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

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

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

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.

• Keywords
• enzyme kinetics, enzyme mechanism, fluorescence resonance energy transfer (FRET), intramembrane proteolysis, membrane reconstitution, rhomboid protease, substrate specificity, transmembrane domain,
• MeSH
• Fluorescent Dyes chemistry   MeSH
• Kinetics MeSH
• Liposomes MeSH
• Peptides metabolism   MeSH
• Peptide Hydrolases metabolism   MeSH
• Fluorescence Resonance Energy Transfer MeSH
• Substrate Specificity MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Fluorescent Dyes MeSH
• Liposomes MeSH
• Peptides MeSH
• Peptide Hydrolases MeSH