Mécanismes de Résilience Cellulaire dans les Processus Normaux et Pathologiques / Mechanisms of Cellular Resilience in Health and Diseases

Séminaire organisé par Anne Dejean (Institut Pasteur, Paris, France) et Anne Bertolotti (MRC-LMB, Cambridge, Royaume-Uni) du 6 au 11 mai 2019

Résumé 

Dix-huit chercheurs et deux post-doctorantes travaillant en Allemagne, aux États-Unis, en France, aux Pays-Bas, au Royaume-Uni et en Suisse se sont réunis à la Fondation des Treilles du 6 au 11 mai 2019 pour discuter des mécanismes de résilience cellulaire. La perte progressive de la résilience cellulaire est à la base du vieillissement et des maladies liées à l’âge. Cette perte de résilience se traduit par l’accumulation de protéines anormales dans le cas des maladies dégénératives ou de dommages à l’ADN affectant également l’homéostasie protéique des cellules vieillissantes ou tumorales. Cellules et organismes ont évolué pour survivre aux conditions difficiles et ont ainsi acquis une résilience à ces dommages. Les cellules consacrent alors une grande partie de leurs ressources à la prévention de leur accumulation, via plusieurs mécanismes majeurs de contrôle qualité des protéines tels que les systèmes de dégradation des protéines, l’ubiquitine et SUMO. Cette conférence unique a réuni des chefs de file travaillant sur les différents mécanismes gouvernant cette santé cellulaire, en lien avec diverses maladies humaines importantes. Biochimistes, chimistes, biologistes cellulaires se sont retrouvés pour présenter à la fois un historique de leurs travaux, remettant ainsi en contexte leurs découvertes séminales, et leurs plus récents projets. L’atmosphère conviviale du colloque a permis des discussions animées, particulièrement au sujet du concept de sérendipité, de l’interdisciplinarité et de l’importance de la recherche fondamentale. Plusieurs présentations ont en effet illustré comment la compréhension de mécanismes moléculaires clés pour le bon fonctionnement des cellules a permis la conversion de ces connaissances en stratégies thérapeutiques, notamment pour de possibles traitements de cancers et de maladies neurodégénératives.

Summary

Eighteen researchers and two post-docs working in France, Germany, the Netherlands, Switzerland, the United Kingdom and the United States got together at the Treilles Foundation from May 6^thto 11^th2019, to discuss the mechanisms of cellular resilience. Gradual loss of cellular resilience is underpinning aging and age-related diseases. Loss of resilience translates into accumulation of abnormal proteins in neurodegenerative diseases as well as DNA damage in cancer and aging cells, DNA alterations that also perturb protein homeostasis. This unique conference brought together key opinion leaders working on diverse mechanisms governing cellular fitness with a link to prominent human diseases. Biochemists, chemists and cellular biologists gathered to present an overview of their work and groundbreaking discoveries, as well as their most recent projects. Cells and organisms have evolved to survive challenging conditions and thereby acquired resilience to damages. Thus, cells devote a large fraction of their resources to prevent accumulation of damages through multiple mechanisms collectively known as quality control systems. Central players of protein quality control are the protein degradation systems, ubiquitin and SUMO. The relaxed atmosphere of the seminar fostered lively discussions, particularly around the concept of serendipity, interdisciplinarity, and the importance of fundamental research. Several presentations have indeed illustrated how the understanding of molecular mechanisms key to the wellbeing of the cells allowed the translation of that knowledge into therapeutic strategies, notably for possible treatments for cancers and neurodegenerative diseases.

Compte-rendu (en anglais):

Brenda Schulman (Max Planck Institute of Biochemistry) presented structural studies of the Cullin-RING family of ubiquitin E3 ligases. Crystal structures of the different subunits in complex have revealed mechanistic insights for the assembly, catalytic activity and specificity of these complexes. Her talk focused on the regulation of Cullin-RING ligases by another, ubiquitin-like, post-translational modification (PTM) called neddylation. NEDD8 covalently and reversibly binds to the Cullin module, playing a key role in the transfer of ubiquitin moieties to target proteins. As these targets have functions in the cell cycle, DNA repair, immunity… E3 ligases can be mutated in diseases or hijacked during virus infections. Understanding of the Cullin-RING mechanism of action at the molecular level would thus allow the design of small molecules for targeted therapies.

Mark Hochstrasser (Yale University) presented two projects from his laboratory on SUMO and ubiquitin proteases. Yeast strains with a deletion for the SUMO-specific protease Ulp2 can adapt to this loss through disomy of two chromosomes, resulting in altered regulation of ribosome biogenesis. Long-term adaptation restored euploidy and was accompanied by loss-of-function mutations in other enzymes of the SUMO pathway, to reduce accumulation of SUMO substrates and restore homeostasis. The second project revealed how the intracellular bacteria Wolbachia, a prevalent infection of insects and arthropods, secretes a toxin containing a deubiquitylase activity resulting in cytoplasmic incompatibility. This phenomenon could be used to control insect populations and reduce vector-borne diseases.

Frauke Melchior (Heidelberg University) highlighted the importance of the reversibility of PTMs, allowing SUMO to act as a molecular switch in signalling pathways. Indeed, cellular stressors alter the SUMO proteome, and she presented unpublished data from her lab showing that EGF induction transiently decreases the SUMOylation of transcriptional repressors, resulting in increased expression of select target genes, thus allowing cells to respond to the stress.

Anne Dejean (Institut Pasteur, Paris) expanded on the role of SUMOylation in transcriptional control, as the vast majority of SUMOylated substrates are chromatin-associated proteins. She first showed a repressive role of SUMO at the interferon β locus, linking this PTM with the repression of innate immunity. SUMOylation inhibition is currently being investigated as a novel cancer treatment strategy in a phase I clinical trial. She then described how SUMO occupies distinct chromatin landscapes in pluripotent versus somatic cells, establishing a role for SUMO in the safeguard of cell identity.

Ron Hay (University of Dundee) discussed the link between ubiquitylation and SUMOylation, through structural studies of the SUMO-targeted ubiquitin ligase RNF4, which can recognize SUMOylated substrates and target them for degradation by the ubiquitin-proteasome pathway. This crosstalk between PTMs notably results in the degradation of the oncogenic PML-RARα, following the very efficient arsenic therapy in cases of acute promyelocytic leukaemia.

Vishva Dixit (Genentech, San Francisco) presented advances in the field of innate immunity, particularly in response to infection by Gram-negative bacteria. Their intracellular lipopolysaccharide activates caspase-11 and a non-canonical inflammasome pathway, resulting in the pyroptotic death of host cells. A genetic screen in mice has allowed identification of several components of this inflammasome, leading in particular to the characterization of Gasdermin-D as a key mediator of the immune response. This and other hits from the screen could serve as potential therapeutic targets against lethal septic shock.

Martin Eilers (University of Würzburg) focused his talk on the mechanism of action of the oncogenic MYCN transcription factor, which drives neuroblastoma tumour progression. MYCN prevents stalling of RNA polymerase II at core promoters and increases transcriptional elongation, by recruiting BRCA1 to suppress R-loop formation. This recruitment is mediated by the ubiquitin protease USP11, whose deubiquitylation activity stabilizes MYCN and BRCA1 levels on chromatin. Binding of USP11 to MYCN is itself regulated by another reversible PTM: dephosphorylation of MYCN at Thr58 is required for its interaction with USP11.  Finally, data was presented on the function of NUAK1 in the PP1/PNUTS complex during spliceosome activity.

Andrew Jackson (The University of Edinburgh) presented the molecular mechanism underlying an auto-inflammatory disease called Aicardi-Goutières syndrome, a genetic mimic of viral infections such as Zika. Mutations in Ribonuclease H2 hinder the proper excision of ribonucleotides incorporated into DNA. Their alternative processing by Topoisomerase I causes replication-associated DNA damage and aberrant mitosis. The ensuing formation and breakdown of micronuclei activate the cGAS/STING pathway, resulting in an inflammatory response. This pathway is also inactivated in many cancers, adding further clinical relevance for the therapeutic targeting of its components.

Peter Walter (University of California, San Francisco) presented data on the link between the integrated stress response (ISR) and cognition. The unfolded protein response network monitors proteins’ folding status and can take corrective action through three distinct molecular pathways, with the ISR resulting in the phosphorylation of eIF2 to repress translation and alleviate stress. The small molecule inhibitor ISRIB can restore translation under active ISR signalling and leads to enhance long-term memory and learning in various mice models of brain injury, thus reversing their cognitive defects.

Jeff Kelly (The Scripps Research Institute, La Jolla) brought structural insights on transthyretin, a tetrameric protein that can aggregate in the heart and brain, causing degenerative amyloid diseases. His lab designed a compound, which prevents this aggregation, and Tafamidis is now used for the treatment of cardiomyopathy due to transthyretin-mediated amyloidosis. He further highlighted the function of autophagy in neurodegenerative diseases, with an emphasis on the systemic nature of these pathologies.

Jason Gestwicki (University of California, San Francisco) showed that client proteins for ubiquitylation can, following cleavage by caspases, bind directly to the E3 ubiquitin ligase CHIP independently of the chaperones Hsp70 and Hsp90. The importance and physiological relevance of this mechanism is supported by interaction studies showing that more than 2700 client proteins might bind CHIP this way and by data suggesting implication of this mechanism in Alzheimer’s disease.

After giving a thorough introduction on TOR and its discovery by his lab, Michael Hall (University of Basel) presented two recent sets of data bringing better understanding on physiopathology of the TOR signalling. The first study demonstrated how mTORC2 signalling leads to hepatic cancer by enhancing expression of the histidine kinase NME1, whose effects on enhancement of p-His levels lead to tumour development when not counterbalanced by the histidine phosphatase LHPP. The second study explored the link between obesity and type II diabetes implicating the hexokinase II.

Ivan Dikic (Goethe University, Frankfurt) first talked about the mechanisms of selective autophagy, a process through which specialized receptors can recognize ubiquitylated bacteria or damaged mitochondria to target them to autophagosomes for degradation. Mutations in the autophagy pathway are linked to familiar cases of ALS and other neuropathies. He also discussed how bacteria could exploit the host’s ubiquitylation machinery to promote its own life cycle, by bringing structural and mechanistic insights on the non-canonical serine ubiquitination of host proteins by the SdeA ligase of Legionella pneumophila.

Thijn Brummelkhamp (Netherlands Cancer Institute) presented a genetic screen approach in haploid cells to assign genes to phenotypes in human cells. He demonstrated how this could be applied to identify new regulators of signal transduction pathways and how further mutagenesis provides mechanistic insights. For example, this approach enabled better understanding of the mechanism by which PD-L1 protects cancer cells against T cells attack, identifying CMTM6 as a transmembrane protein necessary to PD-L1 function. Altogether, the use of this method led to the generation of a growing database assigning regulators to hundreds of molecular phenotypes.

Dario Alessi (University of Dundee) presented data bringing new understanding on the function of LRKK2, one of the genes that when mutated causes familial inherited Parkinson’s. He showed that this kinase phosphorylates a subgroup of 14 Rab proteins, thus affecting the ability of these proteins to interact with cognate effectors. LRKK2 is itself regulated by the Rab29/VPS35 and preliminary results implicate the phosphatase PPM1H in dephosphorylation of LRKK2. Overall, this work could lead the way to better understanding of this form of Parkinson’s disease.

Pierre Genevaux (LMGM-CBI, Toulouse) presented work based on the implication of chaperones in bacterial toxin-antitoxin system. He showed that antitoxins could be rendered dependant to a SecB related chaperone by the presence of a short C-terminal extension that leads to antitoxin aggregation and thus loss of antitoxin function in the absence of the chaperone. The structure of the toxin-antitoxin SecB-like chaperone bound to the chaperone addiction sequence peptide was solved and demonstration of high specificity in this peptide/chaperone binding system was given.

Anne Bertolotti (LMB, Cambridge) presented data showing how selective inhibition of phosphatases can boost protein quality control and lead to potential treatment for degenerative diseases. Identified small molecules can inhibit regulatory subunits of a serine/threonine phosphatase and thus prolong a stress response proteostatic pathway giving the cells more time to cope with stress. These finding strongly challenge the conception of undruggable phosphatases.

Tony Hunter (Salk Institute for Biological Studies, La Jolla) introduced his talk with a history of PTMs, focussing on the now well-understood phosphorylation on tyrosine, which he first discovered 40 years ago. He then presented data on histidine phosphorylation that remains in contrast poorly characterised. Monoclonal antibodies against p-His and a p-His peptide enrichment protocol enabled identification of over 750 p-His proteins in cancer cell lines as well as several new p-His sites, that, together with p-Lys and p-Arg, might account for 20% of all phosphate linked to protein in the cell. Development of a sequence specific p-His antibody implicated p-His in hepatic cancer.

Communications

• Dario ALESSI (Dundee University, UK) – The regulation and function of the Parkinson’s disease LRRK2 protein kinase
• Anne BERTOLOTTI, organisatrice (MRC Laboratory of Molecular Biology, Cambridge, UK) – Selective phosphatase inhibition to boost protein quality control and treat degenerative diseases
• Thijn BRUMMELKAMP (Netherlands Cancer Institute, Amsterdam, The Netherlands) – Studying disease-related networks using haploid genetics
• Lucie CHADELLE (MRC Laboratory of Molecular Biology, Cambridge, UK
• Anne DEJEAN, organisatrice (Institut Pasteur, Paris, France) – Role of SUMO on chromatin
• Ivan DIKIC (Institute of Biochemistry II and Buchmann Institute for Molecular Life Sciences, Goethe University, Frankfurt, Germany) – ER remodelling via selective autophagy
• Vishva DIXIT (Genentech, San Francisco (CA), USA) – Malignancies arising from mutation of tumor suppressor genes display an unexplained tissue proclivity – Why?
• Martin EILERS (University Julius-Maximilians of Würzburg, Germany) – Transcription Stress, MYC and Oncogenic Transformation
• Pierre GENEVAUX (IBCG, CNRS, Université Paul-Sabatier, Toulouse, France) – Growth control by bacterial Toxin-Antitoxin-Chaperone systems
• Jason GESTWICKI (University of California San Francisco, USA) – Targeting Protein Stability to Boost Cellular Resilience
• Michael HALL (Biozentrum, University of Basel, Switzerland) – mTOR signaling in growth and metabolism
• Ronald HAY (Dundee University, School of Life Sciences, UK) – SUMO as a signal for ubiquitin modification
• Mark HOCHSTRASSER (Yale University, Dept. of Molecular Biophysics & Biochemistry, New Haven, USA) – Unexpected roles of ubiquitin and SUMO in cell homeostasis and endosymbiont interactions
• Anthony (Tony) HUNTER (Salk Institute of Biological Sciences, La Jolla, USA) – How important is noncanonical protein phosphorylation in cell physiology?
• Andrew JACKSON (MRC Human Genetics Unit, IGMM, University of Edinburgh, UK) – The enemy within: cell intrinsic surveillance of genome instability
• Jeffery KELLY (The Scripps Research Institute, La Jolla, USA) – Adapting Proteostasis to Ameliorate Degenerative Diseases
• Frauke MELCHIOR (ZMBH, Heidelberg, Germany) – SUMOylation in signal transduction
• Brenda SCHULMAN (Max Planck Institute of Biochemistry, Martinsried, Germany) – Cullin-RING ligases: signaling and mechanisms
• Tatiana TRABOULSI (Institut Pasteur, Paris, France)
• Peter WALTER (University of California San Francisco, Department of Biochemistry and Biophysics, USA) – Targeting the Cell’s Stress Pathways for Therapeutic Benefit