Protein Degradation - Proteasome PDF

PROTEIN DEGRADATION Proteasome INTRODUCTION How to destroy a protein ? UPR UPR is unfolded protein response, there's a cellular response to an increase in unfolded protein, and ER is ER in the endoplasmic reticulum associated with destr...

PROTEIN DEGRADATION Proteasome INTRODUCTION How to destroy a protein ? UPR UPR is unfolded protein response, there's a cellular response to an increase in unfolded protein, and ER is ER in the endoplasmic reticulum associated with destruction. ERAD Ubiquitination, traffic and proteasome proteasome: assembly, structure & function proteasome and chaperon (folding and destruction) 1  PROTEIN DESTRUCTION Spontaneous folding Chaperons Native Protein Aggregation Degradation Eukaryotes ca 30% proteins « misfolded » at one time or another Toxic Aggregates; « dominant negatives » mutations oligomeres Glycosylation is attachment of sugars via specific enzymes. Glycation is non-enzymatic reaction. Glycation is always pathological. It inhibits the activity of certain proteins. postsynthetic damage, e.g., by oxidation, glycation and deamidation 2  Protein Destruction double agents Folding Degradation 3  DEGRADATION & PROTEASOME Questions Extracellular Ex.: trypsin etc Proteolysis intracellular Lysosome intracellular proteolysis. And proteolysis is always a dangerous process, because it can attack also innocent bystanders. proteasome How to protect other cellular components ? (bacteria: no compartment!) How to organize an efficient machinery? (auto-assembly & auto-organisation) How to leave the ER ? 4  PROETIN DEGRADATION CONTENT INTRODUCTION How to destroy a protein ? UPR (Unfolded Protein Response) ERAD (ER associated Destruction) Ubiquitination, traffic and proteasome proteasome: assembly, structure & function proteasome and chaperon (folding and destruction) 5  DEGRADATION & PROTEASOME Folding and quality control are linked Capacity ER = Charge ER Overcharge = That means sending the unfolded proteins to the proteasome. Stress ER Derangement The attenuation, that means Influences the entire cell fewer inhibiting or slowing down protein synthesis Stress: - Mutated Proteins - Increase Synthesis (infection, differentiation) -Metabolic Stress (eg. diabetes) Control of resident proteins 6  DEGRADATION ET PROTEASOME Répliage des protéines et contrôle de qualité II Importance/consequences ERAD Some fifty diseases are known to be linked to UPS/ERAD (folding/destruction) UPS & ERAD can trigger apoptosis -Class I: Loss of function of a protein through mutation (Insulin receptor: diabetes; coagulation factors: hémophilia) -Class II: Accumulation of erroneous proteins (mutations/PGD etc) -Class III: deficit in the UPR -Class IV: deficit distal to the UPR (pe proteasome) Observation CFTRplasmam membrane chloride transport; mucoviscidose CFTR F506; folding necessary before passage ER-Golgi CFTR F506 does not arrive at the plasma membrane (help through small molecule chaperons ?) 7  DEGRADATION ET PROTEASOME UPR Induction oftranscription # « UPR » unfolded protein response # chaperons, Bip, PDI etc UPR: ER enlargement (remember: ER synthesizes most of phospholipides cholestérol) microarrays: >380 genes regulated remodelin biosynthetic pathway ? ER: first step, avoid accumulation 8  DEGRADATION ET PROTEASOME UPR Three known pathways # IRE1 (inositol requiring kinase 1) # ATF6 (activating transcription factor 6) # PERK (RNA activated protein kinase like ER kinase) 9  DEGRADATION AND PROTEASOME ACTIVATION OF CHAPERON EXPRESSION ER LUMEN NORMAL STATE INCREASED FOLDING PROBLEMS Bip, ER CHAPERON FOLDED PROTEIN MOSFOLDED PROTEIN There is BIP, which is a chaperone in the ER. And BIP is also necessary actually to pull the pathway from the transplant. You have the correctly folded protein and the misfolded protein. ormal state here, you have only, or almost only, folded proteins, and you have this chaperone and it doesn't have any proteins freely floating around. Here, if you have a lot of misfolded proteins, most of the BIP, the chaperone, will be actually attracted to these misfolded proteins to help them. So, there is little if any free chaperone in the lumen of the ER. 10 DEGRADATION AND PROTEASOME ACTIVATION OF CHAPERON EXPRESSIOI Chaperon genes  11  DEGRADATION & PROTEASOME ACTIVATION OF CHAPERON EXPRESSION 12 IRE1: 2 ways ATF: regulation? proteolysis?  DEGRADATION & PROTEASOME ACTIVATION OF CHAPERON EXPRESSION Chaperon BiP: (Binding immunoglobulin protein, GRPA5) Bound either to IRE1 or unfolded protéines 13 Binding to IRE1 inhibits dimerisation of IRE1  DEGRADATION & PROTEASOME ACTIVATION OF CHAPERON EXPRESSIONII PERK: Similar to IRE kinase (eIF2-) -/- cells: more sensitive PERK not in yeast, but droso etc Rôle of BiP in activation 14  Dégradation de protéines – le protéasome CONTENU INTRODUCTION How to destroy a protein ? UPR ERAD Ubiquitination, traffic and proteasome proteasome: assembly, structure & function proteasome and chaperon (folding and destruction) 15  DEGRADATION & PROTEASOME Considerations Degradation: dynamic homeostasis, change in structure determines halflife of regulatory proteins immune system: antigen presentation Risks: spatial and temporal control compartmentalisation (lysosomes) prokaryotes: absence of endomembranes (proteasome; archae, pro-, eukaryotes) Needs: unfolding (passage) « reverse chaperons » THE ER ISNOT A DEGRADING COMPARTMENT ! 16  DEGRADATION & PROTEASOME « reverse gear» - solubility (chaperons, glycosylation) - unfolding - channel (sec61? Sec63? Derlin? others?) - »extractor » Cdc48 (ATPase) - escorts (Rad23) - ubiquitination – where? 17  DEGRADATION ET PROTEASOME ERAD CHAPERONES/CHAPERONINES https://youtu.be/jOhNyVjkChM 18  DEGRADATION & PROTEASOME ERAD ER-associated degradation Quality Control Send proteins with folding errors to degradation « proteasome » ERAD: interaction between different systems: induction and degradation induction necessary for dégradation 1) Induction, Atténuation 2) Retrotransport 3) Degradation ER rapidly saturated « A system conceived for ordinary situations » 19 DEGRADATION & PROTEASOME ERAD here they have introduced a mutation in the protein, which then gets blocked in the ER, and you see here the ER is completely swollen there. 20  Dégradation de protéines – le protéasome CONTENU INTRODUCTION How to destroy a protein ? UPR ERAD Ubiquitination, traffic and proteasome proteasome: assembly, structure & function proteasome and chaperon (folding and destruction) 21  UBIQUITIN Nobel Prix 2004 "for the discovery of ubiquitin-mediated protein degradation” Aaron Ciechanover – Technion – Israel Institute of Technology, Haifa, Israel, Avram Hershko – Technion – Israel Institute of Technology, Haifa, Israel and Irwin Rose – University of California, Irvine, USA – Ubiquitination: Signal localisation Destruction of proteins; ERAD; transcription 22 Protein degradation - proteasome examples of biological functions 23  DEGRADATION ET PROTEASOME ubiquitination and protéasome – overview Hypothetical model Ubiquination required for « proteasome » targetting Relation translocon/protzasome proteasome needs first detection of the substrate, tagging by ubiquitination, then recognition and transport, and finally degradation. 24  DEGRADATION & PROTEASOME ubiquitination & proteasome – general view DETECTION (Chaperonnes, « agents doubles ») Tagging RECOGNITION/TRIAGE/TRANSPORT DEGRADATION 26 DEGRADATION & PROTEASOME Ubiquitination - description Posttranslational Modifications -specific proteolysis -glycosylation -lipidation -phosphorylation etc. Ubiquination: Attaching one protein to another! « steric » Negative: steric obstacle Positive: new interactions « Darwin’s phosphate » (possibility of evolution) Ubiquity is a peptide. You attach one peptide to another. 27  DEGRADATION & PROTEASOME Ubiquitination – the chemical reaction  DEGRADATION & PROTEASOME Ubiquitination – the chemical reaction Thioester high energie; therapeutic target It's a grade that you need for entry of carbon into the peptide cycle. Now, the important thing is that these thioesters are high energy. They are highly reactive. « bucket brigade » So, you have two enzymes here, actually, to activate and transfer of Multienzyme complex enzyme complexes, and you have E3 complex, which is Multiple isoforms very important, because that decides which protein will be ubiquitin. -NH2 de Lys E3: spécificité therapeutic target 29  DEGRADATION & PROTEASOME Ubiquitination: Mono, Multi and Poly Ubiquitin, Ubl (ubiquitin like; UBA domains) Desubiquination Cf.: kinases and phosphorylases Specificity:E3 binds to a specific domain on the substrate différent isoforms of E3 Mono/Multi- of polyubiquination Ubiquitin contains 7 LYS Mono/Multi-ubiquination: Endocytosis and re-surfacing (4 Ubi) TGN « 3 dimensional » Signal Polyubiquination: (« network » via different Lys of ubiquitine) Rich in information 3D Protaasome Signalling function Fonction de signalisation 30 (« repair factor for DNA driven errors »)  DEGRADATION & PROTEASOME MONO/MULTI-UBIQUITINATION Internalisation & metabolism/recycling of an RTK EGF recepteur Ub-Ligase Different receptors Ub-Receptor Ubiquitine 31  DEGRADATION ET PROTEASOME MONO/MULTI-UBIQUITINATION Internalisation & metabolism/recycling of an RTK EGF recepteur Ub-Ligase Different receptors Ub-Receptor Ubiquitine 32 33 34 35 (Endosomes/MVB/LYSO) 36 37  DEGRADATION ET PROTEASOME Ubiquitin like 相撲 SUMO (small ubiquitin-related modifier) NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8) Atg (autophagy-related proteins) 38  DEGRADATION ET PROTEASOME UBLs Structural comparison of UBLsStructural comparison of MoaD, ThiS, Ub, SUMO, NEDD8, Atg8 and Atg12 with their respective PDB codes shown below each structure [59–65]. 39 DEGRADATION ET PROTEASOME Cycle de SUMO inactive precursor cleaved by SENP to expose a C-terminal di- glycine motif: mature form activated by the ATP- dependent formation of a thioester bond with the active site of the E1 enzyme passed to the active site cysteine of the E2 conjugating enzyme, Ubc9 transfer of SUMO to the target protein DeSUMOylation is mediated by SENP 40  DEGRADATION ET PROTEASOME MONO/MULTI-UBIQUITINATION NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8) NEDD8 is the most similar UBL to Ub (human NEDD8 58% identical to human Ub) NEDD8 primarily conjugated to and regulates the activity of cullin proteins Cullins: subunits of the largest class of Ub E3 enzymes) Crucial for the control of many important proteins SUMO (small ubiquitin-related modifier) numerous essential functions in eukaryotes multiple isoforms in mammals SUMO is recognized by SIMs (SUMO-interacting motifs) SUMOylation regulates many essential eukaryotic processes Atg12 and Atg8 Autophagy is mediated by >30 autophagy-related (Atg) proteins 2 distinct UBLs (Atg12, Atg8) activated same E1, required for autophagosome formation. Atg8 ligated to a lipid rather than to another protein Atg8 ligation to PE is to mediate delivery of specific cargo to autophagosomes and lysosomes Trafficking machinery 41 DEGRADATION ET PROTEASOME Autres signaux DESTRUCTION BOX Cyclines : RTALGDIGN PEST : PEST 42  DEGRADATION ET PROTEASOME Acétylation - oftent N-terminal - During synthesis (ribosomes), or later - REVERSIBLE 43  DEGRADATION ET PROTEASOME Acétylation - Most proteins are acetylated one moment or another during their life time 44 - Different transferases (Nat: N-acetyl transferases)  DEGRADATION ET PROTEASOME Acétylation - Conséquences 45 Interaction: Folding, Localisation, Degradation DEGRADATION ET PROTEASOME Acétylation Actine 46  DEGRADATION ET PROTEASOME Acetylation - Metabolic regulation 47 METABOLISME: Régulation flux métabolique et expression génique via Acétylation  Dégradation de protéines – le protéasome CONTENU INTRODUCTION How to destroy a protein ? UPR ERAD Ubiquitination, traffic and proteasome proteasome: assembly, structure & function proteasome and chaperon (folding and destruction) 48 Dégradation de protéines – le protéasome CONTENU 49 Dégradation de protéines – le protéasome CONTENU 50  DEGRADATION ET PROTEASOME Le protéasome - introduction - Discovery: the 60ies In situ - REQUIRED ACTIVITIES: - UNFOLDING (reverse chaperons) - RECOGNITION - PROTEOLYSIS -« self compartmentalizing proteases » In the three kingdoms Archae, bacteria, eukaryotes Discovery in bactéria: Sequence comparison (hsIV, prcB) reconstitué From simple to complexe Bacterias: non-essential Yeast: essential Bactéries: sans compartiments Membranaires/vésicules « protéasome sans chapeau » Cible thérapeutique: Bortezomib: multiple myeoloma; 51 peptide-acide boronique DEGRADATION ET PROTEASOME Protéasome: les étapes 52  DEGRADATION ET PROTEASOME Protéasome: les étapes How to obtain structure fonction ?? RX: Rather complex ! Variations ! MODELS HIGH RESOLUTION EM