Activity‐Based Probes for the Study of Proteases: Recent Advances and Developments

Serim, Sevnur; Haedke, Ute; Verhelst, Steven H. L.

doi:10.1002/cmdc.201200057

Cited by 104 publications

(87 citation statements)

References 132 publications

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“…Protease activity profiling (also called activity-based protein profiling of proteases) is an easy and powerful method to monitor the active state of proteases in crude extracts or living organisms (Heal et al, 2011;Serim et al, 2012;Haedke et al, 2013;Willems et al, 2014). Protease activity profiling is based on the use of chemical probes that react covalently with the active site of proteases in an activitydependent manner.…”

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confidence: 99%

Subfamily-Specific Fluorescent Probes for Cysteine Proteases Display Dynamic Protease Activities during Seed Germination

Chandrasekar

Oeljeklaus

et al. 2015

Plant Physiology

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Cysteine proteases are an important class of enzymes implicated in both developmental and defense-related programmed cell death and other biological processes in plants. Because there are dozens of cysteine proteases that are posttranslationally regulated by processing, environmental conditions, and inhibitors, new methodologies are required to study these pivotal enzymes individually. Here, we introduce fluorescence activity-based probes that specifically target three distinct cysteine protease subfamilies: aleurain-like proteases, cathepsin B-like proteases, and vacuolar processing enzymes. We applied protease activity profiling with these new probes on Arabidopsis (Arabidopsis thaliana) protease knockout lines and agroinfiltrated leaves to identify the probe targets and on other plant species to demonstrate their broad applicability. These probes revealed that most commercially available protease inhibitors target unexpected proteases in plants. When applied on germinating seeds, these probes reveal dynamic activities of aleurain-like proteases, cathepsin B-like proteases, and vacuolar processing enzymes, coinciding with the remobilization of seed storage proteins.

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Subfamily-Specific Fluorescent Probes for Cysteine Proteases Display Dynamic Protease Activities during Seed Germination

Chandrasekar

Oeljeklaus

et al. 2015

Plant Physiology

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“…Recently, activity-based probes (ABPs) have been used as a tool for the direct assessment of the activity of these proteases in the context of a native tumor microenvironment [5–7]. These reagents form activity dependent covalent bonds with protease active site nucleophiles, thereby providing readout of the levels of active protease in a cell, tissue, or even whole organisms [8]. In particular, fluorescently quenched ABPs (qABPs) have proven to be powerful tools for the noninvasive optical imaging of cancer and subsequent characterization of the target cathepsins on a histological, cellular, and protein level [9,10].…”

Section: Introductionmentioning

confidence: 99%

Microscopic Detection of Quenched Activity-Based Optical Imaging Probes Using an Antibody Detection System: Localizing Protease Activity

et al. 2014

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Purpose The cysteine cathepsins are a family of proteases that play important physiological roles in both normal cellular physiology and many human diseases. In cancer, the activity of many of the cysteine cathepsins is up-regulated and can be exploited for tumor imaging both in vivo and ex vivo. To characterize the behavior of a topically applied quenched fluorescent activity-based probe, GB119, ex vivo, we developed a basic immunohistochemistry technique to identify unquenched GB119 within tissue. Procedures Immunoblot assays were used to validate the utility of an anit-Cy5 antibody for the detection of unquenched GB119 generated by cathepsin-L. Following validation the anti-Cy5 antibody an immunohistochemical procedure was developed to detect the presence of unquenched GB119 in frozen sections of brain tumors derived from an orthotopic mouse model. Results These studies demonstrate that the anti-Cy5 antibody preferentially recognizes unquenched GB119 and that this differential can be used to identify the regions within the brain and the tumor that contained unquenched GB119. Using H&E staining and antibodies against other biochemical markers it was further determined that unquenched GB119 was localized to the peri-tumor space and co-localized with cathepsin-L expression. Conclusion Our data indicate that this methodology allows high-resolution detection of unquenched GB119 that can be correlated with other immunohistological stains.

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“…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).…”

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confidence: 99%

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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Activity‐Based Probes for the Study of Proteases: Recent Advances and Developments

Cited by 104 publications

References 132 publications

Subfamily-Specific Fluorescent Probes for Cysteine Proteases Display Dynamic Protease Activities during Seed Germination

Subfamily-Specific Fluorescent Probes for Cysteine Proteases Display Dynamic Protease Activities during Seed Germination

Microscopic Detection of Quenched Activity-Based Optical Imaging Probes Using an Antibody Detection System: Localizing Protease Activity

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

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