Rhomboid proteases are ubiquitous intramembrane serine proteases that can cleave transmembrane substrates within lipid bilayers. They exhibit many and diverse functions, such as but not limited to, growth factor signaling, immune and inflammatory response, protein quality control, and parasitic invasion. Human rhomboid protease RHBDL4 has been demonstrated to play a critical role in removing misfolded proteins from the endoplasmic reticulum and is implicated in severe diseases such as various cancers and Alzheimer's disease. Therefore, RHBDL4 is expected to constitute an important therapeutic target for such devastating diseases. Despite its critical role in many biological processes, the enzymatic properties of RHBDL4 remain largely unknown. To enable a comprehensive characterization of RHBDL4's kinetics, catalytic parameters, substrate specificity, and binding modality, we expressed and purified recombinant RHBDL4 and employed it in a Förster resonance energy transfer-based cleavage assay. Until now, kinetic studies have been limited mostly to bacterial rhomboid proteases. Our in vitro platform offers a new method for studying RHBDL4's enzymatic function and substrate preferences. Furthermore, we developed and tested potential inhibitors using our assay and successfully identified peptidyl α-ketoamide inhibitors of RHBDL4 that are highly effective against recombinant RHBDL4. We utilize ensemble docking and molecular dynamics simulations to explore the binding modality of substrate-derived peptides bound to RHBDL4. Our analysis focused on key interactions and dynamic movements within RHBDL4's active site that contributed to binding stability, offering valuable insights for optimizing the nonprime side of RHBDL4 ketoamide inhibitors. In summary, our study offers fundamental insights into RHBDL4's catalytic activities and substrate preferences, laying the foundation for downstream applications such as drug inhibitor screenings and structure-function studies, which will enable the identification of lead drug compounds for RHBDL4.

• Klíčová slova
• endoplasmic reticulum stress, endoplasmic-reticulum-associated protein degradation, enzyme inhibitor, enzyme kinetics, enzyme purification, enzyme structure, protein misfolding, rhomboid protease, serine protease,
• MeSH
• kinetika MeSH
• lidé MeSH
• membránové proteiny * metabolismus   chemie   genetika   antagonisté a inhibitory   MeSH
• rekombinantní proteiny chemie   metabolismus   genetika   MeSH
• rezonanční přenos fluorescenční energie MeSH
• serinové endopeptidasy * chemie   metabolismus   genetika   MeSH
• substrátová specifita MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• membránové proteiny * MeSH
• rekombinantní proteiny MeSH
• serinové endopeptidasy * 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.

• Klíčová slova
• ER-associated degradation, intramembrane protease, membrane transporter, proteostasis, rhomboid,
• 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
• Názvy látek
• ATPázy spojené s různými buněčnými aktivitami MeSH
• bakteriální proteiny MeSH
• endopeptidasy MeSH
• membránové proteiny MeSH

Many membrane proteins are thought to function as dimers or higher oligomers, but measuring membrane protein oligomerization in lipid membranes is particularly challenging. Förster resonance energy transfer (FRET) and fluorescence cross-correlation spectroscopy are noninvasive, optical methods of choice that have been applied to the analysis of dimerization of single-spanning membrane proteins. However, the effects inherent to such two-dimensional systems, such as the excluded volume of polytopic transmembrane proteins, proximity FRET, and rotational diffusion of fluorophore dipoles, complicate interpretation of FRET data and have not been typically accounted for. Here, using FRET and fluorescence cross-correlation spectroscopy, we introduce a method to measure surface protein density and to estimate the apparent Förster radius, and we use Monte Carlo simulations of the FRET data to account for the proximity FRET effect occurring in confined two-dimensional environments. We then use FRET to analyze the dimerization of human rhomboid protease RHBDL2 in giant plasma membrane vesicles. We find no evidence for stable oligomers of RHBDL2 in giant plasma membrane vesicles of human cells even at concentrations that highly exceed endogenous expression levels. This indicates that the rhomboid transmembrane core is intrinsically monomeric. Our findings will find use in the application of FRET and fluorescence correlation spectroscopy for the analysis of oligomerization of transmembrane proteins in cell-derived lipid membranes.

• MeSH
• buněčná membrána metabolismus   MeSH
• dimerizace MeSH
• lidé MeSH
• membránové lipidy metabolismus   MeSH
• membránové proteiny * metabolismus   MeSH
• multimerizace proteinu MeSH
• rezonanční přenos fluorescenční energie * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• membránové lipidy MeSH
• membránové proteiny * MeSH

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 cells, and substantially improved selectivity against human serine hydrolases compared to those of previously known rhomboid inhibitors. Consequently, N-methylene saccharins are promising new templates for the development of rhomboid inhibitors, providing novel tools for probing rhomboid functions in physiology and disease.

• MeSH
• design s pomocí počítače MeSH
• HEK293 buňky MeSH
• inhibitory serinových proteinas chemie   farmakologie   MeSH
• lidé MeSH
• membránové proteiny MeSH
• racionální návrh léčiv * MeSH
• sacharin analogy a deriváty   farmakologie   MeSH
• serinové proteasy metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• inhibitory serinových proteinas MeSH
• membránové proteiny MeSH
• sacharin MeSH
• serinové proteasy 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

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.

• Klíčová slova
• intramembrane protease, rhomboid family, rhomboid protease, structure, substrate recognition,
• 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
• Názvy látek
• chloromethylketony aminokyselin MeSH
• DNA vazebné proteiny MeSH
• endopeptidasy MeSH
• GlpG protein, E coli MeSH Prohlížeč
• membránové proteiny MeSH
• proteiny z Escherichia coli MeSH
• rekombinantní proteiny 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