The three classical mechanisms for protein folding

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The three classical mechanisms for protein folding
(hierarchial)
(Fersht and Daggett, Cell 108, 573-582, 2002)
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Energy landscape of protein folding
rugged landscape multi-state folding
idealized funnel landscape
As the chain forms increasing numbers of
intrachain contacts, and lowers its
internal free energy, its conformational freedom
is also reduced
A rugged landscape with kinetic traps, energy
barriers, and some narrow throughway paths to
native. Folding can be multistate
(K. Dill)
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The folding energy landscape of a protein
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Macromolecular crowding
(approx. 300 mg/ml)
Goodsell 1991, TiBS(16) 203-206
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Major chaperones and their interactions with
substrates
?
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The GroEL-GroES chaperone machine
GroEL proteins (subunits)
GroEL complex (double ring with 7 GroEL/ring)
Active complex
ATP
GroES proteins (subunits)
GroES complex (single ring with 7 GroES/ring)
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Protein folding by GroEL and GroES
GroELGroES with enclosed polypeptide
GroEL with unfolded polypeptide
GroEL
?t 15 sec
The folding activity is ATP-dependent!
U. Hartl
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Conformational changes induced by nucleotide and
GroES (model based on cryo electron microscopy)
H. Saibil
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Hsp70
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Functional cycle of DnaK
Unfolded protein substrate
ATP
DnaJ
GrpE
ADPP
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Substrate binding domain of DnaK
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quality control of misfolded proteins
activity stability control of folded
proteins (Hsp70 Hsp90)
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Hsp70-Hsp90 activation cycle
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The Hsp70-Hsp90 chaperone machinea regulator
for signal transduction and cell cycle
plasma membrane
control of cell proliferation
c-Src
cyclin D
nucleus
Cdk4
control of G1- progression
eIF-2a- kinase
heme
cytosol
control of translation initiation
Hsp70 Hsp90
Ras
plasma membrane
activation of MAP-kinase pathway
Raf-1
stress
nucleus
activation of gene expression
HSF
steroid
nucleus
activation of gene expression
SHR
inactive
active
Mayer Bukau 1999 Curr. Biol.
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Hsp70/Hsp90 clients
Transcription factors Steroid hormone receptors
AR, ER, GR, MR, PR Other nuclear receptors AhR,
RAR Heme activator protein (Hap1) HSF-1 Hypoxia-in
ducible factor-1a MTG8 myeloid leukemia
protein p53 Sim SV40 large T antigen Tumor
promotor-specific binding protein v-erbA
3-Phosphoinositide-dependent kinase-1 Akt Aurora
B Bcr-Abl Calmodulin-regulated eEF-2
kinase Casein kinase II Chk1 c-Mos Death domain
kinase RIP Flt3 Focal adhesion kinase GRK2 Ire1 Ik
B kinases a,b,g,e Kinase suppressor of ras
(KSR) MEK (MAP kinase kinase) MEKK1,
MEKK3 Mik1 MOK, MAK, MRK Phosphatidylinositol
4-kinase Pim-1 Polo mitotic kinase Sevenless
PTK TAK1 TBK1 trkB Wee1, Swe1
Polymerases Telomerase Hepatitis B virus reverse
transcriptase DNA-polymerase a
Kinases Src-family kinases Fps, Fes, Fgr, v-Src,
c-Src, Hck, p56lck, Yes Cycline dependent
kinases Cdc2, Cdk4, Cdk6, Cdk9 Receptor tyrosin
kinases EGFR, ErbB2, IGF-R, Insulin
receptor, PDGFR, VEGFR2 Raf family kinases
v-Raf, c-Raf, B-Raf, Gag-Mil, Ste11 eIF-2a
kinases HRI, Gcn2, Perk, PKR
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Hsp70/Hsp90 clients
Others Aminoacyl t-RNA synthetase Apaf-1 Apoprotei
n B Atrial natriuretic peptide receptor Bid Calpon
in Centrin/centrosome Cna2 (catalytic subunit of
calcineurin) CFTR Ctf13/Skp1 component of
CBF3 Cytoskeletttal proteins actin, tubulin,
myosin eNOS, iNOS, nNOS Erythrocyte membrane
protein (Plasmodium falciparum) Fanconi anemia
group C protein G protein bg Ga0, Ga12 Guanylate
cyclase (b-subunit) HETE binding complex Histones
H1, H2A, H2B, H3, H4 Lysosomal membrane Macrophage
scavenger receptor Mdm2 MMP2 MTG8 NB-LRR
proteins RPM1 and RPS2 Neuropeptide Y
P2X7 purinergic receptor Pancreatic bile
salt-dependent lipase PB2 subunit of influenza
RNA pol. Protease-activated receptor 1
(PAR-1) Proteasome Rab-aGDI Ral-binding protein
1 Reovirus protein s1 SKP2 complexies survivin Tau
protein Thiopurine S-methyltransferase Thyroglobu
lin TLR4/MD-2 complex Vaccinia core protein 4a
http//www.picard.ch/downloads/downloads.htm Wegel
e, et al. 2004 Rev. Physiol. Biochem. Pharmacol.
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Signal transduction pathways related to tumor
progression
Growth Receptor
Cytokines
Survival Receptor
Wnt
Death Factor
EGFR
Cytokine-R
IGF-R
Frizzoed
Grb2 SOS
Src
Fas
Cell Adhesion
PI3K
RAS
Disheveled
Bcl2/Bax
FADD
PDK1
Ral
Cdc42
JAKs
Raf
E-Cadherin
AKT
CytC
Casp 8
GSK-3b
MEK
IKK
Erk
Apaf-1/CytC/ Casp 9
APC
IkB
Elk
NFkB
b-Catenin
Stat3,5
Casp 3
b-Catenin/LEF
a-Catenin
Gene expression
Zhang Burrows 2004 J. Mol. Med.
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Signal transduction pathways related to tumor
progression
Growth Receptor
Cytokines
Survival Receptor
Wnt
Death Factor
EGFR
Cytokine-R
IGF-R
Frizzoed
Grb2 SOS
Src
Fas
Cell Adhesion
PI3K
RAS
Disheveled
Bcl2/Bax
FADD
PDK1
Ral
Cdc42
JAKs
Raf
E-Cadherin
AKT
CytC
Casp 8
GSK-3b
MEK
IKK
Erk
Apaf-1/CytC/ Casp 9
APC
IkB
Elk
NFkB
b-Catenin
Stat3,5
Casp 3
b-Catenin/LEF
a-Catenin
Gene expression
Zhang Burrows 2004 J. Mol. Med.
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ClpB structure
N-terminal domain (NTD)
1st ATPase (D1)
middle domain (M)
2nd ATPase (D2)
Lee et al. Cell 2003
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ClpB Protomers form hexameric rings (in ATP)
3D model
top
side
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Mechanism of ClpB/KJE-mediated protein
disaggregation
ClpB
Weibezahn et al., Cell 2004
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Protein aggregates in neurodegenerative diseases
Alzheimers
Parkinsons
Plaques and tangles
Lewy bodies
PolyQ diseases
Prions
Amyloid plaques
Intranuclear inclusions
Amyotrophic lateral sclerosis
Aggregates
E. Nollen
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Polyglutamine aggregation
Wild type lt 35 residues
Folded protein
Disease gt 35 residues
glutamine residue
other residue
Aggregate
E. Nollen
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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
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C. elegans, a model system to study Dementia
Promotor Unc-54
Qn
GFP
Protein aggregates Huntington
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Length dependent aggregation of polyQ-YFP in C.
elegans
Unc-54
Qn
GFP
Morley et al., 2002
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Q19-expressing animals
Q82-expressing animals
PolyQ of a length of n35 cause - protein
aggregation - drastic impaired mobility
3-4 days old C. elegans
MorleyMorimoto
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Firefly Luciferase
Luciferase
Luciferin ATP O2 Oxoluciferin CO2
Light emission