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.

• Keywords
• endoplasmic reticulum stress, endoplasmic-reticulum-associated protein degradation, enzyme inhibitor, enzyme kinetics, enzyme purification, enzyme structure, protein misfolding, rhomboid protease, serine protease,
• MeSH
• Kinetics MeSH
• Humans MeSH
• Membrane Proteins * metabolism   chemistry   genetics   antagonists & inhibitors   MeSH
• Recombinant Proteins chemistry   metabolism   genetics   MeSH
• Fluorescence Resonance Energy Transfer MeSH
• Serine Endopeptidases * chemistry   metabolism   genetics   MeSH
• Substrate Specificity MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Membrane Proteins * MeSH
• Recombinant Proteins MeSH
• Serine Endopeptidases * MeSH

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.

• Publication type
• Journal Article 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
• Endopeptidases MeSH
• Humans MeSH
• Metalloproteases genetics   metabolism   MeSH
• Mitochondrial Proteins metabolism   MeSH
• Mitophagy MeSH
• Parkinson Disease * drug therapy   MeSH
• Peptide Hydrolases * MeSH
• Protein Kinases metabolism   MeSH
• Ubiquitin-Protein Ligases metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Endopeptidases MeSH
• Metalloproteases MeSH
• Mitochondrial Proteins MeSH
• PARL protein, human MeSH Browser
• Peptide Hydrolases * MeSH
• Protein Kinases MeSH
• Ubiquitin-Protein Ligases MeSH

Membrane-tethered signalling proteins such as TNFα and many EGF receptor ligands undergo shedding by the metalloproteinase ADAM17 to get released. The pseudoproteases iRhom1 and iRhom2 are important for the transport, maturation and activity of ADAM17. Yet, the structural and functional requirements to promote the transport of the iRhom-ADAM17 complex have not yet been thoroughly investigated. Utilising in silico and in vitro methods, we here map the conserved iRhom homology domain (IRHD) and provide first insights into its structure and function. By focusing on iRhom2, we identified different structural and functional factors within the IRHD. We found that the structural integrity of the IRHD is a key factor for ADAM17 binding. In addition, we identified a highly conserved motif within an unstructured region of the IRHD, that, when mutated, restricts the transport of the iRhom-ADAM17 complex through the secretory pathway in in vitro, ex vivo and in vivo systems and also increases the half-life of iRhom2 and ADAM17. Furthermore, the disruption of this IRHD motif was also reflected by changes in the yet undescribed interaction profile of iRhom2 with proteins involved in intracellular vesicle transport. Overall, we provide the first insights into the forward trafficking of iRhoms which is critical for TNFα and EGF receptor signalling.

• Keywords
• ADAM17, Ectodomain shedding, Growth factors, TNF, iRhom, iRhom homology domain,
• MeSH
• Amino Acid Motifs MeSH
• Cell Line MeSH
• EGF Family of Proteins metabolism   MeSH
• Humans MeSH
• RNA, Small Interfering metabolism   MeSH
• Membrane Proteins genetics   metabolism   MeSH
• Mutagenesis MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Half-Life MeSH
• ADAM17 Protein chemistry   metabolism   MeSH
• Protein Domains MeSH
• RNA Interference MeSH
• Signal Transduction MeSH
• Tumor Necrosis Factor-alpha metabolism   MeSH
• Protein Transport MeSH
• Carrier Proteins antagonists & inhibitors   genetics   metabolism   MeSH
• Protein Binding MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• EGF Family of Proteins MeSH
• iRhom1 protein, mouse MeSH Browser
• iRhom2 protein, mouse MeSH Browser
• RNA, Small Interfering MeSH
• Membrane Proteins MeSH
• ADAM17 Protein MeSH
• Tumor Necrosis Factor-alpha MeSH
• Carrier Proteins MeSH

Rhomboid intramembrane proteases regulate pathophysiological processes, but their targeting in a disease context has never been achieved. We decoded the atypical substrate specificity of malaria rhomboid PfROM4, but found, unexpectedly, that it results from "steric exclusion": PfROM4 and canonical rhomboid proteases cannot cleave each other's substrates due to reciprocal juxtamembrane steric clashes. Instead, we engineered an optimal sequence that enhanced proteolysis >10-fold, and solved high-resolution structures to discover that boronates enhance inhibition >100-fold. A peptide boronate modeled on our "super-substrate" carrying one "steric-excluding" residue inhibited PfROM4 but not human rhomboid proteolysis. We further screened a library to discover an orthogonal alpha-ketoamide that potently inhibited PfROM4 but not human rhomboid proteolysis. Despite the membrane-immersed target and rapid invasion, ultrastructural analysis revealed that single-dosing blood-stage malaria cultures blocked host-cell invasion and cleared parasitemia. These observations establish a strategy for designing parasite-selective rhomboid inhibitors and expose a druggable dependence on rhomboid proteolysis in non-motile parasites.

• Keywords
• Plasmodium, Ras-converting enzyme, Toxoplasma, apicomplexan parasites, malaria, presenilin, regulated intramembrane proteolysis, rhomboid protease, serine protease, site-2 protease,
• MeSH
• Amides chemical synthesis   chemistry   pharmacology   MeSH
• Antimalarials chemical synthesis   chemistry   pharmacology   MeSH
• HEK293 Cells MeSH
• Protease Inhibitors chemical synthesis   chemistry   pharmacology   MeSH
• Boronic Acids chemical synthesis   chemistry   pharmacology   MeSH
• Humans MeSH
• Malaria blood   drug therapy   metabolism   MeSH
• Molecular Structure MeSH
• Parasitic Sensitivity Tests MeSH
• Peptides chemical synthesis   chemistry   pharmacology   MeSH
• Plasmodium falciparum drug effects   metabolism   MeSH
• Peptide Hydrolases blood   metabolism   MeSH
• Proteolysis drug effects   MeSH
• Protozoan Proteins antagonists & inhibitors   blood   metabolism   MeSH
• Drug Design * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• Amides MeSH
• Antimalarials MeSH
• Protease Inhibitors MeSH
• Boronic Acids MeSH
• Peptides MeSH
• Peptide Hydrolases MeSH
• Protozoan Proteins MeSH
• ROM4 protein, Plasmodium falciparum MeSH Browser

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.

• Keywords
• Shigella, intramembrane proteolysis, membrane protein complexes, quality control, rhomboid,
• MeSH
• Bacterial Proteins chemistry   metabolism   MeSH
• Endopeptidases chemistry   metabolism   MeSH
• Membrane Proteins metabolism   MeSH
• Protein Domains MeSH
• Proteolysis MeSH
• Shigella sonnei enzymology   metabolism   MeSH
• Substrate Specificity MeSH
• Electron Transport MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Endopeptidases MeSH
• Membrane Proteins 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.

• Keywords
• ER-associated degradation, intramembrane protease, membrane transporter, proteostasis, rhomboid,
• MeSH
• ATPases Associated with Diverse Cellular Activities metabolism   MeSH
• Bacillus subtilis growth & development   metabolism   MeSH
• Bacterial Proteins metabolism   MeSH
• Endopeptidases metabolism   MeSH
• Membrane Proteins metabolism   MeSH
• Proteomics methods   MeSH
• Gene Expression Regulation, Bacterial MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• ATPases Associated with Diverse Cellular Activities MeSH
• Bacterial Proteins MeSH
• Endopeptidases MeSH
• Membrane Proteins 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
• Computer-Aided Design MeSH
• HEK293 Cells MeSH
• Serine Proteinase Inhibitors chemistry   pharmacology   MeSH
• Humans MeSH
• Membrane Proteins MeSH
• Drug Design * MeSH
• Saccharin analogs & derivatives   pharmacology   MeSH
• Serine Proteases metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Serine Proteinase Inhibitors MeSH
• Membrane Proteins MeSH
• Saccharin MeSH
• Serine Proteases 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.

• Keywords
• crystal structure, inhibition, inhibitor, intramembrane protease, ketoamide, mechanism, rhomboid protease, specificity,
• MeSH
• Gram-Negative Bacteria enzymology   MeSH
• Gram-Positive Bacteria enzymology   MeSH
• Serine Proteinase Inhibitors chemical synthesis   chemistry   pharmacology   MeSH
• Molecular Conformation MeSH
• Models, Molecular MeSH
• Peptide Hydrolases metabolism   MeSH
• Drug Design * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Serine Proteinase Inhibitors MeSH
• Peptide Hydrolases 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
• Epithelium metabolism   MeSH
• Epithelial Cells metabolism   MeSH
• Homeostasis * MeSH
• Protein Interaction Domains and Motifs MeSH
• Humans MeSH
• Membrane Proteins metabolism   MeSH
• ADAM10 Protein metabolism   MeSH
• ADAM17 Protein metabolism   MeSH
• Proteolysis MeSH
• Proteome * MeSH
• Proteomics * methods   MeSH
• Amino Acid Sequence MeSH
• Serine Endopeptidases MeSH
• Serine Proteases genetics   metabolism   MeSH
• Substrate Specificity MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Membrane Proteins MeSH
• ADAM10 Protein MeSH
• ADAM17 Protein MeSH
• Proteome * MeSH
• RHBDL2 protein, human MeSH Browser
• Serine Endopeptidases MeSH
• Serine Proteases MeSH