Catalytic Mechanism of Rhomboid Protease GlpG Probed by 3,4-Dichloroisocoumarin and Diisopropyl Fluorophosphonate

Xue, Yi; Ha, Ya

doi:10.1074/jbc.m111.310482

Cited by 50 publications

(92 citation statements)

References 51 publications

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“…In contrast, IC 11 binding showed a loss in signal over time, which is the result of slow hydrolysis of the ester bond between the serine and the IC ring. This is in accordance with recent findings for DCI (30). With better gel resolution, labeling of GlpG by IC 6 shows two distinct protein species (Fig.…”

Section: Compoundsupporting

confidence: 81%

“…As observed in structures with other inhibitors, TM5 moves slightly away from TM2 and L5 lifts upward (23,25,30) (Fig. S3 A and B).…”

Section: Compoundmentioning

confidence: 60%

“…2C), an interaction also observed in a recent structure of GlpG in complex with a Cbz-containing phosphonate inhibitor (28). The aromatic ring of IC 16 stacks against the side chain of W236, which in other inhibitor complexes is rotated away toward the bilayer (23,25,30). Perhaps it is because of the position of the W236 side chain that a hydrophobic cavity postulated as the S2′ substrate binding site (23) is not observed in this new rhomboid-IC structure (Fig.…”

Section: Compoundmentioning

confidence: 69%

“…S3C). The conformations of residues Y205, W236, and H254, however, closely match those of the apoenzyme rather than the conformations observed in the presence of inhibitors (23,25,30) (Fig. S3 D-G).…”

Section: Compoundmentioning

confidence: 95%

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Activity-based probes for rhomboid proteases discovered in a mass spectrometry-based assay

Vosyka

Vinothkumar

Wolf

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Rhomboid proteases are evolutionary conserved intramembrane serine proteases. Because of their emerging role in many important biological pathways, rhomboids are potential drug targets. Unfortunately, few chemical tools are available for their study. Here, we describe a mass spectrometry-based assay to measure rhomboid substrate cleavage and inhibition. We have identified isocoumarin inhibitors and developed activity-based probes for rhomboid proteases. The probes can distinguish between active and inactive rhomboids due to covalent, reversible binding of the active-site serine and stable modification of a histidine residue. Finally, the structure of an isocoumarin-based inhibitor with Escherichia coli rhomboid GlpG uncovers an unusual mode of binding at the active site and suggests that the interactions between the 3-substituent on the isocoumarin inhibitor and hydrophobic residues on the protease reflect S′ subsite binding. Overall, these probes represent valuable tools for rhomboid study, and the structural insights may facilitate future inhibitor design.MALDI screening | covalent inhibition | regulated intramembrane proteolysis P roteolysis controls many important biological processes, such as apoptosis, antigen presentation, and blood coagulation. Selective digestion of protein substrates is possible by a combination of tight posttranslational control of protease activity (1) and the protease's substrate specificity, which generally is governed by the primary sequence around the scissile bond (2). The use of inhibitors and activity-based probes (ABPs) has led to a tremendous gain in understanding the roles of proteases within physiological and pathological processes (3). ABPs are small molecules that bind only to active enzymes, but not to zymogen or inhibitor-bound forms (4). ABPs generally consist of a detection tag, a spacer, and a "warhead." The warhead covalently binds to the target enzyme(s) and often is derived from a mechanism-based inhibitor. In the past, ABPs were used to study the activation, localization, and function of soluble proteases in a variety of organisms and disease models (5).Most proteases are soluble and surrounded by an aqueous environment. However, several families of intramembrane proteases exist (6-8): the metalloprotease family M50 (site-2 protease), the aspartic protease family A22 (signal peptide peptidase and γ-secretase), and the serine protease family S54 [rhomboid; numbering according to the MEROPS database (9)]. Rhomboid was discovered in 2001 as a protease in the EGF receptor signaling pathway in the fruitfly Drosophila melanogaster (10). Interestingly, rhomboid genes occur in all kingdoms of nature and are found in most sequenced organisms (11,12). Rhomboids appear to have a wide range of physiological functions, including bacterial protein export (13) and invasion by apicomplexan parasites (14,15), but the roles of many rhomboids remain to be discovered.Rhomboids catalyze peptide bond hydrolysis using a catalytic dyad formed by a serine residue in transmembrane domain...

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Section: Compoundsupporting

confidence: 81%

“…As observed in structures with other inhibitors, TM5 moves slightly away from TM2 and L5 lifts upward (23,25,30) (Fig. S3 A and B).…”

Section: Compoundmentioning

confidence: 60%

Section: Compoundmentioning

confidence: 69%

Section: Compoundmentioning

confidence: 95%

See 2 more Smart Citations

Activity-based probes for rhomboid proteases discovered in a mass spectrometry-based assay

Vosyka

Vinothkumar

Wolf

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…Crystallography and cryo-EM have given us a look at the structural properties of several IMPs [46,62,64,80,81] and also of multiple inhibitor-rhomboid protease complexes [9,78,[82][83][84][85][86]. However, this has not yet resulted in the design of more potent rhomboid inhibitors.…”

Section: Potency Of Inhibitorsmentioning

confidence: 99%

Intramembrane proteases as drug targets

Verhelst

2017

The FEBS Journal

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Proteases are considered attractive drug targets. Various drugs targeting classical, soluble proteases have been approved for treatment of human disease. Intramembrane proteases (IMPs) are a more recently discovered group of proteolytic enzymes. They are embedded in lipid bilayers and their active sites are located in the plane of a membrane. All four mechanistic families of IMPs have been linked to disease, but currently, no drugs against IMPs have entered the market. In this review, I will outline the function of IMPs with a focus on the ones involved in human disease, which includes Alzheimer's disease, cancer, and infectious diseases by microorganisms. Inhibitors of IMPs are known for all mechanistic classes, but are not yet very potent or selective – aside from those targeting γ‐secretase. I will here describe the different features of IMP inhibitors and discuss a list of issues that need attention in the near future in order to improve the drug development for IMPs.

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Influence of hydrophobic mismatch on the catalytic activity of Escherichia coliGlpG rhomboid protease

et al. 2014

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Rhomboids comprise a broad family of intramembrane serine proteases that are found in a wide range of organisms and participate in a diverse array of biological processes. Highresolution structures of the catalytic transmembrane domain of the Escherichia coli GlpG rhomboid have provided numerous insights that help explain how hydrolytic cleavage can be achieved below the membrane surface. Key to this are observations that GlpG hydrophobic domain dimensions may not be sufficient to completely span the native lipid bilayer. This formed the basis for a model where hydrophobic mismatch Induces thinning of the local membrane environment to promote access to transmembrane substrates. However, hydrophobic mismatch also has the potential to alter the functional properties of the rhomboid, a possibility we explore in the current work. For this purpose, we purified the catalytic transmembrane domain of GlpG into phosphocholine or maltoside detergent micelles of varying alkyl chain lengths, and assessed proteolytic function with a model water-soluble substrate. Catalytic turnover numbers were found to depend on detergent alkyl chain length, with saturated chains containing 10-12 carbon atoms supporting maximal activity. Similar results were obtained in phospholipid bicelles, with no proteolytic activity being detected in longer-chain lipids. Although differences in thermal stability and GlpG oligomerization could not explain these activity differences, circular dichroism spectra suggest that mismatch gives rise to a small change in structure. Overall, these results demonstrate that hydrophobic mismatch can exert an inhibitory effect on rhomboid activity, with the potential for changes in local membrane environment to regulate activity in vivo.

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Catalytic Mechanism of Rhomboid Protease GlpG Probed by 3,4-Dichloroisocoumarin and Diisopropyl Fluorophosphonate

Cited by 50 publications

References 51 publications

Activity-based probes for rhomboid proteases discovered in a mass spectrometry-based assay

Activity-based probes for rhomboid proteases discovered in a mass spectrometry-based assay

Intramembrane proteases as drug targets

Influence of hydrophobic mismatch on the catalytic activity of Escherichia coliGlpG rhomboid protease

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