Integral membrane proteins carry out essential functions in the cell, and their activities are often modulated by specific protein-lipid interactions in the membrane. Here, we elucidate the intricate role of cardiolipin (CDL), a regulatory lipid, as a stabilizer of membrane proteins and their complexes. Using the in silico-designed model protein TMHC4_R (ROCKET) as a scaffold, we employ a combination of molecular dynamics simulations and native mass spectrometry to explore the protein features that facilitate preferential lipid interactions and mediate stabilization. We find that the spatial arrangement of positively charged residues as well as local conformational flexibility are factors that distinguish stabilizing from non-stabilizing CDL interactions. However, we also find that even in this controlled, artificial system, a clear-cut distinction between binding and stabilization is difficult to attain, revealing that overlapping lipid contacts can partially compensate for the effects of binding site mutations. Extending our insights to naturally occurring proteins, we identify a stabilizing CDL site within the E. coli rhomboid intramembrane protease GlpG and uncover its regulatory influence on enzyme substrate preference. In this work, we establish a framework for engineering functional lipid interactions, paving the way for the design of proteins with membrane-specific properties or functions.

• Klíčová slova
• E. coli, lipid binding, mass spectrometry, membrane protein, molecular biophysics, structural biology,
• MeSH
• endopeptidasy metabolismus   chemie   genetika   MeSH
• Escherichia coli metabolismus   genetika   MeSH
• kardiolipiny * metabolismus   chemie   MeSH
• membránové proteiny * metabolismus   chemie   genetika   MeSH
• proteinové inženýrství * MeSH
• proteiny z Escherichia coli * metabolismus   chemie   genetika   MeSH
• simulace molekulární dynamiky MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA vazebné proteiny MeSH
• endopeptidasy MeSH
• GlpG protein, E coli MeSH Prohlížeč
• kardiolipiny * MeSH
• membránové proteiny * MeSH
• proteiny z Escherichia coli * MeSH

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

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
• Názvy látek
• endopeptidasy MeSH
• metaloproteasy MeSH
• mitochondriální proteiny MeSH
• PARL protein, human MeSH Prohlížeč
• proteasy * MeSH
• proteinkinasy MeSH
• ubikvitinligasy 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

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