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Il a récemment été démontré que la nucléocapside du virus de l’hépatite C (VHC) subit l’ajout post-traductionnel d’un groupement acyl gras sur le résidu C172. Cette modification, appelée palmitoylation, est requise pour la production de virions infectieux. Chez les eucaryotes, la palmitoylation est catalysée par les enzymes de la famille DHHC. Notre objectif était d’identifier lesquelles des 23 protéines DHHC présentes chez les mammifère possèdent une activité palmitoyl-acyl transferase (PAT) sur la nucléocapside du VHC. Nous avons d’abord étudié l’expression et la localisation des protéines DHHC dans les hépatocytes humaines. Nous avons ensuite mesuré la variation du niveau de palmitoylation de la nucléocapside lorsque chaque DHHC candidate est surexprimée ou réprimée. Ce criblage a mené à l’identification de 5 enzymes qui exercent une activité PAT sur la nucléocapside du VHC : DHHC 1, 2, 3, 6 et 7. Leur co-localisation avec le substrat viral a été confirmée. / As previously demonstrated, a fatty acid group is post-translationnally added on the residue C172 of Hepatitis C virus (HCV) core protein and this modification, termed palmitoylation, is required for viral assembly. In eukaryotes, palmitoylation is performed by a family of enzymes sharing a closely related DHHC motif. The purpose of this study was to identify which of the 23 mammalian DHHCs could perform palmitoyl acyltransferase activity on the HCV core protein. First, we characterized the expression and localization of DHHC enzymes in human hepatocytes. Then we evaluated the variation in core palmitoylation levels when each cellular DHHC proteins was over-expressed or repressed. This screen identified five enzymes with PAT activity on HCV core protein; DHHC 1, 2, 3, 6 and 7. Their co-localization with the viral protein was also demonstrated. These findings pave the way for future studies on the role of core palmitoylation during the HCV infection.

Palmitoylation is a post-translational modification that has been implicated in the control of multiple proteins, including ion channels. S-Palmitoylation is a lipophilic modification that involves the attachment of palmitate through a thioester linkage to a cysteine residue in a target protein. By increasing the hydrophobicity of the target region, palmitoylation can promote membrane targeting. Here, palmitoylation is shown to play an important role in regulating large conductance calcium- and voltage- activated (BK) potassium channels. The STREX splice variant of the BK channel contains a 58 amino acid insert at the splice site C2 within the intracellular C-terminal RCK1-RCK2 linker that confers increased calcium sensitivity to the channel and determines PKA inhibition of channel activity. The cysteine rich STREX domain was predicted to be palmitoylated, and using an imaging assay STREX was shown to act as a membrane targeting domain through palmitoylation of a di-cysteine motif (C645:C645). A membrane potential assay and electrophysiological analysis demonstrates that palmitoylation at the C645:C646 site in STREX is important in mediating the increased calcium sensitive properties inherent to the STREX channel. Palmitoylation is also shown to modulate PKA channel inhibition. The stability of palmitoylation can often be reliant on the local environment within the protein. Generally in most proteins; lipidated regions, basic domains or transmembrane domains are found adjacent to a palmitoylation site. In STREX, a polybasic domain composed of 11 basic residues just upstream from the C645:C646 palmitoylation site, functions to control the palmitoylation status of the STREX insert. A site directed mutagenesis approach to disrupt the polybasic domain revealed an important role in controlling membrane targeting of the STREX C-terminus, mediating the increased calcium sensitivity inherent to STREX channels and controlling the palmitoylation status of the C645:C646 palmitoylation site using multiple techniques involving electrophysiology, fluorescent imaging and biochemical assays. Further to this, using imaging to examine the membrane association of fluorescently tagged C-terminal proteins, phosphorylation is shown to function as a physiological electrostatic switch to regulate the polybasic region in controlling palmitoylation of the STREX insert. Finally, an additional palmitoylation site that is constitutively expressed in all BK channels was identified to be located in the S0-S1 linker (C53:C54:C56). Mutation of the C53:C54:C56 palmitoylation site in the S0-S1 linker was shown to abolish all palmitoylation in BK channels that did not contain the STREX insert. Palmitoylation allows the S0-S1 linker to associate with the plasma membrane however the mutated de-palmitoylated channels did not affect channel conductance or the calcium/voltage sensitivity of the channel. Palmitoylation of the S0-S1 linker was shown to be a critical determinant of cell surface expression of BK channels, as steady state surface expression levels were reduced by ~55% in the C53:C54:C56 mutant. STREX channels that could not be palmitoylated in the S0-S1 linker also showed decreased surface expression even through STREX insert palmitoylation was unaffected. Palmitoylation is rapidly emerging as an important post-translational mechanism to control ion channel behaviour. This work reveals that palmitoylation of the BK channel can control channel function of the STREX splice variant channel and can regulate cell surface expression in all other channel variants. Palmitoylation appears to be functionally independent at these two distinct sites expressed within the same channel protein.

S-palmitoylation is a reversible post-translational lipid modification of proteins by adding a 16-carbon palmitate onto a cysteine residue. Palmitoylation has been shown to control the trafficking and function of many signalling proteins including ion channels. In this Thesis, palmitoylation is shown to control both the plasma membrane expression and gating properties of large conductance calcium- and voltage- dependent potassium (BK) channels. The BK channel is assembled from four pore-forming α-subunits. Each α-subunit contains seven transmembrane domains (S0-S6), with an extracellular N-terminus and a large intracellular C-terminus. BK channel α-subunit is encoded by a single gene Kcnma1 that undergoes extensive pre mRNA splicing at various splice sites, thus there are a number of alternatively spliced variants of α-subunits. Using quantitative imaging assays, palmitoylation of the intracellular S0-S1 loop controlled trafficking of full length ZERO variant BK channels to the plasma membrane in HEK293 cells as well as neuronal N2a cells. Importantly, all four α-subunits need to be palmitoylated for robust surface expression. Thus, palmitoylation of the S0-S1 loop of the α-subunit is important for surface expression of BK channels. The BK channel may also assemble with auxiliary β-subunits (β1-4) that regulate surface expression and gating properties of BK channels. The N-terminus of the β1- subunit and the C-terminus of the β4-subunit were shown to be palmitoylated using [3H]-palmitate incorporation, respectively. However, mutation of the palmitoylated cysteine (C18 in β1 and C193 in β4) to alanine to generate depalmitoylated β- subunits had no significant effects on the electrophysiological properties resulting from co-expression with the ZERO variant of the BK channel. However, although palmitoylation of the S0-S1 loop does not affect the electrophysiological properties of the ZERO channels alone, it is important for the shift in the V0.5max of ZERO channel when co-expressed with the β1-subunit, but not β4-subunit. These data suggest that palmitoylation of the S0-S1 loop controls the functional coupling between the ZERO α-subunit and β1-subunit. Although palmitoylation of C18 in the N-terminus of the β1-subunit was not required for functional coupling to α-subunits, we identified other critical residues within the short intracellular N-terminus of the β1-subunit that are essential. The functional coupling between BK α- and β1-subunit was predicted to be controlled by the interaction between a non-classic amphipathic α-helix in the β1 subunit N-terminus and the plasma membrane. Deletion, or mutations predicted to disrupt the interaction significantly decreased the β1-subunit induced left shift in the BK channel V0.5max. This suggests that the amphipathic in-plane anchor is critical for functional coupling of β1-subunits with BK channel α-subunits. In this Thesis, we demonstrated: i) palmitoylation of the α-subunit S0-S1 loop controls surface membrane expression of BK channels, and also controls functional regulation by β1, but not β4-subunits; and ii) a potential non-classical amphipathic in-plane anchor in the β1 N-terminus is essential for functional coupling with α- subunits. These studies help us further understand the regulation of BK channels and suggest potential therapeutic targets for various diseases related to dysfunctional BK channels, such as hypertension.

Ces dernières années, il est apparu que les espèces réactives de l'oxygène (ROS) ne sont pas seulement des substances délétères qui induisent des dommages de molécules biologiques, mais qu’elles sont également impliquées dans la signalisation cellulaire. Des niveaux contrôlés de H2O2 sont en particulier impliqués dans le processus de régénération chez différentes espèces. Nous avons donc étudié la régulation de la signalisation de H2O2 au cours de régénération de la nageoire caudale chez le poisson zèbre adulte. Nous avons montré que les nerfs contrôlent les niveaux de H2O2 à la fois dans les tissus normaux et après blessure; ce processus est médié par les cellules de Schwann qui expriment Shh. En plus, H2O2 stimule la croissance des nerfs, ce qui suggère la présence d'une boucle de rétrocontrôle positif.Les niveaux redox sont très dynamiques non seulement lors de la régénération, mais aussi au coursdu développement. Nous avons ensuite examiné le rôle de H2O2 pendant la morphogenèse et plusprécisément, son impact sur la croissance axonale et sa relation avec la signalisation Shh. Nous avons constaté que la réduction des niveaux de H2O2, normalement très élevé au cours de la morphogenèse, altère les projections axonales et que cet effet peut être sauvé par l'activation de la voie de signalisation de Shh.Les cibles de la signalisation redox comprennent des protéines dont l'activité est dépendante d'une cystéine, car l'état d'oxydation de cet acide aminé peut être modifié par les niveaux de H2O2. Le processus de S-acylation, qui est très important pour le processus de croissance des projections axonales et pour la voie de signalisation de Shh, consiste en la fixation covalente d'un acide gras, souvent le palmitate, au group sulfurique d’une cysteine et il est donc une cible potentielle de signalisation de ROS. Nous avons mis en évidence une corrélation entre le niveau de palmitoylationd’une protéine et les niveaux de H2O2 dans la cellule. La pertinence de cette observation estactuellement testée in vivo. / In the recent years it is becoming evident that reactive oxygen species (ROS) are not only deleterious compounds that induce damage of biological molecules, but are also important molecules that can mediate different signaling pathways. Controlled ROS, and in particular H2O2, levels have been found to be involved in the regenerative process of different species. We then focused on the regulation of H2O2 signaling during regeneration of the adult zebrafish caudal finand we showed that nerves control H2O2 levels both in normal tissue and after lesioning; this process is mediated by Schwann cells, through Shh signaling. In addition, there is also a reciprocal action of H2O2 on nerve growth, suggesting the presence of a positive feedback loop.Redox levels are highly dynamic not only during regeneration, but also during development. We then looked at the role of H2O2 during morphogenesis and more specifically, its impact on axonal growth and its relationship with Shh signaling. We found that reduction of H2O2 levels, normally very high during morphogenesis, impairs axon projections and that this effect can be rescued by the activation of Shh signaling. Moreover, we found that different redox levels modify the intracellulardistribution of Shh protein and also its extracellular availability. These results further strengthen the relationship between H2O2 and Shh signaling pathways.It is widely accepted that targets of redox signaling include proteins whose activity is dependent on an active cysteine because the oxidative status of this amino acid can be modified by H2O2 levels.The process of S-acylation, which is very important for both the processes of axonal projections growth and Shh signaling, consists in the covalent attachment of a fatty acid, often palmitate, to a cysteine sulphur and it is then a possible target of ROS signaling. We asked whether a relationship between H2O2 levels and protein S-palmitoylation could exist and we found that augmentation of H2O2 levels downregulates the S-palmitoylation process. The relevance of this observation iscurrently being tested in vivo.

Le développement de bactéries sous la forme de communautés multicellulaires, appelées biofilms, engendre la formation d’un environnement très hétérogène dans lequel les bactéries sont confrontées à différents stress nécessitant un rapide réajustement de leur métabolisme et de leur physiologie. Cette capacité d’adaptation ainsi que des études transcriptomiques, suggèrent fortement que les bactéries vivant sous forme de biofilm puissent présenter des propriétés nouvelles en comparaison à des bactéries planctoniques. Cela nous a conduit à examiner si la formation de biofilm chez Escherichia coli pouvait engendrer des modifications originales de l’enveloppe bactérienne. Nous avons tout d’abord comparé la structure du lipopolysaccharide de bactéries issues de cultures planctoniques et biofilms et mis en évidence que la formation de biofilm chez les bactéries à Gram négatif entraine une augmentation du niveau de palmitoylation du lipide A dépendante de l’enzyme PagP et contribuant à la tolérance des bactéries aux agents antimicrobiens. L’étude des mécanismes sous-jacents a permis de déterminer que cette augmentation du niveau de palmitoylation est, en partie, causée par une induction de l’expression de pagP qui est spécifique au biofilm et regulée par RcsB en réponse à l’osmolarité du biofilm. Nous avons également étudié l’impact de l’adaptation des bactéries au mode de vie biofilm sur la structure de la paroi bactérienne et mis en évidence des différences dans les ponts inter peptidiques présents au sein du peptidoglycane qui pourraient conduire à une augmentation de la rigidité de l’enveloppe bactérienne ainsi qu’à la résistance au stress chez les bactéries. Pour finir, nous avons examiné si les protéines SPFH (QmcA HflK HflC YqiK) associées à des microrégions spécialisées de la membrane -ou radeaux lipidiques- pouvaient jouer un rôle dans la formation de biofilm chez E. coli. Alors que les gènes codant pour ces protéines ne semblent pas être induits en biofilm ni être essentiels à son développement, nous avons identifié plusieurs régulateurs influençant leur expression ainsi que plusieurs fonctions associées aux protéines SPFH que nous sommes en train d’étudier. / The development of bacterial multicellular communities, called biofilms, creates a highly heterogeneous environment, in which bacteria subjected to various stresses need to quickly readjust their metabolism and physiology. This capacity of adaptation, confirmed by many transcriptome analyses, strongly suggest that biofilm bacteria could display novel properties compared to individualized cells. This prompted us to investigate whether biofilm formation could trigger original membrane modifications in Escherichia coli. We first compared the lipopolysaccharide structure of planktonic and biofilm bacteria and demonstrated that biofilms formed by Gram-negative bacteria undergo an increase in lipid A palmitoylation mediated by the PagP enzyme and contributing to bacterial resistance to antimicrobial peptides and host immune defenses. Investigation of the underlying mechanisms of this phenomenon allowed determining that increased lipid A palmitoylation is, at least partially, due to a biofilm-specific and RcsB-dependent induction of pagP expression in response to biofilm osmolarity. We also investigated how bacterial adaptation to the biofilm environment could impact the cell wall structure and identified differences in peptidoglycan linkages that could increase cell rigidity and bacterial resistance to stress. Finally, we investigated the potential role in biofilm formation of E. coli SPFH proteins (QmcA HflK HflC YqiK) associated with dynamic membrane microdomains - or lipid rafts-. Whereas SPFH encoding genes are not differentially expressed in biofilm and are not essential for biofilm formation, we identified several regulators impacting their expression and several functions associated to SPFH proteins that we identified is currently under investigation.

Doctor of Philosophy / Department of Biology / Rollie J. Clem / Members of the protein super family of tetraspanins are best defined by a simple structure comprising four transmembrane domains, two extracellular loops of unequal size, and short cytoplasmic regions. Despite their small size, tetraspanins are able to participate in multiple functions, as diverse as B cell activation, cancer metastasis, and viral infection. To compensate for a lack of intrinsic enzymatic activity, tetraspanins have gained the fascinating ability of associating with numerous different proteins. In addition, tetraspanins interact with each other forming a network on the plasma membrane: the tetraspanin web. In this way, functionally related proteins binding to different tetraspanins can be brought into close vicinity, thereby enhancing signaling pathways. The tetraspanin web is a dynamic environment and its regulation has grasped the attention of several research groups in the past few years. Particularly, several tetraspanins have been found to be palmitoylated, a post-translational modification attaching a palmitic acid to cysteine residues in a reversible manner. Palmitoylation is thought to be important for the integrity of the tetraspanin web. We examined the effect of disrupting putative palmitoylation sites on the tetraspanin CD81 by mutating its juxtamembrane cysteines. By flow cytometry, we observed a decrease in the detection of mutant CD81 at the cell surface. This was not due to defects in protein trafficking or antibody affinity, and might reflect an abnormal CD81 distribution in a membrane environment that prevents the exposure of the epitope recognized by the CD81 antibody. Immunoblotting analysis revealed a novel CD81 processing event that was impaired in the mutant CD81 proteins compared to wild-type. Finally, co-immunoprecipitation assays showed a reduction in binding of tetraspanin CD9 and Ig superfamily member EWI-2 to mutant CD81. Taken together, these results suggest the importance of juxtamembrane cysteines (via palmitoylation or protein conformational changes) in protein interactions of CD81 within the tetraspanin web. Although 33 tetraspanins are expressed in humans, less than half of them have been well studied. Among the “orphan” tetraspanins awaiting further examination is Tspan-2. Here, we provide the first elements for the characterization of mammalian Tspan-2 by investigating expression patterns, N-glycosylation status, and association with other tetraspanins.

Les protéines RGS (Regulator of G-protein Signaling) sont des inhibiteurs des voies de signalisation des protéines G. RGS4 atténue l’activité de protéines G dans plusieurs tissus tel que la diminution de son activité peut accroître la sévérité de la bradycardie, cardiomyopathies liées au diabète, l’invasion de cellule cancéreuse du sein, résistance à l’insuline et intolérance au glucose. RGS4 a été localisé à la membrane plasmique ainsi que dans des compartiments intracellulaires, cependant, son mode de trafique intracellulaire reste méconnu. En utilisant des outils de microscopie confocale sur cellules vivantes et méthode de détection d’activité des voies de signalisation conditionnée par les protéines G, nous avons caractérisé l’importance de deux sites de palmitoylation, ces deux sites : Cys2 et Cys12 montrent des intérêts complémentaires dans le trafic de RGS4 vers la membrane cellulaire. Dans un axe linéaire, nous avons identifié DHHC3 et 7, deux enzymes de palmitoylation participant au trafique intracellulaire de RGS4 et donc à la maximalisation de son activité inhibitrice des voies de signalisation contrôlées par Galphaq. Enfin des marqueurs de membranes endosomales, les protéines rab ont permis de caractériser les voies de trafic intracellulaire empruntée par RGS4, par exemple RGS4 est internalisé de la membrane plasmique par Rab5 et recyclé à la membrane cellulaire par Rab11. L’activation ou inhibition de Rab5 et 11 ont permis d’observer des changements d’activité de RGS4. Ces travaux confèrent une base de données pour des études ultérieures visant à développer des stratégies thérapeutiques à accroître les fonctionnalités de RGS4. / RGS proteins (Regulator of G-protein Signaling) are potent inhibitors of heterotrimeric G-protein signaling. RGS4 attenuates G-protein activity in several tissues such that loss of its function may lead to bradycardia, diabetic cardiomyopathy, breast cancer cell invasion, insulin resistance and glucose intolerance. RGS4 has been localized to both plasma membrane and intracellular pools, however, the nature of its intracellular trafficking remains to be elucidated. G-protein inhibition requires the presence of RGS4 at the plasma membrane. In this work, we characterized the complementary roles of two putative palmitoylation sites on RGS4 to target intracellular compartments and plasma membrane. We identified palmitoylation on Cys2 and 12 respectively important for RGS4 endosomal targeting and plasma membrane localization, when mutations were introduced to the palmitoylation sites, RGS4 capability of inhibiting Gq-mediated signaling was impaired. As a continuum we identified two palmitoylating enzymes, DHHC3 and 7 as modulator of RGS4 localization and function. Knock downs of DHHC3 and 7 impaired RGS4 endosomal and plasma membrane targeting and capability of inhibiting M1-muscarinic receptor signaling. Finally we used live cell confocal microscopy to define RGS4 intracellular trafficking routes. Specifically Rab5 mediated RGS4 trafficking from the plasma membrane to intracellular compartments while Rab11 mediated RGS4 trafficking to the plasma membrane. Activation and inhibition of Rab5 and 11 routes impaired RGS4 capability of inhibiting M1-muscarinic receptor signaling pathway. These novel findings provide a strong rationale for future studies aimed at developing new strategies to increase the function of RGS4.

Regulator of G-protein signalling 4 (RGS4) modulates Gq and Gi signalling at the plasma membrane (PM). It has been demonstrated that the addition of palmitate to cysteine residues is an important regulator of RGS protein localization and function. The family of palmitate transferase enzymes shares a conserved Asp-His-His-Cys (DHHC) motif. We set out to establish the DHHC isoform(s) that affect RGS4 activity in HEK201 cells. Confocal microscopy revealed that overexpression of DHHCs 3 and 7 mobilized RGS4 to the Golgi. Knockdown of either DHHC3 or DHHC7 attenuated RGS4 inhibition of Gαq-coupled Ca2+ release and reduced RGS4 PM localization. Consistent with a role in promoting RGS4 lipid bilayer targeting, dominant negative mutants of the five most highly expressed DHHCs in HEK201 cells also diminished RGS4 PM association. Together, these data suggest that members of the mammalian DHHC family regulate RGS4 localization and function, likely through palmitoylation of its target cysteine residues.

Regulator of G-protein signalling 4 (RGS4) modulates Gq and Gi signalling at the plasma membrane (PM). It has been demonstrated that the addition of palmitate to cysteine residues is an important regulator of RGS protein localization and function. The family of palmitate transferase enzymes shares a conserved Asp-His-His-Cys (DHHC) motif. We set out to establish the DHHC isoform(s) that affect RGS4 activity in HEK201 cells. Confocal microscopy revealed that overexpression of DHHCs 3 and 7 mobilized RGS4 to the Golgi. Knockdown of either DHHC3 or DHHC7 attenuated RGS4 inhibition of Gαq-coupled Ca2+ release and reduced RGS4 PM localization. Consistent with a role in promoting RGS4 lipid bilayer targeting, dominant negative mutants of the five most highly expressed DHHCs in HEK201 cells also diminished RGS4 PM association. Together, these data suggest that members of the mammalian DHHC family regulate RGS4 localization and function, likely through palmitoylation of its target cysteine residues.