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					The three classical mechanisms for protein folding




          (hierarchial)




           (Fersht and Daggett, Cell 108, 573-582, 2002)
          Energy landscape of protein folding
                                                   rugged landscape:
   idealized funnel landscape                      multi-state folding




As the chain forms increasing numbers of       A rugged landscape with kinetic traps,
intrachain contacts, and lowers its internal   energy barriers, and some narrow
free energy, its conformational freedom is     throughway paths to native.
also reduced                                   Folding can be multistate
                                                                                 (K. Dill)
The folding energy landscape of a protein
Macromolecular crowding
    (approx. 300 mg/ml)




  Goodsell 1991, TiBS(16): 203-206
          Major chaperones
and their interactions with substrates




                             ?
    The GroEL-GroES chaperone machine
GroEL proteins           GroEL complex
  (subunits)      (double ring with 7 GroEL/ring)

                                                    Active complex



                                               ATP

GroES proteins           GroES complex
  (subunits)     (single ring with 7 GroES/ring)
Protein folding by GroEL and GroES


                 GroEL with         GroEL:GroES with
GroEL
            unfolded polypeptide   enclosed polypeptide



                                                ∆t = 15 sec




 The folding activity is ATP-dependent!

                                                    U. Hartl
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       Dekompressor “GIF”                Dekompressor “GIF”
             benötigt.                         benötigt.
Conformational changes induced by nucleotide and GroES
      (model based on cryo electron microscopy)




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                                                  H. Saibil
  Cellular roles of Hsp70 chaperone systems

stress-related functions              house-keeping functions

 aggregation prevention                  de novo folding

        disaggregation                   translocation
                           Hsp70
              refolding                  assembly and disassembly

           degradation                   regulation of activity

                                         regulated degradation



                wide range of substrate conformers

     unfolded - folding intermediates - aggregated - native
           Functional cycle of DnaK

     ATPase domain      Substrate binding domain


                                    Unfolded protein
                                    substrate
          ATP    ATP



ADP + P
                                     DnaJ
   GrpE


                ADP·P
      QuickTime™ an d a
     Video d ecompressor
are need ed to see this picture .
Substrate binding domain of DnaK
                 quality control of
                 misfolded proteins


chaperones   regular folding pathways




             activity & stability control
                 of folded proteins
                  (Hsp70 & Hsp90)
                   Hsp70-Hsp90 activation cycle

 Function of
cochaperones:
                                                        Hsp90
  > Targeting factor                            Bag-1
  > Adaptor                 Hsp70         Hip
  > Regulator                        Hsp40
                                                                 Hop
                                                                     Aha1

                   client



          active                    ligand



                                                                       p23
                                                                    cdc37
                                                                immunophilins
       The Hsp70-Hsp90 chaperone machine:
  a regulator for signal transduction and cell cycle

                                                  plasma
                c-Src     90              P       membrane     control of
                          37                                   cell proliferation
                               cyclin D
               Cdk4                               nucleus      control of G1-
                          90
                          37                                   progression
              eIF-2a-          heme               cytosol
              kinase                                           control of translation
                          90              P
                                                               initiation
Hsp70                     37
                               Ras                plasma
Hsp90                                             membrane     activation of
               Raf-1      90              90                   MAP-kinase pathway
                          37              37
                               stress             nucleus      activation of
                HSF
                          90                                   gene expression
                               steroid
                                                  nucleus      activation of
                SHR 2390                                       gene expression
                        inactive         active


        >120 different targets!!!!                           Mayer & Bukau 1999 Curr. Biol.
                             Hsp70/Hsp90 clients
Transcription factors                                   3-Phosphoinositide-dependent kinase-1
Steroid hormone receptors: AR, ER, GR, MR, PR           Akt
Other nuclear receptors: AhR, RAR                       Aurora B
Heme activator protein (Hap1)                           Bcr-Abl
HSF-1                                                   Calmodulin-regulated eEF-2 kinase
Hypoxia-inducible factor-1a                             Casein kinase II
MTG8 myeloid leukemia protein                           Chk1
p53                                                     c-Mos
Sim                                                     Death domain kinase RIP
SV40 large T antigen                                    Flt3
Tumor promotor-specific binding protein                 Focal adhesion kinase
v-erbA                                                  GRK2
                                                        Ire1
Polymerases                                             IkB kinases a,b,g,e
Telomerase                                              Kinase suppressor of ras (KSR)
Hepatitis B virus reverse transcriptase                 MEK (MAP kinase kinase)
DNA-polymerase a                                        MEKK1, MEKK3
Kinases                                                 Mik1
Src-family kinases: Fps, Fes, Fgr, v-Src, c-Src, Hck,   MOK, MAK, MRK
p56lck, Yes                                             Phosphatidylinositol 4-kinase
Cycline dependent kinases: Cdc2, Cdk4, Cdk6, Cdk9       Pim-1
Receptor tyrosin kinases: EGFR, ErbB2, IGF-R,           Polo mitotic kinase
Insulin receptor,                                       Sevenless PTK
PDGFR, VEGFR2                                           TAK1
Raf family kinases: v-Raf, c-Raf, B-Raf, Gag-Mil,       TBK1
Ste11                                                   trkB
eIF-2a kinases: HRI, Gcn2, Perk, PKR                    Wee1, Swe1
                           Hsp70/Hsp90 clients
Others
                                                            P2X7 purinergic receptor
Aminoacyl t-RNA synthetase
                                                            Pancreatic bile salt-dependent lipase
Apaf-1
                                                            PB2 subunit of influenza RNA pol.
Apoprotein B
                                                            Protease-activated receptor 1 (PAR-1)
Atrial natriuretic peptide receptor
                                                            Proteasome
Bid
                                                            Rab-aGDI
Calponin
                                                            Ral-binding protein 1
Centrin/centrosome
                                                            Reovirus protein s1
Cna2 (catalytic subunit of calcineurin)
                                                            SKP2 complexies
CFTR
                                                            survivin
Ctf13/Skp1 component of CBF3
                                                            Tau protein
Cytoskeletttal proteins: actin, tubulin, myosin
                                                            Thiopurine S-methyltransferase
eNOS, iNOS, nNOS
                                                            Thyroglobulin
Erythrocyte membrane protein (Plasmodium falciparum)
                                                            TLR4/MD-2 complex
Fanconi anemia group C protein
                                                            Vaccinia core protein 4a
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                                                 http://www.picard.ch/downloads/downloads.htm
NB-LRR proteins RPM1 and RPS2                        Wegele, et al. 2004 Rev. Physiol. Biochem. Pharmacol.
Neuropeptide Y
 Signal transduction pathways related to tumor progression
                                                          Growth
                               Survival                  Receptor          Cytokines
        Wnt                    Receptor

                                                                                                 Death
                                                                                                 Factor
                                                                    Grb2
                                                   Src              SOS
Cell                                PI3K
Adhesion                                                   RAS
                  Disheveled        PDK1            Ral             Cdc42 JAKs        Bcl2/Bax      FADD
                                                            Raf
                                      AKT
                    GSK-3b                                   MEK                        CytC        Casp 8
                                           IKK
                      APC                                         Erk                Apaf-1/CytC/
                                             IkB
                                                                                       Casp 9
                                                   NFkB             Elk
                   b-Catenin
                                                                           Stat3,5             Casp 3
                                       b-Catenin/LEF
                   a-Catenin
                                                              Gene expression




Zhang & Burrows 2004 J. Mol. Med.
 Signal transduction pathways related to tumor progression
                                                          Growth
                               Survival                  Receptor          Cytokines
        Wnt                    Receptor

                                                                                                 Death
                                                                                                 Factor
                                                                    Grb2
                                                   Src              SOS
Cell                                PI3K
Adhesion                                                   RAS
                  Disheveled        PDK1            Ral             Cdc42 JAKs        Bcl2/Bax      FADD
                                                            Raf
                                      AKT
                    GSK-3b                                   MEK                        CytC        Casp 8
                                           IKK
                      APC                                         Erk                Apaf-1/CytC/
                                             IkB
                                                                                       Casp 9
                                                   NFkB             Elk
                   b-Catenin
                                                                           Stat3,5             Casp 3
                                       b-Catenin/LEF
                   a-Catenin
                                                              Gene expression




Zhang & Burrows 2004 J. Mol. Med.
                ClpB structure




                            N-terminal domain
                                  (NTD)


middle domain (M)                1st ATPase (D1)



                                   2nd ATPase (D2)


                                        Lee et al. Cell 2003
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
Protein aggregates in neurodegenerative diseases
                   Alzheimer’s                     Parkinson’s




                Plaques and tangles                 Lewy bodies

        PolyQ diseases                                       Prions




      Intranuclear inclusions    Amyotrophic lateral     Amyloid plaques
                                     sclerosis




                                      Aggregates                           E. Nollen
                Polyglutamine aggregation

    Wild type < 35 residues



                                       Folded protein



     Disease > 35 residues




glutamine residue
other residue
                                        Aggregate

                                               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



Promotor
Unc-54         Qn           GFP




           Protein aggregates: Huntington
Length dependent aggregation of polyQ-YFP in
                C. elegans


  Unc-54
            Qn         GFP




                                    Morley et al., 2002
      Q19-expressing animals                      Q82-expressing animals



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


                  3-4 days old C. elegans

Morley&Morimoto
               Firefly Luciferase




                       Luciferase
Luciferin + ATP + O2                Oxoluciferin + CO2

                                    Light emission

				
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posted:3/29/2013
language:English
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