFand
dexamethasone (DEX) were used as controls in the experiments focused on protein
turnover.
Alltreatmentsstartedatday7offusion.Catalase(Sigma‐Aldrich,C3155)wasappliedat
3000units/ml, glucose oxidase (Sigma‐Aldrich, G7141)was applied at 10munits/ml,
IGF (Sigma‐Aldrich, Australia) was applied at 20 ng/ml, and DEX (Sigma‐Aldrich,
Australia)wasapplied.All treatmentswererefreshedevery24hruntil thecellswere
harvested.
2.2 Protein extraction
Proteins frommyotubes were extracted using either TCA acetone or a phospho‐safe
methoddependingontheintendeduseofthecells.
2.2.1 TCA acetone extraction
Followingtreatment(Section2.1.4),myotubecultureswerewashedtwicewithPBSand
lysedwith 20% (w/v) trichloroacetic acid (TCA) in acetone to denature, precipitate
proteinsandprevent furtherredoxreactions(Armstrongetal.2011).Asobservedby
30
Armstrong and coworkers (2011), application of the TCA turned the protein pellet
whitebutnotthesupernatantconfirmingtheproteinswerepresentinthepelletnotthe
supernatant.
Afterwashing,700µlof20%TCA/acetonewasaddedtoeachdishandthemyotubes
were detached using a cell scraper (SARSTEDT, 831830). The supernatant and the
resultingpelletthatcontainthemyotubesweretransferredtoa1.5mlmicrocentrifuge
tube and then centrifuged at 10000rpm for 5 min (4°C) in a centrifuge (Eppendorf,
5417R).ToremovetheremainingTCAinthesamples,thesupernatantwasdischarged
andtheproteinpelletswerewashedtwicewith1mlofacetone.Theproteinpelletwas
thenresuspendedin300µlofTrisbuffer(50mMTriswith0.5%SDS).
Tocompletelyresuspendtheproteins,thesamplewassonicatedat40%ampfor2min
onice,followingby30minofvortexing.Thesamplewasthentransferredtoanew1.5
mlmicrocentrifugetubeforproteinquantification(Section2.3.2).
2.2.2 Phospho‐safe extraction
Forwesternblotting,theproteinsfrommyotubeswereextractedusingaphospho‐safe
method.Followingtreatment(Section2.1.4),myotubecultureswerewashedtwicewith
PBS and lysed with a phospho‐safe extraction cocktail consisting of 2.5 ml of
PhosphoSafeExtractionBuffer(Novagen,71296)andaquarterofaproteaseinhibitor
tablet(Roche,04693159001).
After washing with 1 ml of PBS (4°C) twice, 100 µl of cold phospho‐safe extraction
cocktailwasaddedintoeachdishandmyotubesweredetachedusingacellscraper.The
supernatantandtheresultingpelletthatcontainthemyotubesweretransferredtoa1.5
ml microcentrifuge tube and incubated on ice for 20 min. Halfway through the
incubation,thesamplewasvortexedforapproximately5sec.Aftertheincubation,the
samplewasvortexedagainfollowedbycentrifugationat12000gfor10min(4°C)ina
centrifuge (Eppendorf, 5417R). 97 µl of supernatant was transferred to a 0.6 ml
microcentrifuge tube forwesternblottingandremainingsupernatantwas transferred
toanother0.6mlmicrocentrifugetubeforproteinquantification(Section2.3.1).
31
2.3 Protein quantification
Theproteinsampleswerequantifiedbydifferentmethodsdependingonthe intended
useofcells.
2.3.1 Bradford assay
Forwesternblotting, theproteinsampleswerequantifiedusing theBradfordmethod
(Bradford 1976). Following harvesting (Section 2.2.2), the Bio‐Rad Protein Assay Kit
(Bio‐Rad, 500‐0001)was used to quantify the concentration of each sample. Protein
samples were quantified with reference to a Bovine serum albumin (BSA) standard
absorbancecurve.BSAstandardsofknownconcentrations(0,100,200,300,400,and
500µg/ml)preparedbyserialdilutionofBSA(stockconcentration1mg/ml)in0.01M
PBS.
Theproteinsampleswere firstdiluted20‐foldwith0.01MPBSand then10µlof the
dilutedproteinsampleandstandardsweretransferredintriplicatetothewellsofa96‐
well plate. 200 µl of Bio‐Rad reagent was then added to each well. The plate was
incubatedfor10minwithgentleshakingonashakeratroomtemperatureandatthe
endofthisincubationperiod,theabsorbanceofeachwellwasmeasuredat595nmina
platereader(BioTekPowerwaveXSSpectrophotometerwithKC4ver.3.4program).The
concentrationoftheproteinsampleswasextrapolatedfromtheBSAstandardcurve.
2.3.2 Micro BCA assay
ThemicroBCAassaykit(Sigma,QPBCA‐1KT)wasusedtoaccesstotalproteincontent.
Afterharvestingandresuspension(Section2.2.1),proteinsampleswerequantifiedwith
referencetoaBovineSerumAlbumin(BSA)standardabsorbancecurve.BSAstandards
ofknownconcentrations(0,5,10,20,30,and40µg/ml)preparedbyserialdilutionof
BSA (stock concentration 40µg/ml) in Tris buffer (2mM Triswith 0.5% SDS). The
workingreagentwascomposedwith25partsofreagentA,25partsofreagentB,and1
partofreagentC.
1µlofproteinsamplewasdiluted25‐foldwith0.5%SDSandafurther10‐folddilution
withTrisbuffer(2mMTriswith0.5%SDS)ina1.5mlmicrocentrifugetube.Thiswas
32
followed by adding 250 µl of working reagent added into each tube. 200 µl of each
standardwastransferredto1.5mlmicrocentrifugetubesand200µlofworkingreagent
was then added into each tube. Thesemicrocentrifuge tubeswere vortexed and then
incubatedfor1hratat60°C.Themicrocentrifugetubeswerevortexedagainand100
µlofincubatedsolutionwasthenaliquotedintriplicatetoeachwellofa384‐wellplate.
Theabsorbanceofeachwellwasmeasuredat562nmintheBioTekplatereader.
2.3.3 Detergent compatible (DC) protein assay
For 2‐tag labeling, the protein samples were quantified using the DC assay method.
After dual‐labeling (Section 2.7.2), a DC assay kit (Bio‐Rad, 500‐0112) was used to
quantify the concentrations of each sample. Protein samples were quantified with
referencetoaBovineSerumAlbumin(BSA)standardabsorbancecurve.BSAstandards
ofknownconcentrations(0,0.1,0.2,0.3,0.4,0.6,0.8,and1mg/ml)preparedbyserial
dilutionofBSA(stockconcentration1mg/ml)inassaybufferconsistingofTrisbuffer
(0.5MTriswith0.5%SDS)diluted1:1withdistilleddoubledeionized(ddi)water.
Beforeperformingthequantification,thereagentA’wasmadewith1mlofreagentA,
20µlof reagentC, and1.02mlofddiwater.7.5µlofproteinsamplewasdiluted1:1
withTrisbuffer(0.5MTriswith0.5%SDS)andfollowedwithanother1:1dilutionwith
ddiwaterina1.5mlmicrocentrifugetube.30µlofeachstandardwastransferredtoa
1.5mlmicrocentrifuge tube. 105 µl of reagent A’ and 255 µl of reagent Bwere then
added to each microcentrifuge tube. The tubes were vortexed for 5 min and then
incubated for 10 min at room temperature. 100 µl of incubated solution was then
aliquotedintriplicatetoeachwellofa384‐wellplate.Theabsorbanceofeachwellwas
measuredat750nmintheBioTekplatereader.
2.4 Measurement of protein synthesis
Thelevelofproteinsynthesisinmyotubeswasmeasuredusingradioactiveleucine.
2.4.1 Incorporation of radioactive leucine
7‐day‐oldC2C12myotubesweretreatedwithtreatments(catalase,glucoseoxidaseand
IGF)for24hrunderserum‐starvedconditions.Thefollowingday,1µCiofradioactive
33
leucine (leucine,L‐[3,4,5‐3H(N)],PerKinElmer,NET‐460)with treatmentswasapplied
tothesemyotubesfor24hr.
2.4.2 Harvest
After radioactive leucine incorporation and treatment, themyotubeswere harvested.
Prior to harvesting, the medium was removed from each dish and transferred to a
centrifugetube for laterdeterminationofradioactivity.700µlofTCA/acetone(20%)
was added to each dish and myotubes were detached using a cell scraper. The
supernatantandtheresultingpelletthatcontainthemyotubesweretransferredtoa1.5
mlmicrocentrifuge tube centrifuged at 10000 gat room temperature in a centrifuge
(Eppendorf,5415C).Thesupernatantwasdiscardedwithoutdisturbingthepelletand
thepelletwasthenwashedtwicewith500µlofleucinesolution(1mML‐leucinein0.6
M HClO4, Sigma, L8000). The samplewas then resuspended in 300 µl of NaOH (300
mM).Thesamplewasheatedto40°Candvortexeduntilfullysolubilized.
2.4.3 Radiation analysis
100µlofsupernatantandproteinsampleweretransferredintotubescontaining2mlof
scintillation cocktail solution and vortexed for 5 sec. The radioactivity was then
measured in a scintillation counter.The incorporation rateof radioactive leucinewas
obtainedasfollows:
The amount of incorporated radioactive leucine was then obtained by timing the
incorporationrateofradioactiveleucineasfollows:
The amount of non‐radioactive leucine in DMEM was obtained from supplier. The
concentrationofL‐leucineinDMEMwas0.802mMand2mlofDMEMwasusedineach
dish.Therefore,theamountofnon‐radioactiveleucineineachdishwas1.604µmol.The
34
ratio of radioactive leucine in total leucine could thenbe calculatedwith the valueof
non‐radioactiveleucineandradioactiveleucineasfollows:
The ratio of radioactive leucine was then used to covert the amount of radioactive
leucineincorporationtototalleucineincorporationasfollows:
2.5 Measurements of protein degradation
Therateofproteindegradationinmyotubeswasmeasuredusingradioactiveleucine.
2.5.1 Incorporation of radioactive leucine
Radioactivelecuine(1µCi)wasappliedtothe6‐day‐oldC2C12myotubesunderserum‐
containedconditions.Themediumwasremovedthenextdayandtreatments(catalase,
glucoseoxidase, IGF,andDEX)wereappliedtothese labeledmyotubes for48hr.The
treatmentwasrefreshedevery24hrandtheradioactivity levels inthespentmedium
weredetermined.
2.5.2 Harvest
After radioactive leucine incorporation and treatment, themyotubeswere harvested.
Prior to harvesting, the medium was removed from each dish and transferred to a
centrifugetube forradioactivityanalysis.700µlofTCA/acetone(20%)wasaddedto
eachdishandmyotubesweredetachedusinga cell scraper.The supernatantand the
resultingpelletthatcontainthemyotubesweretransferredtoa1.5mlmicrocentrifuge
tube centrifugedat10000gat room temperature ina centrifuge (Eppendorf,5415C).
The supernatantwasdiscardedwithout disturbing thepellet and thepelletwas then
35
washed twicewith500µl of leucine solution (1mML‐leucine in0.6MHClO4, Sigma,
L8000).The samplewas then resuspended in300µl ofNaOH (300mM).The sample
washeatedto40°Candvortexeduntilfullysolubilized.
2.5.3 Radiation analysis
100µlofsupernatantandproteinsampleweretransferredintotubescontaining2mlof
scintillation cocktail solution and vortexed for 5 sec. The radioactivity was then
measured in a scintillation counter. The ratio of radioactive leucine release was as
obtainedasfollows:
UnlikeDMEM, the concentration of leucine in serumwasnot able to bemeasured or
obtained. Therefore, the ratio of radioactive leucine releasewas taken as the rate of
proteindegradation.
For data presentation, the rates of protein degradation of all other treatment groups
werenormalizedtotherateofproteindegradationofuntreatedculturesasfollows:
2.6 Western Blot
Western blotting was used to detect the specific proteins and signaling pathways
affected by catalase and glucose oxidase treatment. Two types of western blotting
procedureswereused.TheSodiumdodecylsulfatepolyacrylamidegelelectrophoresis
(SDS‐PAGE) was used to establish themethod. The Bio‐Rad systemwas used for all
subsequentexperiments.
36
2.6.1 SDS Polyacrylamide Gel Electrophoresis (SDS‐PAGE)
After treatment, themyotubeswere harvested using a phospho‐safemethod (Section
2.2.2)andquantifiedusingtheBradfordassay(Section2.3.1).Thesequantifiedproteins
wereseparatedbySDS‐PAGEon12%(resolving)polyacrylamidegelunderdenaturing
conditions. Protein samples were prepared by adding 3× protein loading buffer
(consisting of 0.19M Tris pH6.8, 6% (w/v) SDS, 30% (v/v) glycerol, 0.03%(w/v)
BromophenolBlueand0.3MDTT)totheproteinsamplesandthenheat‐denaturedat
95°Cfor5min.
SDS‐PAGEgelswerepreparedpriortoelectrophoresis.Theresolvinggel(seeTable2.1)
waspouredintotheglassplateassembly,overlaidwithddiwaterandlefttopolymerise
forapproximately20minatroomtemperature.Followingremovaloftheddiwater,the
stacking gel (see Table 2.1) was then poured on top of the set resolving gel and gel
combswereinsertedimmediately.APSandTEMEDwereaddedtobothsolutionsprior
topouring.
Table2.1CompositionofresolvingandstackinggelReagent 12%Resolvinggel 4%Stackinggel
1.5MTris(pH8.8) 7.5ml
0.5MTris(pH6.8) 3.75ml
10%SDS 300µl 150µl
30%Acrylamide/Bissolution,37:5:1 12ml 1.95ml
ddiwater 9.87ml 8.25ml
10%APS 300µl 75µl
TEMED 30µl 15µl
Oncethestackinggelhadpolymerized,thegelcastingchamberwasthentransferredto
ageltankcontainingcold1×electrodebufferconsistingof6.06gTris,28.83gGlycine,
2gSDSandmadeup to1Lwithddiwater.Thegelcombswereremovedandall the
remainingun‐polymerizedpolyacrylamidewasflushedoutof thewellswithelectrode
buffer. Prepared protein samples and 5 µl of Precision Plus Protein™ Kaleidoscope
Standards (Bio‐Rad, 161‐0375) were loaded in the wells and the gel was
electrophoresedforapproximately2hrat120Vat4°C.
37
While the gel was electrophoresing, polyvinylidene difluoride (PVDF) membrane
(Amersham)wassoakedinmethanolfor5sec.Thesoakedmembranewithfilterpapers
and spongepadswerepre‐soaked in transferbuffer (consistingof3.03 gTris, 14.4 g
Glycine,100mlMethanolandmadeupto1Lwithddiwater)at4°C.
After electrophoresis, the gel was assembled in the transfer cassette (Fig. 2.2) and
placed in the electroblotting tank filled with cold transfer buffer. The proteins were
transferredfromgeltothePVDFmembraneviaelectrophoresisat100Vfor90minat
roomtemperature.
Figure2.2TheassembleoftransfercassetteThepolyacrylamidegelandmembraneweresandwichedbetweenthepre‐soakedspongesandfilterpapers in transfer buffer. Proteins on the gelwere transferred to themembrane from cathode toanode.
After transfer, thePVDFmembranewasremovedandwashedbriefly inTrisBuffered
Saline(TBSpH7.5;consistingof12.1gTris,9gNaCl,madeupto1Lwithddiwaterand
pH with HCl) with 0.1 % (v/v) Tween‐20 (TBS‐T). The washed membrane was
38
incubated in blocking buffer (5% skimmilk in TBS‐T) for 1 hr at room temperature
withgentleshaking.ThiswasfollowedbywashingwithTBS‐Ttwice,10mineachtime,
and incubated with diluted primary antibody at 4� overnight with gentle shaking.
Primaryantibodies(Table2.2)werediluted1:1000inTBS‐Tcontaining5%BSA.The
previouslyoptimizedprotocolsprovidedbysupplier(CellSignaling)wereadopted.
Table2.2PrimaryantibodiesAntibody CatLog No.
Phospho‐4EBP1(Thr37/46)RabbitmAb 2855
Phospho‐eIF2α(Ser51)XP® RabbitmAb 5199
4EBP1Rabbit 9452
eIF2αRabbit 9722
AktRabbit 9272
After the incubation, themembranewaswashedwithTBS‐T twice,10mineach time,
and incubated with secondary antibody (Thermo, 31460) diluted 1:5000 in TBS‐T
containing 5 % skim milk for 1 hr at room temperature with gentle shaking. The
membrane was washed briefly twice with TBS‐T and then incubated with
chemiluminescent substrate solution (Table 2.3) for 5min at room temperature. The
signalwasexposedtofilmandthefilmwasdevelopedinadarkroom.
Table2.3Chemiluminescencesubstratesolutionusedforproteindetection
Product ProviderProtein
abundanceonmembrane
SuperSignalWestPicoChemiluminescentSubstrate
ThermoScientific
High
WesternLightingUltra PerkinElmer Low
LuminataCrescendoWesternHRPsubstrate
Millipore Medium
2.6.2 Bio‐Rad system
Western Blot analysis using Bio‐Rad system was adopted for all subsequent
experiments.
After treatment, themyotubeswere harvested using a phospho‐safemethod (Section
2.2.2)andquantifiedusingtheBradfordassay(Section2.3.1).Thesequantifiedproteins
samples were separated by precast gradient gels (Bio‐Rad, 456‐1086). The gel was
placed in a gel tank containing 1× electrode buffer (diluted 1 in 10 from 10 × Tris/
39
Glycine/SDS,Bio‐Rad,161‐0772)andthegelcombswereremoved.10µgoftheprotein
sampleand4µlofPrecisionPlusProtein™Kaleidoscope™Standardswerethenloaded
inthewellsandthesamplewereelectrophoresedat150Vforapproximately1.5hrat
roomtemperature.
Protein transferwas carriedoutusing theTrans‐Blot®Turbo™Transfer System (Bio‐
Rad, 170‐4155), which involves semi‐dry protein transfer. The gel was sandwiched
between thenitrocellulosemembrane and filter papers from theTrans‐Blot®Turbo™
Mini Nitrocellulose Transfer Packs (Bio‐Rad) and transfer was performed using the
3minproteintransferprogram(turbosetting).
After transfer, themembranewaswashedwithTBS‐Tandblocked inblockingbuffer.
Afterblotting,themembranewaswashedwithTBS‐Tandincubatedindilutedprimary
antibodyat4°Covernightwithgentle shaking.Themembranewas thenwashedwith
TBS‐Tand incubated insecondaryantibodyfor1hratroomtemperaturewithgentle
shaking.ThemembranewaswashedbrieflytwicewithTBS‐Tandthenincubatedwith
chemiluminescencesubstratesolution(Table2.3)for5minatroomtemperature.The
signalwasdetectedandcapturedbyusingChemiDoc™MPSystem(Bio‐Rad,170‐8280)
andImageLab™SoftwareVersion4.0(Bio‐Rad).
2.6.3 Densitometry analysis
Densitometrywasperformedonthewesternblottingresultimages(Sections2.6.1and
2.6.2) using the NIH Image freeware program Image Processing and Analysis in Java
(Image J) (http://rsb.info.nih.gov/ij/). The phosphorylation level of a specific protein
was expressed as the densitometry ratio of the phosphorylated protein to the total
amountofthatprotein,forexample(phosphorylatedeIF2α/totaleIF2α).TotalAktwas
usedasaloadingcontrol.
2.7 Measurements of thiol oxidation‐2 tag labeling
General oxidation and oxidation of specific proteinswere assessed by 2 tag labeling.
This method was developed in our laboratory for labeling animal protein samples
(Armstrongetal.2011)andwasadaptedheretolabelcellculturesamples.
40
2.7.1 Preparation of protein samples
Three dishes of myotubes from each culture batch were treated with catalase and
glucoseoxidase.Aftertreatment,themyotubeswereharvestedwith20%TCA/acetone
(Section 2.2.1) but only one dish was resuspended in Tris buffer for protein
quantification(Section2.3.2).TheremainingdisheswerekeptinTCA/acetonefor2tag
labeling(duallabeling).
2.7.2 Dual labeling of protein thiols with fluorescent tags
ThemyotubesinTCA/acetoneweresonicatedat40%Ampsfor2minonice.Thiswas
followedbytransferring100µgoftheproteinpellettoa1.5mlmicrocentrifugetube.
The tubes were then centrifuged at 10000rpm for 5 min (4°C) in a centrifuge
(Eppendorf, 5417R). The supernatant was discharged and the protein pellet was
washed with cold acetone (150 µl, 4°C). The centrifugation and washing steps were
repeatedtoremoveanyresidualTCAbeforesuspensionandlabeling.
After removal of TCA, the reduced protein thiols in protein samplewere labeled. To
perform this labeling, theproteinpelletwas suspended in50µl ofTris buffer (0.5M
Triswith0.5%SDS,pH7.3)and5µlof5mMBODIPYFL‐N‐(2‐aminoethyl)maleimide
(FLm, Invitrogen, B10250). To fully suspend the protein pellet, the samples were
sonicatedat40%Ampsfor1minonicefollowedbyvortexingandincubationatroom
temperaturefor30minindark.ToremovetheexcessFLm,thesampleswereapplied
andmixedwith200µlofcoldacetoneandthenincubatedovernightat‐20°Cforprotein
precipitation.Theproteinsamplewascentrifugedat10000rpmfor10min(4°C)next
day and resulted protein pellet was washed with 200 µl of cold acetone to remove
unboundFLm.Thiswasfollowedbyanincubationovernightat‐20°C.Onthenextday,
the protein samplewas centrifuged at 10000rpm for 10min (4°C) and the resulting
protein pellet was resuspended in 50 µl of Tris buffer (0.5 M Tris with 0.5 % SDS,
pH7.0). 21 µl of the suspended protein sample was transferred to a 0.6 ml
microcentrifugetube.
As oxidized thiols needed to be reduced before labeling, 4 µl of 25 mM Tris(2‐
carboxyethyl) phosphine hydrochloride (TCEP, Sigma, C4706) was added and mixed
41
with the 21 µl of FLm‐labeled protein sample to give a final TCEP concentration of
4mM.Thiswasfollowedbyincubationfor1hrinthedarkatroomtemperature.
Afterreduction,theproteinsamplewasmixedwith25µlofTrisbuffer(0.5MTriswith
0.5%SDS,pH7.0)and5µlof5mMTEXASRED‐C2‐malemide(TRm,Invitrogen,T6008)
tolabeltheoxidizedthiols.Afterincubationfor1hrinthedarkatroomtemperature,
excessTRmdyewas removedbymixing thesamplewith220µlof coldacetone then
incubated overnight at ‐20°C for protein precipitation. On the next day, the protein
samplewascentrifugedat10000rpmfor10min(4°C)andtheresultingproteinpellet
was resuspended in25µlofTrisbuffer (0.5MTriswith0.5%SDS,pH7.0)and then
mixedwith100µlofcoldacetone.Thesamplewasspundownwithmini‐centrifuging
andthenincubatedovernightat‐20°Cforproteinprecipitation.Theproteinsamplewas
centrifuged at 10000rpm for 10min (4°C) next day and resulting protein pelletwas
resuspendedandincubatedagainovernightat‐20°Cforproteinprecipitation.Afterthe
finalcentrifugation,theresultingproteinpelletwasresuspendedin50µlofTrisbuffer
(0.5MTriswith0.5%SDS,pH7.0).7.5µlofdual‐labeledproteinsamplewastakento
measure the levelofproteincontent (Section2.3.3)and therestwaskept toquantify
theleveloffluorescence(Sections2.7.3and2.7.4).
2.7.3 Fluorescence measurement using a plate reader
Fluorescence measurements of FLm and TRm for the protein samples were
standardized to FLm and TRm standard curves (Table 2.4). To prepare the standard
curves, eachdyewasdiluted from5mMto1.5mMbymixing6µlof5mMdyewith
14 µl of DMSO. The 60 µM dye/ovalbumin stock solution was consisting of 16 µl of
1.5mMdye,160µlof2mMovalbumin(Sigma,A5378)and224µlofTrisbuffer(0.5M
Triswith0.5%SDS,pH7.0).Thissolutionwasincubatedindarkfor30minbeforeuse.
For the TRm standard curve, the dye/ovalbumin stock solution was further diluted
8‐fold.
Afterincubation,allstandardswerediluted10‐foldwith0.1MNaOH,andeachprotein
samplewasdiluted32‐foldwith0.1MNaOH.Alldilutedstandardsandproteinsamples
were aliquoted in triplicate (100 μl/ well) to each well of a 384‐well plate. The
fluorescence of each sample was then measured using a fluorescent plate reader
42
(FluostarOptima)withwavelengthssetat485nmexcitationand520nmemissionfor
FLmand595nmexcitationand610nmemissionforTRm.
Table2.4DilutionforFLmandTRmstandardsFLm(µM) TRm(µM) Stocksolution(µl) Trisbuffer(µl)
0 0 0 100
6 0.75 10 90
12 1.5 20 80
24 3.0 40 60
36 4.5 60 40
48 6.0 80 20
60 7.5 120 0
2.7.4 SDS‐PAGE
After the protein quantification (Section 2.3.3), dual‐labeled protein samples were
separatedwithprecast gradient gel (Section2.6.2). Toquantify reduced andoxidized
thiolsofaspecificproteinbands, in‐gelFLmandTRmstandardcurveswereprepared
(Section 2.7.2) with several modifications. Firstly, the stock solution was made by
mixing4µlof60µMFLm/ovalbumin,1µlof60µMTRm/ovalbuminand95µlof2mM
ovalbuminthatgavea finalconcentrationof2.4µMFLmwith0.6µMTRm.Theblank
ovalbuminwasconsistingof3µlofTrisbuffer(0.5MTriswith0.5%SDS,pH7.0)with
97 µl of 2 mM ovalbumin. The in‐gel standards were then prepared by a range of
dilutionswiththisovalbuminsolution(Table2.5).
Table2.5In‐gelFLm/TRmstandardsFLm/TRm(nmol) Stocksolution(µl) Ovalbumin(µl)
0/0 0 10
0.0048/0.0012 2 8
0.0096/0.0024 4 6
0.0144/0.0036 6 4
0.0192/0.0048 8 2
0.024/0.006 10 0
Afterpreparingthein‐gelstandards,3×proteinloadingbufferwasaddedtotheprotein
samplesandstandardsandthenheat‐denaturedat95°Cfor5min.3µgofproteinand
10 µl of each standard were loaded and the gel was electrophoresed at 150 V for
43
approximately1.5hratroomtemperature.Thefluorescenceofeachlanewasmeasured
using a typhoon gel scanner (GE Healthcare Life Science, Typhoon Trio) with
wavelengthssetat520nmforFLmand610nmforTRm.Followinggelanalysisusing
theImageJsoftware,theamountofreducedandoxidizedthiolsinspecificproteinwas
determinedwithreferencetotheFLm/TRmstandardcurves.
2.8 Statistics
All data were analyzed with one‐way ANOVA with post‐hoc tests (unstacked) on
Statplus(AnalystSoft,U.S.A.).Thep‐valuewasobtainedusingFisher’sLeastSignificant
Difference (Fisher LSD). The stats only performed when experiments were repeated
morethanthreetimes.
44
Chapter 3: Development of methods for the study of protein content in C2C12 myotubes in response to treatment with catalase and glucose oxidase
3.1 Introduction
Sarcopeniaandcachexiaaretwotypesofmusclewasting.Sarcopeniaisareductionin
musclemassandstrengththatoccurswithageingandisassociatedwithareductionin
motorunitnumberandatrophyofmusclefibers,especiallytypeIIafibers.Thelossof
musclemassisclinicallyimportantbecauseitleadstodiminishedstrengthandexercise
capacity as a result of the loss of 5 % of muscle mass per decade of life from 40s
onwardsandmoreafter theageof65 (Lenk et al. 2010). Cachexia iswidely recognized
asseverewastingaccompanyingdiseasestatessuchascancer(Tazi et al. 2010)(Fig.3.1)
or immunodeficiency (Thomas 2007). About 80 % of all cancer patients suffer from
cachexiawhich leads to impairedmobility and accounts directly for around 20% of
cancer‐related deaths (Glass et al. 2010; Mathew 2011; Silverio et al. 2011; Wang et al.
2011; Wysong et al. 2011).
Figure3.1ChangesinmusclemassaccompanyingcancerComparedwithahealthymouse (A), thehindlimbofamousebearingC26coloncarcinoma(B) isseverelyatrophiedat3weeks following transplantation.Reprintedbypermission fromMacmillanPublishersLtd:[BMCCancer],copyright(Aulinoetal.2010;Coletti2013).
Previousstudiesof sarcopeniaandcachexiahaveshown thatan increase inoxidative
stress can induce a decrease in muscle size. In sarcopenia patients, an increase in
A. B.
45
oxidative stresswith age resulting in changes tomitochondrial function is thought to
playanimportantroleinthedeclineofphysiologicfunction.Mitochondrialproduction
of superoxide anions has been proposed to be the primary source of this oxidative
stress (Mansouri et al. 2006) and the oxidative damage of mitochondria has been
demonstrated to increase the generation of hydrogen peroxide in cells (Lass et al.
1998).The increasedgenerationof these reactiveoxygen species (ROS) is thought to
inducetheoxidationofmuscleproteinsandsubsequentlossofmusclemass(Capeletal.
2005).
In cachexia, tumour‐bearinganimals show lossofmuscleweight forup to twoweeks
aftertumourimplantation(Guarnieretal.2010).Thislossofmusclemassisthoughtto
result from an increased rate of protein degradation as regulated by the ubiquitin‐
proteasomeproteolyticpathway(Pennaetal.2010;Eddinsetal.2011;Mathew2011;
Wang et al. 2011). Loss of muscle mass is also thought to be linked to increased
oxidative stress resulting from decreased antioxidase activity and high levels of ROS
generation(Mantovanietal.2002;Mantovanietal.2002;Lenketal.2010;Silverioetal.
2011). Further evidence to support a role of the attenuation of muscle wasting in
tumour‐bearingmiceaftertreatmentwiththeantioxidantEGCGderivedfromgreentea
isfurtherevidencetosupportaroleforoxidativestressinmusclewasting(Wangetal.
2011).
Whileoxidativestress is thought tobeakeycauseofmusclewasting incachexiaand
sarcopenia,itisnotcertainhowthisoxidativestresseffectsproteindegradation.Inthe
C2C12 murine model of muscle wasting, oxidative stress of these myotubes has been
shown to decrease protein levels. When these myotubes were treated with 100 μM
hydrogen peroxide, the rate of ubiquitin conjugation by the ubiquitin‐proteasome
proteolyticpathway increased leading toproteindegradation (Lietal.2003).Further
understanding of the mechanism underlying these myotube changes in response to
changes in hydrogen peroxide induced by catalase and glucose oxidase, however, is
required. This includes the role of protein turnover, signaling pathways, and thiol
oxidation.Tobetterunderstand thesemechanisms,methodsneed tobedeveloped to
measure thesechanges in theC2C12model.This chapterdescribes theoptimizationof
existingmethodstoharvestandquantifyproteinlevelsintheC2C12modelinresponse
tocatalaseandglucoseoxidasetreatment.
46
3.2 Methods
AllmethodsaredescribedindetailinChapter2.
Myotubecultures
The myotubes were cultured in 35mm petri dishes. In the preliminary experiments
which aimed to optimize protein extraction, a single untreated or catalase‐treated
culture sample was used. In the preliminary experiments which aimed to optimize
proteinquantification,differentconcentrationsofbovineserumalbumin(BSA) inTris
bufferwereused. Inallsubsequentexperiments,proteinextractionandquantification
was performed for up to three petri dishes per treatment for every treatment group
(untreated,catalase,andglucoseoxidase).Theseexperimentswererepeateduptofive
timesusingfreshC2C12cultures.
Proteinextraction
Trichloroaceticacid(TCA)wasusedtoprotonateallthiolsandtoprecipitatethecellular
proteins to prevent their subsequent oxidation (Aslund et al. 1999; Delaunay et al. 2000).
To extract proteins from themyotubes, the petri dishes containingmyotube cultures
were washed briefly twice with 1ml phosphate buffered saline (PBS). 20 %
TCA/acetone (w/v) (700 µl) was then added to the petri dishes and the cells were
harvestedwithacellscraper.TheTCA/acetoneandcellsweretransferredtoa1.5ml
microcentrifugetubeandthetubeswerethencentrifugedat10000rpmfor5min(4°C).
Thesupernatantwasdischargedandtheproteinpelletwaswashedwithcoldacetone(1
ml).ThecentrifugationandwashingstepswererepeatedtoremoveanyresidualTCA.
Theproteinpelletwas thensuspended inTrisbuffer (300µl,50mMTriswith0.5%
SDS,pH7.0)andquantifiedusingthemicroBCAassay.
47
Proteinquantification‐microBCAassay
ThecommercialkitfromSigma(QuantiPro™BCAAssayKit,QPBCA‐1KT)wasusedto
quantifythetotalproteincontentforeachtreatmentgroup.1µloftheproteinsample
wasdiluted25‐foldwithSDSbuffer(0.5%SDS), followedbyanother10‐folddilution
withTrisbuffer(2mMTriswith0.5%SDS,pH7.0)ina1.5mlmicrocentrifugetube.250
µl of the cocktail reagent (A:B:C=25:25:1) was added into each diluted sample and
incubated for1hrat60°C.The incubatedsolution (100µl)was then transferred toa
384‐wellplateandtheabsorbancewasanalyzedat562nminaplatereader(BioTek,
PowerWaveHT).
3.3 Results
3.3.1 Modifying the extraction method to extract proteins from C2C12 myotubes
An existing method developed in our laboratory for the extraction of protein from
muscle(Armstrongetal.2011)wasadaptedtosuitC2C12tissueculturesamples.Inthe
existingmuscle tissueprotocol,1mlof20%TCA/acetone isused toextractproteins
from20mgofskeletalmuscle.Sincetheamountofproteinineachtissueculturedishis
much less than in the skeletal muscle samples used in the existing method, the
myotubeswereharvestedwitharangeof20%TCA/acetonevolumes(20µl,40µl,100
µl, 300 µl, 500 µl, and 700 µl). Using this range of TCA/acetone volumes, 100 µl of
TCA/acetonewasfoundtobetheminimumvolumerequiredtocoverthesurfaceofthe
dish.Although300µl and500µlwere sufficient to cover the surfaceof thedish, the
acetonerapidlyevaporatedattheselowvolumes,makingextractionmoredifficultand
reducing protein yields (Fig. 3.2). Therefore, 700 µl TCA/acetone was used in all
subsequentexperiments.
48
Figure3.2ThetotalproteincontentMyotubes (7‐day‐old) were harvested with different volumes of 20 % TCA/acetone. The proteinpelletwaswashedwithacetoneandjustresuspendedwithTrisbuffer.TheproteincontentwasthenmeasuredusingthemicroBCAassay.
AfterresuspensionwithTrisbuffer,mostoftheproteinpelletremainedunsuspended.
Therefore, we presumed that sonication was needed for full suspension. A range of
sonicationtimesweretestedfrom30secto2min.Overthisrangeofsonicationtimes,
theproteinpelletwasfoundtobefullyresuspendedaftersonicationat40%ampfor2
min and protein recovery was increased substantially (Fig. 3.3). Therefore, in all
subsequentexperiments,theproteinpelletsweresonicatedusingtheseconditions.
0
5
10
15
20
25
30
35
700 500 300
Total protein/ dish
(µg)
20% TCA/acetone (µl)
49
Figure3.3ThetotalproteincontentMyotubes (7‐day‐old) were harvested with different volumes of TCA/acetone and washed withacetone.TheproteincontentwasthenmeasuredusingthemicroBCAassay.Theproteinpelletwasresuspendedandsonicatedat40%ampfor2minoniceinTrisbuffer.
3.3.2 Method to quantify protein content in C2C12 myotubes
In the existing method for the extraction of protein frommuscle taken from animal
models,thedetergentcompatibleproteinassay(DCassay,Bio‐rad500‐0112)wasused
to assay the skeletal muscle samples. However, as tissue culture samples generally
produce lower protein levels than animal models, it was predicted that this method
would not be sensitive enough to detect changes in protein levels at these low
concentrations. Preliminary experiments using a range of low concentration bovine
serum albumin (BSA) solutions prepared using Tris buffer were undertaken to
determine the suitability of theDC assay and protein assays for the quantification of
proteinlevelsinC2C12myotubesinresponsetochangesinoxidativestress.
The DC assay is designed for samples suspended in detergent‐based solution. To
determineifthisassayissuitablefortheC2C12cultures,BSAwasfirstdissolvedinTris
buffer (50mM Tris with 0.5% SDS) at 0‐0.4mg/ml and then assayed using the DC
method. As expected and as evident in standard curve, this assay was not sensitive
enough todetect low levelsofBSA(Fig.3.4)and thereforeunlikely tobe thesuitable
methodfortheC2C12tissueculturesamplesinthisstudy.
0
100
200
300
400
500
600
700
800
700 500
Total protein/dish (µg)
20% TCA/acetone (µl)
50
Figure3.4ThestandardcurveofDCassayinTrisbufferBSAinTrisbufferfrom0to0.4mg/mlwasusedtoperformDCassay.Theabsorbancewasanalyzedat750nminaplatereader(BioTek,PowerWaveHT).
TheBradford assay (Bio‐rad, 500‐0006) is another commonmethodused toquantify
theconcentrationofproteinsamples.Toperformthisassay,BSAwasfirstdissolvedin
Tris buffer (50 mM Tris with 0.5 % SDS) from 0‐0.4 mg/ml. After mixing with the
workingreagent,theSDSintheTrisbufferreactedwiththeworkingreagenttoproduce
aprecipitatethatpreventedaccuratemeasurementofabsorbance.Therefore,thisassay
wasnotusedforfurtheranalysisoftheC2C12model.
The micro BCA assay was then tested for protein detection sensitivity using a
commercialKit(Sigma,QuantiPro™BCAAssayKit,QPBCA‐1KT).Thiskitisdesignedto
givealinearresponsefrom0.5to30µg/mlofprotein.Toassessthesuitabilityofthis
kit toproteindetection from tissue culture samples,BSAwasdissolved inTrisbuffer
(50mMTriswith0.5%SDS)from0‐0.04mg/mlandassayed.Asevidentfromstandard
curve (Fig. 3.5‐A), this assay was not sensitive enough to analyze low concentration
proteinsamplesundertheseconditions.
According to the product technical bulletin for the micro BCA assay, the maximum
allowableTris concentration for theassay is50mMand themaximumallowableSDS
concentrationis5%.TotestifTrisorthecombinationofTrisplusSDSwasinterfering
withtheBSAquantification,0‐0.04mg/mlBSAstandardswerethenpreparedinin0.5
y = -0.1122x2 + 0.0926x + 0.063 R² = 0.98001
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0 0.1 0.2 0.3 0.4 0.5
Abs
orb
ance
BSA (mg/ml)
0.5
51
%SDS solution and 2mMTris bufferwith 0.5% SDS. As evident from the standard
curves (Fig. 3.5‐B and Fig. 3.5‐C), a reduction on the Tris concentration allowed low
concentration protein samples to be detected. In all subsequent experiments, the
proteinlevelsofC2C12myotubeswerequantifiedwiththemicroBCAassaywith2mM
Trisand0.5%SDS.
Figure3.5StandardcurveformicroBCAassayusingBSAinvariousbuffersBSA (0 to 0.04 mg/ml) in different buffers assayed using the micro BCA assay. (A) HighconcentrationTrisbuffer(50mMTriswith0.5%SDS)(B)0.5%SDSsolution(C)LowconcentrationTris buffer (2 mM Tris with 0.5 % SDS). Absorbance was analyzed at 750 nm in a plate reader(BioTek,PowerWaveHT).
3.3.3 Measuring the level of protein content in C2C12 myotubes in response to catalase and glucose oxidase
To investigate the changes in protein levels in response to changes in hydrogen
peroxide and oxidative stress levels, the myotubes were treated with catalase and
glucoseoxidaseinserum‐starvedconditionsfor48hrand72hr,withtreatmentsbeing
refreshedevery24hr.Thismyotubeswerethenharvestedwith20%TCA/acetoneand
theprotein levelsquantifiedusingthemicroBCAassay.Asexpected, the levelof total
protein was significantly increased after treatment with catalase for 72 hr and
significantly decreased after treatment with glucose oxidase for 48 hr (Fig. 3.6).
Evidenceofmyotubedeathwasapparentinthe72hsampleslikelyduetotheageofthe
cultures. To investigate changes in protein turnover, signaling pathways, and thiol
oxidation in response to catalase and glucose oxidase treatment, all subsequent
experimentswereanalyzedafter48hroftreatment.
y = -6.8722x2 + 1.0375x + 0.084 R² = 0.98945
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0 0.01 0.02 0.03 0.04 0.05
Ab
sorb
ance
BSA (mg/ml)
y = -60.682x2 + 13.766x + 0.0873 R² = 0.99883
0
0.1
0.2
0.3
0.4
0.5
0.6
0 0.01 0.02 0.03 0.04 0.05
Abs
orb
anc
e
BSA (mg/ml)
y = -72.267x2 + 24.693x + 0.1025 R² = 0.99874
0
0.2
0.4
0.6
0.8
1
1.2
0 0.01 0.02 0.03 0.04 0.05
Abs
orba
nce
BSA (mg/ml)
A. B. C.
52
Figure3.6TotalproteinlevelsinC2C12myotubesinresponsetocatalaseandglucoseoxidasetreatment
Myotubes(7‐day‐old)wereleftuntreatedortreatedwithcatalase(3000units/ml;Cat.),orglucoseoxidase(10munits/ml;GluO.)for48hrinserum‐starvedconditions.Proteinsampleswerecollectedas described in Chapter 2.2.1 and quantified as described in Chapter 2.3.2. (A) This data was anaverageoffiveexperimentsforeachtreatmentgroup(2‐3dishes/treatmentgroup)(B)Thisdatawasanaverageoffourexperimentsforeachtreatmentgroup(2‐3dishes/treatmentgroup).72hrdataisnotshownbecausemyotubedeathwasevidentatthistime‐point.Dataisshownasmean±SEM.
3.4 Discussion
In the present chapter, the impact of catalase and glucose oxidase on total protein
content was examined using protein extraction and quantification methods. These
methodswerefirstlyoptimizedformyotubeculturesamplesandthisisthefirstreport
of changes in total protein content in C2C12 myotubes in response to catalase and
glucoseoxidasetreatment.
Usingproteinextractionandquantificationmethods, theleveloftotalprotein inC2C12
myotubes was found to be increased after treatment with catalase for 72 hr and
decreased after treatment with glucose oxidase for 48 hr. This finding is similar to
previousstudiesofpatientswithmuscleloss(Thomas2007;Lenketal.2010;Tazietal.
2010).Whileoxidativestressisthoughttoinducetheselossesinmusclemass(Lasset
al.1998;Mantovanietal.2002;Mantovanietal.2002;Capeletal.2005;Mansourietal.
2006;Lenketal.2010;Silverioetal.2011), theexactmechanismsthatunderly these
0
200
400
600
800
1000
1200
1400
1600
1800
2000
Total p
rotein
(µg/dish)
0
200
400
600
800
1000
1200
1400
1600
1800
2000
Total p
rotein
(µg/dish)
*
* A. B.
53
losses are not clear yet but an imbalance between the level of protein synthesis and
degradationisthoughttobeinvolved(Balagopaletal.1997).InChapter4,methodsto
investigate changes in protein turnover bymeasure the changes in protein synthesis
anddegradationinC2C12myotubesaredeveloped.
54
Chapter 4: Development of methods for the measurement of protein synthesis and degradation in C2C12 myotubes in response to treatment with catalase and glucose oxidase
4.1 Introduction
Thebalancebetweenproteinsynthesisanddegradationcontrolsproteinlevelswithina
cell organism (Bassell et al. 1997). Whenmusclewasting occurs, there is a decrease in
proteinsynthesisand/oranincreaseinproteindegradation,whichleadstoadecrease
in total protein content. Oxidative stress is thought to be a key intermediary in
promoting muscle wasting (Muller et al. 2006; Arthur et al. 2008). In this study, an
inducer of oxidative stress, hydrogen peroxide, is used to investigate the effects of
oxidativestressinmyotubes.
InChapter3,anincreaseinthetotalproteincontentwasobservedinmyotubestreated
with catalase, which reduces cellular hydrogen peroxide levels (Jones et al. 1968;
Boverisetal.1972;Orretal.1994;Dayetal.1997).Adecreaseintotalproteincontent
inmyotubestreatedwithglucoseoxidasewasalsoobserved.Glucoseoxidaseincreases
the level of hydrogen peroxide in cells (Weiss et al. 1981; Starkebaum et al. 1986;
Salazaretal.1997).Whilethemechanismsthatinducedthesechangesintotalprotein
in these myotubes are not clear, hydrogen peroxide mediated changes in protein
synthesishavebeenreportedinotherstudies.
Inyeastcells,thelevelofproteinsynthesishasbeenshowntodecreaseinresponseto
up to 2mMhydrogen peroxide (Shenton et al. 2003; Shenton et al. 2006). In Clone9 cell, a
cell culturemodel of normal liver epithelial cells, the rate of protein degradation has
been shown to increase in response up to 1mM hydrogen peroxide or a continuous
hydrogenperoxidefluxgeneratedbytheglucose/glucoseoxidasereaction(Gruneetal.
1995;Gruneetal.1997).Whenhemoglobinispre‐treatedwith0.5to50mMhydrogen
peroxide,therateofproteindegradationhasalsobeenfoundtoincrease(Fligieletal.
1984).Inmusclecells,proteinsynthesishasalsobeenshowntodecreaseinresponseto
100 µM hydrogen peroxide (Orzechowski et al. 2002) and the expression of
arogin1/MAFbx, the ubiquitin ligase gene that mediates muscle atrophy, is also
enhancedinresponsetohydrogenperoxide(Lietal.2005).
55
Given the apparent importance of hydrogen peroxide in protein synthesis and
degradation, the present study set out to investigate changes in the level of protein
synthesis and the rate of protein degradation inmyotubes treatedwith catalase and
glucoseoxidasebyadaptingestablishedmethods(Pollard1996;Reinheckeletal.2000;
Casey et al. 2002; Catalgol et al. 2009). As insulin growth factor (IGF) is known to
increase the level of protein synthesis and decrease the rate of protein degradation
(Pham et al. 2000; Brink et al. 2001; Li et al. 2004; Sacheck et al. 2004; Zhao et al. 2007;
McGilchrist et al. 2011; Chen et al. 2012; Clemmons 2012), IGFwas used as the control for
theproteinsynthesisanddegradationexperiements.Astumournecrosisfactor(TNF)is
knowntoincreaseproteindegradationrates(Lietal.2000;Lietal.2005;Leckeretal.
2006),itwasusedasacontrolfortheinitialproteindegradationstudies.TNFwaslater
replaced by dexamethasone (DEX) which is known to increase protein degradation
rates(Sacheck et al. 2004; Sandri et al. 2004).
4.2 Methods
AllmethodsaredescribedindetailinChapter2.
Myotubecultures
The myotubes were cultured in 35mm petri dishes. In the preliminary experiments
whichaimedtooptimizethetimepointoftheincorporationofradioactivity,threepetri
dishes per treatment for every treatment group (untreated, catalase, TNF) from one
single C2C12 culture was used. In all subsequent experiments, two petri dishes per
treatment for every treatment group (untreated, catalase, glucose oxidase, IGF, and
dexamethasone).TheseexperimentswererepeateduptoeighttimesusingfreshC2C12
cultures. In this study, all myotubes were treated under serum‐starved condition to
avoidtheanypossibleeffectofenzymesintheserum.
Proteinsynthesis
Protein synthesiswasassessedbymeasuring the incorporationof radioactive leucine
(leucine, L‐[3,4,5‐3H(N)] into protein. The experimentswere carried out in 7‐day‐old
C2C12myotubecultures(Section2.4) treatedwithcatalase,glucoseoxidase,or IGF for
24hr.Therefreshedtreatmentplusradioactiveleucine(0.5µCi/ml)werethenapplied
totheculturesforafurther24hr.Thelevelofradioactiveleucineintheproteinpellet
56
and supernatant was then analyzed using a scintillation counter. Protein synthesis
results are expressed as the total leucine incorporation per dish per 24 hr
(µmol/dish/24hr)andstatisticalanalysiswasperformedusingStatPlus.
Proteindegradation
Protein degradation was assessed by measuring the release of radioactive leucine
(leucine,L‐[3,4,5‐3H(N)]fromcells.Theexperimentswerecarriedoutin6‐day‐oldC2C12
myotube cultures (Section 2.5) pre‐labeled for 24 hr with radioactive leucine (0.5
µCi/ml, leucine (L‐[3,4,5‐3H(N)]). The pre‐labeled myotubes were then treated with
catalase,glucoseoxidase,IGF,orDEXfor48hr,andtreatmentwasrefreshedevery24
hr.Thelevelofradioactiveleucineinproteinpelletandsupernatantwasthenanalyzed
using a scintillation counter. Protein degradation results are presented as the
percentage of the radioactive leucine release per dish per 24 hr (%/dish/24hr) and
statisticalanalysiswasperformedusingStatPlus.
4.3 Results
4.3.1 Establishment of method for measuring protein synthesis in C2C12 myotubes
TomeasurethelevelofproteinsynthesisinC2C12myotubes,theamountofradioactive
leucine (leucine, L‐ [3,4,5‐3H(N)]) incorporated into newly synthesized proteins was
assessed.Toestablishthismethod,apreliminaryexperimentwasundertakenapplying
radioactiveleucine(1µCi/ml,initialactivity)andcatalaseto7‐day‐oldmyotubesfor24
hr. The protein pellets and supernatantwere collected and the leucine incorporation
wasassessed.
This preliminary experiment showed there was no significant changes in protein
synthesis in the catalase‐treated myotubes compared to the untreated cultures,
however, the incorporationofradioactive leucinewithin thecatalase treatmentgroup
was highly variable (Fig 4.1‐A). This high level of variability in leucine incorporation
suggeststhatthemeasurementmightbeaffectedbysomemetabolicdisturbancesinthe
catalasetreatmentgroup.
To minimize metabolic disturbance (Pollard 1996), all myotubes were subsequently
labeledinpre‐conditionedmediapriortotheadditionofradioactiveleucinebytreating
57
the7‐day‐oldmyotubeswith/withouttreatmentfor24hrinserum‐starvedconditions.
Themediawith/withouttreatmentwasthenrefreshedandthemyotubeswereexposed
toradioactiveleucine(0.5µCi/ml,initialactivity)forafurther24hr.Thisresultedina
reductioninthevariabilityofleucineincorporationandanincreaseinleucineuptake,in
boththecatalase‐treatedgroupanduntreatedgroup(Fig.4.1‐B).
Figure4.1LeucineincorporationinC2C12myotbestreatedwithcatalase(A) Myotubes (7‐day‐old) were left untreated or treated with catalase (3000 units/ml; +Cat.)together with radioactive leucine (1 µCi/ml, initial activity) for 24 hr under serum‐starvedconditions.(B)Myotubes(7‐day‐old)werepre‐conditionedwithcatalase(3000units/ml;+Cat.),orinsulingrowthfactor(30ng/ml;+IGF)for24hrunderserum‐starvedconditionsRadioactiveleucine(0.5µCi/ml,initialactivity)andfreshcatalasewerethenappliedtothesepre‐conditionedmyotubesfor a further 24 hr under serum‐starved conditions. The total leucine incorporation in eachtreatmentgroupwasexpressedasmean±SEM(n=3/treatmentgroup).ThesupernatantandproteinpelletwerecollectedandassayedsimultaneouslyforradioactivityasdescribedinChapter2,section2.4.3.
All subsequent protein synthesis measurements used this pre‐conditionedmedia. As
levels of unincorporated radioactive leucine were also high, the initial levels of
radioactiveleucineweredecreasedfrom1µCi/mlto0.5µCi/mlforallsubsequenttests.
4.3.2 Establishment of method for measuring protein degradation in C2C12 myotubes
Tomeasure therateofproteindegradation in theC2C12myotubes, thereleaserateof
radioactive leucine (leucine,L‐ [3,4,5‐3H(N)])wasassessed.Toestablish thismethod,
0
5
10
15
20
25
Untreated +Cat. +IGF
Leucine
inco
rpora
tion
(µmol/d
ish/24
hr)
0.0
0.5
1.0
1.5
2.0
Untreated +Cat.
Leucine incorporation (µmol/dish/24hr)
A. B.
58
catalaseortumournecrosisfactor(TNF)wasappliedto7‐day‐oldmyotubesfor24hr.
Radioactive leucine (0.5µCi/ml, initial activity) and fresh catalase or TNFwere then
applied to these pre‐treated myotubes for another 48 hr, with refreshment of these
treatmentsafterthefirst24hr.Theproteinpelletsandsupernatantwerecollectedand
thereleaseof radioactive leucinewasassessed.Therewasnosignificantdifference in
protein degradation in either the catalase‐ or TNF‐treated group compared to the
untreatedthegroup(Fig.4.2).
Figure4.2RadioactiveleucinereleasefromC2C12myotubestreatedwithcatalaseandTNF
Myotubes (7‐day‐old) were left untreated or treated with catalase (3000 units/ml; +Cat.) andtumournecrosisfactor(20ng/ml;+TNF)for24hr.FreshcatalaseorTNFwithradioactiveleucine(0.5µCi/ml,initialactivity)wereappliedtothesemyotubesforafurther24hr.FreshcatalaseorTNFwereappliedtotheselabeledandtreatedmyotubesforanother24hr.Thepercentageofthereleaseof radioactive leucine fromeach treatment groupwas expressed asmean± SEM (n=3 /treatmentgroup). The supernatant and protein pellets were collected and assayed simultaneously forradioactivity as described in Chapter2, section 2.5.3. The myotubes were cultured under serum‐starvedconditions.
As shown in Fig. 4.1‐B, the amount of leucine incorporation was different between
treatments,whichmayhaveimpactedontheaccuratemeasurementofleucinerelease.
Toequalizetheamountofincorporatedleucinepriortomeasurerelease,the6‐day‐old
myotubeswerepre‐labeledwithradioactiveleucine(0.5µCi/ml, initialactivity)under
serum‐containedconditionfor24hrpriortoanytreatment.Thesemyotubeswerethen
treated with IGF, DEX, and TNF for 48 hr under serum‐starved conditions, with the
0
10
20
30
40
50
60
Untreated +Cat. +TNF
Radioactive leucine
release (%/dish/24hr)
59
treatmentsrefreshedafterthefirst24hr.AsevidentinFig.4.3,undertheseconditions,
changeswereobservedintheIGFandDEXtreatmentgroupsandthevariabilitywithin
treatmentsgroupswaslow.However,afterseveralroundsofexperiments,nochangein
protein degradationwas observed in the TNF‐treatedmyotubes. Therefore, DEX and
IGFwereusedascontrolsinsteadofTNFinallsubsequentexperiments.
Figure4.3Radioactiveleucinereleasefrompre‐labeledC2C12myotubeswithvarioustreatments
Myotubes (6‐day‐old) were pre‐labeled with radioactive leucine (0.5 µCi/ml, initial activity) inserum‐containing medium (2 % horse serum) for 24 hr. These labeled myotubes were then leftuntreatedortreatedwithinsulingrowthfactor(30ng/ml;+IGF),dexamethasone(40ng/ml;+DEX),ortumournecrosisfactor(20ng/ml;+TNF)inserum‐freemediumfor48hr.Freshtreatmentswerereplacedafter24hr.Thepercentage releaseof radioactive leuine fromeach treatmentgroupwasnormalizedwithuntreatedgroupandexpressedasmean±SEM[n=4culturebatches(+IGF,+DEX);n=5culturebatches(+TNF)](unpublisheddata).ThesupernatantandproteinpelletswerecollectedandassayedsimultaneouslyforradioactivityasdescribedinChapter2,Section2.5.3.*p<0.05
4.3.3 Measuring protein synthesis in C2C12 myotubes
Usingtheconditionsestablishedinsection4.1,themyotubeswerepre‐conditionedand
thentreatedwithcatalase,glucoseoxidaseandIGF.Thelevelofproteinsynthesiswas
significantlydecreasedaftercatalasetreatment(Fig.4.4‐A).Whileasignificantdecrease
inproteinsynthesiswasnotobservedafterglucoseoxidasetreatment(Fig.4.4‐B),there
was a significant increase with IGF treatment, indicating the system is capable of
detectingchangesofproteinsynthesisinC2C12myotubes.
0
20
40
60
80
100
120
Radioactive leucine
release (%/dish/24hr)
*
*
60
Figure4.4ProteinsynthesisinC2C12Myotubeswithcatalaseandglucoseoxidasetreatment
(A)Myotubes(7‐day‐old)wereleftuntreatedortreatedwithcatalase(3000units/ml;+Cat.)orIGF(30ng/ml;+IGF)for24hr.Radioactiveleucine(0.5µCi/ml,initialactivity)withfreshcatalaseorIGFtogetherwerethenappliedtothesepre‐conditionedmyotubesforafurther24hr.(B)Myotubes(7‐day‐old) were left untreated or treated with glucose oxidase (10 munits/ml; +GluO.) for 24 hr.Radioactive leucine (0.5 µCi/ml, initial activity) with fresh glucose oxidase together were thenapplied to these pre‐conditionedmyotubes for a further 24 hr. The total leucine incorporation ineach treatment group was expressed as mean ± SEM [n=8 culture batches (+Cat.); n=4 culturebatches (+IGF, +GluO.)]. The supernatant and protein pellets were collected and assayedsimultaneouslyforradioactivityasdescribedinChapter2,Section2.4.3.Themyotubesin(A)and(B)wereculturedunderserum‐starvedconditions.*p<0.05,**p<0.005
4.3.4 Measuring protein degradation in C2C12 myotubes
Usingtheconditionsestablishedinsection4.2,themyotubeswerepreconditionedand
then treated with catalase, glucose oxidase, IGF and DEX. As expected, the rate of
protein degradation was significantly decreased after catalase treatment. While a
significantincreaseinproteindegradationratewasnotobservedafterglucoseoxidase
treatment(Fig.4.5).TheIGFsignificantlydecreasedtherateofproteindegradationand
theDEXsignificantlyincreasedtherateofproteindegradation,indicatingthissystemis
capableofdetectingchangesofproteindegradationinC2C12myotubes.
0
5
10
15
20
25
Untreated +Cat. +IGF
Leu
cin
e in
corp
ora
tio
n (
µm
ol/2
4h
r)
0
5
10
15
20
25
Untreated +GluO. L
eu
cin
e in
co
rpo
rati
on
(µ
mo
l/24h
r)
*
* *A. B.
61
Figure4.5RadioactiveleucinereleasefromC2C12myotubeswithvarioustreatments
Myotubes (6‐day‐old) were pre‐labeled with radioactive leucine (0.5 µCi/ml, initial activity) inserum‐contained medium (with 2 % horse serum) for 24 hr. These labeled myotubes were leftuntreatedortreatedwithcatalase(3000units/ml;+Cat.),glucoseoxidase(10munits/ml;+GluO.),insulingrowthfactor(30ng/ml;+IGF),ordexamethasone(40ng/ml;+DEX)inserum‐freemediumfor48hr.Freshtreatmentswererefreshedatthefirst24hr.Thepercentagereleaseofradioactiveleuine fromeachtreatmentgroupwasnormalizedwithuntreatedgroupandexpressedasmean±SEM [n=5culturebatches (+Cat.); n=4 culturebatches (+GluO,+IGF, +DEX)].The supernatant andproteinpelletswerecollectedandassayedsimultaneouslyforradioactivityasdescribedinChapter2,Section2.5.3.*p<0.05
4.4 Discussion
Incorporation of radioactive amino acids into proteins is frequently used tomeasure
changes in protein synthesis and degradation (Ratan et al. 1994; Grune et al. 1995;
Pollard1996;Reinheckeletal.2000;Siwiketal.2001;Caseyetal.2002;Shentonetal.
2003; Shenton et al. 2006; Catalgol et al. 2009). To measure changes in protein
synthesis and degradation in C2C12myotubes, twonovel controlswere used: IGF and
DEX. Using these controls, the methods developed in this study were found to be
effectiveinmeasuringproteinturnoverinC2C12myotubes.
0
20
40
60
80
100
120
140
Untre
ated
+Cat
+Glu
O
+IG
F
+DEX R
adioac
tive
leucine
degrad
atio
n (%
/24hr)
*
*
*
62
Thesemethodswerethenusedtoshow,forthefirsttime,howproteinturnoverinC2C12
myotubesisaffectedbycatalaseandglucoseoxidasetreatment.Whenproteinturnover
in C2C12 myotubes was measured after expose to catalase, there was a significant
decreaseinproteinsynthesisanddegradation.Thisisconsistentwiththeknownaction
ofcatalase,whichdecreasesoxidativestress incellsbydecreasinghydrogenperoxide
levels,andtheincreaseintotalnetproteinobservedinthesemyotubesafterexposure
to catalase in Chapter 3. However, in response to glucose oxidase treatment, protein
synthesisanddegradationintheC2C12myotubeswasnotchangedsignificantlydespitea
significant decrease in total net protein observed in response to this treatment in
Chapter3.Eventhoughthechangesinproteinturnoverafterglucoseoxidasetreatment
werenotsignificant,decreasedproteinsynthesisandincreasedproteindegradationis
often indicative of a decrease in protein levels. According to Finkel and coworkers, a
decrease in oxidative stress might in itself stress to the cell, resulting in decreased
proteinsynthesis.
Arangeofconflictingresultshavebeenobservedinotherstudies focusingonprotein
turnoverindifferentcelltypesexposedtohydrogenperoxide.Insomestudies,protein
synthesis decreased and protein degradation increased in response to hydrogen
peroxide (Grune et al. 1997; Orzechowski et al. 2002; Shenton et al. 2003; Shenton et al.
2006).Inastudyofcardiacfibroblasts,collagensynthesislevelswereshowntodecrease
after exposure to hydrogen peroxide, but the level of total protein synthesis did not
change(Siwik et al. 2001).
Furtherstudiesofthesignalingpathwaythatinducechangesinproteinsynthesismay
provideabetterunderstandingofhowmusclecellsresponsetohydrogenperoxide.In
skeletalmuscle,regulationofproteinsynthesisoccursprimarilyattheinitiationphase
of protein translation, which involves at least 13 initiation factors, many of which
assemble from numerous subunits (Syntichaki et al. 2006; Tisdale 2009). As these
eventsarecoordinatedbyinitiationfactorseIF4EandeIF2B(Syntichakietal.2006),the
mechanismbywhichcatalaseandglucoseoxidaseaffecttheseinitiationfactorsinC2C12
myotubeswasaddressedinChapter5.
63
Chapter 5: Development of methods for the study of signaling pathway on protein synthesis in C2C12 myotubes in response to treatment with catalase and glucose oxidase
5.1 Introduction
Changes in total protein content (Chapter 3) and protein turnover (Chapter 4) were
observed in C2C12 myotubes after catalase and glucose oxidase treatment. The
mechanism that induces these changes inprotein synthesis, however, isnot clearbut
mayinvolvechangestosignalingpathwaysinresponsetohydrogenperoxideexposure.
Previousstudiessuggestthe4EBP1signalingpathwaymaybeinvolvedinmediationof
proteinsynthesis.Whenphosphorylationofeukaryotictranslationinitiationfactor4E‐
bindingprotein1(4EBP1)decreases,activityofeukaryoticinitiationfactor4E(eIF4E)
is suppressed leading to a reduction in protein synthesis. In PC12 cells from
phaeochromocytomaoftheratadrenalmedulla,phosphorylationof4EBP1decreasesin
responsetoupto2mMhydrogenperoxideinadose‐dependentmanner.Moreover,this
reduction can be attenuated by pre‐treating the cells with 5 mM of reactive oxygen
species (ROS) scavenger, N‐acetyl‐cysteine (NAC) (Chen et al. 2010). A decrease in
phosphorylationof4EBP1inresponsetoelevatedlevelofhydrogenperoxidehasalso
been observed in a range of cell types, such as aged muscle cells, human lung
adenocarcinomacells,mouseembryonic fibroblastsandcardiacmyocytes (Pateletal.
2002;Zhangetal.2009;Wuetal.2010;Emaraetal.2012).
Otherstudieshaveshownthatanothersignalingpathway,theeIF2αpathway,mayalso
be involved in the mediation of protein synthesis. When phosphorylation on the α
subunit of eukaryotic initiation factor 2 (eIF2α) increases, activity of the eukaryotic
initiationfactor2B(eIF2B)isattenuatedleadingtoareductioninproteinsynthesis.In
PC12 cells, phosphorylation of eIF2α increases in response to 1‐3 mM of hydrogen
peroxideinadose‐dependentmanner.Moreover,thisinductioncouldbeabolishedby
pre‐treatingthecellswith10mMofNAC(O'Loghlenetal.2003).ThisincreaseineIF2α
phosphorylationhasalsobeenobservedinarangeofcelltypes,suchasyeastcellsand
agedskeletalmuscle(Shenton et al. 2006; Mascarenhas et al. 2008; Wu et al. 2010).
64
Giventheapparentimportanceofhydrogenperoxideinphosphorylationof4EBP1and
eIF2α,thepresentstudysetouttoinvestigatethechangesinphosphorylationof4EBP1
and eIF2α with catalase and glucose oxidase. Methods are developed and used to
determinetheeffectsinC2C12myotubes.
5.2 Methods
AllmethodsaredescribedindetailinChapter2.
Myotubecultures
The myotubes were cultured in 35mm petri dishes. In the preliminary experiments
whichaimedtoestablishwesternblotmethod,twotothreeuntreatedortreatedculture
sampleswereused. Inall subsequentexperiments, twopetridishesper treatment for
every treatment group (untreated, catalase, and glucose oxidase). These experiments
wererepeateduptothreetimesusingfreshC2C12cultures.
Proteinextraction
Phospho‐safeextractioncocktailwasusedtoextractallproteinsandtopreventtheloss
of phosphorylated protein during the process of extraction. To extract proteins from
myotubecultures,thepetridishescontainingthemyotubecultureswereplacedonice
andwashedbrieflytwicewith1mlofcold(4°C)phosphatebufferedsaline(PBS).100µl
of phospho‐safe extraction cocktail was then added to the petri dishes and the cells
wereharvestedwithacellscraper.Thephospho‐safeextractioncocktailandcellswere
transferredtoa1.5mlmicrocentrifugetube.Thetubeswereplacedonicefor20min
andvortexedfor5secevery10min.Thetubeswerethencentrifugedat10000rpmfor
5min(4°C).Themajorityofthesupernatant(96µl)waskeptforelectrophoresis,and
theremainingsupernatantwaskeptforproteinquantification.
Proteinquantification
TheBio‐RadProteinAssayKitwasusedtoquantifytheproteinineachmyotubesample.
Protein sampleswere quantifiedwith reference to a BSA standard absorbance curve.
65
BSAstandardsofknownconcentrations (0,100,200,300,400,and500µg/ml)were
preparedbyserialdilutionofBSA(stockconcentration1mg/ml)in0.01MPBS.
Theprotein sampleswere first diluted20‐foldwith0.01MPBS and then10µl of the
dilutedproteinsampleandstandardsweretransferredintriplicatetothewellsofa96‐
well plate. 200 µl of Bio‐Rad reagent was then added to each well. The plate was
incubatedfor10minwithgentleshakingonashakeratroomtemperatureandatthe
endofthisincubationperiod,theabsorbanceofeachwellwasmeasuredat595nmina
plate reader. After protein quantification, the concentration of each sample was
calculatedandtheproteinswerethenseparatedbyelectrophoresis.
WesternBlot‐SDSPolyacrylamideGelElectrophoresis(SDS‐PAGE)system
Self‐made 12 %, 1.5 mm thick acrylamide gels were used in the preliminary
experiments. 20 µg of the protein sample and 4 µl of Precision Plus Protein™
Kaleidoscope™Standards(Bio‐Rad,161‐0375)wereloadedinthewellsandthesample
wereelectrophoresedforapproximately2.5hrat120Vat4°C.Theseparatedsamples
werethentransferredat4°CfromthegeltoaPVDFmembraneovernightat30mA.
Themembranewas thenwashed briefly twicewithTBS‐T buffer and incubatedwith
blockingbuffer (5% skimmilk inTBS‐T) for1hr at room temperature.Theblocked
membranewas thenwashed twice,10mineach time,withTBS‐Tand incubatedwith
dilutedprimaryantibodyat4°Covernightwithgentleshaking.Themembranewasthen
washed again with TBS‐T twice, 10 min each time, and incubated with diluted
secondary antibody for 1 hr at room temperature with shaking. Themembranewas
then briefly washed twice with TBS‐T and then incubated with chemiluminescence
substratesolutionfor5minatroomtemperature.Thesignalwasexposedtofilmand
thefilmwasthendevelopedinadarkroom.
66
WesternBlot‐Bio‐Radsystem
Precastgradientgels(Bio‐Rad,456‐1086)wereusedinallsubsequentexperiments.10
µgoftheproteinsampleand4µlofPrecisionPlusProtein™Kaleidoscope™Standards
(Bio‐Rad,161‐0375)wereloadedinthewellsandthesamplewereelectrophoresedfor
approximately 1.5 hr at room temperature. The separated samples were then
transferredfor7minfromthegeltoamembrane(Bio‐Rad,170‐4158)withTrans‐Blot®
Turbo™ Transfer System (Bio‐Rad, 170‐4155) on turbo setting. The membrane was
thenwashedbriefly twicewithTBS‐Tbufferand incubatedwithblockingbuffer(5%
skimmilk in TBS‐T) for 1 hr at room temperature. The blockedmembranewas then
washed twice, 10 min each time, with TBS‐T and incubated with diluted primary
antibodyat4°Covernightwithgentleshaking.Themembranewasthenwashedagain
withTBS‐Ttwice,10mineachtime,andincubatedwithdilutedsecondaryantibodyfor
1hratroomtemperaturewithshaking.Themembranewasthenbrieflywashedtwice
withTBS‐Tandthenincubatedwithchemiluminescencesubstratesolutionfor5minat
room temperature. The signal was detected and captured by using ChemiDoc™ MP
System(Bio‐Rad,170‐8280)andImageLab™SoftwareVersion4.0(Bio‐Rad).Imagesof
thesemembraneswerethenanalyzedusingImageJ.
Aktwasusedas the loading control and is commonlyusedbyour laboratory for this
purpose(Tan2013;Tanetal.2015).
Antibodies
The primary antibodies used in this research (p‐eIF2α at Ser51 (5199), p‐4EBP1 at
Thr37/46 (2855), eIF2α (9722), 4EBP1 (9452), andAkt (9272))were obtained from
Cell Signaling (Danvers, U.S.A). All primary antibodieswere diluted 1:1000with 5%
BSAinTBS‐T.Thesecondaryanti‐rabbitantibody(Thermo,31460)wasdiluted1:5000
with5%skimmilkinTBS‐T.
67
5.3 Results
5.3.1 Optimization of method for measuring 4EBP1 phosphorylation in C2C12 myotubes
Tomeasurephosphorylationon4EBP1inC2C12myotubes,westernblottingwasusedto
detect phosphorylated 4EBP1 (p4EBP1) and total 4EBP1 (4EBP1). Preliminary
experimentswereperformedtodeterminetherequiredantibodydilutionfactorsandto
optimizetheblottingmethodforthemyotubes.
20µgofproteinsamples fromthemyotubecultureswereseparatedandthesignalof
p4EBP1 and 4EBP1 was detected using western blot‐SDS‐PAGE system. The dilution
factor of primary antibodies was 1:1000 with 5 % BSA in TBS‐T, and 1:10000 for
secondaryantibodywith5%skimmilk inTBS‐T.Themembranewas incubatedwith
chemiluminescencesubstrate(Millipore,WBLUR0100)for5minatroomtemperature.
Using thisprotocol, thep4EBP1and4EBP1bandswere clearly visible and therewas
little background (Fig. 5.1). As this protocol was shown to be suitable for the
measurement of phosphorylation of 4EBP1 in C2C12 myotubes it was used for all
subsequentexperiments.
Figure5.1Detectionofphosphorylated4EBP1andtotal4EBP1Myotubes(7‐day‐old)were left inserum‐starvedconditions(Untreated24hr)orserum‐containedconditions(+2%HS24hr)for24hr.ProteinsampleswerecollectedasdescribedinChapter2.2.2andquantifiedasdescribedinChapter2.3.1.Westernblotwasperformedwith20µgC2C12myotubessamplesasdescribedaboveand60µganimalsample (muscle frommice,obtained fromAssociateProfessorTeaShavlakadze)todetectthesignalofp4EBP1and4EBP1.Theexposuretimeofp4EBP1was1min,and4EBP1was4min.TheprocessofwesternblotwasasdescribedinChapter2.6.1.
p4EBP1
4EBP1
68
5.3.2 Optimization of method for measuring eIF2αphopsphorylation in C2C12 myotubes
TomeasurephosphorylationoneIF2αinC2C12myotubes,westernblottingwasusedto
detect phosphorylated eIF2α (peIF2α) and total eIF2α (eIF2α). As with the previous
4EBP1detectionmethod, theantibodydilution factorsandblottingconditionsneeded
tobeoptimized,soaseriesofpreliminaryexperimentswereconducted.
Usingthesamedilutionfactorsandconditionsfordetectionof4EBP1,westernblotwas
performed for peIF2α and eIF2α. For eIF2α, the bands could be detected using this
protocol (Fig. 5.2). However, these conditions were not suitable for peIF2α as the
backgroundstainingoftheblotobscuredthepeIF2αbands(datanotshown).Toreduce
thebackground stainingof thepeIF2α blot, thepeIF2α primaryantibodywasdiluted
with5%skimmilk inTBS‐T.However, thisdidnot reduce thebackground(datanot
shown).
Figure5.2DetectionoftotaleIF2αMyotubes(7‐day‐old)werecollectedorleftindifferentconditionsthatmyotubeswereincubatedinserum‐starved conditions (Untreated), serum‐starved conditions with catalase (3000 units/ml;+Cat.), serum‐starved conditions with glucose oxidase (10 munits/ml; +GluO.), serum‐containingconditions(+2%HS),orserum‐containingconditionswithcatalase(3000units/ml;+2%HS+Cat.)for48hr.ProteinsampleswerecollectedasdescribedinChapter2.2.2andquantifiedasdescribedinChapter2.3.1.Westernblotwasperformedwith20µgC2C12myotubessamplesasdescribedabovetodetectthesignalofeIF2α.TheeIF2αprimaryantibodywasdiluted1:1000with5%BSAinTBS‐T.Thefilmwasexposedtosignalfor5secanddevelopedfor5sec.TheprocessofwesternblotwasasdescribedinChapter2.6.1.
eIF2α
69
Low affinity between the antibody and peIF2α on the membrane may have been
responsible for the high level of background staining. To increase affinity of this
antibody, the primary antibody was used at a higher concentration by reducing the
dilution factor. The blotting process was repeated with a range of primary antibody
dilutions (1:250 to 1:1000)with 5% skimmilk in TBS‐T. High levels of background
stainingwerestillpresentandthepeIF2αbandswerebarelydistinguishableontheblot
evenatthedilutionfactorof1:250(Fig.5.3).
Figure5.3DetectionofphosphorylatedeIF2αMyotubes(7‐day‐old)werecollectedorleftindifferentconditionsthatmyotubeswereincubatedinserum‐starved conditions (Untreated), serum‐starved conditions with catalase (3000 units/ml;+Cat.), serum‐starved conditions with glucose oxidase (10 munits/ml; +GluO.), serum‐containingconditions(+2%HS),orserum‐containingconditionswithcatalase(3000units/ml;+2%HS+Cat.)for48hr.ProteinsampleswerecollectedasdescribedinChapter2.2.2andquantifiedasdescribedinChapter2.3.1.Westernblotwasperformedwith20µgC2C12myotubessamplesasdescribedabovetodetectthesignalofpeIF2α.ThepeIF2αprimaryantibodywasdiluted1:250with5%skimmilkinTBS‐T.Thefilmwasexposedtosignalfor5minanddevelopedfor5sec.TheprocessofwesternblotwasasdescribedinChapter2.6.1.
To reduce the background staining, 5 % BSA in TBS‐T was used as blocking buffer
insteadof5%skimmilk inTBS‐T.Asshown inFig.5.4, thebackgroundstainingwas
reducedbutwasstillhigh.
peIF2α
70
Figure5.4DetectionofphosphorylatedeIF2αMyotubes(7‐day‐old)werecollectedorleftindifferentconditionsthatmyotubeswereincubatedinserum‐starved conditions (Untreated), serum‐starved conditions with catalase (3000 units/ml;+Cat.), serum‐starved conditions with glucose oxidase (10 munits/ml; +GluO.), serum‐containingconditions(+2%HS),orserum‐containingconditionswithcatalase(3000units/ml;+2%HS+Cat.)for48hr.ProteinsampleswerecollectedasdescribedinChapter2.2.2andquantifiedasdescribedinChapter2.3.1.20µgC2C12myotubessamplesasdescribedabovewereusedtoperformwesternblotasdescribed inChapter2.6.1butwith5%BSA inTBS‐Tasblockingbuffer todetect thesignalofpeIF2α.ThepeIF2αprimaryantibodywasdiluted1:250with5%skimmilkinTBS‐T.Thefilmwasexposedtosignalfor5minanddevelopedfor5sec.
Tosolvethe lowdilutionandbackgroundstainingissue, thepeIF2αprimaryantibody
was replaced with a hypersensitive antibody (5199, Cell Signaling) and the blotting
processassection5.1wasrepeated.Asshown inFig.5.5,using thisantibody,peIF2α
bandswere clear and the background stainingwas greatly reduced.Measurement of
phosphorylation of eIF2α in C2C12myotubes in all subsequent experiments used this
antibodythatdilutedwith5%BSAinTBS‐Tand5%skimmilkinTBS‐Twasusedas
blockingbuffer.
Figure5.5DetectionofphosphorylatedeIF2αMyotubes(7‐day‐old)wereleftuntreatedortreatedwithcatalase(3000units/ml;+Cat.)for48hrinserum‐starved conditions. Protein samples were collected as described in Chapter 2.2.2 andquantified asdescribed inChapter 2.3.1.Westernblotwasperformedwith20µgC2C12myotubessamplesasdescribedabovetodetectthesignalofpeIF2αbyusingChemiDoc™MPSystem(Bio‐Rad,170‐8280)andtheexposuretimewas1sec.TheprocessofwesternblotwasasdescribedinChapter2.6.1.
peIF2α
peIF2α
71
5.3.3 Measuring the rate of phosphorylation on 4EBP1 in C2C12 myotubes
InChapter3and4, thechanges intotalproteincontentandproteinturnoverinC2C12
myotubes in response to catalase and glucose oxidase treatment suggest the
phosphorylation on 4EBP1 may change in response to different levels of hydrogen
peroxide.Totestthis,thesemyotubesweretreatedwithcatalaseandglucoseoxidasein
serum‐starvedconditions for48hrand the levelsofphosphorylated4EBP1, the total
4EBP1, and the total Akt were assessed. The phosphorylation on 4EBP1 was not
significantlychangedineitherthecatalase‐treatedorglucoseoxidase‐treatedmyotubes
(Fig.5.6).
Figure5.64EBP1phosphorylationinC2C12myotubesaftercatalaseandglucoseoxidasetreatment
Myotubes(7‐day‐old)wereleftuntreatedortreatedwithcatalase(3000units/ml;+Cat.),orglucoseoxidase (10 munits/ml; +GluO.) for 48 hr in serum‐starved conditions. Protein samples werecollectedasdescribedinChapter2.2.2,quantifiedasdescribedinChapter2.3.1,andtheprocessofwestern blot was as described in Chapter 2.6.2. (A) Phosphorylated 4EBP1(Thr37/46) and total4EBP1with totalAktusedas the loadingcontrol.Themembrane imagehadbeencroppedtoonlyshow the relevant treatments. (B)Phosphorylation levels.Thisdata representsanaverageof fourexperimentsforeachtreatmentgroup(twodishes/treatmentgroup)andisshownasmean±SEM.
5.3.4 Measuring eIF2α phosphorylation in C2C12 myotubes
As the eIF2α pathway is another pathway that may modulates the rate of protein
synthesis, the myotubes were treated with catalase and glucose oxidase in serum‐
starved conditions for 48 hr, then harvested, and quantified. Western blotting was
undertaken forphosphorylatedeIF2α, totaleIF2α, and totalAkt.Thephosphorylation
0
2
4
6
8
10
12
14
16
18A
B
72
on eIF2αwas significantly increased in these C2C12myotubes in response to catalase
andglucoseoxidasetreatment(Fig.5.7).
Figure5.7eIF2αphosphorylationinC2C12myotubesaftercatalaseandglucoseoxidasetreatment
Myotubes(7‐day‐old)wereleftuntreatedortreatedwithcatalase(3000units/ml;+Cat.),orglucoseoxidase (10 munits/ml; +GluO.) for 48 hr in serum‐starved conditions. Protein samples werecollectedasdescribedinChapter2.2.2,quantifiedasdescribedinChapter2.3.1,andtheprocessofwestern blotwas as described in Chapter 2.6.2. (A) Phosphorylated eIF2α(Ser51) and total eIF2αwith total Akt as the loading control. The membrane image had been cropped to only show therelevanttreatments.(B)Phosphorylationlevels.Thisdatarepresentsanaverageoffourexperimentsforeachtreatmentgroup(twodishes/treatmentgroup)andisshownasmean±SEM.
5.4 Discussion
Although protein synthesis is known to be modulated by signaling pathways, how
catalaseandglucoseoxidasemodulatechangesinproteinsynthesisisunknown.Inthe
present chapter, the impact of catalase and glucose oxidase on signaling pathways in
C2C12myotubeswasexaminedwithwesternblotting.
Phosphorylation of 4EBP1 was not found to be significantly changed by catalase or
glucose oxidase treatment. This suggests that 4EBP1 might not be involved in the
changesinproteinsynthesisinducedbycatalaseandglucoseoxidaseinC2C12myotubes.
This isoncontrasttopreviousstudiesinothercelltypesthathaveshownthat4EBP1
phosphorylation decreases in response to increased hydrogen peroxide (Patel et al.
2002; Zhang et al. 2009; Chen et al. 2010; Wu et al. 2010; Emara et al. 2012).
73
OtherstudiessuggestthattheeIF2αpathwayisresponsibleforthechangesinducedby
hydrogen peroxide in various cells (O'Loghlen et al. 2003; Shenton et al. 2006;
Mascarenhas et al. 2008; Wu et al. 2010). In C2C12 myotubes, we found the rate of
phosphorylation on eIF2α was increased significantly in response to catalase and
glucose oxidase. This may account for the changes in protein synthesis in C2C12
myotubesinresponsetocatalaseandglucoseoxidaseinChapter4.
In addition to the impact that hydrogen peroxide has on protein content, protein
turnoverandsignallingpathways, thisROShasbeenshown toenhanceor inhibit the
formation of disulfide bonds in select proteins in a dose‐dependent manner. These
disulfide bonds are formed while the thiol (–SH) groups of redox sensitive cysteine
residuesareoxidizedand leads to changes inprotein function (Bienert et al. 2006; Rhee
2006),however,howthioloxidationchangesinresponsetocatalaseandglucoseoxidase
treatmentisunknown.InChapter6,toinvestigatechangesinthioloxidation,amethod
tomeasurechangesinthiolgroupsinC2C12myotubesisdeveloped.
74
Chapter 6: Development of a method to measure thiol oxidation in C2C12 myotubes in response to treatment with catalase and glucose oxidase
6.1 Introduction
Changes in total protein content (Chapter 3), protein turnover (Chapter 4) and the
eIF2αpathway(Chapter5)wereobservedinC2C12myotubesaftercatalaseandglucose
oxidasetreatment.Themechanismsthatinducedthesechanges,however,arenotclear
butmayinvolvethioloxidationinresponsivetohydrogenperoxideexposure(Baty et al.
2005).
Thiols contain sulfhydryl (‐SH) groups that are readily oxidized to form stable
disulphide bonds and are important in cellular antioxidant defences and redox
signalling (Mulier et al. 1998; Baty et al. 2005; Terrill et al. 2013). Previous studies suggest
these thiol groups may be oxidized in response to hydrogen peroxide. In Jurkat T‐
lymphocytecells,structuralchangesinsomethiolgroup‐containingproteinshavebeen
observed in response to 200 µM hydrogen peroxide (Baty et al. 2005). Similar results
havebeenfoundinarangeofcells,suchasyeastcells(Delaunay et al. 2000; Imlay 2008).
Giventheapparentimportanceofhydrogenperoxideincellfunction,thepresentstudy
set out to adapt amethod established to investigate changes in the thiol oxidation of
total protein, myosin and actin in muscle tissue (Armstrong et al. 2011) to C2C12
culturedcells.
6.2 Methods
AllmethodsaredescribedindetailinChapter2.
Myotubecultures
The myotubes were cultured in 35mm petri dishes. In the preliminary experiments
which aimed to optimize precipitation efficiencywith ethanol or acetone, onedish of
untreated and catalase‐treated myotubes were used. In all subsequent experiments,
three petri dishes per treatment for every treatment group (untreated, catalase, and
glucose oxidase) were tested. One dish was used for protein quantification and the
75
remainingtwodisheswereusedfor2taglabeling.Theseexperimentswererepeatedup
tofourtimesusingfreshC2C12cultures.
Proteinextraction
Trichloroaceticacid(TCA)wasusedtoprotonateallthiolsandtoprecipitatethecellular
proteins to prevent their subsequent oxidation (Aslund et al. 1999; Delaunay et al. 2000).
To extract proteins from themyotubes, the petri dishes containingmyotube cultures
were washed briefly twice with 1ml phosphate buffered saline (PBS). 20 %
TCA/acetone (w/v) (700 µl) was then added to the petri dishes and the cells were
harvestedwithacellscraper.TheTCA/acetoneandcellsweretransferredtoa1.5ml
microcentrifugetube.Onesamplewasthencentrifugedat10000rpmfor5min(4°C)in
preparation for protein quantification. The supernatantwas then discharged and the
protein pelletwaswashedwith cold acetone (1ml). The centrifugation andwashing
steps were repeated to remove any residual TCA. The protein pellet was then
suspended in Tris buffer (300µl, 50mMTriswith 0.5% SDS, pH7.0) and quantified
usingthemicroBCAassay.Thesamplesfor2taglabelingwereleftinTCA/acetone.
Proteinquantification‐microBCAassay
ThecommercialkitfromSigma(QuantiPro™BCAAssayKit,QPBCA‐1KT)wasusedto
quantifythetotalproteincontentforeachtreatmentgroup.1µloftheproteinsample
wasdiluted25‐foldwithSDSbuffer(0.5%SDS), followedbyanother10‐folddilution
withTrisbuffer(2mMTriswith0.5%SDS,pH7.0)ina1.5mlmicrocentrifugetube.250
µl of the cocktail reagent (A:B:C=25:25:1) was added into each diluted sample and
incubated for1hrat60°C.The incubatedsolution (100µl)was then transferred toa
384‐wellplateandtheabsorbancewasanalyzedat562nminaplatereader(BioTek,
PowerWaveHT).
2taglabeling
76
Samplesfor2taglabelingwereleftin20%TCA/acetone,andsonicatedat40%Amps
for 2 min on ice. 100 µg of the protein pellet was then transferred to a 1.5 ml
microcentrifugetube.TheexcessTCAwasremovedfortheprocessoflabelling.
AfterremovalofTCA,theproteinsampleswerelabelledwithfirsttag,BODIPYFL‐N‐(2‐
aminoethyl)maleimide(FLm,Invitrogen,B10250).Thistaglabelsthereducedformof
inproteinsamples.Toperformthislabelling,50µlofTrisbuffer(0.5MTriswith0.5%
SDS,pH7.3)with5µlofFLm(5mM)wasaddedtotheproteinpellet,whichwasthen
got sonicatedat40%Amps for1minon ice.The resulting labelledsamplewas then
vortexed and incubated at room temperature for 30 min in dark. Excess FLm was
removed by two rounds of precipitation. Each time, 200 µl of ice‐cold acetone was
addedto thesample followedby incubationovernightat ‐20°Candcentrifugation the
followingmorning.Theresultingproteinpelletwas thenresuspended in50µlofTris
buffer(0.5MTriswith0.5%SDS,pH7.0).
Theoxidizedthiolswerethenlabelledwiththesecondtag,Texasredmaleimide(TRm,
Invitrogin,T6008).Toperformthislabelling,21µlofFLm‐labelledproteinsamplewas
then taken andmixed with 4 µl of TCEP (25 mM) in a 0.6 ml microcentrifuge tube,
followedbyincubationatroomtemperaturefor1hrinthedark.Thesampleswerethen
mixedwith25µlTrisbuffer(0.5MTriswith0.5%SDS,pH7.0)and5µlofTRm(5mM),
vortexedbrieflyand incubatedatroomtemperature for1hr in thedark.ExcessTRm
wasremovedbytheapplicationof220µlice‐coldacetoneandincubationovernightat‐
20°C.Theproteinpelletwascentrifuged,resuspendedwith25µlTrisbuffer(0.5MTris
with0.5%SDS,pH7.0)thenprecipitatedagainovernightwith100µlofice‐coldacetone
at‐20°.Thiscentrifugation,resuspension,andprecipitationstepwasrepeatedandthe
resulting2taglabelledproteinpelletwasthenresuspendedin50µlofTrisbuffer(0.5
MTriswith0.5%SDS,pH7.0).
77
Proteinquantification‐DCassay
TheDCassaykit(Bio‐Rad,500‐0112)wasusedtoquantifytheproteincontentofthe2
taglabelledproteinsamples.Trisbuffer(0.25MTris,0.25%SDS,pH7.0)wasusedas
theassaybuffer.7.5µlofeachproteinsamplewasdilutedtwo‐foldwithTrisbuffer(0.5
MTriswith 0.5% SDS, pH7.0) followed by a further two‐fold dilutionwithDDi. The
sampleswerethentreatedaccordingtothekitinstructionsusingthesuppliedworking
reagents. The final product was aliquoted in triplicate (100 µl/well) into a 384‐well
plateandtheabsorbancewasanalyzedat750nminaplatereader(BioTek,PowerWave
HT).
FLmandTRmquantification
The rate of thiol oxidation was measured using a fluorescent assay and gel
electrophoresis.
Theanalysisof fluorescentmeasurementwasstandardizedtoFLmandTRmstandard
curves. The standard curve for FLm was prepared from 0 to 60 µM with 60 µM
FLm/ovalbumin stock solution. For TRm, it was prepared from 0 to 7.5 µM with
TRm/ovalbuminstocksolution.Allpreparedstandardswerediluted10‐foldwith0.1M
NaOHand10µlofproteinsampleswerediluted32‐foldwith0.1MNaOH.Thediluted
standards and protein sampleswere aliquoted in triplicate (100µl/well) into a 384‐
wellplate.Thefluorescenceofeachsamplewasthenmeasuredusingafluorescentplate
reader (Fluostar Optima) with wavelengths set at 485 nm excitation and 520 nm
emissionforFLmand595nmexcitationand610nmemissionforTRm.
ThegelanalysiswasperformedbyelectrophoresiswithprecastgelsfromBio‐Rad.To
quantify the reduced and oxidized thiols of specific protein, in‐gel Flm and TRm
standardswerepreparedas for the fluorescentplate readermeasurement.The in‐gel
FLmstandardswerepreparedfrom0to0.024nmol,and0to0.006nmolforin‐gelTRm
standards. After electrophoresis, the fluorescence of each lanewasmeasured using a
typhoongelscanner(GEHealthcareLifeScience,TyphoonTrio)withwavelengthssetat
520nmforFLmand610nmforTRm.FollowinggelanalysisusingtheImageJsoftware,
78
the amount of reduced and oxidized thiols in specific protein was determined with
referencetotheFLm/TRmstandardcurves.
6.3 Results
6.3.1 Optimize the 2 tag method for C2C12 myotubes model
An existing method developed in our laboratory for 2 tag labelling in muscle tissue
(Armstrong et al. 2011)wasadaptedtosuittheC2C12tissueculturesamples.Thismethod
requires100µgofmyotubeprotein.Whilemusclesamplescanbeeasilyweighed,itis
difficulttoweighmyotubetissueculturesamples.Astheproteinlevelswithinthesame
treatmentgroupswerefoundtobesimilarinpreviousexperiments(seeChapter3),it
wasassumedthatproteinamountswouldbeapproximatelythesamewithinthesame
treatmentgroupsand the samples couldbeweighed inTCA/acetone.Therefore, after
protein quantification of one representative myotube sample, the remainder of the
samplesweresonicatedinTCA/acetoneat40%Ampsfor2minonicethenthevolume
representing100µgofsamplewasaliquotedintoafreshtube.ResidualTCAwasthen
removedfromthis100µgsamplebywashingtwicewith300µlice‐coldacetone.
Accordingtotheoriginalprotocol,theproteinpelletshouldbeprecipitatedrepeatedly
withice‐cold(‐20°C)ethanolafterFLmandTRmlabelling.Totestiftheproteinsamples
fromthemyotubescouldbeprecipitatedusinganethanolsolvent, twoseparated100
µgproteinaliquotsweretakenfromonesample,centrifuged,resuspendedwith50µlof
Trisbuffer(0.5MTriswith0.5%SDS,pH7.3)andthenprecipitatedwitheitherice‐cold
(‐20°C) ethanol or acetone. As shown in Fig. 6.1, therewas a 50%protein losswith
ethanolprecipitationbut little losswithacetoneprecipitation.Therefore,acetonewas
usedforprecipitationinallsubsequentexperiments.Ashigherproteinconcentrations
were believed to increase precipitation efficiency based on precipitation kinetics
(Devidal et al. 1997), the protein pellet was resuspended in lower amounts of Tris
buffer than original protocol recommended. The labelled myotube protein samples
were suspended with 25 µl of Tris buffer during the removal of excess TRm and
resuspendedwith50µlofTrisbufferforthefinalanalysis.
79
Figure6.1Totalproteinlevelsafterprecipitationwithethanolandacetone100µgsamplesfromC2C12myotubesweresuspendedin50µlofTrisbuffer(0.5MTriswith0.5%SDS,pH7.3),sonicated,andprecipitatedwithice‐coldethanolorice‐coldacetoneovernightat‐20°C.ProteincontentwasthenmeasuredusingthemicroBCAassay.
6.3.2 Measuring total thiol oxidation in fluorescently labeled C2C12 myotubes using a fluorescent plate reader
InChapter3 to5, the changes inC2C12myotubes in response to catalase andglucose
oxidasetreatmentsuggestthatthioloxidationmaybechangedinresponsetodifferent
levelsofhydrogenperoxide.Totestthis,thesemyotubesweretreatedwithcatalaseand
glucose oxidase in serum‐starved conditions for 48 hr and the fluorescence of the
labelledsampleswasassessed.Thethioloxidationintotalproteinwasnotsignificantly
changedineitherthecatalase‐treatedorglucoseoxidase‐treatedmyotubes(Fig.6.2).
0
20
40
60
80
100
Ethanol Acetone
Total protein
(µg/sample)
Precipitation solvent
80
Figure6.2ThioloxidationinC2C12myotubesinresponsetocatalaseandglucoseoxidasetreatment
Myotubes(7‐day‐old)wereleftuntreatedortreatedwithcatalase(3000units/ml;+Cat.),orglucoseoxidase (10 munits/ml; +GluO.) for 48 hr in serum‐starved conditions. The fluorescence wasmeasuredafterlabellingthethiolsintheproteinsamples.Thisdatawasanaverageof4experimentsfor each treatment group (2 dishes/treatment group) and was shown as mean ± SEM. Proteinsampleswerecollectedasdescribed inChapter2.2.1and labelledasdescribed inChapter2.7.Thelabelled protein content was quantified as described in Chapter 2.3.3 and fluorescence wasmeasuredasdescribedinChapter2.7.3.
6.3.3 Measuring thiol oxidation in C2C12 myotubes on actin and myosin by gel electrophoresis
Althoughthethioloxidationwasnotsignificantlychangedintotalprotein,itislikelyto
havesignificantchangesinspecificproteinssuchasmyosinandactin.Totestthis,the
labelledproteinsampleswereseparatedbyelectrophoresisandthegelswerescanned
usingatyphoonfluorescencescanner.AsshowninFig.6.3and6.4,thethioloxidations
of myosin and actin were not significantly changed in either the catalase‐treated or
glucoseoxidase‐treatedmyotubes.
26.5
27.0
27.5
28.0
28.5
29.0
29.5
30.0
30.5
31.0
31.5
32.0
Untreated +Cat. +GluO.
Thiol oxidat
ion
(%)
81
Figure6.3ThioloxidationonmyosininC2C12myotubesinresponsetocatalaseandglucoseoxidasetreatment
Myotubes(7‐day‐old)wereleftuntreatedortreatedwithcatalase(3000units/ml;+Cat.),orglucoseoxidase (10 munits/ml; +GluO.) for 48 hr in serum‐starved conditions. Protein samples werecollectedasdescribedinChapter2.2.1andlabelledasdescribedinChapter2.7.3µglabelledproteinwasloadedtothegelandthefluorescencewasthendetectedwithtyphoonfluorescencescanner.(A)Imageof scanned gel. (B)Average thiol oxidationonmyosin of 4 experiments for each treatmentgroup (2 dishes/treatment group) represented asmean ± SEM. The labelled protein contentwasquantified as described in Chapter 2.3.3 and fluorescencewasmeasured as described in Chapter2.7.4
Figure6.4ThioloxidationonactininC2C12myotubesinresponsetocatalaseandglucoseoxidasetreatment
Myotubes(7‐day‐old)wereleftuntreatedortreatedwithcatalase(3000units/ml;+Cat.),orglucoseoxidase (10 munits/ml; +GluO.) for 48 hr in serum‐starved conditions. Protein samples werecollectedasdescribedinChapter2.2.1andlabelledasdescribedinChapter2.7.3µglabelledproteinwasloadedtothegelandthefluorescencewasthendetectedwithtyphoonfluorescencescanner.(A)Imageof scanned gel. (B)Average thiol oxidationonmyosin of 4 experiments for each treatmentgroup (2 dishes/treatment group) represented asmean ± SEM. The labelled protein contentwasquantified as described in Chapter 2.3.3 and fluorescencewasmeasured as described in Chapter2.7.4
BA
0
5
10
15
20
25
30
35
40
45
50
Untreated +Cat. +GluO.
Thiol o
xida
tion
(%)
BA
19.0
19.5
20.0
20.5
21.0
21.5
22.0
22.5
23.0
Untreated +Cat. +GluO.
Thiol o
xida
tion
(%)
82
6.4 Discussion
Afteradaptingamethod tomeasure thiol oxidation inmuscle tissue tomeasure thiol
oxidationinC2C12cellcultures,itwasfoundthattotalthioloxidationandthioloxidation
of myosin and actin were not significantly changed by catalase or glucose oxidase
treatmentof thesemyotubes.This is incontrast topreviousstudiesof increasedthiol
oxidation of proteins from yeast cells and human umbilical vein endothelial cells in
responsetohydrogenperoxidetreatment(Delaunayetal.2000;Imlay2008).However,
anotherstudythatsubsequentlyusedthismethodhasbeenabletodetectanincreasein
thioloxidationinC2C12myotubesinresponsetooxidanttreatmentusingdiamide(Tan
et al. 2015), suggesting this system is capable of measuring thiol oxidation in C2C12
myotubes.
Thedurationofoxidanttreatmentisthoughttobethepossiblereasonfortheabsence
ofthioloxidationobservedinpresentstudy.Inyeastcells,thioloxidationwasobserved
aftertreatingthecellswithhydrogenperoxidefor2.5min,butwasnotevidentafter1
hr hydrogen peroxide treatment (Delaunay et al. 2000). In A548 cells treated with 0.1
mMhydrogen peroxide, an initial decrease in intracellular non‐protein thiols (NPSH)
wasobservedfrom0‐2hrandfollowedbyasubsequentrecoveryby8hrofhydrogen
peroxidetreatment.ThesechangesinNPSHwereattributedtothioloxidationsincethe
levelofNPSHincreasedwithantioxidanttreatment(2mMNAC)forupto2hr(Mulier et
al. 1998).
Inthepresentstudy,themyotubesweretreatedwithcatalaseandglucoseoxidasefora
period of 48 hr to investigate longer term down‐stream effects, like changes in the
signallingpathways.Astheeffectsofthesetreatmentsonthioloxidationmayhavebeen
transient and early in treatment, this study may not have been able to detect them.
StudiesofC57BL/6Jfemalemicehaveshownthatage‐relatedthioloxidationcannotbe
detectedingastrocnemiusmusclesofthesemicefrom3‐29monthsofage(Tohmaetal.
2014),furthersuggestingthattheeffectsofhydrogenperoxideonmuscleproteinthiols
maybyearlyand transientandunlikely tobedetected in longer‐termmodels.Future
studies could focus on assessing thiol oxidation in these myotubes over a range of
shorter,morefrequenttimepoints.
83
Chapter 7: General discussion
7.1 Introduction
Oxidativestressisdefinedasanimbalancebetweenthegenerationofreactiveoxygen
species (ROS) and a reduction in protective mechanism such as antioxidase activity.
This imbalance leads to damage in biomoleculeswith potential impact on thewhole
organism(Reuteretal.2010).ProteinsinparticularareeasilyattackedbyROSresulting
in changes in structure and enzyme activity. Oxidative stress can also affect the
activation of transaction factors, can alter signalling pathways and can damage
membranes(Klaunigetal.1998).
Oxidative stress and oxidative damage to tissues are thought to play a key role in a
range of chronic diseases and conditions including cancer, diabetes, and ageing
(Jefferson1980;Rooyackersetal.1996;Aruoma1998;Wei1998;Baynesetal.1999;
Brownetal.2001;Atalayetal.2002;Ryazanovetal.2002;Martinez‐Vicenteetal.2005;
Valko et al. 2006). In cancer cells, an increase in the generation of ROS enhances
metabolicstressandproliferativecapacity(Chenetal.2008;Reuteretal.2010).Inboth
typesofdiabetes,elevatedglucose levels induce thegenerationofmitochondrialROS,
nonenzymatic glycation of protein, and glucose autooxidation (Evans et al. 2002;
Maritim et al. 2003; Robertson 2004; Rolo et al. 2006; Scheuner et al. 2008). The
generationrateofhydrogenperoxidefrommitochondriaisincreased(Sohaletal.1996;
Wei et al. 2002) inducing the accumulation of irreversibly modified proteins
(Tavernarakis 2008). These oxidized proteins have been implicated in a number of
agingprocessesanddiseases,mostnotablyAlzheimer’sdisease(Klaunigetal.2010).
Inthisstudy,theresponsesofC2C12myotubestocatalaseandglucoseoxidasetreatment
wereassessed to investigate themechanismsofmusclewasting inducedbyoxidative
stress.
7.2 Muscle wasting
Musclewasting involves lossofmuscleproteinmass and function.Cachexia iswidely
recognized as severe wasting accompanying disease states. Up to 80% of advanced
cancer patients have cachexia and 20 % of cancer‐related deaths are thought to be
linkedtothiscondition(Glassetal.2010;Tazietal.2010;Mathew2011;Silverioetal.
84
2011;Wang et al. 2011;Wysong et al. 2011). In elderlywithundiagnosed/diagnosed
diabetes,declines inmusclemassandtotalbodymasshavealsobeenassociatedwith
cachexia(Parketal.2007;Parketal.2009).Sarcopenia,isthelossofmusclemasswith
ageingandcausesdiminishedstrengthandexercisecapacity (Phillips et al. 2005; Lenk et
al. 2010).
Sinceoxidativestressisthoughttoinducetheselossesinmusclemass(Lassetal.1998;
Mantovanietal.2002;Mantovanietal.2002;Capeletal.2005;Mansourietal.2006;
Lenketal.2010;Silverioetal.2011),itwashypothesizedthatthetotalproteincontent
in C2C12myotubeswould change in response to changes in hydrogen peroxide levels
modulatedbycatalaseandglucoseoxidasetreatment.Overall,thetotalproteincontent
was increasedwith catalase treatment for 72 hr and decreasedwith glucose oxidase
treatment for 48 hr (Fig. 3.6). These findings validated the use of C2C12model for
investigatingthemechanismsthatunderlymyotubeproteinresponsestocatalaseand
glucoseoxidasetreatment.
7.3 Protein turnover
Animbalancebetweenproteinsynthesisandproteindegradationleadstoadecreasein
totalproteincontentandresultsinmusclewasting(Balagopaletal.1997;Evans2010).
Incachexiapatients,abnormalitiesinproteinmetabolismhavebeenlinkedtodecreases
in protein synthesis and increases in protein degradation in skeletalmuscle (Tisdale
2001).Otherstudiesofcancerpatientswithcachexiahavefoundthatproteinsynthesis
issignificantlydecreased(Emeryetal.1984;Smithetal.1999)andthishasalsobeen
observedindiabetespatientsandelderlypatientswithmusclewasting(Millwardetal.
1976;Jefferson1980;Gelfandetal.1987;Rooyackersetal.1996;Balagopaletal.1997;
Anthony et al. 2002). Increases in protein degradation are also observed in cachexia
patientswithcancerordiabetes(Gelfandetal.1987;Smithetal.1999;Tisdale2001;
Bachetal.2005),andactinandmyosinhavebeenfoundtobeselectivelytargetedfor
degradationincachexiapatients(Evans2010).
Basedon theseprevious studiesand the changesof totalproteincontentobserved in
this study in response tocatalaseandglucoseoxidase treatment, itwashypothesized
that catalasewould increaseprotein synthesis anddecreaseproteindegradation, and
glucoseoxidasewoulddecreaseproteinsynthesisandincreaseproteindegradationin
85
C2C12myotubes.However,asdescribedinChapter4,thelevelofproteinsynthesiswas
significantlydecreasedinresponsetocatalasetreatmentandtherewaslittlechangein
proteinsynthesisafterglucoseoxidasetreatment(Fig.4.4).Inaddition,whiletherateof
proteindegradationwassignificantlydecreasedinresponsetocatalasetreatment,there
waslittlechangeafterglucoseoxidasetreatment(Fig.4.5).
7.4 Signalling pathways
Previousstudieshaveshownthattheregulationofproteinsynthesisinskeletalmuscle
occurs primary at the initiation phase of protein translation and the initiation factor
eIF4E and eIF2 are involved in this regulation (Syntichaki et al. 2006; Tisdale 2009).
eIF4E, as described in Chapter 1, regulates protein synthesis via its reversible
associationwith4E‐bindingproteins,including4EBP1,throughthemTORpathway.The
protein synthesis is increased when 4EBP1 is phosphorylated, and a decrease in
phosphorylationof4EBP1hasbeen linked toageing, canceranddiabetes (Shahet al.
2000;Syntichaki et al.2006;Armengol et al. 2007;Eleyet al. 2007;Drummondet al.
2008;Handsetal.2009).
Basedonthesestudies,thechangesintotalprotein(Chapter3)andinproteinsynthesis
(Chapter4)observedinthisstudy,itwashypothesizedthatphosphorylationof4EBP1
would decreasewith both catalase and glucose oxidase treatment in C2C12myotubes.
However, phosphorylation of 4EBP1 was not found to be significantly changed with
eithercatalaseorglucoseoxidasetreatment(Fig.5.6),suggestingthat4EBP1mightnot
beinvolvedinproteinsynthesischangesinresponsetocatalaseandglucoseoxidase.
eIF2,asdescribedinChapter1,decreasesproteinphosphorylationbyphosphorylation
of its α subunit through the PERK/eIF2α pathway. As a link between eIF2α
phosphorylationandweightlosshasbeenobservedincanceranddiabetesstudies(Kim
etal.2000;Hardingetal.2001;Ozcanetal.2004;Eleyetal.2007;Scheuneretal.2008),
itwashypothesizedthateIF2αphosphorylationinC2C12myotubeswouldincreaseafter
catalase and glucose oxidase treatment. As expected, increases in eIF2α
phosphorylationwereobservedforbothtreatments(Fig.5.7),suggestingthateIF2α is
involvedintheregulationofproteinsynthesisinC2C12myotubes.
86
7.5 Thiol oxidation
Proteins can respond to ROS in different ways, including the formation of disulfide
bonds from thiol groups containing cysteine residues (Atalay et al. 2002; Poon et al.
2004;Valkoetal.2006).The formationof thesedisulfidebondsare thought toplaya
key role in cancer (Toyokuni et al. 1995;Klaunig et al. 1998;Kumar et al. 2008) and
bothtypesofdiabetes(Maritimetal.2003;Robertson2004;Roloetal.2006).
As significant changes on level of total protein content, protein turnover, and eIF2α
pathwaywereobservedinC2C12myotubesinresponsetocatalaseandglucoseoxidase
treatmentinthepresentstudy,itwashypothesizedthatthioloxidationwoulddecrease
with catalase treatment and increasewith glucose oxidase treatment. However, thiol
oxidation was not found to be significantly changed by either catalase or glucose
oxidase. Subsequent studies undertaken in this laboratory support this observation
(Tohmaetal.2014;Tanetal.2015).
7.6 Future studies
Overall, thisstudyshowsthattheC2C12myotubemodelcanbeusedtoinvestigatethe
relationship between oxidative stress andmuscle wasting. Changes in protein levels,
protein synthesis, protein degradation and eIF2α phosphorylation were observed in
responsetocatalaseandglucoseoxidasetreatmentinthesemyotubes,butnochanges
werefoundinthioloxidationinthecurrentstudy.Theextendeddurationoftreatment,
chosentoexplorethelong‐termdown‐streameffectsofchangesinhydrogenperoxide
levels, might be the reason why thiol oxidation was not detected. Further studies
focused on earlier and more frequent sampling of the cultures may give a better
indicationoftheroleofthioloxidationinthisprocess.
While it is unknown if exogenous enzymes can be taken up by C2C12 myotubes, the
changes observed in total protein levels, protein turnover and signalling pathway
observed in this study suggest glucose oxidase and catalase mediate these changes,
probablyaftercellularuptakefromthemedia.Futurestudiescouldalsobeundertaken
tocompareintracellularandextracellularlevelsofhydrogenperoxidefollowingglucose
oxidase and catalase treatment to confirm the changes observed were a result of
intracellularhydrogenperoxideproduction(Beersetal.1952;Picketal.1980;Picket
87
al. 1981). Hydrogen peroxide could potentially be used as a control, although direct
application is likely to cause significant cell death. Alternatively, uptake of these
enzymescouldbemonitoredbypackagingtheminfluorescentlylabellednanoparticles
suchasliposomes.However,previousresearchinourlaboratoryandothershasshown
the activity of enzymesdecreases in a time‐dependentmanner (Tse et al. 1987;Kho
2010). Therefore, in addition of being time‐consuming and difficult, the expected
reduction in enzyme activity may also make the results of this potential approach
difficulttointerpret.
Further studiesmay also focuson theotherpossibleprotein target sites thatmaybe
affectedbyoxidativestress,suchasthebackboneandothersidechains.Ultimately,itis
hoped that a better understanding of the mechanisms underlying muscle wasting
inducedbyoxidativestresswillleadtonewtherapiesformusclewasting.
88
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Appendices
Posterpresentedat21stAnnualCombinedBiologicalSciencesMeetings,TheUniversity
Club, University of Western Australia, 26th August 2011.
114
PosterpresentedatJointAuPS/ASCEPT/HBPRCAMeeting,PerthConventionExhibition
Centre(PCEC),4th‐7thDecember2011.
115
Poster presented at Development, Function and Repair of the Muscle Cell, Kimmel
Center, New York University, New York, NY, USA, 4th ‐8th June 2012.