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Ribosomal RNA contains many posttranscriptionally modified nucleosides, particularly in the functional parts of the ribosome. The distribution of these modifications varies from one organism to another. In Bacillus subtilis ,the model organism for Gram-positive bacteria, mass spectrometry experiments revealed the presence of 7-methylguanosine (m7G) at position 2574 of the 23S rRNA, which lies in the A-site of the peptidyl transferase center of the large ribosomal subunit. Testing several m7G methyltransferase candidates allowed to identify the RlmQ enzyme, encoded by the ywbD open reading frame, as the MTase responsible for this modification. The enzyme methylates free RNA and not ribosomal 50S or 70S particles, suggesting that modification occurs in the early steps of ribosome biogenesis., SCOPUS: le.j, info:eu-repo/semantics/published

Human Immunodeficiency Virus type 1 (HIV-1) infection can be managed but not cured by the current combinatory antiretroviral therapy (cART). The replication rate of HIV-1, which is directly correlated to the rate of disease progression to Acquired Immunodeficiency Syndrome (AIDS), is controlled primarily at the transcription level of viral genes, which is not targeted by cART. The arrest of the cART is associated with a rapid viremia rebound due to the persistence of long-term reservoirs of virally infected cells, which cannot be eliminated by cART corresponding to cell types or tissue compartments where virally infected cells are maintained. These reservoirs are heterogeneous and principally composed of CD4+ T cells and myeloid cells, the latter understudied and poorly represented in cure-based efforts. Importantly, each reservoir harbors a unique regulation of HIV-1 gene expression. Even though most transcriptional studies on HIV-1 have only focused on the viral promoter 5’long terminal repeat (5’LTR), our laboratory has previously reported an intragenic cis-regulatory region (IRR), which the complete functional unit forming the HIV-1 IRR is composed, from 5’ to 3’, of three subdomains: the 5103 fragment, the hypersensitive site 7 (HS7), and the 5105 fragment. In this context, further deciphering the specific molecular mechanisms underlying HIV-1 gene expression in all host cell types (including myeloid cells) and from each transcriptional regulatory element (including the IRR) is crucial for a deeper understanding of HIV-1 biology and pathogenesis.In the first part of my PhD thesis, we determined the in vivo epigenetic landscape of the IRR in latent and reactivated conditions and in both T-lymphoid and myeloid-infected cells, representing the two major cellular contexts of HIV-1 infection. We demonstrated that the IRR harbored the characteristic features of an enhancer in these two physiological contexts. Second, we explored the contribution of the intragenic lin, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

Bovine Leukemia Virus (BLV) and Human T-Lymphotropic Virus type I (HTLV-1) are two oncogenic retroviruses characterized by viral latency, thereby allowing escape from the host immune surveillance and the development of BLV- and HTLV-1-associated diseases. In this PhD thesis, we demonstrated for the first time the role of the cellular factor UHRF1 (Ubiquitin-like with PHD and RING Finger domains 1) in the transcriptional repression of BLV and HTLV-1. We showed the in vitro binding and in vivo recruitment of UHRF1 to the BLV and HTLV-1 transcriptional promoters located in the viral 5’ Long Terminal Repeat (5’LTR). We demonstrated the repressive role of UHRF1 in BLV and HTLV-1 gene expression in both basal and transactivated conditions using transient transfection assays. Moreover, we showed that UHRF1 epigenetically repressed the HTLV-1 5’LTR using RNA interference and that UHRF1 interacted with the HTLV-1 Tax transactivator. Finally, our results using primary cells from HTLV-1-infected individuals suggested the involvement of UHRF1 in the tumoral development observed in the HTLV-1-infected individuals. In conclusion, this PhD thesis provides a better understanding of BLV and HTLV-1 transcriptional regulations and offers new perspectives for developing innovative therapeutic strategies against BLV and HTLV-1 infections and their associated diseases., Doctorat en Sciences, info:eu-repo/semantics/nonPublished

RelA/SpoT Homologs (RSH) enzymes control the bacterial stringent response through the synthesis and hydrolysis of the alarmone (p)ppGpp. Current biochemical and structural data is not sufficient to correctly understand RSH enzymatic activity as well as their regulation and interaction with molecular partners. This doctoral thesis has demonstrated the implication of Rel ACT domain in the regulation of its enzymatic activity and its interaction with the EIIANtr protein in Caulobacter crescentus. In addition, it has also confirmed the presence of a dedicated (p)ppGpp allosteric binding site in the catalytic domain of Rel in Bacillus subtilis and characterized the regulation of the latter’s activity during potassium starvation, notably by the resolution of the crystallographic structure of Rel complexed with the DarB protein. Through the discovery of these novel results, this thesis deepens the fundamental workings of RSH activity regulation., Doctorat en Sciences, info:eu-repo/semantics/nonPublished

Toxin-antitoxin (TA) systems are small genetic modules that are widespread in prokaryotes. While initially discovered on plasmids, which they stabilize, TA systems are also abundantly found on bacterial chromosomes. Their functions are highly debated and have been examined in this thesis. First, we show that TA systems do not seem to play a role in stress response or antibiotic tolerance, as previously reported in the literature. Secondly, our single cell experiments demonstrate that the activation of TA systems promotes cell death in cells when a TA-encoding plasmid is lost, indicating that TA activation leads to death and not to a state of stress tolerance. We therefore conclude that TA systems are addictive genes that promote their own retention and that of the genetic elements that encode them., Doctorat en Sciences, info:eu-repo/semantics/nonPublished

Long RSHs are enzymes that synthetize and hydrolyse a secondary messenger molecule, (p)ppGpp. This molecule acts as a global regulator targeting metabolic processes to ensure bacterial survival upon environmental challenges. Because (p)ppGpp is linked with bacterial virulence and resistance to antibiotics, a fundamental understanding of its regulation is key to develop novel antimicrobial treatments. Long RSH have two N-terminal domains, one for hydrolysis of (p)ppGpp (HD-domain) and one for synthesis of (p)ppGpp (SYNTH-domain), and four regulatory domains at the C-terminus that control catalysis by the N-terminal domains. A combination of structure determination and biochemical assays were used to study the long RSHs at the molecular level and determine the bases of the allosteric mechanisms that control the activities of the enzymes. The data demonstrate an allosteric conformational switch between the N-terminal catalytic domains of these enzymes that prevent futile catalytic cycles. Indeed, the data shows binding of substrates to one catalytic domain allosterically inhibits substrate-binding and catalysis by the opposing catalytic domain. Furthermore, feedback activation of (p)ppGpp-synthesis, the molecular basis of which was unknown, in context of amino-acid starvation was studied. The allosteric positive feedback regulation of (p)ppGpp-synthesis was shown to exploit the conformational switch between the two catalytic states to modulate both activities. An allosteric binding-pocket was located at the linker-region between the two catalytic domains at which (p)ppGpp-synthesis is auto-induced allosterically while (p)ppGpp-hydrolysis is inhibited. Lastly, the conformational landscape involved in the regulation of these enzymes was explored using Nanobodies to discover a previously undetected regulatory hotspot. Based on the conformational switch as well, this regulatory hotspot modulates catalytic activity allosterically. The newly discovered hotspot might be impor, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

Chromosomal translocations that involve the nucleoporin NUP98 are recurrent in childhood therapy-related acute myeloid leukemias, characterized by disruption of normal hematopoiesis and bone marrow failure. Previous experiments showed that the expression of Nup98 fusion proteins leads to changes in nuclear envelope architecture and HOXA9 overexpression. My work provided new data supporting a mechanical function of RB in the nuclear architecture of NUP98 fusions, disclosing a possible mechanism of action involving the chromatin regulators. Furthermore, new pieces of evidence on HOXA9 regulation mediated by NUP98-related leukemia were revealed, which might be interesting for new target identification for the treatment of HOXA9-overexpressing diseases, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

Toutes les cellules de l’organisme relarguent de façon constitutive des nucléotides dans l’espace extracellulaire. Ces nucléotides extracellulaires agissent comme des molécules de signalisation, entre autres, via les récepteurs P2Y (P2YRs) qui sont des récepteurs couplés aux protéines G (GPCRs). Les P2YRs sont exprimés, pour la majorité, de façon ubiquitaire dans l’organisme. L’analyse phénotypique des lignées de souris knockout pour les P2YRs (P2YR-/-) a permis de confirmer l’importance de cette voie de communication dans différents processus physio/pathologiques. Au sein de notre laboratoire, grâce à l’utilisation de souris P2YR-/-, nous étudions depuis quelques années le rôle de ces récepteurs dans la biologie des cellules stromales mésenchymateuses multipotentes (MSCs). Ces cellules se caractérisent par une faible immunogénicité et surtout par la propriété de multipotence, i.e. capacité de se différencier en plusieurs types cellulaires (adipocytes, chondroblastes, ostéoblastes). Cette dernière propriété suscite l’intérêt de la médecine régénérative qui vise à exploiter la multipotence de ces cellules en thérapie cellulaire. Toutefois, leur utilisation à des fins thérapeutiques est encore limitée, notamment parce que les mécanismes régulateurs de ces processus de différenciation sont encore mal compris. Il est donc fondamental de continuer à étudier les voies de signalisation régulant les processus de différenciation de ces cellules pour leurs applications cliniques.Dans ce travail de recherche, nous avons investigué l’implication de la signalisation dépendant des P2YR dans le modèle expérimental des MSCs dérivées du tissu adipeux sous-cutané inguinal (ATMSCs) murin. D’abord, nous avons montré que parmi les 7 sous-types connus de récepteur P2Ys, les ATMSCs expriment le P2Y2R (récepteur à l’ATP et l’UTP) et le P2Y6R (récepteur à l’UDP) et que ces récepteurs sont fonctionnels. Par une approche combinant l’analyse quantitative de leur expression, l’effet de leur act, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

The high conservation of nucleotides of the T-loop, including their chemical identity, are hallmarks of tRNAs from organisms belonging to the three Domains of Life. These structural characteristics allow the T-loop to adopt a peculiar intraloop conformation able to interact specifically with other conserved residues of the D-loop, which ultimately folds the mature tRNA in a unique functional canonical L-shaped architecture. Paradoxically, despite the high conservation of modified nucleotides in the T-loop, enzymes catalyzing their formation depend mostly on the considered organism, attesting for an independent but convergent evolution of the post-transcriptional modification processes. The driving force behind this is the preservation of a native conformation of the tRNA elbow that underlies the various interactions of tRNA molecules with different cellular components., SCOPUS: re.j, info:eu-repo/semantics/published

Human Onchocerciasis is a neglected tropical disease of debilitating consequences caused by the nodule dwelling nematode Onchocerca volvulus. Excretory Secretory Products (ESPs) help the parasite to establishing infection in an immunocompetent host and thus make O. volvulus ESPs potential sources of diagnostic, therapeutic or immunomodulatory markers. Here, a blend of bioinformatics, biochemical, analytical and immunologic methods were employed to identify one such ESP as a GM2 activator protein orthologue. The cDNA of OvGM2AP was detected in most parasite stages and the protein was detected in ESPs of adult males, adult females and L3 stage. Recombinant OvGM2AP was expressed in Sf21 insect cells and was found to be glycosylated at Asn 173 by mass spectrometry analysis. The recombinant OvGM2AP could significantly differentiate infected from non-infected individuals on total IgG ELISA. No improvement in diagnostic indices was observed when IgG sub-classes were used to detect OvGM2AP by ELISA. A drop in IgG titre was observed to correlate with increased rounds of ivermectin administration. However, as observed from alignment of OvGM2AP sequences with other nematode species, recombinant OvGM2AP was found to cross react with sera from patients with other nematode infections such as Loa loa, Mansonella perstans and Brugia malayi. GM2 degradation to GM3 could not be mediated by OvGM2AP in the presence of human β-hexosaminidase A. However, when examined for competitive inhibition with artificial GM2 substrates such as MUG and MUGs, OvGM2AP was found to competitively inhibit the degradation of MUG by β-hexosaminidase A as opposed to the human GM2AP. Due to the genetic intractability of O. volvulus, Caenorhabditis elegans was used as a model to study the function of OvGM2AP. The C. elegans orthologous gene cpp-1 was found to be expressed predominantly in the hypoderm, with a paralogous form confined to specialized hypodermis such as seam cells. CRISPR-CAS9 mediated knock-out, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

To improve and complete our knowledge of archaeal tRNA modification patterns, we have identified and compared the modification pattern (type and location) in tRNAs of three very different archaeal species, Methanococcus maripaludis (a mesophilic methanogen), Pyrococcus furiosus (a hyperthermophile thermococcale), and Sulfolobus acidocaldarius (an acidophilic thermophilic sulfolobale). Most abundant isoacceptor tRNAs (79 in total) for each of the 20 amino acids were isolated by two-dimensional gel electrophoresis followed by in-gel RNase digestions. The resulting oligonucleotide fragments were separated by nanoLC and their nucleotide content analyzed by mass spectrometry (MS/MS). Analysis of total modified nucleosides obtained from complete digestion of bulk tRNAs was also performed. Distinct base- and/or ribose-methylations, cytidine acetylations, and thiolated pyrimidines were identified, some at new positions in tRNAs. Novel, some tentatively identified, modifications were also found. The least diversified modification landscape is observed in the mesophilic Methanococcus maripaludis and the most complex one in Sulfolobus acidocaldarius Notable observations are the frequent occurrence of ac4C nucleotides in thermophilic archaeal tRNAs, the presence of m7G at positions 1 and 10 in Pyrococcus furiosus tRNAs, and the use of wyosine derivatives at position 37 of tRNAs, especially those decoding U1- and C1-starting codons. These results complete those already obtained by others with sets of archaeal tRNAs from Methanocaldococcus jannaschii and Haloferax volcanii., SCOPUS: ar.j, info:eu-repo/semantics/published

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 releva, Option Biologie moléculaire du Doctorat en Sciences, info:eu-repo/semantics/nonPublished

While ammonium is an excellent nitrogen source for microorganisms and plants, it is known as acytotoxic metabolite and for its critical role in acid/base homeostasis in animals. Ammonium transportinside the cells is ensured by proteins of the Mep-Amt-Rh superfamily, which are conserved frombacteria to humans.The main objective of the thesis is to refine the understanding of the regulation of the three ammoniumtransport proteins Mep1, Mep2 and Mep3 from Saccharomyces cerevisiae. The three Mep proteins areregulated by the Npr1 kinase and the conserved TORC1 signaling pathway. While the activity of Mep2is regulated by phosphorylation of the C-terminal 457 serine, the activity of Mep1 and Mep3 is inhibitedby the factor Amu1 / Par32. In the presence of a poor nitrogen source, Npr1 induces phosphorylation ofAmu1 which appears mainly cytosolic and, Mep1 and Mep3 are active. On the other hand, in thepresence of a good nitrogen source, the activity of TORC1 induces the inhibition of Npr1 and thereforethe dephosphorylation of Amu1 which accumulates at the cell surface and inactivates Mep1 and Mep3.In order to further study the regulation of Mep1 / 3, a genetic screen was performed to isolate suppressorsrecovering Mep1-dependent ammonium transport in the absence of Npr1. Several mutations, insertionsand deletions have been identified in the MEP1 and AMU1 genes allowing Mep1 to be activeindependently of Npr1. This work shows that all the point mutations in Mep1 delimit an area at theinterface between the hydrophobic body of Mep1 and the cytosol, and that part of the C-terminus (CTR)is required for optimal activity of Mep1 but appears dispensable for regulation by Amu1 and Npr1. Thegenetic screen also shows that the last 15 amino acids of Amu1 are required to inactivate Mep1. Finally,the isolation of suppressors showing no mutation in MEP1 and AMU1 could reveal new factors involvedin the control of Mep1.The results indicate that Mep1 is inactivated in the presence of glutamine, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

The dimorphic transition from the yeast to the filamentous form of growth allows cells to explore their environment for more suitable niches and is often crucial for the virulence of pathogenic fungi. In contrast to their Mep1/3 paralogues, fungal Mep2-type ammonium transport proteins of the conserved Mep-Amt-Rh family have been assigned an additional receptor role required to trigger the filamentation signal in response to ammonium scarcity. Here, genetic, kinetic, expression and structure-function analyses were used to shed light on the poorly characterized signaling role of Saccharomyces cerevisiae Mep2. We show that Mep2 variants lacking the C-terminal tail conserve the ability to induce filamentation, revealing that signaling can proceed in the absence of exclusive binding of putative partners to the largest cytosolic domain of the protein. Our data support that filamentation signaling requires the conformational changes accompanying substrate translocation through the pore crossing the hydrophobic core of Mep2. pHluorin reporter assays show that the transport activity of Mep2 and of non-signaling Mep1 differently affect yeast cytosolic pH in vivo, and that the unique pore variant Mep2H194E, with apparent uncoupling of transport and signaling functions, acquires increased ability of acidification. Functional characterization in Xenopus oocytes reveals that Mep2 mediates electroneutral substrate translocation while Mep1 performs electrogenic transport. Our findings highlight that the Mep2-dependent filamentation induction is connected to its specific transport mechanism, suggesting a role of pH in signal mediation. We also show that the signaling process is conserved for the Mep2 protein from the human pathogen Candida albicans. Our results allow to propose a model for the sensing function of Mep2 where pH and calcium are key players., Doctorat en Sciences, info:eu-repo/semantics/nonPublished

The M42 aminopeptidases are a family of dinuclear aminopeptidases widely distributed in Prokaryotes. They are potentially associated to the proteasome, achieving complete peptide destruction. Their most peculiar characteristic is their quaternary structure, a tetrahedron-shaped particle made of twelve subunits. The catalytic site of M42 aminopeptidases is defined by seven conserved residues. Five of them are involved in metal ion binding which is important to maintain both the activity and the oligomeric state. The sixth conserved residue, a glutamate, is the catalytic base deprotonating the water molecule during peptide bond hydrolysis. The seventh residue is an aspartate whose function remains poorly understood. This aspartate residue, however, must have a critical role as it is strictly conserved in all MH clan enzymes. It forms some kind of catalytic triad with the histidine residue and the metal ion of the M2 binding site. We assess its role in TmPep1050, an M42 aminopeptidase of Thermotoga maritima, through a mutational approach. Asp-62 was substituted with alanine, asparagine, or glutamate residue. The Asp-62 substitutions completely abolished TmPep1050 activity and impeded dodecamer formation. They also interfered with metal ion binding as only one cobalt ion is bound per subunit instead of two. The structure of Asp62Ala variant was solved at 1.5 Å showing how the substitution has an impact on the active site fold. We propose a structural role for Asp-62, helping to stabilize a crucial loop in the active site and to position correctly the catalytic base and a metal ion ligand of the M1 site., SCOPUS: ar.j, info:eu-repo/semantics/published