The three classical mechanisms for protein folding

(Fersht and Daggett, Cell 108, 573-582, 2002)

(hierarchial)

Energy landscape of protein folding

rugged landscape: multi-state folding

A rugged landscape with kinetic traps, energy barriers, and some narrow throughway paths to native. Folding can be multistate

idealized funnel landscape

(K. Dill)

As the chain forms increasing numbers of intrachain contacts, and lowers its internalfree energy, its conformational freedom is also reduced

The folding energy landscape of a protein

Goodsell 1991, TiBS(16): 203-206

Macromolecular crowding

(approx. 300 mg/ml)

Major chaperones and their interactions with substrates

?

The GroEL-GroES chaperone machine

GroEL proteins(subunits)

GroEL complex (double ring with 7 GroEL/ring)

Active complex

GroES complex (single ring with 7 GroES/ring)

GroES proteins(subunits)

ATP

Protein folding by GroEL and GroES

GroELGroEL with

unfolded polypeptideGroEL:GroES with

enclosed polypeptide

The folding activity is ATP-dependent!

U. Hartl

∆t = 15 sec

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H. Saibil

Conformational changes induced by nucleotide and GroES(model based on cryo electron microscopy)

Hsp70

Cellular roles of Hsp70 chaperone systems

aggregation prevention

disaggregation

refolding

degradation

stress-related functions

de novo folding

translocation

assembly and disassembly

regulation of activity

regulated degradation

house-keeping functions

wide range of substrate conformers

unfolded - folding intermediates - aggregated - native

Functional cycle of DnaK

ATP

ADP + P

ATP

ADP·P

Unfolded protein substrate

ATPase domain Substrate binding domain

DnaJGrpE

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Substrate binding domain of DnaK

activity & stability controlof folded proteins(Hsp70 & Hsp90)

regular folding pathways

quality control of misfolded proteins

chaperones

Hsp70-Hsp90 activation cycle

ligandactive

Hsp90

client

Hsp70

Function of cochaperones:

> Targeting factor> Adaptor> Regulator Hsp40

Hip

Bag-1

Hop

Aha1

p23

immunophilinscdc37

The Hsp70-Hsp90 chaperone machine:a regulator for signal transduction and cell cycle

Mayer & Bukau 1999 Curr. Biol.

c-Src control of cell proliferation

plasma membrane90

37

stressHSF

nucleus

activation ofgene expression

steroid

SHRnucleus

Cdk4

cyclin D

control of G1-progression

nucleus

hemeP

eIF-2-kinase control of translation

initiation

cytosol

Ras

Raf-1activation ofMAP-kinase pathway

plasmamembrane

P

9037

9037

9037

9037

90

9023

inactive active

activation ofgene expression

Hsp70Hsp90

>120 different targets!!!!

Transcription factorsSteroid hormone receptors: AR, ER, GR, MR, PROther nuclear receptors: AhR, RARHeme activator protein (Hap1)HSF-1Hypoxia-inducible factor-1aMTG8 myeloid leukemia proteinp53SimSV40 large T antigenTumor promotor-specific binding proteinv-erbA

PolymerasesTelomeraseHepatitis B virus reverse transcriptaseDNA-polymerase

3-Phosphoinositide-dependent kinase-1AktAurora BBcr-AblCalmodulin-regulated eEF-2 kinaseCasein kinase IIChk1c-MosDeath domain kinase RIPFlt3Focal adhesion kinaseGRK2Ire1IB kinases ,,,Kinase suppressor of ras (KSR)MEK (MAP kinase kinase)MEKK1, MEKK3Mik1MOK, MAK, MRKPhosphatidylinositol 4-kinasePim-1Polo mitotic kinaseSevenless PTKTAK1TBK1trkBWee1, Swe1

KinasesSrc-family kinases: Fps, Fes, Fgr, v-Src, c-Src, Hck, p56lck, YesCycline dependent kinases: Cdc2, Cdk4, Cdk6, Cdk9Receptor tyrosin kinases: EGFR, ErbB2, IGF-R, Insulin receptor,PDGFR, VEGFR2Raf family kinases: v-Raf, c-Raf, B-Raf, Gag-Mil, Ste11eIF-2 kinases: HRI, Gcn2, Perk, PKR

Hsp70/Hsp90 clients

OthersAminoacyl t-RNA synthetaseApaf-1Apoprotein BAtrial natriuretic peptide receptorBidCalponinCentrin/centrosomeCna2 (catalytic subunit of calcineurin)CFTRCtf13/Skp1 component of CBF3Cytoskeletttal proteins: actin, tubulin, myosineNOS, iNOS, nNOS Erythrocyte membrane protein (Plasmodium falciparum)Fanconi anemia group C proteinG protein G0, G12Guanylate cyclase (-subunit)HETE binding complexHistones H1, H2A, H2B, H3, H4Lysosomal membraneMacrophage scavenger receptorMdm2MMP2MTG8NB-LRR proteins RPM1 and RPS2Neuropeptide Y

P2X7 purinergic receptor Pancreatic bile salt-dependent lipasePB2 subunit of influenza RNA pol.Protease-activated receptor 1 (PAR-1)ProteasomeRab-GDIRal-binding protein 1Reovirus protein s1SKP2 complexiessurvivinTau proteinThiopurine S-methyltransferaseThyroglobulinTLR4/MD-2 complexVaccinia core protein 4a

http://www.picard.ch/downloads/downloads.htmWegele, et al. 2004 Rev. Physiol. Biochem. Pharmacol.

Hsp70/Hsp90 clients

E-Cadherin

Frizzo

ed

IGF

-R EG

FR

Cyt

oki

ne-

R

Fas

Wnt

Cell Adhesion

Survival Receptor

Growth Receptor Cytokines

Death Factor

Disheveled

GSK-3

APC

-Catenin

-Catenin

-Catenin/LEF

PI3K

PDK1

AKT

IKK

IB

NFB

Src Grb2SOS

RAS

RalRaf

Cdc42

MEK

Erk

Elk

JAKs

Stat3,5

Bcl2/Bax FADD

CytC Casp 8

Apaf-1/CytC/Casp 9

Casp 3

Gene expression

Zhang & Burrows 2004 J. Mol. Med.

Signal transduction pathways related to tumor progression

E-Cadherin

Frizzo

ed

IGF

-R EG

FR

Cyt

oki

ne-

R

Fas

Wnt

Cell Adhesion

Survival Receptor

Growth Receptor Cytokines

Death Factor

Disheveled

GSK-3

APC

-Catenin

-Catenin

-Catenin/LEF

PI3K

PDK1

AKT

IKK

IB

NFB

Src Grb2SOS

RAS

RalRaf

Cdc42

MEK

Erk

Elk

JAKs

Stat3,5

Bcl2/Bax FADD

CytC Casp 8

Apaf-1/CytC/Casp 9

Casp 3

Gene expression

Zhang & Burrows 2004 J. Mol. Med.

Signal transduction pathways related to tumor progression

ClpB structure

N-terminal domain (NTD)

1st ATPase (D1)

2nd ATPase (D2)

middle domain (M)

Lee et al. Cell 2003

ClpB Protomers form hexameric rings (in ATP)

top side

3D model

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Mechanism of ClpB/KJE-mediated protein disaggregation

Weibezahn et al., Cell 2004

ClpB

Protein aggregates in neurodegenerative diseases

Plaques and tangles Lewy bodies

Amyloid plaques

Aggregates

Intranuclear inclusions

Alzheimer’s Parkinson’s

PolyQ diseases Prions

Amyotrophic lateral sclerosis

E. Nollen

Polyglutamine aggregation

glutamine residue

other residue

Disease > 35 residues

Wild type < 35 residues

Aggregate

Folded protein

E. Nollen

Caenorhabditis elegans

*lives in the ground *size 1mm*has 959 cells*has a transparent body, observable in light microscope*life span: 20 days

Perfect tool: - easy genetic manipulation

C. elegans, a model system to study Dementia

GFPQn

Protein aggregates: Huntington

Promotor

Unc-54

Length dependent aggregation of polyQ-YFP in C. elegans

GFPQn

Unc-54

Morley et al., 2002

Q19-expressing animals Q82-expressing animals

0

20

40

60

80

100

Morley&Morimoto

PolyQ of a length of n≥35 cause:- protein aggregation- drastic impaired mobility

3-4 days old C. elegans

Luciferin + ATP + O2 Oxoluciferin + CO2 Luciferase

Light emission

Firefly Luciferase