Light-induced protein degradation : a chemical biology approach Quentin Delacour To cite this version: Quentin Delacour. Light-induced protein degradation : a chemical biology approach. Chemical Physics [physics.chem-ph]. Université Pierre et Marie Curie - Paris VI, 2015. English. NNT : 2015PA066347. tel-01557326 HAL Id: tel-01557326 https://tel.archives-ouvertes.fr/tel-01557326 Submitted on 6 Jul 2017 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la difusion de documents entifc research documents, whether they are pub- scientifques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Université Pierre et Marie Curie Ecole doctorale 388 : Chimie Physique et Chimie Analytique de Paris Centre Laboratoire PASTEUR Pôle de Chimie Bio-Physique Light-induced protein degradation A chemical biology approach Présentée par Quentin Delacour Thèse de doctorat de Chimie-Physique Dirigée par Arnaud GAUTIER et Ludovic JULLIEN Présentée et soutenue publiquement le vendredi 25 septembre 2015 Devant un jury composé de : M. Dr. Arnaud GAUTIER Directeur de thèse M. Prof. Ludovic JULLIEN Directeur de thèse Mme Dr. Sandrine SAGAN Examinateur M. Dr. Alexandre SPECHT Rapporteur M. Dr. Christoph TAXIS Rapporteur Mme Prof. Sophie VRIZ Examinateur 2 Light-induced protein degradation A chemical biology approach Présentée par Quentin Delacour Thèse de doctorat de Chimie-Physique S.D.G. 3 List of abbreviations a.a.: amino-acid InsP6: Inositol-6 Phosphate ABA: Abscissic acid IRES: Internal ribosomal entry site AID: Auxin inducible degron Jα: C-ter helix of AtLOV2 AFB: Auxin signaling F-box protein KikG/R: Kikume green/red APC/C: Anaphase-promoting complex E3 LOV: Light oxygen voltage ARF: Auxin response factors LRR: Leucine rich repeats At: Arabidopsis thaliana M: Mock sample ATCC: American type culture collection NA: numerical aperture B-LID: Blue light inducible degradation NAA: Naphtalene acetic acid Cdk: Cyclin-dependent kinase NLS: Nuclear localization signal CHIP: C-ter of Hsc70 interacting protein NPE: 1-(2-nitrophenyl)-ethanol CHO: Chinese Hamster ovarian N-ter: N-terminus of a protein CID: Chemically-inducible dimerization ODC: Ornithine decarboxylase CMV: Cytomegalo Virus Os: Oryza sativa CRL: Cullin-RING ligase PA-IAA: Photoactivatable IAA Cry: AtCryptochrome PBS: Phosphate saline buffer C-ter: C-terminus of a protein PDB: Protein data dank Cul: Cullin POI: Protein of interest DBP: Designer binding protein PQC: Protein quality control DD: Destabilizing degron psd: photosensitive degron DHFR: Dihydrofolate reductase PROTAC: Proteolysis targeting chimeric D-MEM: Dulbecco's modified eagle protein medium ROI: Region of interest DMNB:4,5-Dimethoxy-2-nitrobenzyl SAC: Spindle assembly checkpoint DMSO: Dimethyl sulfoxyde Sc: Saccharomyces cerevisiae DUB: Deubiquitin enzyme SD: Standard deviation EGFP: Enhanced green fluorescent protein shAID: short Auxin-inducible degron E1: Ubiquitin activating enzyme siRNA: small interfering RNA E2: Ubiquitin conjugating enzyme Skp1: S-phase kinase-associated protein E3: Ubiquitin ligase enzyme TEV: Tobacco-etch Virus ER: endoplasmic reticulum TIPI: TEV protease-induced protein FKBP: FK506 binding-protein inactivation FMN: Flavin mononucleotide TIR1: Transport inhibitor response 1 FP: Fluorescent protein tRNA: transfer RNA GA: Gibberellic acid ts: temperature sensitive GFP: Green fluorescent protein UAA: Unnatural amino-acid HA: Haemaglutinin Ub: Ubiquitin HEK293: Human embryonic kidney UAE: Ubiquitin activating enzyme HPLC: High-performance liquid UBD: Ubiquitin-binding domain chromatography UCD: Ubiquitin-conjugating domain Hs: Homo sapiens UCE: Ubiquitin conjugating enzyme E2 hν: light-illumination UPS: Ubiquitin proteasome system HSP: Heat-shock protein UV: Ultra violet IAA: Indole acetic acid YFP: Yellow fluorescence protein IB: Immunoblotting 2,4 D: 2,4 Dichlorophenoxyacetic acid 4 Contents List of abbreviations ................................................................................................................................4 Foreword ............................................................................................................................................... 15 PART A: INTRODUCTION .................................................................... 17 Chapter 1: Strategies for the control of protein degradation 1.0 Motivation: development of a generic perturbation method ...................................................... 19 1.1 The Ubiquitin-Proteasome System allows the selective degradation of protein substrates ..... 22 1.1.1 The Proteasome is an efficient proteolytic complex in eukaryotes ......................................... 22 1.1.1.1 The Proteasome is a protein complex with differentiated subunits ................................ 22 1.1.1.2 Interaction with Proteasome is sufficient for proteolysis ................................................ 23 1.1.1.3 Conclusion ........................................................................................................................ 24 1.1.2 Polyubiquitination of protein substrates is a proteasomal degradation addressing tag ........ 24 1.1.2.1 Ubiquitin conjugation is an important post-translational mechanism ............................. 24 1.1.2.2 Polyubiquitination controls the degradation of protein substrates ................................. 25 1.1.3 Perturbation of the UPS pathway by small molecules, a chemical biology approach ............ 27 1.1.3.1 Motivation ......................................................................................................................... 27 1.1.3.2 Perturbation of the Proteasome activity globally impairs the UPS .................................. 27 1.1.3.3 Perturbation of the activity of UPS enzymes .................................................................... 28 1.1.3.4 Targeting E3 Ubiquitin Ligases can trigger specific substrate degradation ...................... 28 1.1.4 Complementary signaling can implement substrate selection for proteasomal degradation ...................................................................................................................................... 29 1.2 The UPS Protein Quality Control (PQC) pathway allows the degradation of defective protein substrates ............................................................................................................................................. 30 1.2.1 N-end rule pathway: lessons from a destabilization pathway ................................................. 30 1.2.1.1 The N-end rule pathway: from instability observations to chimeric destabilization ... 33 1.2.1.2 Towards the conditional degradation of a POI by N-end rule application ....................... 33 5 1.2.2 Misfolded proteins can be degraded by the PQC pathway ...................................................... 35 1.2.2.1 Misfolded proteins are addressed by the UPS pathway in vivo ........................................ 36 1.2.2.2 A chemical biology approach: conditional control of POI by small molecules ................. 37 1.3 The UPS allows the selective regulation of functional proteins .................................................. 40 1.3.1 Cullin-Ring Ligases (CRL) present a generic, yet modulable strategy for selective degradation of protein substrates ........................................................................................................................ 40 1.3.1.1 CRL is a common strategy for substrate ubiquitination in eukaryotes ............................. 40 1.3.1.2 SCF strategy rely on specific substrate recognition .......................................................... 42 1.3.2 A chemical biology approach to POI depletion: hijacking the UPS selectivity by chimeric bipartite molecules ............................................................................................................ 44 1.3.2.1 Chimeric bipartite F-box proteins target POI degradation ............................................... 44 1.3.2.2 Chimeric bipartite adapter proteins rely on specific POI binding domains ...................... 45 1.3.2.3 PROTACs: a small-molecule approach to conditional POI depletion ................................ 47 1.4 Auxin-induced degradation, a conditional small-molecule system for specific protein depletion ............................................................................................................................................................... 49 1.4.1 Nuclear Auxin-Response in plant involves controlled degradation strategies ........................ 49 1.4.1.1 The phytohormone auxin regulates genetic expression by controlled degradation ........ 49 1.4.1.2 Auxin directly mediates heterodimerization of TIR1 and Aux/IAA ................................... 50 1.4.2 Auxin-Inducible-Degron (AID), a chemical biology methodology for conditional POI degradation ....................................................................................................................................... 51 1.4.2.1 The Auxin-Inducible degron allows conditional degradation in yeast .............................. 51 1.4.2.2 The AID methodology can be extended to mammalian culture cells ............................... 53 1.4.2.3 Fine regulation of auxin-mediated response by combinatorial variants .......................... 53 1.5 Conclusion ....................................................................................................................................... 55 Chapter 2: Strategies for the photocontrol of protein activity in vivo 2.1. Photosensible protein modules trigger effector responses upon light- illumination ................. 57 2.1.1. Description of photosensible protein modules ....................................................................... 57 2.1.2. Examples of photosensible protein modules .......................................................................... 58 2.1.2.1 LOV-domain modules ........................................................................................................ 58 2.1.2.2 Cryptochromes based modules ......................................................................................... 59 2.1.2.3 Phytochrome modules ...................................................................................................... 60 2.1.2.4 FP Dronpa monomeric state is reversibly controllable by light ........................................ 60 2.1.3. Classification of optogenetic systems based on photosensible protein modules ................. 61 2.1.3.1 By physiological function target ....................................................................................... 61 6 2.1.3.2 By actuation mechanism .................................................................................................. 62 2.1.4. Conclusion ............................................................................................................................... 62 2.2 Chemical actuators enable photocontrol of physiological activities ............................................ 63 2.2.1 Classification of chemical actuators ......................................................................................... 63 2.2.1.1 Photoswitchable effectors ................................................................................................ 63 2.2.1.2 Caged effectors ................................................................................................................. 64 2.2.1.3 Genetically encoded caged proteins: chemical genetics approaches ............................... 65 2.2.2 Specification of a good caged compound ................................................................................ 65 2.2.2.1 The caged compound must be inactivate in the dark ....................................................... 66 2.2.2.2The caged compound must be activatable upon light illumination................................... 66 2.2.2.3The caged compound must be compatible with cellular physiology ................................. 67 PART B: RESULTS ............................................................................................................ 69 Chapter 3: Development and Optimization of AID depletion in mammalian cells 3.1 Specifications for the implementation of an AID methodology in mammalian cells .................. 71 3.1.1 AID methodology necessitates dual expression of heterologous proteins .............................. 72 3.1.1.1 Bicistronic vector strategy allow dual protein expression ................................................ 72 3.1.1.2 A single plasmid strategy for AID implementation ........................................................... 72 3.1.2 Specification of cell line model for evaluation of the AID methodology ................................. 73 3.1.2.1 Mammalian cell line and culture medium selection ......................................................... 73 3.1.2.2 Culture medium must be auxin-free ................................................................................. 73 3.1.2.3 Culture medium must be compatible with light illumination ........................................... 73 3.1.3 Validation of the AID methodology in a mammalian cell line ................................................. 74 3.1.3.1 Implementation of auxin-conditional control of POI in CHO cells ................................... 74 3.1.3.2 Implementation of auxin-conditional control of POI in HEK293 cells ............................... 75 3.2 Optimization of the AID methodology for efficient POI depletion in HEK293 cells ..................... 76 3.2.1 Subcellular addressing is an important parameter for efficient POI depletion ...................... 76 3.2.1.1 Slow POI depletion is observed upon auxin addition in HEK293 cells .............................. 76 3.2.1.2 Heterologous OsTIR1 and AID-GFP-NLS are not colocalized in HEK cells ......................... 77 3.2.2 Co-localization of TIR1 and AID-GFP-NLS increases the degradation rate .............................. 78 3.2.2.1 OsTIR1 can be addressed to the nucleus by NLS fusion .................................................... 78 7 3.2.2.2 Co-localization of TIR1 and AID-GFP-NLS increases the rate of degradation twofold ...... 78 3.2.2.3 Observations ..................................................................................................................... 79 3.2.2.4 Discussion .......................................................................................................................... 80 3.2.3 Probing F-box/Skp1 fusions for AID optimization .................................................................... 81 3.2.3.1 F-box/Skp1 fusions were reported to enhance AID depletion in yeast ............................ 81 3.2.3.2 F-box/Skp1 fusion enhance AID depletion in mammalian cells ........................................ 81 3.3 Acceleration of POI depletion kinetics using AID F-box variants .................................................. 82 3.3.1 TIR1 homologs allow auxin-conditional degradation in heterologous system ....................... 82 3.3.1.1 TIR1 homologs exhibit differential range of auxin-response depletion in plants ............ 82 3.3.1.2 Development of mammalian AID methodologies based on TIR1 homologs ................... 83 3.3.1.3 Kinetic comparison of F-box AtAFB2 and OsTIR1 for auxin-induced degradation ............ 84 3.3.1.4 Conclusion ......................................................................................................................... 84 3.3.2 Point mutations in TIR1 accelerate auxin-conditional degradation ........................................ 84 3.3.2.1 Identification of TIR1 mutants for enhanced Aux/IAA degradation in yeast .................... 84 3.3.2.2 Introduction of point mutations in TIR1 variants in mammalian AID methodology ........ 85 3.3.2.3 OsTIR1 mutants were selected as potential targets for enhanced AID ............................ 86 3.3.2.4 Introduction of D170E mutation to OsTIR1 enhances the degradation rate .................... 86 3.4 Conclusion ....................................................................................................................................... 87 Supplement 3.5 Development of AID methodology for generic application in mammalian cells .... 88 3.5.1 Development of a shorter AID tag ........................................................................................... 88 3.5.1.1 Motivation ......................................................................................................................... 88 3.5.1.2 Identification of essential functionalities in AID and construction of a short degron ..... 88 3.5.1.3 The short AID is functional but less-efficient than full-length AID .................................... 89 3.5.1.4 Discussion .......................................................................................................................... 90 3.5.1.5 Validation in literature ..................................................................................................... 90 3.5.2 Use of esterase-sensitive auxin for enhanced permeability ................................................... 91 3.5.2.1 Motivation ......................................................................................................................... 91 3.5.2.2 Me-IAA can trigger AID depletion in mammalian cells ..................................................... 91 3.5.3 Development of cytoplasmic AID depletion is hindered by the nuclear localization of AID ... 92 3.5.3.1 Heterologous AID-EGFP fusions are adressed to the nucleus in HEK293 cells ................ 93 3.5.3.2 Endogenous NLS sequences are present in AID ............................................................... 93 8 Chapter 4: Design of Photo-activatable auxins 4.1 Introduction: structural analysis of auxin perception by TIR1 enables the rational design of caged auxins ......................................................................................................................................... 95 4.1.1 Auxin perception in plants ....................................................................................................... 95 4.1.2 Caged auxins in plants ........................................................................................................... 97 4.2 Development of carboxylate-conjugated DMNB caged-auxin...................................................... 98 4.2.1 First-generation ....................................................................................................................... 98 4.2.1.1 Motivation ......................................................................................................................... 98 4.2.1.2 Synthesis ............................................................................................................................ 98 4.2.1.3 Photochemical characteristics .......................................................................................... 98 4.2.1.4 In vivo stability ................................................................................................................... 99 4.2.2 Discussion .............................................................................................................................. 100 4.2.3 Second generation ................................................................................................................. 100 4.2.3.1 Synthesis .......................................................................................................................... 100 4.2.3.2 Stability ............................................................................................................................ 101 4.2.4 Third generation .................................................................................................................... 102 4.2.4.1 Synthesis .......................................................................................................................... 102 4.2.4.2 Photochemical characterization ...................................................................................... 102 4.2.4.3 In vivo stability ................................................................................................................. 103 4.2.5 Conclusion ............................................................................................................................. 103 4.3-Alternative strategies for the development of caged auxins .................................................... 104 4.3.1 Coumarin-based caging groups ............................................................................................. 104 4.3.2 NH-protection strategy .......................................................................................................... 105 4.3.2.1 Motivation ....................................................................................................................... 105 4.3.2.2 Model for reactivity ......................................................................................................... 106 4.3.2 NH-protection with DMNB ..................................................................................................... 106 4.3.2.1 Synthesis .......................................................................................................................... 106 4.3.2.2 Photochemical evaluation ............................................................................................... 106 4.4 Conclusion .................................................................................................................................... 106 9