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Matthew Freeman - One of the best experts on this subject based on the ideXlab platform.
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bacterial Rhomboid proteases mediate quality control of orphan membrane proteins
The EMBO Journal, 2020Co-Authors: Guangyu Liu, Kvido Strisovsky, Matthew Freeman, Stephen E Beaton, Adam Graham Grieve, Rhiannon M Evans, Miranda Rogers, Fraser A Armstrong, Rachel M Exley, Christoph M TangAbstract:Although multiprotein membrane complexes play crucial roles in bacterial physiology and virulence, the mechanisms governing their quality control remain incompletely understood. In particular, it is not known how unincorporated, orphan components of protein complexes are recognised and eliminated from membranes. Rhomboids, the most widespread and largest superfamily of intramembrane proteases, are known to play key roles in eukaryotes. In contrast, the function of prokaryotic Rhomboids has remained enigmatic. Here, we show that the Shigella sonnei Rhomboid proteases GlpG and the newly identified Rhom7 are involved in membrane protein quality control by specifically targeting components of respiratory complexes, with the metastable transmembrane domains (TMDs) of Rhomboid substrates protected when they are incorporated into a functional complex. Initial cleavage by GlpG or Rhom7 allows subsequent degradation of the orphan substrate. Given the occurrence of this strategy in an evolutionary ancient organism and the presence of Rhomboids in all domains of life, it is likely that this form of quality control also mediates critical events in eukaryotes and protects cells from the damaging effects of orphan proteins.
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Quantitative proteomics screen identifies a substrate repertoire of Rhomboid protease RHBDL2 in human cells and implicates it in epithelial homeostasis
Scientific Reports, 2017Co-Authors: Nicholas Johnson, Jana Březinová, Emma Burbridge, Colin Adrain, Matthew Freeman, Elaine Stephens, Kvido StrisovskyAbstract:Rhomboids are intramembrane serine proteases conserved in all kingdoms of life. They regulate epidermal growth factor receptor signalling in Drosophila by releasing signalling ligands from their transmembrane tethers. Their functions in mammals are poorly understood, in part because of the lack of endogenous substrates identified thus far. We used a quantitative proteomics approach to investigate the substrate repertoire of Rhomboid protease RHBDL2 in human cells. We reveal a range of novel substrates that are specifically cleaved by RHBDL2, including the interleukin-6 receptor (IL6R), cell surface protease inhibitor Spint-1, the collagen receptor tyrosine kinase DDR1, N-Cadherin, CLCP1/DCBLD2, KIRREL, BCAM and others. We further demonstrate that these substrates can be shed by endogenously expressed RHBDL2 and that a subset of them is resistant to shedding by cell surface metalloproteases. The expression profiles and identity of the substrates implicate RHBDL2 in physiological or pathological processes affecting epithelial homeostasis.
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Substrates and physiological functions of secretase Rhomboid proteases.
Seminars in cell & developmental biology, 2016Co-Authors: Viorica Lastun, Adam G. Grieve, Matthew FreemanAbstract:Rhomboids are conserved intramembrane serine proteases with widespread functions. They were the earliest discovered members of the wider Rhomboid-like superfamily of proteases and pseudoproteases. The secretase class of Rhomboid proteases, distributed through the secretory pathway, are the most numerous in eukaryotes, but our knowledge of them is limited. Here we aim to summarise all that has been published on secretase Rhomboids in a concise encyclopaedia of the enzymes, their substrates, and their biological roles. We also discuss emerging themes of how these important enzymes are regulated.
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Intramembrane proteolysis by Rhomboids: catalytic mechanisms and regulatory principles.
Current opinion in structural biology, 2013Co-Authors: Kutti R Vinothkumar, Matthew FreemanAbstract:Rhomboids are intramembrane serine proteases that cleave membrane proteins within the bilayer, and which control a wide variety of biological processes. Recent structures of Escherichia coli Rhomboids in complex with mechanism-based inhibitors provide insight into their catalytic mechanism. The inhibitor structures also reveal potential substrate-binding sites within the enzyme and provide a template for modeling substrate binding at the active site. The regulation of Rhomboid activity exploits the different membrane compartments in cells to segregate enzyme and substrate. Catalytically inactive Rhomboid-like proteins called iRhoms provide another form of regulation, by interacting with Rhomboid substrates and preventing their cleavage. Extramembranous domains of Rhomboids may play an as yet unexplored role in substrate recognition and regulation
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structure of Rhomboid protease in complex with β lactam inhibitors defines the s2 cavity
Structure, 2013Co-Authors: Kutti R Vinothkumar, O A Pierrat, Jonathan M Large, Matthew FreemanAbstract:Rhomboids are evolutionarily conserved serine proteases that cleave transmembrane proteins within the membrane. The increasing number of known Rhomboid functions in prokaryotes and eukaryotes makes them attractive drug targets. Here, we describe structures of the Escherichia coli Rhomboid GlpG in complex with β-lactam inhibitors. The inhibitors form a single bond to the catalytic serine and the carbonyl oxygen of the inhibitor faces away from the oxyanion hole. The hydrophobic N-substituent of β-lactam inhibitors points into a cavity within the enzyme, providing a structural explanation for the specificity of β-lactams on Rhomboid proteases. This same cavity probably represents the S2′ substrate binding site of GlpG. We suggest that the structural changes in β-lactam inhibitor binding reflect the state of the enzyme at an initial stage of substrate binding to the active site. The structural insights from these enzyme-inhibitor complexes provide a starting point for structure-based design for Rhomboid inhibitors.
Kvido Strisovsky - One of the best experts on this subject based on the ideXlab platform.
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bacterial Rhomboid proteases mediate quality control of orphan membrane proteins
The EMBO Journal, 2020Co-Authors: Guangyu Liu, Kvido Strisovsky, Matthew Freeman, Stephen E Beaton, Adam Graham Grieve, Rhiannon M Evans, Miranda Rogers, Fraser A Armstrong, Rachel M Exley, Christoph M TangAbstract:Although multiprotein membrane complexes play crucial roles in bacterial physiology and virulence, the mechanisms governing their quality control remain incompletely understood. In particular, it is not known how unincorporated, orphan components of protein complexes are recognised and eliminated from membranes. Rhomboids, the most widespread and largest superfamily of intramembrane proteases, are known to play key roles in eukaryotes. In contrast, the function of prokaryotic Rhomboids has remained enigmatic. Here, we show that the Shigella sonnei Rhomboid proteases GlpG and the newly identified Rhom7 are involved in membrane protein quality control by specifically targeting components of respiratory complexes, with the metastable transmembrane domains (TMDs) of Rhomboid substrates protected when they are incorporated into a functional complex. Initial cleavage by GlpG or Rhom7 allows subsequent degradation of the orphan substrate. Given the occurrence of this strategy in an evolutionary ancient organism and the presence of Rhomboids in all domains of life, it is likely that this form of quality control also mediates critical events in eukaryotes and protects cells from the damaging effects of orphan proteins.
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discovery and validation of 2 styryl substituted benzoxazin 4 ones as a novel scaffold for Rhomboid protease inhibitors
Bioorganic & Medicinal Chemistry Letters, 2018Co-Authors: Parul Goel, Kvido Strisovsky, Anežka Ticha, Isabella Ogorek, Martin Hubalek, Claus U Pietrzik, Thorsten Jumpertz, Boris Schmidt, David C Mikles, Sascha WeggenAbstract:Abstract Rhomboids are intramembrane serine proteases with diverse physiological functions in organisms ranging from archaea to humans. Crystal structure analysis has provided a detailed understanding of the catalytic mechanism, and Rhomboids have been implicated in various disease contexts. Unfortunately, the design of specific Rhomboid inhibitors has lagged behind, and previously described small molecule inhibitors displayed insufficient potency and/or selectivity. Using a computer-aided approach, we focused on the discovery of novel scaffolds with reduced liabilities and the possibility for broad structural variations. Docking studies with the E. coli Rhomboid GlpG indicated that 2-styryl substituted benzoxazinones might comprise novel Rhomboid inhibitors. Protease in vitro assays confirmed activity of 2-styryl substituted benzoxazinones against GlpG but not against the soluble serine protease α-chymotrypsin. Furthermore, mass spectrometry analysis demonstrated covalent modification of the catalytic residue Ser201, corroborating the predicted mechanism of inhibition and the formation of an acyl enzyme intermediate. In conclusion, 2-styryl substituted benzoxazinones are a novel Rhomboid inhibitor scaffold with ample opportunity for optimization.
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Quantitative proteomics screen identifies a substrate repertoire of Rhomboid protease RHBDL2 in human cells and implicates it in epithelial homeostasis
Scientific Reports, 2017Co-Authors: Nicholas Johnson, Jana Březinová, Emma Burbridge, Colin Adrain, Matthew Freeman, Elaine Stephens, Kvido StrisovskyAbstract:Rhomboids are intramembrane serine proteases conserved in all kingdoms of life. They regulate epidermal growth factor receptor signalling in Drosophila by releasing signalling ligands from their transmembrane tethers. Their functions in mammals are poorly understood, in part because of the lack of endogenous substrates identified thus far. We used a quantitative proteomics approach to investigate the substrate repertoire of Rhomboid protease RHBDL2 in human cells. We reveal a range of novel substrates that are specifically cleaved by RHBDL2, including the interleukin-6 receptor (IL6R), cell surface protease inhibitor Spint-1, the collagen receptor tyrosine kinase DDR1, N-Cadherin, CLCP1/DCBLD2, KIRREL, BCAM and others. We further demonstrate that these substrates can be shed by endogenously expressed RHBDL2 and that a subset of them is resistant to shedding by cell surface metalloproteases. The expression profiles and identity of the substrates implicate RHBDL2 in physiological or pathological processes affecting epithelial homeostasis.
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Production of Recombinant Rhomboid Proteases
Methods in Enzymology, 2017Co-Authors: Elena Arutyunova, Kvido Strisovsky, Ramakanta Panigrahi, M.j. LemieuxAbstract:Rhomboid proteases are intramembrane enzymes that hydrolyze peptide bonds of transmembrane proteins in the lipid bilayer. They play a variety of roles in key biological events and are linked to several disease states. Over the last decade a great deal of structural and functional knowledge has been generated on this fascinating class of proteases. Both structural and kinetic analyses require milligram amounts of protein, which may be challenging for membrane proteins such as Rhomboids. Here, we present a detailed protocol for optimization of expression and purification of three Rhomboid proteases from Escherichia coli (ecGlpG), Haemophilus influenzae (hiGlpG), and Providencia stuartii (AarA). We discuss the optimization of expression conditions, such as concentration of inducing agent, induction time, and temperature, as well as purification protocol with precise details for each step. The provided protocol yields 1–2.5 mg of Rhomboid enzyme per liter of bacterial culture and can assist in structural and functional studies of intramembrane proteases.
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Activity Assays for Rhomboid Proteases.
Methods in Enzymology, 2016Co-Authors: Elena Arutyunova, Kvido Strisovsky, M.j. LemieuxAbstract:Abstract Rhomboids are ubiquitous intramembrane serine proteases that are involved in various signaling pathways. This fascinating class of proteases harbors an active site buried within the lipid milieu. High-resolution structures of the Escherichia coli Rhomboid GlpG with various inhibitors revealed the catalytic mechanism for Rhomboid-mediated proteolysis; however, a quantitative characterization was lacking. Assessing an enzyme's catalytic parameters is important for understanding the details of its proteolytic reaction and regulatory mechanisms. To assay Rhomboid protease activity, many challenges exist such as the lipid environment and lack of known substrates. Here, we summarize various enzymatic assays developed over the last decade to study Rhomboid protease activity. We present detailed protocols for gel-shift and FRET-based assays, and calculation of K M and V max to measure catalytic parameters, using detergent solubilized Rhomboids with TatA, the only known substrate for bacterial Rhomboids, and the model substrate fluorescently labeled casein.
Sinisa Urban - One of the best experts on this subject based on the ideXlab platform.
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a spatially localized Rhomboid protease cleaves cell surface adhesins essential for invasion by toxoplasma
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Fabien Brossier, Travis J Jewett, David L Sibley, Sinisa UrbanAbstract:Apicomplexan parasites cause serious human and animal diseases, the treatment of which requires identification of new therapeutic targets. Host-cell invasion culminates in the essential cleavage of parasite adhesins, and although the cleavage site for several adhesins maps within their transmembrane domains, the protease responsible for this processing has not been discovered. We have identified, cloned, and characterized the five nonmitochondrial Rhomboid intramembrane proteases encoded in the recently completed genome of Toxoplasma gondii. Four T. gondii Rhomboids (TgROMs) were active proteases with similar substrate specificity. TgROM1, TgROM4, and TgROM5 were expressed in the tachyzoite stage responsible for the disease, whereas TgROM2 and TgROM3 were expressed in the oocyst stage involved in transmission. Although both TgROM5 and TgROM4 localized to the cell surface in tachyzoites, TgROM5 was primarily at the posterior of the parasite, whereas adhesins were sequestered in internal micronemes. Upon microneme secretion, as occurs during invasion, the MIC2 adhesin was secreted to the apical end and translocated to the posterior, the site of cleavage, where it colocalized only with TgROM5. Moreover, only TgROM5 was able to cleave MIC adhesins in a cell-based assay, indicating that it likely provides the key protease activity necessary for invasion. T. gondii Rhomboids have clear homologues in other apicomplexans including malaria; thus, our findings provide a model for studying invasion by this deadly pathogen and offer a target for therapeutic intervention.
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reconstitution of intramembrane proteolysis in vitro reveals that pure Rhomboid is sufficient for catalysis and specificity
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Sinisa Urban, Michael S WolfeAbstract:Intramembrane proteolysis is a new paradigm in biology that controls signaling events throughout evolution. Hydrolysis of peptide bonds is thought to occur within the normally hydrophobic membrane environment, but insights into this unusual activity have been lacking because of difficulty in recapitulating activity in vitro. We have reconstituted intramembrane proteolysis with a pure recombinant substrate and Rhomboid proteins in both detergent micelles and artificial membrane environments. Rhomboid proteins from diverse organisms including two model bacteria, a pathogen, an extremophile, and an animal were robustly active in pure form, proving that Rhomboids are a new class of enzymes and do not require cofactors to catalyze intramembrane proteolysis. Rhomboid proteins directly recognized their substrates in vitro by the top of the substrate transmembrane domain, displaying specificity apparently reciprocal to that of γ-secretase, the only other activity known to cleave type-I transmembrane domains. Rhomboid proteases represent a different evolutionary path to a serine protease mechanism and exhibited an inhibitor profile unlike other serine proteases. Intriguingly, activity was dramatically modulated by different membrane phospholipid environments, suggesting a mechanism for regulating these proteases. This analysis promises to help reveal the biochemical mechanisms and biological roles of this most widely conserved membrane protein family.
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Diverse Substrate Recognition Mechanisms for Rhomboids: Thrombomodulin Is Cleaved by Mammalian Rhomboids
Current biology : CB, 2004Co-Authors: Olli Lohi, Sinisa Urban, Matthew FreemanAbstract:The Rhomboids are a recently discovered family of intramembrane proteases that are conserved across evolution. Drosophila was the first organism in which they were characterized, where at least Rhomboids 1-3 activate EGF receptor signaling by releasing the active forms of EGF-like growth factors. Subsequent work has begun to shed light on the role of these proteases in bacteria and yeast, but nothing is known about the function of Rhomboids in vertebrates beyond evidence that the subclass of mitochondrial Rhomboids is conserved. Here, we report that the anticoagulant cell-surface protein thrombomodulin is the first mammalian protein to be a Rhomboid substrate in a cell culture assay. The thrombomodulin transmembrane domain (TMD) is cleaved only by vertebrate RHBDL2-like Rhomboids. Thrombomodulin TMD cleavage is directed not by sequences within the TMD, as is the case with Spitz but by its cytoplasmic domain, which, at least in some contexts, is necessary and sufficient to determine cleavage by RHBDL2. These data suggest that thrombomodulin could be a physiological substrate for Rhomboid. Moreover, the discovery of a second mode of substrate recognition by Rhomboids implies mechanistic diversity in this family of intramembrane proteases.
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Substrate Specificity of Rhomboid Intramembrane Proteases Is Governed by Helix-Breaking Residues in the Substrate Transmembrane Domain
Molecular cell, 2003Co-Authors: Sinisa Urban, Matthew FreemanAbstract:Rhomboid intramembrane proteases initiate cell signaling during Drosophila development and Providencia bacterial growth by cleaving transmembrane ligand precursors. We have determined how specificity is achieved: Drosophila Rhomboid-1 is a site-specific protease that recognizes its substrate Spitz by a small region of the Spitz transmembrane domain (TMD). This substrate motif is necessary and sufficient for cleavage and is composed of residues known to disrupt helices. Rhomboids from diverse organisms including bacteria and vertebrates recognize the same substrate motif, suggesting that they use a universal targeting strategy. We used this information to search for other Rhomboid substrates and identified a family of adhesion proteins from the human parasite Toxoplasma gondii, the TMDs of which were efficient substrates for Rhomboid proteases. Intramembrane cleavage of these proteins is required for host cell invasion. These results provide an explanation of how Rhomboid proteases achieve specificity, and allow some Rhomboid substrates to be predicted from sequence information.
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Conservation of Intramembrane Proteolytic Activity and Substrate Specificity in Prokaryotic and Eukaryotic Rhomboids
Current biology : CB, 2002Co-Authors: Sinisa Urban, Daniel Schlieper, Matthew FreemanAbstract:Abstract Rhomboid is an intramembrane serine protease responsible for the proteolytic activation of Drosophila epidermal growth factor receptor (EGFR) ligands [1]. Although nothing is known about the function of the ∼100 currently known Rhomboid genes conserved throughout evolution, a recent analysis suggests that a Rhomboid from the pathogenic bacterium Providencia stuartii is involved in the production of a quorum-sensing factor [2]. This suggests that an intercellular signaling mechanism may have been conserved between prokaryotes and metazoans [3]. However, the function of prokaryotic Rhomboids is unknown. We have examined the ability of eight prokaryotic Rhomboids to cleave the three Drosophila EGFR ligands. Despite their striking sequence divergence, Rhomboids from one Gram-positive and four Gram-negative species, including Providencia , specifically cleaved Drosophila substrates, but not similar proteins such as Transforming Growth Factor α (TGFα) and Delta. Although the sequence similarity between these divergent Rhomboids is very limited, all contain the putative serine catalytic triad residues, and their specific mutation abolished protease activity. Therefore, despite low overall homology, the Rhomboids are a family of ancient, functionally conserved intramembrane serine proteases, some of which also have conserved substrate specificity. Moreover, a function for Rhomboids in activating intercellular signaling appears to have evolved early.
Steven H. L. Verhelst - One of the best experts on this subject based on the ideXlab platform.
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An internally quenched peptide as a new model substrate for Rhomboid intramembrane proteases.
Biological Chemistry, 2018Co-Authors: Elena Arutyunova, Ayodeji N. Kulepa, Steven H. L. Verhelst, Howard S. Young, Zhenze Jiang, Anthony J. O’donoghue, Jian Yang, M. Joanne LemieuxAbstract:: Rhomboids are ubiquitous intramembrane serine proteases that cleave transmembrane substrates. Their functions include growth factor signaling, mitochondrial homeostasis, and parasite invasion. A recent study revealed that the Escherichia coli Rhomboid protease EcGlpG is essential for its extraintestinal pathogenic colonization within the gut. Crystal structures of EcGlpG and the Haemophilus influenzae Rhomboid protease HiGlpG have deciphered an active site that is buried within the lipid bilayer but exposed to the aqueous environment via a cavity at the periplasmic face. A lack of physiological transmembrane substrates has hampered progression for understanding their catalytic mechanism and screening inhibitor libraries. To identify a soluble substrate for use in the study of Rhomboid proteases, an array of internally quenched peptides were assayed with HiGlpG, EcGlpG and PsAarA from Providencia stuartti. One substrate was identified that was cleaved by all three Rhomboid proteases, with HiGlpG having the highest cleavage efficiency. Mass spectrometry analysis determined that all enzymes hydrolyze this substrate between norvaline and tryptophan. Kinetic analysis in both detergent and bicellular systems demonstrated that this substrate can be cleaved in solution and in the lipid environment. The substrate was subsequently used to screen a panel of benzoxazin-4-one inhibitors to validate its use in inhibitor discovery.
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stable and functional Rhomboid proteases in lipid nanodiscs by using diisobutylene maleic acid copolymers
Journal of the American Chemical Society, 2018Co-Authors: Marta Barniolxicota, Steven H. L. VerhelstAbstract:Rhomboid proteases form a paradigm for intramembrane proteolysis and have been implicated in several human diseases. However, their study is hampered by difficulties in solubilization and purification. We here report on the use of polymers composed of maleic acid and either diisobutylene or styrene for solubilization of Rhomboid proteases in lipid nanodiscs, which proceeds with up to 48% efficiency. We show that the activity of Rhomboids in lipid nanodiscs is closer to that in the native membrane than Rhomboids in detergent. Moreover, a Rhomboid that was proteolytically unstable in detergent turned out to be stable in lipid nanodiscs, underlining the benefit of using these polymer-stabilized nanodiscs. The systems are also compatible with the use of activity-based probes and can be used for small molecule inhibitor screening, allowing several downstream applications.
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Discovery and Biological Evaluation of Potent and Selective N-Methylene Saccharin-Derived Inhibitors for Rhomboid Intramembrane Proteases.
Biochemistry, 2017Co-Authors: Parul Goel, Steven H. L. Verhelst, Anežka Ticha, Martin Hubalek, Thorsten Jumpertz, David C Mikles, Minh T.n. Nguyen, Darren C. Johnson, Daniel A. Bachovchin, Isabella OgorekAbstract:Rhomboids are intramembrane serine proteases and belong to the group of structurally and biochemically most comprehensively characterized membrane proteins. They are highly conserved and ubiquitously distributed in all kingdoms of life and function in a wide range of biological processes, including epidermal growth factor signaling, mitochondrial dynamics, and apoptosis. Importantly, Rhomboids have been associated with multiple diseases, including Parkinson’s disease, type 2 diabetes, and malaria. However, despite a thorough understanding of many structural and functional aspects of Rhomboids, potent and selective inhibitors of these intramembrane proteases are still not available. In this study, we describe the computer-based rational design, chemical synthesis, and biological evaluation of novel N-methylene saccharin-based Rhomboid protease inhibitors. Saccharin inhibitors displayed inhibitory potency in the submicromolar range, effectiveness against Rhomboids both in vitro and in live Escherichia coli ...
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Inhibitor Fingerprinting of Rhomboid Proteases by Activity-Based Protein Profiling Reveals Inhibitor Selectivity and Rhomboid Autoprocessing.
ACS Chemical Biology, 2015Co-Authors: Eliane V. Wolf, Annett Zeissler, Steven H. L. VerhelstAbstract:Rhomboid proteases were discovered almost 15 years ago and are structurally the best characterized intramembrane proteases. Apart from the general serine protease inhibitor 3,4-dichloro-isocoumarin (DCI) and a few crystal structures of the Escherichia coli Rhomboid GlpG with other inhibitors, there is surprisingly little information about inhibitors of Rhomboids from other species, probably because of a lack of general methods to measure inhibition against different Rhomboid species. We here present activity-based protein profiling (ABPP) as a general method to screen Rhomboids for their activity and inhibition. Using ABPP, we compare the inhibitory capacity of 50 small molecules against 13 different Rhomboids. We find one new pan Rhomboid inhibitor and several inhibitors that display selectivity. We also demonstrate that inhibition profile and sequence similarity of Rhomboids are not related, which suggests that related Rhomboids may be selectively inhibited. Finally, by making use of the here discovered...
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Inhibitor Fingerprinting of Rhomboid Proteases by Activity-Based Protein Profiling Reveals Inhibitor Selectivity and Rhomboid Autoprocessing.
ACS chemical biology, 2015Co-Authors: Eliane V. Wolf, Annett Zeissler, Steven H. L. VerhelstAbstract:Rhomboid proteases were discovered almost 15 years ago and are structurally the best characterized intramembrane proteases. Apart from the general serine protease inhibitor 3,4-dichloro-isocoumarin (DCI) and a few crystal structures of the Escherichia coli Rhomboid GlpG with other inhibitors, there is surprisingly little information about inhibitors of Rhomboids from other species, probably because of a lack of general methods to measure inhibition against different Rhomboid species. We here present activity-based protein profiling (ABPP) as a general method to screen Rhomboids for their activity and inhibition. Using ABPP, we compare the inhibitory capacity of 50 small molecules against 13 different Rhomboids. We find one new pan Rhomboid inhibitor and several inhibitors that display selectivity. We also demonstrate that inhibition profile and sequence similarity of Rhomboids are not related, which suggests that related Rhomboids may be selectively inhibited. Finally, by making use of the here discovered...
M. Joanne Lemieux - One of the best experts on this subject based on the ideXlab platform.
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PARL Protease: A Glimpse at Intramembrane Proteolysis in the Inner Mitochondrial Membrane
Journal of molecular biology, 2020Co-Authors: Laine Lysyk, Raelynn Brassard, Nicolas Touret, M. Joanne LemieuxAbstract:Intramembrane proteolysis, although once a controversial concept, is a widely studied field. Four classes of intramembrane proteases have been identified and are classified by their catalytic mechanism of peptide bond hydrolysis: metallo, glutamyl, aspartyl, and serine proteases. One of the most studied of these classes is the Rhomboid superfamily of serine intramembrane proteases. Rhomboids consist of six or seven transmembrane segments that form a helical bundle within the membrane and are involved in a multitude of cellular processes. These proteases are characterized by a catalytic dyad composed of a serine and a histidine residue, which distinguishes them from classical serine proteases wherein a catalytic triad is utilized. Of all currently identified Rhomboid proteases, one that is of great interest is the mammalian mitochondrial Rhomboid protease PARL. Most well known for its processing of the kinase PINK1 and potential link to Parkinson's disease, PARL has been shown to cleave a variety of substrates within the cell including PGAM5, Smac, TTC19, and others. While recent proteomic studies have provided insight on new potential substrates of PARL, its regulation, activity, and role in maintaining mitochondrial homeostasis remain largely unknown.
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An internally quenched peptide as a new model substrate for Rhomboid intramembrane proteases.
Biological Chemistry, 2018Co-Authors: Elena Arutyunova, Ayodeji N. Kulepa, Steven H. L. Verhelst, Howard S. Young, Zhenze Jiang, Anthony J. O’donoghue, Jian Yang, M. Joanne LemieuxAbstract:: Rhomboids are ubiquitous intramembrane serine proteases that cleave transmembrane substrates. Their functions include growth factor signaling, mitochondrial homeostasis, and parasite invasion. A recent study revealed that the Escherichia coli Rhomboid protease EcGlpG is essential for its extraintestinal pathogenic colonization within the gut. Crystal structures of EcGlpG and the Haemophilus influenzae Rhomboid protease HiGlpG have deciphered an active site that is buried within the lipid bilayer but exposed to the aqueous environment via a cavity at the periplasmic face. A lack of physiological transmembrane substrates has hampered progression for understanding their catalytic mechanism and screening inhibitor libraries. To identify a soluble substrate for use in the study of Rhomboid proteases, an array of internally quenched peptides were assayed with HiGlpG, EcGlpG and PsAarA from Providencia stuartti. One substrate was identified that was cleaved by all three Rhomboid proteases, with HiGlpG having the highest cleavage efficiency. Mass spectrometry analysis determined that all enzymes hydrolyze this substrate between norvaline and tryptophan. Kinetic analysis in both detergent and bicellular systems demonstrated that this substrate can be cleaved in solution and in the lipid environment. The substrate was subsequently used to screen a panel of benzoxazin-4-one inhibitors to validate its use in inhibitor discovery.
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Reversible Unfolding of Rhomboid Intramembrane Proteases.
Biophysical journal, 2016Co-Authors: Rashmi Panigrahi, Elena Arutyunova, Pankaj Panwar, Katharina Gimpl, Sandro Keller, M. Joanne LemieuxAbstract:Denaturant-induced unfolding of helical membrane proteins provides insights into their mechanism of folding and domain organization, which take place in the chemically heterogeneous, anisotropic environment of a lipid membrane. Rhomboid proteases are intramembrane proteases that play key roles in various diseases. Crystal structures have revealed a compact helical bundle with a buried active site, which requires conformational changes for the cleavage of transmembrane substrates. A dimeric form of the Rhomboid protease has been shown to be important for activity. In this study, we examine the mechanism of refolding for two distinct Rhomboids to gain insight into their secondary structure-activity relationships. Although helicity is largely abolished in the unfolded states of both proteins, unfolding is completely reversible for HiGlpG but only partially reversible for PsAarA. Refolding of both proteins results in reassociation of the dimer, with a 90% regain of catalytic activity for HiGlpG but only a 70% regain for PsAarA. For both proteins, a broad, gradual transition from the native, folded state to the denatured, partly unfolded state was revealed with the aid of circular dichroism spectroscopy as a function of denaturant concentration, thus arguing against a classical two-state model as found for many globular soluble proteins. Thermal denaturation has irreversible destabilizing effects on both proteins, yet reveals important functional details regarding substrate accessibility to the buried active site. This concerted biophysical and functional analysis demonstrates that HiGlpG, with a simple six-transmembrane-segment organization, is more robust than PsAarA, which has seven predicted transmembrane segments, thus rendering HiGlpG amenable to in vitro studies of membrane-protein folding.
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Functional Implications of Domain Organization Within Prokaryotic Rhomboid Proteases.
Advances in experimental medicine and biology, 2015Co-Authors: Rashmi Panigrahi, M. Joanne LemieuxAbstract:Intramembrane proteases are membrane embedded enzymes that cleave transmembrane substrates. This interesting class of enzyme and its water mediated substrate cleavage mechanism occurring within the hydrophobic lipid bilayer has drawn the attention of researchers. Rhomboids are a family of ubiquitous serine intramembrane proteases. Bacterial forms of Rhomboid proteases are mainly composed of six transmembrane helices that are preceded by a soluble N-terminal domain. Several crystal structures of the membrane domain of the E. coli Rhomboid protease ecGlpG have been solved. Independently, the ecGlpG N-terminal cytoplasmic domain structure was solved using both NMR and protein crystallography. Despite these structures, we still do not know the structure of the full-length protein, nor do we know the functional role of these domains in the cell. This chapter will review the structural and functional roles of the different domains associated with prokaryotic Rhomboid proteases. Lastly, we will address questions remaining in the field.
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Oligomeric state study of prokaryotic Rhomboid proteases.
Biochimica et biophysica acta, 2012Co-Authors: Padmapriya Sampathkumar, Michelle W. Mak, Sarah J. Fischer-witholt, Emmanuel Guigard, Cyril M. Kay, M. Joanne LemieuxAbstract:Rhomboid peptidases (proteases) play key roles in signaling events at the membrane bilayer. Understanding the regulation of Rhomboid function is crucial for insight into its mechanism of action. Here we examine the oligomeric state of three different Rhomboid proteases. We subjected Haemophilus influenzae, (hiGlpG), Escherichia coli GlpG (ecGlpG) and Bacillus subtilis (YqgP) to sedimentation equilibrium analysis in detergent-solubilized dodecylmaltoside (DDM) solution. For hiGlpG and ecGlpG, Rhomboids consisting of the core 6 transmembrane domains without and with soluble domains respectively, and YqgP, predicted to have 7 transmembrane domains with larger soluble domains at the termini, the predominant species was dimeric with low amounts of monomer and tetramers observed. To examine the effect of the membrane domain alone on oligomeric state of Rhomboid, hiGlpG, the simplest form from the Rhomboid class of intramembrane proteases representing the canonical Rhomboid core of six transmembrane domains, was studied further. Using gel filtration and crosslinking we demonstrate that hiGlpG is dimeric and functional in DDM detergent solution. More importantly co-immunoprecipitation studies demonstrate that the dimer is present in the lipid bilayer suggesting a physiological dimer. Overall these results indicate that Rhomboids form oligomers which are facilitated by the membrane domain. For hiGlpG we have shown that these oligomers exist in the lipid bilayer. This is the first detailed oligomeric state characterization of the Rhomboid family of peptidases.
