Rhomboids are unique membrane proteins that use a serine protease hydrolysis mechanism to cleave a transmembrane substrate within the lipid bilayer. This remarkable proteolytic activity is achieved by a core domain comprised of 6 transmembrane segments that form a hydrophilic cavity submerged in the membrane. In addition to this core domain, many rhomboids also possess aqueous domains of varying sizes at the N- and/or C-terminus, the sequences of which tend to be rhomboid-type specific. The functional role of these extramembranous domains is generally not well understood, although it is thought that they may be involved in regulation of rhomboid activity and specificity. While extramembranous domains may be important for rhomboid activity, they are absent in all x-ray crystal structures available. For this reason, we have focused on uncovering the structural and functional relationship between the rhomboid cytoplasmic domain and its catalytic transmembrane core. To investigate the structure and function of the bacterial rhomboid cytoplasmic domain, full-length rhomboids from Escherichia coli and Pseudomonas aeruginosa were studied using solution nuclear magnetic resonance (NMR) spectroscopy, mutation and activity assays. The P. aeruginosa rhomboid was purified in a range of membrane-mimetic media, evaluated for its functional status in vitro and investigated for its NMR spectroscopic properties. Results from this study suggested that an activity-modulating interaction might occur between the catalytic core transmembrane domain and the cytoplasmic domain. Further investigation of this hypothesis with the E. coli rhomboid revealed that protease activity relies on a short but critical sequence N-terminal to the first transmembrane segment. This sequence was found to have a direct impact on the rhomboid active site, and should be included in future structural studies of this catalytic domain. The structure of the cytoplasmic domain from the E. coli rhomboid was also determined by solution NMR. We found that it forms slowly-exchanging dimers through an exchange of secondary structure elements between subunits, commonly known as three-dimensional domain swapping. Beyond this rare example of domain swapping in a membrane protein extramembranous domain, we found that the rate of exchange between monomeric and dimeric states could be accelerated by transient interactions with large detergent micelles with a phosphocholine headgroup, but not by exposure to other weakly denaturing conditions. This novel example of micelle-catalyzed domain swapping interactions raises the possibility that domain swapping interactions might be induced by similar interactions in vivo. Overall, the results of this thesis have identified detergent conditions that preserve the highest level of activity for bacterial rhomboids, defined the minimal functional unit beyond what had been identified in available x-ray crystal structures, and characterized a novel micelle-catalyzed domain-swapping interaction by the cytoplasmic domain.

Rhomboids are unique membrane proteins that use a serine protease hydrolysis mechanism to cleave a transmembrane substrate within the lipid bilayer. This remarkable proteolytic activity is achieved by a core domain comprised of 6 transmembrane segments that form a hydrophilic cavity submerged in the membrane. In addition to this core domain, many rhomboids also possess aqueous domains of varying sizes at the N- and/or C-terminus, the sequences of which tend to be rhomboid-type specific. The functional role of these extramembranous domains is generally not well understood, although it is thought that they may be involved in regulation of rhomboid activity and specificity. While extramembranous domains may be important for rhomboid activity, they are absent in all x-ray crystal structures available. For this reason, we have focused on uncovering the structural and functional relationship between the rhomboid cytoplasmic domain and its catalytic transmembrane core. To investigate the structure and function of the bacterial rhomboid cytoplasmic domain, full-length rhomboids from Escherichia coli and Pseudomonas aeruginosa were studied using solution nuclear magnetic resonance (NMR) spectroscopy, mutation and activity assays. The P. aeruginosa rhomboid was purified in a range of membrane-mimetic media, evaluated for its functional status in vitro and investigated for its NMR spectroscopic properties. Results from this study suggested that an activity-modulating interaction might occur between the catalytic core transmembrane domain and the cytoplasmic domain. Further investigation of this hypothesis with the E. coli rhomboid revealed that protease activity relies on a short but critical sequence N-terminal to the first transmembrane segment. This sequence was found to have a direct impact on the rhomboid active site, and should be included in future structural studies of this catalytic domain. The structure of the cytoplasmic domain from the E. coli rhomboid was also determined by solution NMR. We found that it forms slowly-exchanging dimers through an exchange of secondary structure elements between subunits, commonly known as three-dimensional domain swapping. Beyond this rare example of domain swapping in a membrane protein extramembranous domain, we found that the rate of exchange between monomeric and dimeric states could be accelerated by transient interactions with large detergent micelles with a phosphocholine headgroup, but not by exposure to other weakly denaturing conditions. This novel example of micelle-catalyzed domain swapping interactions raises the possibility that domain swapping interactions might be induced by similar interactions in vivo. Overall, the results of this thesis have identified detergent conditions that preserve the highest level of activity for bacterial rhomboids, defined the minimal functional unit beyond what had been identified in available x-ray crystal structures, and characterized a novel micelle-catalyzed domain-swapping interaction by the cytoplasmic domain.

Rhomboids are unique membrane proteins that use a serine protease hydrolysis mechanism to cleave a transmembrane substrate within the lipid bilayer. This remarkable proteolytic activity is achieved by a core domain comprised of 6 transmembrane segments that form a hydrophilic cavity submerged in the membrane. In addition to this core domain, many rhomboids also possess aqueous domains of varying sizes at the N- and/or C-terminus, the sequences of which tend to be rhomboid-type specific. The functional role of these extramembranous domains is generally not well understood, although it is thought that they may be involved in regulation of rhomboid activity and specificity. While extramembranous domains may be important for rhomboid activity, they are absent in all x-ray crystal structures available. For this reason, we have focused on uncovering the structural and functional relationship between the rhomboid cytoplasmic domain and its catalytic transmembrane core. To investigate the structure and function of the bacterial rhomboid cytoplasmic domain, full-length rhomboids from Escherichia coli and Pseudomonas aeruginosa were studied using solution nuclear magnetic resonance (NMR) spectroscopy, mutation and activity assays. The P. aeruginosa rhomboid was purified in a range of membrane-mimetic media, evaluated for its functional status in vitro and investigated for its NMR spectroscopic properties. Results from this study suggested that an activity-modulating interaction might occur between the catalytic core transmembrane domain and the cytoplasmic domain. Further investigation of this hypothesis with the E. coli rhomboid revealed that protease activity relies on a short but critical sequence N-terminal to the first transmembrane segment. This sequence was found to have a direct impact on the rhomboid active site, and should be included in future structural studies of this catalytic domain. The structure of the cytoplasmic domain from the E. coli rhomboid was also determined by solution NMR. We found that it forms slowly-exchanging dimers through an exchange of secondary structure elements between subunits, commonly known as three-dimensional domain swapping. Beyond this rare example of domain swapping in a membrane protein extramembranous domain, we found that the rate of exchange between monomeric and dimeric states could be accelerated by transient interactions with large detergent micelles with a phosphocholine headgroup, but not by exposure to other weakly denaturing conditions. This novel example of micelle-catalyzed domain swapping interactions raises the possibility that domain swapping interactions might be induced by similar interactions in vivo. Overall, the results of this thesis have identified detergent conditions that preserve the highest level of activity for bacterial rhomboids, defined the minimal functional unit beyond what had been identified in available x-ray crystal structures, and characterized a novel micelle-catalyzed domain-swapping interaction by the cytoplasmic domain.

Rhomboids are unique membrane proteins that use a serine protease hydrolysis mechanism to cleave a transmembrane substrate within the lipid bilayer. This remarkable proteolytic activity is achieved by a core domain comprised of 6 transmembrane segments that form a hydrophilic cavity submerged in the membrane. In addition to this core domain, many rhomboids also possess aqueous domains of varying sizes at the N- and/or C-terminus, the sequences of which tend to be rhomboid-type specific. The functional role of these extramembranous domains is generally not well understood, although it is thought that they may be involved in regulation of rhomboid activity and specificity. While extramembranous domains may be important for rhomboid activity, they are absent in all x-ray crystal structures available. For this reason, we have focused on uncovering the structural and functional relationship between the rhomboid cytoplasmic domain and its catalytic transmembrane core. To investigate the structure and function of the bacterial rhomboid cytoplasmic domain, full-length rhomboids from Escherichia coli and Pseudomonas aeruginosa were studied using solution nuclear magnetic resonance (NMR) spectroscopy, mutation and activity assays. The P. aeruginosa rhomboid was purified in a range of membrane-mimetic media, evaluated for its functional status in vitro and investigated for its NMR spectroscopic properties. Results from this study suggested that an activity-modulating interaction might occur between the catalytic core transmembrane domain and the cytoplasmic domain. Further investigation of this hypothesis with the E. coli rhomboid revealed that protease activity relies on a short but critical sequence N-terminal to the first transmembrane segment. This sequence was found to have a direct impact on the rhomboid active site, and should be included in future structural studies of this catalytic domain. The structure of the cytoplasmic domain from the E. coli rhomboid was also determined by solution NMR. We found that it forms slowly-exchanging dimers through an exchange of secondary structure elements between subunits, commonly known as three-dimensional domain swapping. Beyond this rare example of domain swapping in a membrane protein extramembranous domain, we found that the rate of exchange between monomeric and dimeric states could be accelerated by transient interactions with large detergent micelles with a phosphocholine headgroup, but not by exposure to other weakly denaturing conditions. This novel example of micelle-catalyzed domain swapping interactions raises the possibility that domain swapping interactions might be induced by similar interactions in vivo. Overall, the results of this thesis have identified detergent conditions that preserve the highest level of activity for bacterial rhomboids, defined the minimal functional unit beyond what had been identified in available x-ray crystal structures, and characterized a novel micelle-catalyzed domain-swapping interaction by the cytoplasmic domain.

Rhomboid intramembrane serine proteases cleave polypeptide chains within lipid bilayer. Rhomboid proteases were originally discovered in Drosophila melanogaster where they regulate ontogenesis of the fly, but they are present in all domains of life. Nowadays, various diseases, such as malaria, amoebiasis, Parkinson's disease, various tumour malignancies, and diabetes, have been linked with rhomboid proteases. However, natural substrates and function of most rhomboids remain elusive. Cell biology tools are needed for unravelling functions of rhomboids, as well as for potential pharmacological applications, and this together fuels the effort to develop specific rhomboid inhibitors. The inhibitors known to date always bear an electrophilic warhead attacking the nucleophilic serine of the atypical serine-histidine catalytic dyad of rhomboid. From the various developed inhibitors, peptidyl -ketoamides substituted at the ketoamide nitrogen by hydrophobic groups, discovered in our laboratory, hold the biggest potential. They are potent, reversible, selective, tunable, and are built around a pharmacophore already approved for medical use. Here, I set out to improve peptidyl -ketoamides by exploring the chemical space in the active site of rhomboid and testing substituents of the ketoamide nitrogen of increasing...

Membrane proteins of the rhomboid-family are evolutionarily widely conserved and include rhomboid intramembrane serine proteases and rhomboid-like proteins. The latter have lost their catalytic activity in evolution but retained the ability to bind transmembrane helices. Rhomboid-family proteins play important roles in intercellular signalling, membrane protein quality control and trafficking, mitochondrial dynamics, parasite invasion and wound healing. Their medical potential is steeply increasing, but in contrast to that, their mechanistic and structural understanding lags behind. Rhomboid protease GlpG from E.coli has become the main model rhomboid-family protein and the main model intramembrane protease - it was the first one whose X-ray structure was solved. GlpG cleaves single-pass transmembrane proteins in their transmembrane helix, but how substrates bind to GlpG and how is substrate specificity achieved is still poorly understood. This thesis investigates the importance of the transmembrane helix of the substrate in its recognition by GlpG using mainly enzyme kinetics and site-directed mutagenesis. We find that the transmembrane helix of the substrate contributes significantly to the binding affinity to the enzyme, hence to cleavage efficiency, but it also plays a role in cleavage site...

L'enveloppe cellulaire bactérienne joue plus qu'un rôle de barrière d'échange. Elle est au coeur des processus cellulaires essentiels comme la morphogenèse et la division. Cette structure abrite environ un quart des protéines codées par le génome. Le but de mon travail a été de mieux comprendre le rôle de deux protéines membranaires dans la construction et la dynamique de l'enveloppe chez Bacillus subtilis. GluP est une protéase intramembranaire rhomboïde. Ces protéases clivent des segments transmembranaires dans la membrane afin de moduler l'activité de diverses protéines. Elles participent à de nombreux processus cellulaires chez les eucaryotes. Cependant, les fonctions biologiques des rhomboïdes procaryotes sont pour l'heure presque totalement inconnues. Nos résultats suggèrent que GluP participe au contrôle qualité des protéines membranaires à la manière des pseudo-rhomboïdes associées au système ERAD eucaryote. Elle forme un complexe avec FtsH, une protéase majeure du contrôle qualité des protéines. Ce complexe est impliqué dans la dégradation d'un substrat de rhomboïde. Le rôle de GluP serait de permettre la dislocation du segment transmembranaire et faciliter la prise en charge du substrat par FtsH. Le second projet auquel j'ai participé a consisté à comprendre le rôle de la protéine CmmB dans la morphogenèse. Son absence conduit à une morphologie cellulaire élargie. CmmB semble faire partie de la machinerie de synthèse du peptidoglycane au cours de l'élongation de la paroi. Elle serait nécessaire au bon fonctionnement d'une ou de plusieurs penicillin-binding proteins (PBPs). En particulier, nous proposons que CmmB est un cofacteur de la transpeptidase PBP2a. / The bacterial cell envelope is an obligatory barrier. It is a fundamental component in essential cellular processes such as morphogenesis and cell division. It hosts about a quarter of the proteins encoded in the genome. My work was aimed at understanding the function of two membrane proteins in the building and the dynamics of the cell envelope in the model bacterium Bacillus subtilis.GluP is a rhomboid intramembrane protease. Usually, rhomboids cleave transmembrane segments within the membrane to modulate protein functions. In eukaryotes, they participate in many cellular processes and their dysfunction lead to several pathologies. However, prokaryotic rhomboid functions remain almost totally unknown. Our results suggest that GluP is involved in bacterial membrane protein quality control, in a process akin to pseudo-rhomboid dependent endoplasmic reticulum associated protein degradation in eukaryotes. GluP forms a complex with FtsH, a major protease in protein quality control. That complex is not involved in the cleavage of a membrane substrate but in its degradation. We propose that GluP is required for the dislocation of the transmembrane segment, thus facilitating full-length substrate degradation by FtsH in the cytoplasm. My thesis second objective was to understand the role of the CmmB protein in morphogenesis. The absence of CmmB leads to slightly enlarged cells. CmmB seems to belong to the peptidoglycan synthesis machinery for cell-wall elongation. Our data support the idea that it is required for the proper activity of one or several penicillin-binding proteins (PBPs). In particular, we propose that CmmB is a cofactor of the PBP2a transpeptidase.

Intramembrane proteases from the rhomboid-like superfamily are enzymes widely distributed and conserved in all domains of life. They participate in many important processes such as membrane protein quality control or mitochondrial dynamics. Their activity is also linked with diseases like Parkinson's disease or cancer. This makes them potential therapeutic targets. In this work we tried to elucidate in more detail the mechanism of action of the main model intramembrane protease, GlpG from E. coli. We also focused on the mechanism of eukaryotic rhomboid RHBDL2, one of the four mammalian rhomboids, function of which is poorly understood. To acquire more detailed information about substrate-enzyme interaction, we synthesized a series of novel peptidyl-chloromethylketone inhibitors derived from natural rhomboid substrate TatA from P. stuartii. Crystal structure of the complex of GlpG with these inhibitors revealed four substrate binding subsites (S1 to S4) of the enzyme and explained its observed substrate specificity structurally. This study showed that substrate cleavage rate can be dramatically modified by changing the substrate sequence in positions P1 to P5. This helped us develop fluorogenic transmembrane peptide substrates for rhomboid proteases, which are usable in detergent and liposomes, and...

Regulated intramembrane proteolysis is an interesting process involved in a multitude of cellular pathways. Enzymes which catalyse this are termed intramembrane proteases (IMPRs), cleaving proteins passing through the membrane within their transmembrane domain. Rhomboid proteases are serine IMPRs. They are widely distributed among organisms and evolutionarily conserved, but despite many efforts, their physiological roles are largely unexplored. RHBDL4 is a mammalian rhomboid protease localised to the endoplasmic reticulum. It is involved in the development of colorectal cancer, which makes it an important focus of research, but its physiological function is not well understood. In order to explore it, I established and employed a proximity proteomics approach, termed APEX2. It is based on biotinylation of proteins in the spatial proximity of the target in the physiological environment of intact living cells. Labelled proteins are subsequently purified, identified and quantified by mass spectrometry. Exploring the physiological vicinity of RHBDL4, its interaction partners and substrates can be revealed and the detailed subcellular compartment, where RHBDL4 resides, can thus be inferred. During three independent experiments in HCT116 cell line, three proteins emerged repeatedly in the RHBDL4...

Rhomboids are intramembrane serine proteases that belong to the evolutionarily widespread rhomboid superfamily. Rhomboids developed a slightly different catalytic mechanism compared to classical serine proteases; they utilise a catalytic dyad (Ser/His) instead of the common triad (Ser/His/Asp), and the rhomboid active site is buried in the membrane. This, coupled with their hydrophobicity, makes them quite difficult to study. Therefore, even though they are known to be involved in several important biological processes it is still not clear how exactly most of them are involved in the regulation of or in the pathologies of diseases related to these processes (such as malaria, Parkinson's disease or cancer). Our understanding is hindered by the lack of tools for their characterisation both in vitro and in vivo. In my thesis I present new fluorogenic substrates based on the LacYTM2 sequence, which is hydrolysed by several different rhomboid proteases. Using Förster resonance energy transfer (FRET)-based methods, these substrates are suitable for continuous monitoring of rhomboid activity in vitro. Modifications in the P5-P1 residues can improve selectivity for a specific rhomboid, the choice of FRET pair of fluorophores that absorbes light of longer wavelengths makes them suitable for high throughput...