CHMI 4237 E Special topics in Biochemistry

CHMI 4237 ECHMI 4237 E

Special topics in Special topics in BiochemistryBiochemistry

Eric R. Gauthier, Ph.D.Dept. Chemistry-Biochemistry

Laurentian University

Stress signaling

1CHMI 4237 E - Winter 2010

2

Cell stressesCell stresses

CHMI 4237 E - Winter 2010

Chemicals

Radiation

Osmotic pressure

Nutrient

Heat

Viruses

Hypoxia

MechanicalOxidative

3

Cellular responses to stressCellular responses to stress


?

Cell cyclearrest

Death

Adaptation

4



?

Cell cyclearrest

Death

Adaptation

- Metabolic rewiring

- Differentiation

- Plasticity

- Motility

- Defense

- Aging

5



Stress Adaptation Example

Oxidative stress Metabolic rewiringIncrease in NAPH-producing pathways

Hypoxia DifferentiationIncrease in red blood cells

Increased exercise PlasticityChange in muscle fiber type

Wound Motility Wound closure

Foreign RNA Translation arrest Viral infection

Food shortage Lifespan increase Caloric restriction

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1. Response to nutrient starvation1. Response to nutrient starvation


Food shortage

Cell cycleProtein synthesis

Alternative food sourceOR

Commit suicide

7

1. Response to nutrient starvation1. Response to nutrient starvation Blocking ATP-vorous processes Blocking ATP-vorous processes


http://8e.devbio.com/image.php?id=187


http://8e.devbio.com/im

age.php?id=188


http://8e.devbio.com/im

age.php?id=188

10

eIF2 phosphorylation leads to eIF2 phosphorylation leads to translation arresttranslation arrest


http://deverlab.nichd.nih.gov/graphics/fig3a.gif

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GCN2 – linking amino acid GCN2 – linking amino acid availability with translationavailability with translation


http://deverlab.nichd.nih.gov/sub_rec.htm

The EMBO Journal Vol. 20 No. 6 pp. 1425-1438, 2001

12

ATF4 translation is INCREASED ATF4 translation is INCREASED when eIF2 is phosphorylated…when eIF2 is phosphorylated…


Trends in Endocrinology & MetabolismVolume 20, Issue 9, November 2009, Pages 436-443

uORF = - upstream open reading frame (i.e. upstream of the ATF4 ORF)- uORF1 encodes for a 3-amino acid protein- uORF2 overlaps with the ATF4 ORF but is out of frame.

13

ATF4 translation is limited when ATF4 translation is limited when amino acids are plentyfulamino acids are plentyful



14

ATF4 translation is limited when ATF4 translation is limited when amino acids are plentyfulamino acids are plentyful



15

ATF4 gene targetsATF4 gene targets



TranscriptionInitiation machinery

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ATF4 gene targetsATF4 gene targets


Tren

ds in

En

do

crino

log

y & M

etabo

lismV

olume 20, Issue 9, N

ovember 2009, P

ages 436-443

Gene Function

ASNS Amino acid synthesis

SNAT2CAT-1

Amino acid transporters

4EBP-1 Translation arrest

ATF3CHOP

Transcription factors

TRB3 AKT inhibitor

17

GCN2/eIF2 and feeding behaviorGCN2/eIF2 and feeding behavior


Mo

lecu

lar

Cel

l, V

ol.

18, 1

41–1

48, A

pri

l 15,

200

5

mTOR: the art of sensing mTOR: the art of sensing nutrientsnutrients

mTOR:◦ Protein serine kinase◦ Same family as ATM

Inhibited by Rapamycin◦ Rapamycin: secreted by

Streptomyces hygroscopicus◦ From Rapa Nui (aka Easter Island)

Senses:◦ Glucose◦ Amino acids◦ Energy levels

Triggers: ◦ AKT activation◦ Translation◦ Proliferation

CHMI 4237 E - Winter 2010 18

HEAT repeats: protein interaction domainFAT domain: domain structure shared by FRAP, ATM and TRRAP

NA

TU

RE

RE

vIEW

S | M

olecu

lar cell Bio

log

y vOlU

ME

10 | MA

y 2009 | 307

mTORC1: targeting translationmTORC1: targeting translation

CHMI 4237 E - Winter 2010 19

Oncogene (2006) 25, 6416–6422

mTORC1: targeting translationmTORC1: targeting translation

CHMI 4237 E - Winter 2010 20

NATURE REvIEWS | Molecular cell Biology vOlUME 10 | MAy 2009 | 307

mTORC1

p70 S6K

S6 ribosomal

protein

eIF4B

↑ eIF4A helicaseactivity↑ translation

efficiency

mTORC1: wide ranging effectsmTORC1: wide ranging effects

CHMI 4237 E - Winter 2010 21

http://www.cellsignal.com/reference/pathway/mTor.html

PRAS40PRAS40

mTORC1 regulationmTORC1 regulation

CHMI 4237 E - Winter 2010 22

TSC 1/2: ◦ GTPase activating enzyme

targets Rheb◦ Mutated in patients with

Tuberous Sclerosis

Rheb: ◦ small G protein◦ In GTP-bound form: activates

mTOR

NATURE REVIEWS | DRUG DISCOVERY VOLUME 4 | DECEMBER 2005 | 989

mTOR

RaptorGL

AKTAKT

mTOR

RaptorGL

PRAS40PRAS40PO4

PRAS40◦ Binds mTORC and inhibits

it;◦ Dissociates form mTORC

upon phosphorylation by AKT

mTORC1 regulationmTORC1 regulation

CHMI 4237 E - Winter 2010 23http://www.lsi.umich.edu/files/ken_research_fig1.jpg


Glucose and mTORC1Glucose and mTORC1

CHMI 4237 E - Winter 2010 24


http://www.lsi.umich.edu/files/ken_research_fig1.jpg

When glucose in plenty…When glucose in plenty…

CHMI 4237 E - Winter 2010 25

ATP

GLUCOSEGLUCOSE

Glycolysis

Acetyl-CoA

TCA

ATP

NADH

CO2

Citrate

Fatty acids

ETC

ATP

Energyreserves

Glycogen

ATP ADP

Cellular processes

ETC

When glucose in scarce…When glucose in scarce…

CHMI 4237 E - Winter 2010 26

ATP

GLUCOSEGLUCOSE

Glycolysis

Acetyl-CoA

TCA

ATP

NADH

CO2

Citrate

Fatty acids

ETC

ATP

Energyreserves

Glycogen

Glutamine

But, how are glucose levels But, how are glucose levels sensed?sensed?

CHMI 4237 E - Winter 2010 27

ATP ADP

ADP KinaseADP Kinase

ADPAMP

But, how are glucose levels But, how are glucose levels sensed?sensed?

CHMI 4237 E - Winter 2010 28

ATP ADP

ADP KinaseADP Kinase

ADPAMP

AMPK

Adaptation

AMPK and mTORC1AMPK and mTORC1

CHMI 4237 E - Winter 2010 29

http://www.cellbiol.net/ste/alpobesity4.php

Other targets of AMPKOther targets of AMPK

CHMI 4237 E - Winter 2010 30

http

://w

ww

.cel

lsig

nal.c

om/r

efer

ence

/pat

hway

/AM

PK

.htm

l

Other targets of AMPKOther targets of AMPK- carbohydrate metabolism- carbohydrate metabolism

CHMI 4237 E - Winter 2010 31

Other targets of AMPKOther targets of AMPK- lipid metabolism- lipid metabolism

CHMI 4237 E - Winter 2010 32

Other targets of AMPKOther targets of AMPK- cell cycle- cell cycle

CHMI 4237 E - Winter 2010 33

Other triggers of AMPKOther triggers of AMPK

CHMI 4237 E - Winter 2010 34

Other triggers of AMPKOther triggers of AMPK- adrenalin- adrenalin

CHMI 4237 E - Winter 2010 35

Adrenalin



CHMI 4237 E - Winter 2010 36



CHMI 4237 E - Winter 2010 37


CHMI 4237 E - Winter 2010 38

Oth

er

trig

gers

of

Oth

er

trig

gers

of

AM

PK

AM

PK

- In

su

lin

- In

su

lin

http://stke.sciencemag.org/content/sigtrans/vol2006/issue346/im

ages/large/3462006re7F

1.jpeg

Amino acids and mTORAmino acids and mTOR

CHMI 4237 E - Winter 2010 39http://www.efdeportes.com/efd131/leucine-stimulates-mtor-01.jpg

Amino acids and mTORAmino acids and mTOR

CHMI 4237 E - Winter 2010 40

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2677387/pdf/nihms103321.pdf

Autophagy – cell cannibalismAutophagy – cell cannibalism

CHMI 4237 E - Winter 2010 41

nature cell biology volume 9 | number 10 | O CTOBER 2007 5 NOVEMBER 2004 VOL 306 SCIENCE

Autophagy – cell cannibalismAutophagy – cell cannibalism

CHMI 4237 E - Winter 2010 42Biochem. J. (2005) 391, 191–202

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Autophagy: autophagosome Autophagy: autophagosome formationformation

Autophagosome

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Atg9

9

Membrane transport?

1 13

1

Atg1/ULK1

13

Atg13

mTOR

Starvation

Vps34

Vps34

PI3P

PhagophoreNucleation

2

Atg2/18

Atg23/27

PhagophoreElongation

Atg16

Atg12 Atg5LC3-I

Atg10 GlyLC3-IIAtg4

Atg3

LC3-II

PtdE

Atg7

LC3 – a marker of autophagyLC3 – a marker of autophagy

CHMI 4237 E - Winter 2010 44

http://www.cellsignal.com/products/4108.html

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Autophagy: fusion with lysosomeAutophagy: fusion with lysosome

Autophagosome

Lysosome

Atg22

Cathepsins

H+

H+

ADP+ Pi

ATP

Autolysosome

Aminoacids

46

Autophagy - modulationAutophagy - modulation

Inhibitingautophagy

Lysosome

H+

H+

ADP+ Pi

ATP

Bafilomycin A

Chloroquine

Atg9

9

Lipid transport?

1 13

113

mTOR

Rapamycin

Increasing autophagy

PI3P

3-MA

Vps34

Beclin UVRAC

p150

Vps34

BeclinUVRAC

p150

Bcl2 Bcl2

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CHMI 4237 E Special topics in Biochemistry