Assist other proteins to achieve a functionally active 3D structure Molecular chaperones Assist other proteins to achieve a functionally active 3D structure They catalyze protein folding or unfolding in energy-dependent reactions Most of these molecules are heat shock proteins (formed during thermal damage)- protect against denaturation Some of them are associated with proteases (protein degradation)

Protein quality control in the cell Interplay of molecular chaperones and proteases in the cell. Substrate proteins are shown in red; an ATP-dependent chaperone, such as GroEl, is shown in bluse; a Clp chaperone is purple and is associated with a compartmentalized protease shown in green. Molecular chaperones play a critical role in protein quality control during the course of cell growth as well as during stress conditions. Normal protein synthesis produces nascent unfolded proteins. Although some nascent polypeptides are able to fold spontaneously (1), others require the action of molecular chaperones, icluding members of the DnaK/Hsp70 and GroEL/Hsp60 families, to facilitate folding (2). Unfolded and misfolded proteins also arise in cells as a result of environmental stresses, such as heat shock, or pathologic conditions, such as inflammation, tissue damage, infection, and genetic diseases involving mutant proteins. Molecular chaperones are able to refold and reactive some misfolded proteins (2). Ther irreversibly misfolded proteins are recognized by the proteasome. These multicomponent proteases use associated chaperones to unfold and deliver damaged proteins to the proteases for degradation (3). Finally, proteins that are neither refolded nor degraded form insoluble aggregates in the cell (4). Aggregates are not always an end product, but can be dissolved by molecular chaperones.

Misfolded proteins are normally detected and cleared from cell (or stored in aggresomes) Regulation of protein folding in the ER. Many newly synthesized proteins are translocated into the ER, where they fold into their three-dimensional structures with the help of a series of molecular chaperones and folding catalysts (not shown). Correctly folded proteins are then transported to the Golgi complex and then delivered to the extracellular environment. However, incorrectly folded proteins are detected by a quality-control mechanism and sent along another pathway (the unfolded protein response) in which they are ubiquitinated and then degraded in the cytoplasm by proteasomes. CM Dobson, “Protein folding and misfolding”, Nature, 426, 884-890 (2003)

General mechanism of aggregation to form amyloid fibrils Unfolded or partially unfolded proteins associate with each other to form small, soluble aggregates that undergo further assembly into protofibrils or protofilaments (a) and then mature fibrils (b). The fibrils often accumulate in plaques or other structures such as the Lewy bodies associated with Parkinson’s disease (c). Some of the early aggregates seem to be amorphous or micellar in nature, although others form ring-shaped species with diameters of approximately 10 nm (d). CM Dobson, “Protein folding and misfolding”, Nature, 426, 884-890 (2003)

M Stefani and CM Dobson, “Protein aggregation and aggregate toxicity: new insights into protein folding, misfolding diseases and biological evolution”, J. Mol. Med 81:678-699 (2003)

Figure 6-89. Protein aggregates that cause human disease Figure 6-89. Protein aggregates that cause human disease. (A) Schematic illustration of the type of conformational change in a protein that produces material for a cross-beta filament. (B) Diagram illustrating the self-infectious nature of the protein aggregation that is central to prion diseases. PrP is highly unusual because the misfolded version of the protein, called PrP*, induces the normal PrP protein it contacts to change its conformation, as shown. Most of the human diseases caused by protein aggregation are caused by the overproduction of a variant protein that is especially prone to aggregation, but because this structure is not infectious in this way, it cannot spread from one animal to another. (C) Drawing of a cross- filament, a common type of protease-resistant protein aggregate found in a variety of human neurological diseases. Because the hydrogen-bond interactions in a  sheet form between polypeptide backbone atoms (see Figure 3-9), a number of different abnormally folded proteins can produce this structure. (D) One of several possible models for the conversion of PrP to PrP*, showing the likely change of two a-helices into four b-strands. Although the structure of the normal protein has been determined accurately, the structure of the infectious form is not yet known with certainty because the aggregation has prevented the use of standard structural techniques. (C, courtesy of Louise Serpell, adapted from M. Sunde et al., J. Mol. Biol. 273:729 739, 1997; D, adapted from S.B. Prusiner, Trends Biochem. Sci. 21:482 487, 1996.) Alberts et al 2002

ACIDES AMINES - PROTEINES boîte magique Houba! structure native structure désorganisée chaperonne Comment se replient in vivo les polypeptides en cours de synthèse? Si beaucoup de protéines peuvent se replier seules, d’autres ont besoin d’aide. Cette aide est apportée par d’autres protéines que l’on appelle chaperonnes.

http://swissmodel.expasy.org/course/text/chapter4.htm

MOLECULAR CHAPERONES: Definition A large group of unrelated protein families whose role is to stabilize unfolded proteins, unfold them for translocation across membranes or for degradation, and/ or to assist in their correct folding and assembly. Properties • Molecular chaperones interact with unfolded or partially folded protein subunits, e.g. nascent chains emerging from the ribosome, or extended chains being translocated across subcellular membranes. • They stabilize non-native conformation and facilitate correct folding of protein subunits. • They do not interact with native proteins, nor do they form part of the final folded structures. • Some chaperones are non-specific, and interact with a wide variety of polypeptide chains, but others are restricted to specific targets. • They often couple ATP binding/hydrolysis to the folding process. • Essential for viability, their expression is often increased by cellular stress. Main role: They prevent inappropriate association or aggregation of exposed hydrophobic surfaces and direct their substrates into productive folding, transport or degradation pathways

FAMILIES OF MOLECULAR CHAPERONES Small heat shock proteins (hsp25) [holders] •protect against cellular stress •prevent aggregation in the lens (cataract) Hsp60 system (cpn60, GroEL) ATPase [(un)folders] •protein folding Hsp70 system (DnaK, BiP) ATPase [(un)folders] •stabilization of extended chains •membrane translocation •regulation of the heat shock response Hsp90 [holder] •binding and stabilization/regulation of steroid receptors, protein kinases Hsp100 (Clp) ATPase [unfolder] •thermotolerance, proteolysis, resolubilization of aggregates Calnexin, calreticulin •glycoprotein maturation in the ER •quality control Folding catalysts: PDI, PPI [folders] Prosequences: alpha-lytic protease, subtilisin (intramolecular chaperones) [folders]

Disulfide isomerization (PDI) Protein disulfide isomerase Human protein databank http://www.hprd.org/protein/07181 Cysteine residues can be "tagged" with iodoacetate

Structure cristalline de GroEL avec une résolution de 30Å ACIDES AMINES - PROTEINES Structure cristalline de GroEL avec une résolution de 30Å

Different sites of action Location of chaperone is very important: cytosol? membrane? organelle? extracellular? periplasmic? e.g., calnexin; must be near polypeptide entry? ribosome-bound? soluble? associated with particular structures? must bear sequence tag to target it there chaperonin required for its own folding e.g., clusterin binds large number of extracellular proteins e.g., PapD/FimC is required for pilus folding/assembly

Co-localization chaperones can co-localize with: other chaperones protein degradation machinery different substrates etc. Example: - misfolded proteins may end up in aggresomes (e.g., CFTR) - aggresomes contain various molecular chaperones, including Hsp70 and Hsp40, as well as proteasome components This can potentially cause problems: - researchers expressed mutant CFTR - they then expressed mutant GFP that is normally broken down - saw GFP fluorescence (green) in the cytosol (i.e., it wasn’t degraded) - has implications for proteins that aggregate in cell and cause diseases

Two families of molecular chaperone for protein folding: DnaK/DnaJ/GrpE (or hsp70) family: bind to growing polypeptide chains while they are being synthesized by ribosomes and prevent premature folding (co-translational) Chaperonin family (GroE chaperonin): assist correct folding at a later stage (posttranslational)

Chaperones: Variations on a theme

dnaJ, dnaK, grpE (foldosome) Cytoplasmic (ER homologues) Interaction between the J-domain of DnaJ and the N-terminal ATPase domain of DnaK stimulates ATP hydrolysis. A second interaction mediated by zinc center II locks DnaK on substrate. The nucleotide exchange factor GrpE then exchanges ADP with ATP and unlocks DnaK.

Chaperonins Are protein molecules that assist in the proper folding of other proteins Hollow cylinder Cap Chaperonin (fully assembled) Steps of Chaperonin Action: An unfolded poly- peptide enters the cylinder from one end. The cap attaches, causing the cylinder to change shape in such a way that it creates a hydrophilic environment for the folding of the polypeptide. The cap comes off, and the properly folded protein is released. Correctly folded protein Polypeptide 2 1 3 Figure 5.23

(double ring with 7 GroEL/ring) (single ring with 7 GroES/ring) 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)

GroEL (hsp60) Chaperonin 7-fold symmetry two rings

GroEL (Hsp60)

GroEL/GroES complex

GroEl/GroES complex Free GroEL binds denatured proteins very tightly. GroEL undergoes an allosteric transition from its tight-binding state to a weaker binding state on the cooperative binding of nucleotides (ATP/ADP) and GroES. GroES modulates binding of GroEL 7 ATP molecules bind to GroEL ATP hydrolysis drives transition back to tight binding state

Protein folding by GroEL and GroES GroEL with unfolded polypeptide GroEL:GroES with enclosed polypeptide GroEL ∆t = 15 sec The folding activity is ATP-dependent! U. Hartl

Nascent-chain binding chaperone: TF Trigger Factor (TF) - most effective peptidyl prolyl isomerase (PPIase) - behaves as a conventional molecular chaperone, i.e., can bind non-native proteins - ribosome-bound (interacts with RNA in the 50S ribosome subunit, but some of it is cytosolic) - interacts with large fraction of nascent polypeptides (as determined by cross-linking) - only occurs bacteria (where it is ubiquitous), although other eukaryal/archaeal proteins have FKBP domains - deletion is not lethal(!) However, deletion is lethal when knock out bacterial Hsp70, which also binds nascent chains crystal structure suggests that it forms a ‘pocket’ for chains exiting the ribosome (recall the ‘crouching Dragon’ structure presented in class) • how do the chaperone binding site and PPIase cooperate? • what is the exact nature of the polypeptide binding site?

Folding of newly synthesized proteins in E. coli TF TF: Trigger Factor assisted by ribosomal chaperones K: DnaK (Hsp70) J: DnaJ E: GrpE EL: GroEL ES: GroES

Nascent-chain binding chaperone: NAC Nascent polypeptide Associated Complex (NAC) - eukaryotic protein consists of alpha and beta subunits; archaea have only beta subunit - as with TF, bound to ribosome - does not contain domain resembling a PPIase Primary function: - prevents inappropriate targeting of nascent polypeptides by SRP - if ER signal sequence is present, SRP binds it, causes translation arrest, and docking occurs; co-translational insertion of protein then takes place, and the sequence is cleaved - if ER sequence is not present, NAC prevents SRP from binding to the nascent chain - evidence suggests it may help targeting to mitochondria

Nascent-chain binding chaperone: Hsp70 Found in nearly all compartments where protein folding takes place: - cytosol of eukaryotes (Hsp70) and bacteria (DnaK) - mitochondria (mt-Hsp70) - chloroplast (cp-Hsp70) - endoplasmic reticulum (BiP) - in yeast and nematodes, there are at least 14 different Hsp70’s One surprising exception: - not found in all archaea; this has been viewed as a paradox - reason is that it has been shown to bind nascent polypeptides: - it can be cross-linked to nascent chains in eukaryotes and bacteria - another reason is that it is important for de novo protein folding

Hsp70 in de novo protein biogenesis Hsp70 is believed to bind and stabilize nascent polypeptides early in their synthesis--preventing misfolding and aggregation Hsp70 binding and release, in an ATP-dependent manner, may help proteins fold to the native state OR Hsp70 may ‘transfer’ non-native proteins to other chaperones for folding (e.g., chaperonins) Hsp70 is also important during cellular stresses (thermotolerance), and has numerous other functions in the cell apart from assisting de novo protein folding. It often works in collaboration with other chaperones, especially Hsp40

Structure of Hsp70 chaperone Structure of entire molecule (~70 kDa) has not been solved flexible linkage between ATPase and peptide-binding domains, and different conformations of molecule possible polypeptide-binding domain consists of beta-sheet scaffold; loops possess hydrophobic residues that contact peptide domain also has an alpha-helical ‘lid’ that is regulated by the ATPase activity ATPase domain (homology with actin, which also binds ATP) Polypeptide binding domain with bound peptide ‘substrate’

Bacterial DnaK functional cycle DnaJ (Hsp40 homologue) has affinity for unfolded proteins, and can deliver a substrate to DnaK DnaK has fast on- and off-peptide binding rate when ATP is bound DnaJ helps accelerate DnaK’s ATPase DnaK has slow on- and off-peptide binding rate when in ADP conformation (i.e., it binds stably) GrpE is a nucleotide exchange factor; it ‘opens’ up DnaK’s nucleotide binding site to help it release ADP and re-bind ATP Released proteins may then be folded or might re-bind DnaJ/DnaK for another round of folding, or may interact with a chaperonin

DnaJ (Hsp40) Hsp40 may bind nascent polypeptides directly, passing these on to Hsp70 although it is a molecular chaperone in its own right, it seems to operate mostly in conjunction with Hsp70 there are numerous Hsp40 homologues in eukaryotes and bacteria; some are specific for the different Hsp70’s, and some actually modulate the function or localization of Hsp70’s There also exists a number of additional chaperone cofactors that modulate the activity of Hsp70’s: - e.g., Hip and Bag; these affect ATPase activity of Hsp70 in yeast, zuotin is an RNA-binding Hsp40 chaperone that is ribosome-bound; a cytosolic Hsp70 interacts with it to bind to nascent polypeptides

Nascent-chain binding chaperone: prefoldin Discovery - a group performed a screen for yeast genes that were synthetically lethal in combination with a gamma-tubulin mutation - found 5 genes that when disrupted, resulted in cytoskeleton defects • actin: sensitivity to osmotic stress, latrunculin-A; disrupted actin filaments • tubulin: sensitivity to benomyl; disrupted microtubules - another lab independently purified a bovine protein complex containing 6 proteins that could bind unfolded actin and tubulin; the yeast complex was later purified and shown to possess the same 6 orthologous proteins as the bovine complex Characterization - synthetic lethality with various actin and tubulin mutants, as well as mutants involved in microtubule processes (i.e., cofactors A-E) - may cooperate with cytosolic chaperonin (CCT) in actin and tubulin biogenesis

Prefoldin subunit structure Predicting coiled coils in proteins: - a number of web-based programs are available - rely on the repeating unit of the coiled coil - a and d positions in a-g heptad repeat are usually hydrophobic - the a and d positions form the apolar interface between the two helices; because of alpha helices normally have 3.6 residues/turn, the 3.5 residues/turn of the coiled coil induces a strain on the helix Some coiled coils can have three or more helices

Prefoldin quaternary structure most of surface is hydrophilic in character inside tips of the coiled coils and ‘bottom’ of cavity display some hydrophobic character Structure of archaeal prefoldin hexamer oligomerization domain is a double beta-barrel structure coiled coils are ~80A long and would be expected to behave independently

Protein Degradation Some protein degradation pathways are nonspecific - randomly cleaved proteins seem to be rapidly degraded However, there is also a selective, ATP-dependent pathway for degradation - the ubiquitin-mediated pathway Ubiquitin is a highly-conserved, 76 residue (8.5 kD) protein found widely in eukaryotes Proteins are committed to degradation by conjugation with ubiquitin Degradation is very important in maintaining order in the cell--imagine the junk that would build up (like our space junk) It is a key component of regulation--in some cases, degradation is the only way to stop the activity of a protein Proteolysis occurs in lysosomes, organelles where non-specific cleavage occurs, or in proteosomes, another type of structure (non organellar) which is sequestered from the cell

Ubiquitin and Degradation Three proteins involved: E1, E2 and E3 E1 is the ubiquitin-activating enzyme - it forms a thioester bond with C-terminal Gly of ubiquitin Ubiquitin is then transferred to a Cys-thiol of E2, the ubiquitin-carrier protein Ligase (E3) selects proteins for degradation. the E2-S~ubiquitin complex transfers ubiquitin to these selected proteins More than one ubiquitin may be attached to a protein target First step goes through an adenylate intermediate like tRNA aminoacylation does Lys e-amino groups are most often targeted by the ligase (E3). Proteins that are selected 1) have free amino terminus 2) not Met, Ser, Ala, Thr, Val, Gly, Cys Most susceptible proteins are secreted proteins, with end being exposed after transport into the ER PEST sequence-proteins are also degraded readily (Pro, Glu, Ser, Thr)

In this depiction, the 26S proteosome is the 20S plus two caps--19S caps 20S proteosome is 7 different a and 7 different b subunits The caps control the specificity of the proteosome--only ubiquitinated substrates will be allowed in. (u biquitin only eucaryotic)

Catalogue des Protéines La course……. 2007 1-5 protéines/jour 2000/semaines

MS-based protein identification : general concept experimental In silico Protein sample Protein sequence(s) Specific protease e.g. trypsin software Protein fragments (5-30 AA peptides) Protein fragment sequences (same protease specificity) MS software Exact masses of peptides Fragmentation (MS/MS) spectrum of each peptide Calculated exact masses of peptides Calculated fragmentation spectrum of each peptide software Best Match(es)

Automated LC- MS/MS run Chromatogram : Total ion current vs. time 25 30 35 40 45 50 55 60 65 70 75 80 Time, min 0.00 5.00e4 1.00e5 421.68 653.32 464.17 582.25 722.28 501.73 507.75 740.41 595.29 700.34 1 2 3 4 Full scan : +TOF MS: m/z, amu 400 450 500 550 600 650 700 750 800 850 900 200 518.82 777.8176 536.80 662.76 531.47 507.1541 1553 (2+) 1553 (3+) MS/MS peptide 4 : +TOF Product (662.8): 10.0 14.0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 m/z, amu 0.0 5.0 235.08 166.06 147.10 262.10 120.06 409.18 207.09 663.3252 110.04 614.2136 138.06

Mol. CelL Proteomics In press. 2007 The combined analysis of 45 gel slices resulted in the acquisition of 516,649 tandem mass spectra, which yielded 35,963 unique peptide identifications

Problématique Interactome Organisation supramoléculaire de la cellule Identification de la fonction des gènes Techniques de détection des interactions protéine-protéine Double-hybride chez la levure Purification de complexes Génétique Prédictions in silico

LE DOUBLE-HYBRIDE DANS LA LEVURE, le test DA Proie Y DF Appât X Gène rapporteur La protéine appât (dont on veut identifier les partenaires) est fusionnée au domaine de fixation à l'ADN (DF) d'un facteur de transcription. Les protéines proies (partenaires potentiels) sont fusionnées au domaine d'activation de la machinerie basale de transcription (DA) d'un facteur de transcription. Les protéines fusions sont exprimées dans des cellules de levure contenant un gène rapporteur dont l'expression est placée sous le contrôle du site de fixation pour le domaine de fixation à l'ADN (DF).

LE DOUBLE-HYBRIDE DANS LA LEVURE, le test DA Proie Y ARN rapporteur DA Proie Y DF Appât X Gène rapporteur Lorsque la protéine proie Y est capable d'interagir avec la protéine appât X, le domaine d'activation se retrouve à proximité du promoteur du gène rapporteur et la transcription a lieu.

Interactions qui n'existent pas physiologiquement  Faux-positifs DF Appât X DA Proie Y Appât A Appât B Appât C La proie collante (interagit avec un très grand nombre d'appâts) DF Appât X Gène rapporteur ARN rapporteur L'appât auto-activateur (activation de la transcription en absence d'interacteur)

Le double-hybride systématique Idée: identifier toutes les interactions protéine-protéine dans une cellule. 1/ prendre tous les gènes d’un organisme comme ‘appâts’. cribler tous ces appâts contre une banque de ‘proies’. 2/ cribler toutes les paires de gènes possibles Possibilite de rajouter l’etude sur C. elegans Nécessité du haut débit, automatisation 2 études dans la levure

Avantages/Inconvénients de l’approche double hybride Protéines chimères Protéines hétérologues 2 protéines à la fois Compartiment cellulaire=noyau de la levure Les faux positifs (auto-activateurs, protéines ‘collantes’) Les faux négatifs Conditions fixes 90% d’interactions déjà connues non retrouvées! les moins In vivo Interactions transitoires ou instables Indépendant du niveau d’expression naturel des protéines Haut-débit Avec une banque, définition de domaines d’interaction (SID dans l’étude sur Helicobacter) les plus

Purification de complexes protéiques par affinité * choisir une technique de co-purification par affinité. * construire une protéine recombinante étiquetée pour chaque gène d’intérêt. * purifier le complexe. * identifier les composants par spectrométrie de masse.

Tandem Affinity Purification Rigaut et al. (1999) Nat. Biotech. Méthode 2 étapes de purification par affinité Conditions d’élution douces Pas de surproduction de la protéine étiquetée. Etiquette TAP Calmodulin Binding Peptide (5kDa) Site de coupure par la protéase TEV 2 domaines de fixation aux IgG de la protéine A (20kDa) TEV IgG beads CBP-TEV-Prot.A

Purifications systématiques de complexes Idée: prendre tous les gènes d’un organisme comme ‘appâts’. construire toutes les souches recombinantes étiquetées purifier tous les complexes identifier tous les partenaires par spectrométrie de masse Nécessité du haut débit, automatisation 2 études dans la levure (méthodes TAP) Voir articles Gavin et al. et Krogan et al., 2006, Nature Possibilite de rajouter l’etude sur C. elegans

Avantages/Inconvénients de l’approche par purification Protéines recombinantes Expression faible ou artificielle Haut-débit possible mais lourd Contaminants (30% douteux!) Interactions faibles perdues Les moins Complexes en conditions natives (caractérisation biochimique) Dans l’organisme d’intérêt Complexes avec plus de 2 composants, interactions stabilisées Définition d’un réseau d’ordre supérieur entre les complexes Les plus

Données issues du transcriptome Synexpression = * coexpression i.e. pour que des protéines puissent interagir ensemble, il faut qu’elles soient coexprimées. * corégulation i.e. les groupes de gènes corégulés (mêmes variations du nombre de transcrits en fonction des conditions étudiées) sont enrichis en gènes codant des protéines interagissant ensemble.

Purification par affinité Croisement des différents résultats dans la levure superposition et complémentarité Nombre d’interactions détectées dans des cribles en double-hybride sur la levure Ito et al., 4081 (92) Uetz et al., 1032 (179) Fromont-Racine et al., 357 (25) 146 (40) 4 (2) 8 (3) Purification par affinité protéines purifiées pour 93 appâts communs TAP 444 Flag 744 133 (10%) 577 877 Gavin et al. (2002) Ho et al. (2002) Explications possibles de ces résultats: Les expériences ne sont pas à saturation. Il y a une énorme proportion de faux positifs. Des biais pour certains types d’interactions. nombre d’interactions détectées Double-hybride 1403 TAP 3222 54 (2-4%) Gavin et al. (2002) Uetz et al. (2000) D’après les données de Ito et al. (2002) Mol. Cell. Proteomics; Salwinski et Eisenberg (2003) Curr. Op. Struct. Biol.

à l’échelle du proteome? Conclusion Carte d’interaction à l’échelle du proteome? Faisabilité? * En tout, 10000 interactions connues. Etudes haut-débit: 30000 au moins (3 partenaires/prot. en moyenne) * Modifications des patterns d’interaction en fonction du développement, ou des conditions extérieures. * On voudrait: Maximum d’interactions Minimum de faux positifs (50%!) Utilité? Outil impressionnant mais…. Il sera toujours nécessaire de valider les interactions par une approche biologique spécifique ou de croiser des cartes obtenues par des méthodes différentes.

Challenge III the modification PTM identification?? PTM modeling, dependency ?? PTM and interaction ??

Challenge II the building blocks Domain Boundaries?? Domain Interaction ?? Domain and Function ??