La spectrométrie de masse - un couteau suisse pour disséquer la structure et la fonction du spermatoprotéasome

Mass spectrometry – Swiss Army knife to dissect the spermatoproteasome structure and function

• Auteur / Autrice : Dusan Zivkovic
• Direction : Marie-Pierre Bousquet, Julien Marcoux
• Type : Thèse de doctorat
• Discipline(s) : Biologie structurale et fonctionnelle
• Date : Soutenance le 16/07/2021
• Etablissement(s) : Toulouse 3
• Ecole(s) doctorale(s) : École doctorale Biologie Santé Biotechnologies (Toulouse)
• Partenaire(s) de recherche : Laboratoire : Institut de pharmacologie et de biologie structurale (Toulouse ; 1996-....)
• Jury : Président / Présidente : Christel Lutz
• Jury : Rapporteurs / Rapporteuses : Olivier Coux, Frank Sobott, Silke Meiners

Mots clés

Mots clés contrôlés

Spectrométrie de masse

Protéasome

Spermatogenèse

Mots clés libres

Spermatogénèse

Protéasome

Spermatoprotéasome

Protéomique

Spectrometrie de masse structurale

Spermatogenesis

Proteasome

Spermatoproteasome

Proteomics

Structural mass spectrometry

Résumé

Le protéasome est le complexe enzymatique protéolytique principal de la cellule. Son cœur catalytique (20S) est formé de quatre anneaux heptamériques. Son activité et sa spécificité de substrat peuvent être régulées par les complexes 19S, PA28αβ, PA28γ et PA200 ainsi que par des sous-unités 20S alternatives. La spermatogenèse est un processus de différenciation des cellules germinales mâles: les spermatogonies (SPG) se transforment en spermatocytes (SPC), en spermatides (SPT) puis en spermatozoïdes. Ce processus requière une protéolyse intense. Le spermatoprotéasome (spt20S) est spécifique des gamètes en développement et essentiel à la spermatogenèse. Il diffère du protéasome standard (std20S) par la sous-unité α4s qui remplace la sous-unité constitutive α4. Le spt20S joue un rôle important avec PA200 dans la progression de la méiose, mais les mécanismes qui le rendent différent du std20S restent inconnus.  Nous avons établi des stratégies protéomiques complémentaires pour caractériser les complexes du protéasome immunopurifiés à partir de testicules. L’analyse Top-Down de protéasome purifié, nous a permis de montrer pour la première fois qu’α4s et α4 portent les mêmes MPTs. La protéomique Bottom-Up, nous a permis de comparer les immunopurifications (IPs) de protéasomes totaux avec celles obtenues avec un anticorps spécifique du stp20S que nous avons développé. Nous avons établi qu’α4 et α4s ne coexistent pas dans le même 20S, bien qu'ils soient presque également abondants dans les testicules. Nous avons également trouvé plus de 19S et de PA200 liés au spt20S qu’au std20S. Les autres protéines préférentiellement associées au spt20S incluent PI31 et Fbxo7 qui sont cruciales pour la fertilité et d’importants médiateurs du transport du protéasome et de l'ancrage des E3 ligases - deux processus qui semblent cruciaux pour la fonction du spt20S pendant la spermatogenèse. Nous avons ensuite obtenu des cellules germinales à différents stades de la différenciation et l'analyse protéomique des lysats ainsi que des IPs, nous a permis d’établir un interactome dynamique du protéasome tout au long de la spermatogenèse. Nous avons observé un changement total du std20S au spt20S entre les SPG pré-méiotiques et les SPC/SPT méiotiques et post-méiotiques. Un changement d'expression semble responsable, plutôt qu’une incorporation préférentielle d’α4s. En entrant dans la méiose, l'association de PA200 avec le protéasome a augmenté 7 fois, confirmant son importance dans le développement des gamètes. Bien que PA200 soit d’après la littérature le principal activateur du spt20S, nous montrons que le 19S est est en réalité majoritaire, lié à 60% des 20S dans les SPC - une activation du protéasome sans précédent. De nombreux partenaires du spt20S sont identifiés à la fois dans les cellules germinales et dans les testicules entiers, montrant la robustesse de nos méthodes

The proteasome is the main enzymatic complex for targeted proteolysis in the cell. Its core complex (20S) consists of four stacked heptameric rings and requires activator complexes: 19S, PA28αβ, PA28γ and PA200, which regulate 20S activity and substrate specificity. Alternative 20S subunits exist to further modulate the proteasome activity. Spermatogenesis is a process of male germ cell differentiation, where spermatogonia (SPG) transform through spermatocyte (SPC), then spermatid (SPT) stages, to become spermatozoa. This process requires intense proteolysis. The spermatoproteasome (spt20S) is specific to the developing gametes and essential for spermatogenesis. It differs from the standard proteasome (std20S) by only one subunit – α4s, which replaces the constitutive α4 subunit. Together with PA200, the spt20S plays an important role in meiosis progression, however, the mechanisms that make it different compared to std20S remain unknown. We established complementary proteomic pipelines for characterisation of proteasome complexes in the testes, combining immunopurification (IP) and mass spectrometry (MS) analysis. Our Top-Down analysis of purified proteasome, showed for the first time that both α4 and α4s carry the same PTMs. Using Bottom-Up proteomics we compared the interactome of total proteasomes with that of the spt20S only, obtained using a specific antibody we developed for this purpose. We established that α4 and α4s do not co-exist in the same 20S, although they are almost equally abundant in the testes. We also measured that 19S and PA200 regulators were bound in higher ratios to the spt20S compared to std20S. Among other preferentially-associated proteins of spt20S were PI31 and Fbxo7, both shown to be crucial for fertility. They mediate proteasome transport and docking of E3 ligases – both processes that could be crucial for spt20S function. We then obtained germ cells at different differentiation stages and performed a proteomics analysis of both lysates and IP-ed proteasome complexes, to establish a dynamic image of the proteasome throughout spermatogenesis. We observed a complete shift from std20S to spt20S between pre-meiotic SPG and meiotic and post-meiotic SPC and SPT cells. We explained this by a shift in expression, rather than preferential incorporation of α4s. Upon entering meiosis, the PA200 association with core proteasome increased 7-fold, marking its importance in gamete development. Although PA200 was represented in literature as the main spt20S interactor, we show that 19S was undoubtedly stoichiometrically dominant, occupying 60% of the existing 20S in SPCs – an unprecedented proteasome activation. Identified spt20S-interacting proteins largely correlated with previous interactome analysis on the whole testes, showing robustness of our methods. We identified synaptonemal proteins bound exclusively to spt20S and numerous proteins involved in ubiquitylation, cell cycle and meiotic progression as well as cellular transport, which fits the current model of spt20S role, proposed by earlier work. The shift from α4 to α4s in meiosis was shown to be crucial, but what is the molecular basis for this transition? The hydrogen-deuterium exchange experiment coupled to MS helped us to show for the first time that the two proteasomes exhibit different binding interfaces: α4s contains regions that are more flexible compared to α4. We further supported this finding with pull-down assays, which showed that 19S binds more strongly to the spt20S than to std20S, which would explain the increase in proteolytic activity required during meiosis. The spt20S showed a higher tryptic activity compared to the std20S in vitro, which might reflect a particular need for histone degradation. Altogether, our data reveal a more complex process of spt20S regulation than previously suggested and set the basis for structural and functional differences between the spt20S and std20S.