SRCD Physiology Talk 2009

8/4/2019 SRCD Physiology Talk 2009

1/69

Health Innovations Research Institute,School of Medical Science, RMIT University

Biophysical aspects of albumin: implications to

renal physiology

or

The Application of SRCD to Physiology:

Reaching for the Higher-Hanging Fruit.

Dr Len Pattenden


2/69


Acknowledgements

Prof Philip Poronnik - RMIT (Project Leader)

Dr Greg Tesch - Monash (CIB)

Dr Darren Kelly - St Vincents

Dr David Nickolic-Paterson - Monash Medical Centre

Prof Giuliano Siligardi - Diamond & Liverpool, UK

Dr Rohanah Hussain - Diamond & Liverpool, UK

Assoc. Prof. Josephine Forbes - Baker IDI

Dr. Matthew A. Perugini - Bio21 Institute

Jarrod Voss - Bio21 Institute

Dr Frank Wien - Soleil, France


3/69

medulla

cortex

Kidney Glomerulus

Proximaltubule

distaltubule

Afferent

Efferent

Fenestrated

endothelium

Basementmembrane

Foot process

Podocyte

capillary

SlitDiaphragm


4/69

QuickTime and adecompressor

are needed to see this picture.

Fenestrated

endothelium

Basementmembrane

Glomerulus is size and charge selective barrier for filtration ofwater, ions, and low mw proteins but virtuallyimpermeable toplasma albumin (2-20mg/day).

Haraldsson et al., Properties of

the glomerular barrier and

mechanisms of proteinuria.

Physiol Rev 88:451-87, 2008.


5/69

QuickTime and adecompressorare needed to see this picture.

250nm

FP

GBM

E

150nm

SD

SD

10nm

PMPM

FP = Foot Process E = EndotheliumSD = Slit DiaphragmPM = Podocyte membrane

GBM = Glomerular BasementMembrane

If, SD is a size-selective filter, thenwhy does not clog?

-ve glycosaminoglycans GBM, PM

Lahdenkari et al., J Am Soc Nephrol 2004;15:2611-8. Wartiovaara et al., J Clin Invest 2004;114:1475-83.

QuickTime and a

decompressorare needed to see this picture.

40nm


6/69


End-Stage Renal Failure (ESRF)

major health problem

Patients who progress to ESRF require life-long dialysis or kidneytransplantation (50% die of cardiovascular disease)

>16,000 patients currently on renal replacement therapy, diabetic

nephropathy is single most common cause of ESRF.

In 2005 the cost of ESRF treatment in Australia was $1.2 billion

projected to rise rapidly in the coming years due to the epidemic of

type 2 diabetes.


7/69

Albuminuria

first clinical sign of kidney disease in diabetic patients

(mg/day: 2-20 norm, 30-300 micro, >300 macro)

major indpt risk factor for cardiovascular disease important factor in diabetic patient management

Currently dont understand the strong associationbetween albuminuria and cardiovascular disease,

which is especially strong in diabetic patients.


8/69

What is Albumin?

Produced by the liver and secreted directly into the circulation.The half-life of albumin in the circulation is about 20 days

Physiological roles:

maintenance of oncotic pressure (albumin provides 80% of theplasma oncotic pressure - pulls liquids in - intravascularcompartments and body tissues). transport of small molecules such as calcium, unconjugated

bilirubin, free fatty acids, cortisol and thyroxine. binds drugs in the serum, eg warfarin, phenylbutazone andclofibrate.

Lipids - muscle strength Family


9/69

82.7 (8.27nm)

78.14 (7.8nm)

3.7nm

4.4nm

Heart Shape


10/69

QuickTime and a

decompressorare needed to see this picture.

200nm

200nm

100nm 100nm

Textbook View of the Mechanism of albuminuria Assumed increase urinaryexcretion albuminuria is due toa breakdown in the barrierfunction (leaky glomerulus).Largely unknown.

Congenital nephrotic syndrome

Haraldsson et al., Properties of the glomerular

barrier and mechanisms of proteinuria. PhysiolRev 88:451-87, 2008.

The key data supporting

the theory of kidney

filtration by charge

selectivity: Retarded

clearance of dextran

sulphate compared to

neutral dextran.

Must reinterpret

since it has been

observed thatdextran is taken

up and de-

sulphated duringrenal clearance!!


11/69


Albuminuria

Basis of the Research (driver):

It is important to identify the mechanism of

albuminuria in diabetes to develop new therapeutic

strategies and earlier detection of disease.


12/69

For ConsiderationRadiolabelled albumin demonstrated that much larger amounts of

albumin are excreted in the urine than previously thought.Comper et al., Disease-dependent mechanisms of albuminuria.Am J Physiol Renal Physiol 295:1589-1600, 2008.

Due to significant amounts of very small peptides (


13/69

ProximalTubule

Bowmans Capsule


14/69

An Alternative Mechanism of Microalbuminuria in DiabeticNephropathy

Thus, diabetes induced lysosomal dysfunction may be a common

mechanistic link between albuminuria and CVD. However, a majorcriticism of these studies is that the chemical labelling methods

employed produce changes in albumin structure which could alter its

handling in vivo, and therefore the presence of albumin-derived peptides

may be an artifact of the labelling methods.Norden et al. Quantitative amino acid and proteomic analysis: very low excretion of polypeptides >750 Da in normal urine.Kidney Int66: 1994-2003, 2004.

Hypothesis:

The reduction in albumin degradation is due to the recognisedreduction in tubular lysosomal activity in diabetes. Therefore,

diabetes induced lysosomal dysfunction is a common

mechanism for albuminuria and endothelial dysfunction in

cardiovascular disease.


15/69

Lobe 1 translate forward

Lobe 2 translate back

Linker translateforward

disulfides

Torsion Hypothesis

Lobe movements, and/orlinker, and or disulfide

breakage could changeAlbumin structure fromheart to cigar.

Implication 1: differentshaped Albumin may be

recognised andprocessed differently aspart of normal biology.

Implication 2: labelling ormodifications in disease

states may influence orlock a particularconformation.

If correct, in cigar form thestructure may be as small as

3.7nm.


16/69

Lobe 1

Lobe 2

Linker

disulfides

Close up of region


17/69

Lysine residue locations58 Lys residues in total

Torsion region

bivalve region

Linker region

Ligand region


18/69

Close up of torsionRegion. A Lys(359)follows C357, C358.

Total of 5 Lys inregion which mayinfluence folding

h


19/69

Approach

Biophysical/Structural: Are different forms ofAlbumin handled differently? Can we identify

correct/incorrect structures of albumin? Can we study

the structure of albumin in the endosomal

environment?

Physiological: Measuring the nature (intact or

degraded) of albumin excreted in the urine can detect

the presence of diabetic kidney disease at an earlier

stage than current clinical albumin assays which detect

intact albumin only?


20/69

Sedimentation velocity experiment

Proteins sediment based on their size and shape (the size resolution is approxthe square of the particle radii), and by adjusting the rotor speed alters the sizerange that is covered. Hence with different size/shapes of a protein there is adifferent sedimentation profile and from the profile the size/shape of a proteincan be determined.

Samples were centrifuged at 40,000rpm and the data was collected at a single

wavelength (230nm in continuous mode), using a time interval of 300s and a step-size

of 0.003cm without averaging.

temp = 20C. sample (~0.15 mg/ml)

Buffers: 50 mM NaH2PO4, pH 7 D = 1.0053g/mL, vis = 1.013g/mL

50 mM NaC2H3O2, pH 4 D = 1.0007 g/mL vis = 1.027g/mL

Analytical Ultracentrifugation (AUC)


21/69

Sedimentation velocity analysis of pH 4

Glycated Albumin.

Top Residuals plotted as a function of radial

position (cm) from the axis of rotation.

Panel A The absorbance at 230 nm is plotted

as a function of radius (cm) at 6 min intervals.

The raw absorbance data (symbols) is overlaid

with the nonlinear least-squares best-fits (solid

lines) to the c(s) model (Schuck, 2000).

Panel B Continuous size, c(s), distribution

plotted as a function of sedimentation coefficient

(S). Continuous size-distribution analysis was

performed using the program SEDFIT (Schuck,

2000). The c(s) distribution best fit yielded anrmsd = 0.0063 and Runs test Z = 5.02.

C i f Alb i t H 7 0 d H 4 0 M th l t d ( H 4 0)


22/69

QuickTime and a

BMP decompressor

are needed to see this picture.

pH 7

pH 4pH 4 -CH3

Shows the loss of globularity in

albumin at pH 4.0 and most

significantly under methylated

conditions.

Comparison of Albumin at pH 7.0, and pH 4.0 vs Methylated (pH 4.0)


23/69

QuickTime an d a

BMP decompressorare needed to see this picture.

pH 7 Glycated

pH 4 Glycated

No significant change

in sedimentation

coefficient for f/foindicates no shape

change in the

glycosylated samples.

Comparison of glycated albumin at pH 4.0 vs pH 7.0.


24/69

Hydrodynamic properties of Albumin Species

Sample Mon S20,w1

(Svedberg, S)

Dim S20,w1

(Svedberg, S)

Monf/f02

Mon a/b3

pH 7 4.3 6.8 1.36 3.75

pH 4 4.0 6.1 1.46 5.05

pH 7 Glycated 4.6 7.0 1.28 2.63

pH 4 Glycated 4.5 6.9 1.30 2.98

pH 4 CH3 2.8 4.1 2.09 15.0

1 Standardized sedimentation coefficient taken from the ordinate maximum of the c(s)

distribution (data not shown).2Frictional coefficient of the dimer (Laue et al., 1992)

3Axial ratio (a/b) assuming a prolate elli psoidal structure (Laue et al., 1992).


25/69

pH 7a/b 3.75

pH 4a/b 5.05

pH 7 Glycateda/b 2.63


Expected:~3

Expected:~5.3


26/69

pH 4a/b 5.05

pH 4 -CH3a/b 15.0


Expected:

~5.3


27/69

What is a Synchrotron?

Sample

Sample


28/69


29/69


30/69

QuickTime and aMPEG-4-video decompressorare needed to see this picture.


31/69

QuickTime and a

MPEG-4-video decompressorare needed to see this picture.


32/69

QuickTime and a

MPEG-4-video decompressorare needed to see this picture.


33/69

What is Circular Dichroism (CD)?

chr 2 str of biopolymers.

abs in the UV, vis and IR regions determinethe absolute configuration of molecules in

solution.


34/69

CD Spectra

Different secondary structure types have characteristic CD spectra


35/69

SRCD Advantages

Lower wavelength data (higher structuralinformation content)

Higher signal-to-noise (smaller samplerequirements)

Rapid measurements (lower requirement forsignal averaging)

Can analyse samples in high concentration& absorbing buffers (& membrane samples)


36/69

SRCD vs CDHigher photon flux (energy)

+Extended wavelength

Opens up enormous opportunities

Synergy with X-ray & NMR to answer questionsin structural and functional genomics


37/69

Laboratory-based CD Spectra


38/69

Synchrotron CD Spectra

Lab CD


39/69

Increased Structural Data - Accuracy

WavelengthRange (nm)

IndependentVariables

200-260 2190-260 3-4

178-260 5

168-260 6

160-260 7-8

Accuracy of curve fitting is dependent on wavelength range

Helix

Turns (4 types)

Other

Sheet (parallel &

antiparallel)

Fold motifs?


40/69

SRCD Photon Flux

AustralianSRCD


41/69

Dimming augmented by absorption (quartz optics, oxygen, sample,buffer)

Limiting collection of meaningful data at wavelengths below 190 nm.

The HT dynode voltage of the photo-multiplier tube indicates such noise.


42/69


43/69


44/69

150mM NaF

10mM NaF

150mM NaF

PrP(106-126)


45/69

Structural GenomicsFold Recognition

High Helical Content

Myoglobin (79%)

Mechanosensitive Channel (52%)

ConA (46%)

Ferric Enterobactin Recpt (49%)

High Sheet Content


46/69


47/69

H li 8 i Li id


48/69

-30000

-20000

-10000

0

10000

20000

30000

40000

50000

170 180 190 200 210 220 230 240 250 260

Wavelength (nm)

H2O 26.2 19.8

PCPI(4) 16.4 29.4

PCPI 71.2 3.3PCPG 52 9.4PC 52.6 7

Helix SheetLGKKFKKYFLQLLKYIPP

G305

KL L

K LK

P

KFLF

YK Q

YI

P

ITI

CI AYFN NC

L NPLF YG

F

286

Helix 8

20

16

12

8

4

0

-4

-8

-12

Helix 8 in Lipids


49/69

-40000

-20000

0

20000

40000

60000

170 180 190 200 210 220 230 240 250 260

Wavelength (nm

LFY Peptide in LipidsH2O 40.7 18.9

PCPI(4) 66.2 7.1

PCPI 66.1 3PCPG 70.6 2.9PC 62.6 6.9

Helix SheetLFYGFLGKKFKKYFLQLLKYI

PCPI(4,5)P2 46.8 13.3

DDM 5XCMC 76.4 1.5

G305

K

LL

K LK

P

KF

LF

YK Q

YI

P

ITI

CI AYFN NC

L NPLF YG

F

286

Helix 8

18

12

6

0

-6

-12


50/69

-7

-5

-3

-1

1

3

5

170 190 210 230 250 270

Wavelength

Elipticity

OCD Atvs PCPG(1:1) 1:25 HBS

AT vs PCPG(1:1) 1:25 in HBS

AT vs PCPG(1:1) 1:25 in 50mM PO4

AT vs PCPG(1:4) 1:25 in PO4

Liposome

C M CD Bi di E i t


51/69

CaM CD Binding Experiments

-10

-5

0

5

10

15

180 200 220 240 260 280

wavelength (nm)

DeltaEp

silon


52/69


53/69

SRCD Beamline Design:

The challenges of building your own instruments

Instrumentation Purged withHigh vacuumCaF2 or LiF for optical windows and sample

ll t t 140 (H O 168)


54/69

Instrumentation gdry nitrogen(removeoxygen/ozone)

High vacuumcells, measurements to ~140 nm, (H2O ~168)


55/69

Can take a heat-load, polished, surface material and coating(?)


56/69


57/69


58/69


59/69


60/69


61/69


62/69

Reversibleradiation-induced

Thermal denaturationof Albumin


63/69

QuickTime and a

decompressorare needed to see this p icture.

pHpH 4 data at non-damaging synchrotronclosely matches pH 7 data at a damaging

synchrotron


64/69

Lipodrugs binding to Albumin


65/69

Lipodrugs Stabilise Albumin

Pronase enzymatic degradation of albumin fatty acid free at37C as a function of time monitored by CD spectroscopy.

-4000

-2000

0

2000

4000

200 220 240

Wavelength (nm)

t=120

t=90

t=75

t=60

t=45

t=30

t=15

t=0

-4000

-2000

0

2000

4000

200 220 240

Wavelength (nm)

=(

L-R)(M-1cm-1)

HSAff + Pronase (1:100 w/w) [HSAff+gabaC8 (1:6)]+ Pronase (1:100w/w)

30


66/69

-20

-10

0

10

20

190 200 210 220 230 240 250 260 270

AlbuminAGE Albumin-CH3 Albumin

pH 7

Sample Mon S20w Mon a/b

pH 7 Alb 4.3 3.75

pH 7 AGE 4.6 2.63

pH 4 Alb 4.0 5.05

pH4 AGE 4.5 2.98

pH 4 -CH3 2.8 15.0

50


67/69

-30

-20

-10

0

10

20

30

40

190 200 210 220 230 240 250 260 270

pH 4 Albumin

pH 4 AGE Albumin

pH 4 methylated Albumin

region showing helicity

Sample Mon S20w Mon a/b

pH 7 Alb 4.3 3.75

pH 7 AGE 4.6 2.63

pH 4 Alb 4.0 5.05

pH4 AGE 4.5 2.98

pH 4 -CH3 2.8 15.0

pH 4

AlbuminAGE Albumin-CH3 Albumin

Summary


68/69

Summary

Albumin can adopt different conformations(heart and cigar) - likely part of the naturalbiological function

Glycated (diabetic) forms have a defined andstable structure - little change under endosomalconditions. ??? Lysosomal dysregulation???

Labelled Albumin does not necessarily have thesame structure/biophysical properties and maybe handled differently by the kidneys.

Health Innovations Research Institute


69/69


Acknowledgements

Prof Philip Poronnik - RMIT (Project Leader)

Dr Greg Tesch - Monash (CIB)

Dr Darren Kelly - St Vincents

Dr David Nickolic-Paterson - Monash Medical Centre

Prof Giuliano Siligardi - Diamond & Liverpool, UK

Dr Rohanah Hussain - Diamond & Liverpool, UK

Assoc. Prof. Josephine Forbes - Baker IDI

Dr. Matthew A. Perugini - Bio21 InstituteJarrod Voss - Bio21 Institute

Dr Frank Wein - Soleil, France

Documents

SRCD Physiology Talk 2009