Rhomboid-family intramembrane serine proteases are evolutionarily widespread. Their functions in different organisms are gradually being uncovered and already suggest medical relevance for infectious diseases and cancer. In contrast to these advances, selective inhibitors that could serve as efficient tools for investigation of physiological functions of rhomboids, validation of their disease relevance or as templates for drug development are lacking. In this review I extract what is known about rhomboid protease mechanism and specificity, examine the currently used inhibitors, their mechanism of action and limitations, and conclude by proposing routes for future development of rhomboid protease inhibitors.

• MeSH
• cílená molekulární terapie * MeSH
• enzymatické testy MeSH
• inhibitory proteas chemie   terapeutické užití   MeSH
• lidé MeSH
• membránové proteiny antagonisté a inhibitory   metabolismus   MeSH
• substrátová specifita MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Magnesium homeostasis is essential for life and depends on magnesium transporters, whose activity and ion selectivity need to be tightly controlled. Rhomboid intramembrane proteases pervade the prokaryotic kingdom, but their functions are largely elusive. Using proteomics, we find that Bacillus subtilis rhomboid protease YqgP interacts with the membrane-bound ATP-dependent processive metalloprotease FtsH and cleaves MgtE, the major high-affinity magnesium transporter in B. subtilis. MgtE cleavage by YqgP is potentiated in conditions of low magnesium and high manganese or zinc, thereby protecting B. subtilis from Mn2+ /Zn2+ toxicity. The N-terminal cytosolic domain of YqgP binds Mn2+ and Zn2+ ions and facilitates MgtE cleavage. Independently of its intrinsic protease activity, YqgP acts as a substrate adaptor for FtsH, a function that is necessary for degradation of MgtE. YqgP thus unites protease and pseudoprotease function, hinting at the evolutionary origin of rhomboid pseudoproteases such as Derlins that are intimately involved in eukaryotic ER-associated degradation (ERAD). Conceptually, the YqgP-FtsH system we describe here is analogous to a primordial form of "ERAD" in bacteria and exemplifies an ancestral function of rhomboid-superfamily proteins.

• MeSH
• ATPázy spojené s různými buněčnými aktivitami metabolismus   MeSH
• Bacillus subtilis růst a vývoj   metabolismus   MeSH
• bakteriální proteiny metabolismus   MeSH
• endopeptidasy metabolismus   MeSH
• membránové proteiny metabolismus   MeSH
• proteomika metody   MeSH
• regulace genové exprese u bakterií MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Intramembrane serine proteases of the rhomboid family are widespread, and their gradually uncovered functions in different organisms already suggest medical relevance for infectious diseases and cancer. However, selective inhibitors that could serve as research tools for rhomboids, for validation of their disease relevance, or as templates for drug development are lacking. Here I summarize the current knowledge about rhomboid protease mechanism and specificity, overview the currently used inhibitors, and conclude by proposing avenues for future development of rhomboid protease inhibitors.

• MeSH
• endopeptidasy chemie   metabolismus   MeSH
• inhibitory proteas chemie   MeSH
• lidé MeSH
• membránové proteiny antagonisté a inhibitory   chemie   MeSH
• substrátová specifita MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Although activity-based protein profiling (ABPP) has been used to study a variety of enzyme classes, its application to intramembrane proteases is still in its infancy. Intramembrane proteolysis is an important biochemical mechanism for activating proteins residing within the membrane in a dormant state. Rhomboid proteases (intramembrane serine proteases) are embedded in the lipid bilayers of membranes and occur in all phylogenetic domains. The study of purified rhomboid proteases has mainly been performed in detergent micelle environments. Here we report on the reconstitution of rhomboids in liposomes. Using ABPP, we have been able to detect active rhomboids in large and giant unilamellar vesicles. We have found that the inhibitor profiles of rhomboids in micelles and liposomes are similar, thus validating previous inhibitor screenings. Moreover, fluorescence microscopy experiments on the liposomes constitute the first steps towards activity-based imaging of rhomboid proteases in membrane environments.

• MeSH
• enzymatické testy metody   MeSH
• inhibitory proteas farmakologie   MeSH
• micely MeSH
• proteasy metabolismus   MeSH
• unilamelární lipozómy chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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.

• MeSH
• benzoxaziny chemická syntéza   chemie   MeSH
• chymotrypsin chemie   MeSH
• DNA vazebné proteiny antagonisté a inhibitory   chemie   genetika   MeSH
• Drosophila chemie   MeSH
• endopeptidasy chemie   genetika   MeSH
• enzymatické testy MeSH
• Escherichia coli enzymologie   MeSH
• inhibitory serinových proteinas chemická syntéza   chemie   MeSH
• katalytická doména MeSH
• lidé MeSH
• membránové proteiny antagonisté a inhibitory   chemie   genetika   MeSH
• mutace MeSH
• objevování léků MeSH
• proteiny Drosophily metabolismus   MeSH
• proteiny z Escherichia coli antagonisté a inhibitory   chemie   genetika   MeSH
• serin chemie   MeSH
• simulace molekulového dockingu MeSH
• skot MeSH
• styreny chemická syntéza   chemie   MeSH
• transformující růstový faktor alfa metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Calcium influx through plasma membrane calcium release-activated calcium (CRAC) channels, which are formed of hexamers of Orai1, is a potent trigger for many important biological processes, most notably in T cell-mediated immunity. Through a bioinformatics-led cell biological screen, we have identified Orai1 as a substrate for the rhomboid intramembrane protease RHBDL2. We show that RHBDL2 prevents stochastic calcium signaling in unstimulated cells through conformational surveillance and cleavage of inappropriately activated Orai1. A conserved disease-linked proline residue is responsible for RHBDL2's recognizing the active conformation of Orai1, which is required to sharpen switch-like signaling triggered by store-operated calcium entry. Loss of RHBDL2 control of CRAC channel activity causes severe dysregulation of downstream CRAC channel effectors, including transcription factor activation, inflammatory cytokine expression, and T cell activation. We propose that this surveillance function may represent an ancient activity of rhomboid proteases in degrading unwanted signaling proteins.

• MeSH
• aktivace lymfocytů MeSH
• buněčná membrána metabolismus   MeSH
• Drosophila melanogaster MeSH
• gating iontového kanálu MeSH
• HEK293 buňky MeSH
• konformace proteinů MeSH
• lidé MeSH
• membránové proteiny metabolismus   MeSH
• mutace MeSH
• proteasy chemie   MeSH
• protein ORAI1 chemie   MeSH
• serinové endopeptidasy metabolismus   MeSH
• signální transdukce MeSH
• stochastické procesy MeSH
• vápník metabolismus   MeSH
• vápníková signalizace fyziologie   MeSH
• vápníkové kanály chemie   MeSH
• vazba proteinů MeSH
• výpočetní biologie MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The mechanisms of intramembrane proteases are incompletely understood due to the lack of structural data on substrate complexes. To gain insight into substrate binding by rhomboid proteases, we have synthesised a series of novel peptidyl-chloromethylketone (CMK) inhibitors and analysed their interactions with Escherichia coli rhomboid GlpG enzymologically and structurally. We show that peptidyl-CMKs derived from the natural rhomboid substrate TatA from bacterium Providencia stuartii bind GlpG in a substrate-like manner, and their co-crystal structures with GlpG reveal the S1 to S4 subsites of the protease. The S1 subsite is prominent and merges into the 'water retention site', suggesting intimate interplay between substrate binding, specificity and catalysis. Unexpectedly, the S4 subsite is plastically formed by residues of the L1 loop, an important but hitherto enigmatic feature of the rhomboid fold. We propose that the homologous region of members of the wider rhomboid-like protein superfamily may have similar substrate or client-protein binding function. Finally, using molecular dynamics, we generate a model of the Michaelis complex of the substrate bound in the active site of GlpG.

• MeSH
• chloromethylketony aminokyselin chemická syntéza   farmakologie   MeSH
• DNA vazebné proteiny antagonisté a inhibitory   chemie   genetika   metabolismus   MeSH
• endopeptidasy chemie   genetika   metabolismus   MeSH
• Escherichia coli chemie   enzymologie   genetika   MeSH
• katalytická doména MeSH
• krystalografie rentgenová MeSH
• membránové proteiny antagonisté a inhibitory   chemie   genetika   metabolismus   MeSH
• molekulární modely * MeSH
• mutace MeSH
• proteiny z Escherichia coli antagonisté a inhibitory   chemie   genetika   metabolismus   MeSH
• Providencia chemie   MeSH
• rekombinantní proteiny MeSH
• simulace molekulární dynamiky * MeSH
• substrátová specifita MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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.

• MeSH
• epitel metabolismus   MeSH
• epitelové buňky metabolismus   MeSH
• homeostáza * MeSH
• interakční proteinové domény a motivy MeSH
• lidé MeSH
• membránové proteiny metabolismus   MeSH
• protein ADAM10 metabolismus   MeSH
• protein ADAM17 metabolismus   MeSH
• proteolýza MeSH
• proteom * MeSH
• proteomika * metody   MeSH
• sekvence aminokyselin MeSH
• serinové proteasy genetika   metabolismus   MeSH
• substrátová specifita MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Rhomboid intramembrane proteases are the enzymes that release active epidermal growth factor receptor (EGFR) ligands in Drosophila and C. elegans, but little is known about their functions in mammals. Here we show that the mammalian rhomboid protease RHBDL4 (also known as Rhbdd1) promotes trafficking of several membrane proteins, including the EGFR ligand TGFα, from the endoplasmic reticulum (ER) to the Golgi apparatus, thereby triggering their secretion by extracellular microvesicles. Our data also demonstrate that RHBDL4-dependent trafficking control is regulated by G-protein coupled receptors, suggesting a role for this rhomboid protease in pathological conditions, including EGFR signaling. We propose that RHBDL4 reorganizes trafficking events within the early secretory pathway in response to GPCR signaling. Our work identifies RHBDL4 as a rheostat that tunes secretion dynamics and abundance of specific membrane protein cargoes.

• MeSH
• endoplazmatické retikulum metabolismus   MeSH
• exozómy metabolismus   MeSH
• Golgiho aparát metabolismus   MeSH
• membránové proteiny sekrece   MeSH
• myši MeSH
• transformující růstový faktor alfa sekrece   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The mitochondrial rhomboid protease PARL regulates mitophagy by balancing intramembrane proteolysis of PINK1 and PGAM5. It has been implicated in the pathogenesis of Parkinson's disease, but its investigation as a possible therapeutic target is challenging in this context because genetic deficiency of PARL may result in compensatory mechanisms. To address this problem, we undertook a hitherto unavailable chemical biology strategy. We developed potent PARL-targeting ketoamide inhibitors and investigated the effects of acute PARL suppression on the processing status of PINK1 intermediates and on Parkin activation. This approach revealed that PARL inhibition leads to a robust activation of the PINK1/Parkin pathway without major secondary effects on mitochondrial properties, which demonstrates that the pharmacological blockage of PARL to boost PINK1/Parkin-dependent mitophagy is a feasible approach to examine novel therapeutic strategies for Parkinson's disease. More generally, this study showcases the power of ketoamide inhibitors for cell biological studies of rhomboid proteases.

• MeSH
• endopeptidasy MeSH
• lidé MeSH
• metaloproteasy genetika   metabolismus   MeSH
• mitochondriální proteiny metabolismus   MeSH
• mitofagie MeSH
• Parkinsonova nemoc * farmakoterapie   MeSH
• proteasy * MeSH
• proteinkinasy metabolismus   MeSH
• ubikvitinligasy metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH