Rhomboid proteases play a variety of physiological roles, but rhomboid protease inhibitors have been mostly developed for the E. coli model rhomboid GlpG. In this work, we screened different electrophilic scaffolds against the human mitochondrial rhomboid PARL and found 4-oxo-β-lactams as submicromolar inhibitors. Multifaceted computations suggest explanations for the activity at the molecular scale and provide models of covalently bound complexes. Together with the straightforward synthesis of the 4-oxo-β-lactam scaffold, this may pave the way toward selective, nonpeptidic PARL inhibitors.

• Publikační typ
• časopisecké články 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.

• Klíčová slova
• CRAC channel, Orai1, RHBDL2, T cell, calcium, rhomboid protease, signalling, transmembrane,
• 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
• Názvy látek
• membránové proteiny MeSH
• ORAI1 protein, human MeSH Prohlížeč
• Orai1 protein, mouse MeSH Prohlížeč
• proteasy MeSH
• protein ORAI1 MeSH
• RHBDL2 protein, human MeSH Prohlížeč
• RHBDL2 protein, mouse MeSH Prohlížeč
• serinové endopeptidasy MeSH
• vápník MeSH
• vápníkové kanály MeSH

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.

• Klíčová slova
• Shigella, intramembrane proteolysis, membrane protein complexes, quality control, rhomboid,
• MeSH
• bakteriální proteiny chemie   metabolismus   MeSH
• endopeptidasy chemie   metabolismus   MeSH
• membránové proteiny metabolismus   MeSH
• proteinové domény MeSH
• proteolýza MeSH
• Shigella sonnei enzymologie   metabolismus   MeSH
• substrátová specifita MeSH
• transport elektronů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• endopeptidasy MeSH
• membránové proteiny MeSH

Rhomboid-family intramembrane proteases regulate important biological processes and have been associated with malaria, cancer, and Parkinson's disease. However, due to the lack of potent, selective, and pharmacologically compliant inhibitors, the wide therapeutic potential of rhomboids is currently untapped. Here, we bridge this gap by discovering that peptidyl α-ketoamides substituted at the ketoamide nitrogen by hydrophobic groups are potent rhomboid inhibitors active in the nanomolar range, surpassing the currently used rhomboid inhibitors by up to three orders of magnitude. Such peptidyl ketoamides show selectivity for rhomboids, leaving most human serine hydrolases unaffected. Crystal structures show that these compounds bind the active site of rhomboid covalently and in a substrate-like manner, and kinetic analysis reveals their reversible, slow-binding, non-competitive mechanism. Since ketoamides are clinically used pharmacophores, our findings uncover a straightforward modular way for the design of specific inhibitors of rhomboid proteases, which can be widely applicable in cell biology and drug discovery.

• Klíčová slova
• crystal structure, inhibition, inhibitor, intramembrane protease, ketoamide, mechanism, rhomboid protease, specificity,
• MeSH
• gramnegativní bakterie enzymologie   MeSH
• grampozitivní bakterie enzymologie   MeSH
• inhibitory serinových proteinas chemická syntéza   chemie   farmakologie   MeSH
• molekulární konformace MeSH
• molekulární modely MeSH
• proteasy metabolismus   MeSH
• racionální návrh léčiv * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• inhibitory serinových proteinas MeSH
• proteasy 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 endopeptidasy 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
• Názvy látek
• membránové proteiny MeSH
• protein ADAM10 MeSH
• protein ADAM17 MeSH
• proteom * MeSH
• RHBDL2 protein, human MeSH Prohlížeč
• serinové endopeptidasy MeSH
• serinové proteasy MeSH

Rhomboid proteases are increasingly being explored as potential drug targets, but their potent and specific inhibitors are not available, and strategies for inhibitor development are hampered by the lack of widely usable and easily modifiable in vitro activity assays. Here we address this bottleneck and report on the development of new fluorogenic transmembrane peptide substrates, which are cleaved by several unrelated rhomboid proteases, can be used both in detergent micelles and in liposomes, and contain red-shifted fluorophores that are suitable for high-throughput screening of compound libraries. We show that nearly the entire transmembrane domain of the substrate is important for efficient cleavage, implying that it extensively interacts with the enzyme. Importantly, we demonstrate that in the detergent micelle system, commonly used for the enzymatic analyses of intramembrane proteolysis, the cleavage rate strongly depends on detergent concentration, because the reaction proceeds only in the micelles. Furthermore, we show that the catalytic efficiency and selectivity toward a rhomboid substrate can be dramatically improved by targeted modification of the sequence of its P5 to P1 region. The fluorogenic substrates that we describe and their sequence variants should find wide use in the detection of activity and development of inhibitors of rhomboid proteases.

• Klíčová slova
• enzyme kinetics, enzyme mechanism, fluorescence resonance energy transfer (FRET), intramembrane proteolysis, membrane reconstitution, rhomboid protease, substrate specificity, transmembrane domain,
• MeSH
• fluorescenční barviva chemie   MeSH
• kinetika MeSH
• liposomy MeSH
• peptidy metabolismus   MeSH
• proteasy metabolismus   MeSH
• rezonanční přenos fluorescenční energie MeSH
• substrátová specifita MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fluorescenční barviva MeSH
• liposomy MeSH
• peptidy MeSH
• proteasy 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 metabolismus   MeSH
• myši MeSH
• transformující růstový faktor alfa metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• membránové proteiny MeSH
• Rhbdl3 protein, mouse MeSH Prohlížeč
• transformující růstový faktor alfa MeSH

In this issue of The EMBO Journal, mechanistic analyses of substrate cleavage by rhomboid intramembrane proteases suggest that catalytic efficiency towards natural, transmembrane substrates is allosterically stimulated by initial substrate interaction with an intramembrane exosite, whose formation depends on rhomboid dimerisation. In the realm of intramembrane proteolysis, dimerisation and allosteric cooperativity represent new concepts that, once confirmed more broadly, should radically alter our view of how these proteases work.

• MeSH
• alosterická regulace * MeSH
• buněčná membrána enzymologie   MeSH
• Escherichia coli enzymologie   MeSH
• Haemophilus influenzae enzymologie   MeSH
• membránové proteiny metabolismus   MeSH
• Providencia enzymologie   MeSH
• serinové proteasy metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• komentáře MeSH
• práce podpořená grantem MeSH
• Názvy látek
• membránové proteiny MeSH
• serinové proteasy MeSH