Diabetes is the seventh leading cause of death in the United States, and nearly 34% of U.S. adults are prediabetic. CTRP3 is an adipose secreted protein that has shown to play a key role in glucose metabolism and insulin sensitivity, however, the research on CTRP3 total levels and its relationship to type 2 diabetes is controversial. The oligomeric state (protein structure) of CTRP3 in relation to metabolic dysfunction has not been studied. This study will be the first analysis of the circulating forms of CTRP3 in human blood. Hypothesis: The relative circulating amounts of the three oligomeric states of CTRP3 will differ in patients with type 2 diabetes. Methods: Human serum samples are analyzed using western blotting under native, reduced non-denaturing, and denaturing conditions. Results: In reducing non-denaturing conditions, three oligomeric states of CTRP3 were visualized in human serum: the high molecular weight (HMW) oligomer, the low molecular weight (LMW) oligomer, and the trimer. Conclusion: Reduced, non-denaturing conditions appear to yield the most effective separation of the three oligomeric states of CTRP3, and further studies aim to observe a difference in oligomeric state with a diabetic phenotype. Investigating the relationship of CTRP3’s oligomeric state with diabetic phenotype could present novel understanding of this protein’s possible protective effects against certain metabolic disorders.

Les protéines membranaires représentent environ 2/3 des cibles thérapeutiques. Le développement de nouveaux médicaments est toutefois limité par l'absence de données structurales pour de nombreuses protéines. Les protéines membranaires s'avèrent en effet difficiles à manipuler et à maintenir en solution ce qui complique leur étude structurale. Les protéines sont en général solubilisées grâce à des surfactants comme les détergents, les amphipols, les hémifluorés et les peptergents. Il est aussi possible de les étudier dans des conditions plus physiologiques en les insérant dans des membranes lipidiques telles que des liposomes, des bicelles, ou des nanodisques.Les nanodisques sont des particules protéolipidiques autoassemblées, composées de protéines d'assemblages et de lipides, qui constituent un système de membranes modèles très prometteur permettant de solubiliser des protéines membranaires dans un milieu dépourvu de détergent. D'autres avantages sont aussi la variabilité de la constitution en lipides et l'accessibilité des deux côtés de la membrane.Dans le cadre de ma thèse, j'ai mis au point l'insertion de plusieurs protéines membranaires en nanodisques afin de permettre leur caractérisation fonctionnelle, biophysique et structurale. Nous avons en particulier étudié le transporteur ABC BmrA impliqué dans la résistance aux antibiotiques et cherché à identifier les changements conformationnels de la protéine en nanodisques par microscopie électronique. Les interactions de la protéine YedZ, un homologue de NADPH oxydases, avec ses partenaires solubles potentiels ont été étudiés par différentes méthodes telles que le pontage chimique, la résonance plasmonique de surface et la spectrométrie de masse native. En parallèle, le mécanisme d'assemblage des nanodisques a été investigué. Une interaction entre les protéines d'assemblages et des cations divalents a été mise en évidence. Cette interaction a un effet sur l'oligomérisation de la protéine d'assemblage mais également sur l'homogénéité des nanodisques. Ces observations nous ont permis d'améliorer les conditions de préparation des nanodisques, condition déterminante pour le succès de nombreuses approches structurales. Nous avons pu en particulier explorer la possibilité de cristalliser ces particules en vue d'études cristallographiques. / Membrane proteins represent around 2/3 of therapeutic targets. However, the development of new drugs is hampered by the lack of structural data for many proteins. Membrane proteins are indeed difficult to handle and to maintain stable in solution, which complicates their study by structural methods. Proteins are usually stabilized by surfactants like detergents, amphipols, hemifluorinated compounds and peptergents. It is also possible to study those proteins in an environment mimicking their native conditions by incorporating them in lipid membranes such as liposomes, bicelles or nanodiscs.Nanodiscs are self-assembled proteolipidic particles, composed of a scaffold protein and lipids. This technology is a top-notch model membrane system, which provides a detergent free environment to study membrane proteins in solution. Further advantages are the possibility to vary the lipid composition and the accessibility of the incorporated protein from both sides of the membrane.During my PhD project, I have achieved the insertion of several membrane proteins into nanodiscs for functional, biophysical and structural characterizations. In particular, we have studied Bmra, an ABC transporter involved in multidrug resistance and tried to identify the conformational changes of the protein in nanodiscs by electron microscopy. The interaction of YedZ, a NADPH oxidase homologue, with potential soluble partners has been studied by various methods such as cross-linking, surface plasmon resonance and native mass spectrometry. In parallel, the mechanism of nanodiscs assembly has been investigated. An interaction between the scaffold protein and divalent cations has been revealed. This interaction influences the oligomerization of the scaffold protein but also the nanodiscs homogeneity. Those observations allowed us to improve the preparation of the nanodiscs, which was an essential step torward the success of many structural approaches. In particular, we were able to explore their accessibility to protein crystallography.

M42 aminopeptidases are dinuclear enzymes widely found in prokaryotes but completely absent from eukaryotes. They have been proposed to hydrolyze peptides downstream the proteasome or other related proteolytic complexes. Their description relies mainly on the pioneering work on four M42 aminopeptidases from Pyrococcus horikoshii. Their quaternary structure consists of twelve subunits adopting a tetrahedral-shaped structure. Such a spatial organization allows the compartmentalization of the active sites which are only accessible to unfolded peptides. The dodecamer assembly results from the self-association of dimers under the control of the metal ion cofactors. Both oligomers have been shown to co-exist in vivo and heterododecamers with broadened substrate specificity may even occur. Yet, the molecular determinants behind the dodecamer assembly remain unknown due the lack of a high-resolution structure of a stable dimer. In addition, the bacterial M42 aminopeptidases are still ill-described due to the paucity of structural studies. This work focuses mainly on the characterization of TmPep1050, an M42 aminopeptidase from Thermotoga maritima. As expected, TmPep1050 adopts the genuine tetrahedral-shaped structure with twelve subunits. It also displays a leucyl-aminopeptidase activity requiring Co2+ as a cofactor. In addition to its catalytic function, Co2+ has a role in the enzyme thermostability and oligomerization. The absence of Co2+ provokes the disassembly of active TmPep1050 dodecamers into inactive dimers. The process, however, is reversible since Co2+ triggers the self-association of dimers into dodecamers, as shown by native MS. The main achievement of this work is the determination of the first high-resolution structure of a dimer, allowing to better understand the dimer-dodecamer transition. Several structural motifs involved in oligomerization are displaced or highly flexible in the TmPep1050 dimer structure. Furthermore, a loop bringing two catalytic relevant residues is displaced outside the catalytic site. These residues are the catalytic base and a ligand involved in the Co2+ binding at the M1 site. The metal ion binding sites have been further investigated to define how they influence the oligomerization of TmPep1050. A mutational study shows that the M1 site strictly controls the dodecamer formation while the M2 site contributes only partly to it. A strictly conserved aspartate residue of the M2 site second shell also plays an important structural role in maintaining the active site integrity. Indeed, its substitution prevents the formation of dodecamer probably due to the lack of stabilization of the active site loop. The characterization of TmPep1050 supports that bacterial M42 aminopeptidases probably share the quaternary structures and dodecamer assembly with their archaeal counterparts. The dimer structure highlights several structural modifications occurring in the dimer-dodecamer transition. Yet, based on current knowledge, no general rules can be drawn for the role of the M1 and M2 sites in oligomerization. Besides, the physiological function of the M42 aminopeptidases is under-examined albeit the proposed link to the proteasome. In this work, this has been investigated using the Escherichia coli M42 aminopeptidases as a model. Yet, no phenotype has been associated to the deletion of their coding genes. Preliminary results have shown that the three enzymes (i) display a redundant substrate specificity, (ii) could be localized partly to the membrane, and (iii) form heterocomplexes. Further experiments are still required to crack the function of these M42 aminopeptidases. / Option Biologie moléculaire du Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Le passage sélectif de molécules à travers la membrane interne des mitochondries est essentiel aux processus métaboliques des cellules eucaryotes. Cette communication cellulaire est assurée par des protéines transmembranaires de la famille des transporteurs mitochondriaux (MCF). Le transporteur ADP/ATP (AAC) est le membre le plus connu et le mieux caractérisé de cette famille. Il est responsable de l'import d'ADP dans la matrice mitochondriale et de l'export d'ATP après synthèse vers le cytosol. La structure d'AAC est connue mais plusieurs questions restent ouvertes concernant le mécanisme du transport, la sélectivité et l'état oligomérique, controversé, de la protéine. Pendant plusieurs années des études biochimiques réalisées sur la protéine solubilisée en détergent étaient en faveur d'une organisation dimérique du transporteur, mais la structure d'AAC, monomérique a remis en cause ce dogme. Afin de caractériser l'organisation oligomérique d'AAC in vivo, nous avons combiné plusieurs approches. Nous avons réalisé des expériences de FRET (Fluorescence Resonance Energy Transfer) directement sur des cellules mammifères ou bactériennes (E. coli) surexprimant la protéine AAC fusionnée avec des sondes FRET. En parallèle, nous avons mis au point des tests fonctionnels afin de contrôler l'état des mitochondries et l'activité du transporteur dans ces cellules. Enfin nous avons étudié la stoechiométrie de liaison de l'inhibiteur carboxyatractyloside grâce à des mesures de respiration sur des mitochondries extraites de foie de rat et placées dans différents états métaboliques. L'ensemble des résultats présentés dans ce manuscrit ont permis de montrer que 1) l'unité fonctionnelle d'AAC est monomérique 2) l'organisation structurale d'AAC dans les membranes natives dépend de l'état métabolique des mitochondries et peut être associée à des phénomènes de régulation. / The transport of small molecules through the inner mitochondrial membrane is essential in eukaryotic metabolism and is selectively controlled by a family of integral membrane proteins, the Mitochondrial Carrier Family (MCF). The ADP/ATP carrier (AAC), which is responsible for the import of ADP to the matrix of mitochondria and the export of newly synthesized ATP toward the cytosol, is the best-known and characterized MCF member. Although its structure sheds light on several aspects of the carrier activity, additional investigations are still required to decipher the whole transport mechanism, to understand the specificity and to characterize the controversial oligomeric state of the protein. For many years, based on studies mainly carried on detergent solubilized AAC the general consensus has been in favor of a dimeric organization of the carrier. The AAC three-dimensional structure, monomeric, broke this dogma. In order to get a precise insight into the in vivo oligomeric organization of AAC we combined several approaches. Fluorescence resonance energy transfer (FRET) measurements were performed directly on mammalian and E.coli cells expressing AAC labeled with several types of FRET probes. In parallel, different functional assays were established to control the state of the mitochondria in these cells and the transport activity of these AAC fusions. Lastly, measurements of the respiration rate coupled to the titration of the inhibitory effect of carboxyatractyloside on isolated rat liver mitochondria were used to investigate the organization of AAC in native mitochondria within two regimes of oxidative phosphorylation. Taken together the results described herein revealed that 1) AAC can function mechanistically as a monomer, 2) the organization of AAC in native membranes might be related to the state of the mitochondria and be involved in regulation.

Bacterial DNA replication can stall at DNA lesions, leading to cell death if the damage fails to be repaired. To circumvent this, bacteria possess a mechanism called translesion DNA synthesis (TLS) to allow DNA damage bypass. The ImuABC TLS mutasome comprises the RecA domain-containing protein ImuA, the inactive polymerase ImuB, and the error-prone polymerase ImuC. ImuA and ImuB are necessary for the mutational function of ImuC that can lead to antimicrobial resistance (AMR) as seen in high-priority pathogens Pseudomonas aeruginosa and Mycobacterium tuberculosis. Understanding how ImuA and ImuB contribute to this function can lead to new targets for antimicrobial development. This research aims to discover the molecular functions of ImuA and ImuB homologs from Myxococcus xanthus through structural modelling and biochemical analyses. ImuA was discovered to be an ATPase whose activity is enhanced by DNA. Based on predicted structural models of the ATPase active site, I identified the critical residues needed for ATP hydrolysis, and found that the ImuA C-terminus regulates ATPase activity. Further, ImuA and ImuBNΔ34 (a soluble truncation of ImuB) display a preference for longer single-stranded DNA and overhang DNA substrates, and their affinity for DNA was quantified in vitro. To better understand how ImuA and ImuB assemble in the TLS mutasome, bacterial two-hybrid assays determined that ImuA and ImuB can self-interact and bind one another. Mass photometry revealed that ImuA is a monomer and ImuBNΔ34 is a trimer in vitro. ImuA and ImuBNΔ34 binding affinity was quantified in vitro at 1.69 μM ± 0.21 by microscale thermophoresis, and removal of the ImuA C-terminus weakens this interaction. Lastly, ImuA and ImuBNΔ34 secondary structures were quantified using circular dichroism spectroscopy, and ImuA was modified to enable crystallization for future structural studies. Together, this research provides a better understanding of ImuABC-mediated TLS, potentially leading to novel antibiotics to reduce the clinical burden of AMR. / Thesis / Master of Science (MSc) / The antimicrobial resistance (AMR) crisis is fueled by the emergence of multi-drug resistant microbes, posing a major threat to global health and disease treatment. Bacteria can develop resistance to antibiotics through mutations in the genome. When the genome becomes damaged, bacteria can acquire these mutations by an error-prone replication mechanism called translesion DNA synthesis (TLS). In some bacteria, TLS involves a specialized enzyme complex, consisting of proteins ImuA, ImuB and ImuC, allowing replication past bulky DNA damage and lesions. The goal of this thesis is to investigate how the ImuA and ImuB proteins contribute to the functioning of this mistake-making machinery. I used biochemical and biophysical methods to identify ImuA and ImuB interactions with each other and themselves. I discovered that ImuA is an enzyme that uses energy to enhance its binding to DNA, and determined the specific amino acids involved in this function.