The Voltage-Dependent Anion Channel (VDAC) is a channel located in the outer mitochondrialmembrane of nearly all eukaryotic cells. It is responsible for the passage of numerous ions andmetabolites in and out of the mitochondria but is also involved in the regulation of the cell functionthrough interactions with other proteins. Its activity is known to be modulated by lipids. Experimentson Phaseolus coccineus VDAC32 (PcVDAC32) have notably suggested a direct interaction betweendioleoylphosphatidylethanolamine (DOPE) head group and the bean VDAC32. Moreover, moleculardynamic simulations have proposed that charged residues of the mouse VDAC1 could be involved indirect interaction with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) polar head.During this PhD thesis, we first constructed and optimised expression systems for production ofPcVDAC32 and of Saccharomyces cerevisiae VDAC1 (ScVDAC1) in yeast and assessed those forcomplementation in yeasts lacking their endogenous VDAC1. We then tested the effect of a polyhistidine-tag placed in C-terminal of the ScVDAC1 on its electrophysiological properties to validate itas a tool for VDAC study. We further analysed the effect of the lipid environment on the ScVDAC1and compared it with the results obtained for PcVDAC32. Finally, we assessed the effect of thesuppression of the Glu185 charge on the ScVDAC1 conductance, selectivity and voltage dependence.We found that expression of PcVDAC32 could complement the growth deficiency of the yeast lackingendogenous VDAC. We also showed that the presence of calcium ions in the experimental solutionsallowed the ScVDAC1 closed states to reach lower conductances. We observed that preincubation withergosterol greatly enhanced the reconstitution of ScVDAC1 in soy extract PLB. We demonstrated thatpH and salt concentration influence the ScVDAC1 functional characteristics and that, according tothose parameters, the presence of a 6xHis-tag can influence VDAC functions. Finally, we showed thatthe ScVDAC1 Glu185 located in the loop between β-strand 12 and 13 is involved in ScVDAC1selectivity and that its substitution by a glutamine residue decreases the ScVDAC1 sensitivity tomembrane curvature stress. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

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EN This Ph.D. thesis focuses on the heme containing gas sensor proteins. These proteins are predominantly present in bacteria, in which play an important role in processes like, sporulation, antibiotic resistance and so on. Heme containing sensor proteins composed of two domains. First one is a globin domain, which contains the heme molecule. Interaction of heme with gas molecule acts as a signal for the activation/inactivation of the second functional domain. Part of this thesis is formed by a review, which summarized the current knowledge about heme containing sensor proteins. In the next part of this thesis we focused on three representatives from the group of oxygen sensor proteins - histidine kinase AfGcHK, diguanylate cyclase YddV and phosphodiesterase EcDOS. The main aim of this thesis was to solve the mechanism of interdomain/intraprotein signal transduction in two oxygen sensor proteins with globin fold of their sensor domain (AfGcHK, YddV). For this purpose, we used the kinetic analysis of their functional domain activity and the methods of structural biology. We also studied the mechanism of interprotein signal transduction in AfGcHK and its cognate partner RR protein. It was also tested, how the presence of sodium disulfide affects the functional properties of oxygen sensor proteins...

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Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

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Signal transduction networks enable organisms to respond to environmental stimuli. Bacteria utilize two-component systems (TCSs) and phosphorelays as their primary means of signal transduction. Histidine kinase (HK) and response regulator (RR) proteins comprise these TCSs and phosphorelays. Previously, signal transduction within TCSs and phosphorelays was thought to only occur through a linear series of phosphotransfers between HKs and RRs. Recently multikinase networks have been shown to be involved in TCS and phosphorelay signal transmission. A multikinase network that includes the HKs RetS and GacS controls the switch between the motile invasive lifestyle and the sessile biofilm lifestyle of the opportunistic human pathogen Pseudomonas aeruginosa. GacS promotes the sessile biofilm lifestyle, while RetS promotes the motile invasive lifestyle via the inhibition of GacS. This inhibition occurs through three distinct mechanisms. Two of the mechanisms are dephosphorylating mechanisms and the third mechanism is a direct interaction between RetS and GacS which results in the inhibition of GacS autophosphorylation. This study examines the direct binding interaction between RetS and GacS using structural biology. We observed a heterodimeric RetS-GacS complex in which the canonical homodimerization interface was replaced with a heterodimeric interface. Heterodimerization between bacterial HKs is currently a novel observation, but it is likely that other HKs heterodimerize. The RetS-GacS direct interaction can serve as a model for HK-HK binding in multikinase networks. / Doctor of Philosophy / The way in which bacteria assess and respond to their environment is of great interest to microbiologists. Bacteria transmit environmental signals via protein interactions. Some of these interactions involve the transfer of phosphate groups, and some involve a direct binding interaction between proteins. We are investigating a direct binding interaction between two proteins, RetS and GacS. These proteins control whether Pseudomonas aeruginosa, an opportunistic pathogen of humans, causes an acute infection, which is characterized by motility and invasiveness, or a chronic infection, which is characterized by a sessile biofilm lifestyle, in a human host. Through the use of structural biology techniques we have visualized the three-dimensional structure of the complex between RetS and GacS. This complex has provided insight into the role of the RetS-GacS interaction in controlling the infection state of P. aeruginosa.

Recent studies suggest that factor (F) XII plays a key role in thrombus stabilization and growth but is dispensable for hemostasis. We have previously shown that histidine-rich glycoprotein (HRG), a protein present in platelets and plasma, binds FXIIa and inhibits FXII autoactivation and FXIIa-mediated activation of FXI, thereby downregulating thrombosis. HRG binds various ligands, including FXIIa, fibrin(ogen), nucleic acids and polyphosphate (polyP). Studies have shown that polyP, released from activated platelets, and artificial surfaces like catheters, can promote FXII activation. This suggests that HRG can downregulate the activation of the contact system. This thesis aims to determine the potential mechanisms by which HRG modulates platelet function and thrombosis induced by polyP or catheters. We show that HRG binds polyP with high affinity and inhibits the procoagulant, prothrombotic and cardiotoxic effects of polyP via at least two mechanisms. First, HRG binds polyP and neutralizes its procoagulant activities and cytotoxic effects. Second, HRG binds FXIIa and attenuates its capacity to promote autoactivation and activate FXI. Also, we identify that HRG serves as a molecular brake for the contact system by attenuating the procoagulant activity of FXIIa regardless of whether FXII activation is triggered systemically with polyP or occurs locally on the surface of catheters. Our studies have identified HRG as a novel ligand for platelet receptor GPIbα on resting platelets, and upon activation, it competes with fibrinogen for binding to GPIIb/IIIa integrin, thereby inhibiting platelet aggregation. These findings suggest that HRG may modulate coagulation as well as platelet function. Therefore, supplementation with HRG or HRG analogs may serve as a potential therapeutic option to attenuate polyP or catheter-induced thrombosis without perturbing hemostasis. / Dissertation / Doctor of Philosophy (PhD)

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