Small Molecule Affinity Fingerprinting

Greenbaum, Doron C.; Arnold, W. David; Lu, Felice; Hayrapetian, Linda; Baruch, Amos; Krumrine, Jennifer R.; Toba, Samuel; Chehade, Kareem A. H.; Brὂmme, Dieter; Kuntz, Irwin D.; Bogyo, Matthew

doi:10.1016/s1074-5521(02)00238-7

Cited by 162 publications

(84 citation statements)

References 31 publications

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“…To determine the protease component of this activity, affinity labeling was performed with the cysteine protease activity probe known as DCG-04 ( Fig. 1a) (14,15). Affinity labeling at various stages in the purification demonstrated enrichment of a DCG-04-labeled 27-kDa protease band (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…In this study the protease responsible for PE-cleaving activity in chromaffin granules was identified by using an activity-based probe for cysteine proteases (14,15) combined with mass spectrometry (MS) for peptide sequencing. Results identified secretory vesicle cathepsin L as the enzyme responsible for the previously described PTP cysteine protease activity involved in enkephalin and neuropeptide production (7)(8)(9)(10).…”

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confidence: 99%

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Cathepsin L in secretory vesicles functions as a prohormone-processing enzyme for production of the enkephalin peptide neurotransmitter

Yasothornsrikul

Greenbaum

Medzihradszky

et al. 2003

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

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Multistep proteolytic mechanisms are essential for converting proprotein precursors into active peptide neurotransmitters and hormones. Cysteine proteases have been implicated in the processing of proenkephalin and other neuropeptide precursors. Although the papain family of cysteine proteases has been considered the primary proteases of the lysosomal degradation pathway, more recent studies indicate that functions of these enzymes are linked to specific biological processes. However, few protein substrates have been described for members of this family. We show here that secretory vesicle cathepsin L is the responsible cysteine protease of chromaffin granules for converting proenkephalin to the active enkephalin peptide neurotransmitter. The cysteine protease activity was identified as cathepsin L by affinity labeling with an activity-based probe for cysteine proteases followed by mass spectrometry for peptide sequencing. Production of T he biosynthesis of enkephalin opioid peptides as well as numerous peptide neurotransmitters and hormones requires proteolytic processing of respective proprotein precursors within regulated secretory vesicles (1-4). The mature, processed enkephalin peptide is stored within these vesicles and undergoes stimulated secretion to mediate neurotransmission and cell-cell communication in the regulation of analgesia, behavior, and immune-cell functions. Secretory vesicles of neuroendocrine chromaffin cells (also known as chromaffin granules) contain enkephalin and its precursor proenkephalin (PE) (5, 6), with relevant prohormone convertases for converting PE into active enkephalin.The primary PE-cleaving activity in chromaffin granules has been characterized as a cysteine protease complex known as ''prohormone thiol protease'' (PTP) (7-10). The cysteine protease activity cleaves PE and enkephalin-containing peptide substrates at paired basic residues, as well as at certain monobasic residues, to generate appropriate enkephalin-related peptide products. Cellular inhibition of PTP by a cysteine protease inhibitor results in reduced production of enkephalin (11). Molecular identification of the protease component responsible for this cysteine protease activity will facilitate our understanding of multiple proteolytic enzymes that produce active peptides including the opioid [Met]enkephalin (ME) (12,13).In this study the protease responsible for PE-cleaving activity in chromaffin granules was identified by using an activity-based probe for cysteine proteases (14, 15) combined with mass spectrometry (MS) for peptide sequencing. Results identified secretory vesicle cathepsin L as the enzyme responsible for the previously described PTP cysteine protease activity involved in enkephalin and neuropeptide production (7-10). Cathepsin L generated the active peptide ME by cleaving enkephalin-containing peptide substrates at native dibasic and monobasic sites. Notably, cathepsin L colocalized with ME in the regulated secretory pathway of chromaffin cells. In cathepsin L gene knockout (KO) mice (16-1...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Cathepsin L in secretory vesicles functions as a prohormone-processing enzyme for production of the enkephalin peptide neurotransmitter

Yasothornsrikul

Greenbaum

Medzihradszky

et al. 2003

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

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198

223

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“…Active Site Labeling of Cysteine Proteinases-Active site labeling experiments of cysteine proteinases were performed as previously described (19,30) with some modifications. Briefly, lysosomal fractions from MDMs were incubated with or without Mu-Leu-Hph-VS-Ph (1 nM or 1 M), a highly potent irreversible cathepsin S inhibitor (31), with Mu-Np2-Hph-VS-2Np (10 nM), a potent general cathepsin but very weak cathepsin K inhibitor (32, 33) (both inhibitors were kindly provided by Celera Corp., South San Francisco, CA), or with CA074 (100 nM; Calbiochem, La Jolla, CA), a selective cathepsin B inhibitor, at 37°C for 1 h prior to incubation with a biotin and tyrosine residue containing E-64 derivative (DCG-04) (50 M) for 1 h at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Cathepsin V, a Novel and Potent Elastolytic Activity Expressed in Activated Macrophages

Yasuda

Greenbaum

et al. 2004

Journal of Biological Chemistry

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Atherosclerosis is characterized by a thickening and loss of elasticity of the arterial wall. Loss of elasticity has been attributed to the degradation of the arterial elastin matrix. Cathepsins K and S are papain-like cysteine proteases with known elastolytic activities, and both enzymes have been identified in macrophages present in plaque areas of diseased blood vessels. Here we demonstrate that macrophages express a third elastolytic cysteine protease, cathepsin V, which exhibits the most potent elastase activity yet described among human proteases and that cathepsin V is present in atherosclerotic plaque specimens. Approximately 60% of the total elastolytic activity of macrophages can be attributed to cysteine proteases with cathepsins V, K, and S contributing equally. From this 60%, two-thirds occur extracellularly and one-third intracellularly with the latter credited to cathepsin V. Ubiquitously expressed glycosaminoglycans (GAGs) such as chondroitin sulfate specifically inhibit the elastolytic activities of cathepsins V and K via the formation of specific cathepsin-GAG complexes. In contrast, cathepsin S, which does not form complexes with chondroitin sulfate is not inhibited; thus suggesting a specific regulation of elastolytic activities of cathepsins by GAGs. Because the GAG content is reduced in atherosclerotic plaques, an increase of cathepsins V and K activities may accelerate the destruction of the elastin matrix in diseased arteries.Atherosclerosis is characterized by arterial intimal enlargement and subsequent lipid deposition leading to the formation of blood stream obstructing plaques. The infiltration of monocyte-derived macrophages (MDMs) 1 and smooth muscle cells (SMCs) into the intima of the inflamed artery contributes to the formation of atherosclerotic lesions. Atherosclerotic lesions resident MDMs and SMCs produce a large number of extracellular matrix-degrading enzymes (1), such as cysteine proteases (2-5) and matrix metalloproteinases (MMPs) (6 -8).Elastin and collagen are the two major extracellular matrix components that provide elasticity and tensile strength to the arterial wall. The destruction of elastin and collagen causes a weakening and rupture of blood vessels (9, 10). MMPs, serine, and cysteine proteases have been identified as major elastolytic proteases in arteries (11, 12). Among cysteine proteases, cathepsins S and K have been considered as the most potent elastolytic activities with cathepsin K exhibiting a slightly higher activity than cathepsin S (13, 14). However, cathepsin K-deficient human macrophages derived from patients with pycnodysostosis were shown to retain their high cysteine proteasedependent elastolytic activities (15). This finding implied that additional cathepsins may contribute to the overall elastolytic activity in human macrophages.We and others (16 -18) have identified and partially characterized a novel human cysteine protease closely related to cathepsin L that was named cathepsin V (also known as cathepsin L2). Cathepsin V shares 80% protein s...

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“…Labeling of this protein could be blocked by pretreatment with the pan-cathepsin inhibitor, JPM-565, as well as the cathepsin B specific inhibitor CA074 (Supplemental Fig 1) suggesting it was in fact cathepsin B. This was a surprising result since profiling studies performed on the cysteine cathepsins had previously demonstrated that these proteases do not tolerate negatively charged amino acid residues in the P2 position in either substrates or epoxide-based inhibitors [12,14,16].…”

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confidence: 89%

“…For each sub-library either the P2, P3 or P4 position was held constant as a single amino acid while the remaining positions were coupled with an equal mixture of 19 amino acids (all 20 natural amino acids minus cysteine and methionine to avoid dimerization and oxidation problems and plus norleucine as a structural analog for methionine) as has been reported previously [12]. Scanning of the natural amino acid sequences through each of the P2-P4 positions provided a specificity fingerprint for legumain that could then be used to select optimal residues for the design of legumain-directed probes.…”

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confidence: 99%

Design of cell-permeable, fluorescent activity-based probes for the lysosomal cysteine protease asparaginyl endopeptidase (AEP)/legumain

Sexton

Witte

Blum

et al. 2007

Bioorganic & Medicinal Chemistry Letters

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Asparaginyl endopeptidase (AEP), also known as legumain, is a cysteine protease that has been ascribed roles in antigen presentation yet its exact role in human biology remains poorly understood. We report here the use of a positional scanning combinatorial library of peptide AOMKs containing a P1 aspartic acid to probe the P2, P3 and P4 subsite specificity of endogenous legumain. Using inhibitor specificity profiles of cathepsin B and legumain, we designed fluorescent ABPs that are highly selective, cell permeable reagents for monitoring legumain activity in complex proteomes.Asparaginyl Endopeptidase (AEP), or legumain, was originally identified in leguminous seeds as a cysteine protease with specificity for asparagine residues in the P1 position [1]. The mammalian legumain homologue is a lysosomal cysteine protease that is a member of the clan CD protease family which includes the caspases, separase and the gingipains [2]. Mammalian legumain has been ascribed a role in the initiation of invariant chain processing during MHC class II mediated antigen presentation [3,4]. Although the nature of this activity remains controversial, legumain is undoubtedly a key player in lysosomal proteolysis, contributing to the processing of antigenic peptides as well as the processing of the papain family cathepsins [5].Like all endocytic proteases, legumain is synthesized as an inactive zymogen, and its activity is regulated by post-translational activation events. Therefore, tools that can be used to monitor legumain's activity are necessary in order to understand its functional role. Activity based probes (ABPs) are reagents that can specifically label active proteases, thus allowing their activity, and more importantly their regulation, to be directly monitored [6,7]. Our laboratory recently reported a synthesis strategy based on the general solid phase methods developed by Ellman and co-workers [8] for the production of peptidyl acyloxymethyl ketone ABPs for diverse cysteine protease activities [9].We have previously demonstrated that the biotinylated ABP b-hex-D-AOMK efficiently labels endogenous legumain in 816 B cell lysates [9]. However this reagent lacks cell permeability and its overall selectivity towards legumain had not been extensively examined. We therefore Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. (Fig. 1a, b). Both ABPs potently labeled a ∼38 kDa protein in acidic proteomes from 816 B cells or RAW264.7 monocytes. This activity was confirmed to be legumain by immunoprecipitation using antisera specific for legumain. In addition to the ∼38 kDa predominant ...

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Small Molecule Affinity Fingerprinting

Cited by 162 publications

References 31 publications

Cathepsin L in secretory vesicles functions as a prohormone-processing enzyme for production of the enkephalin peptide neurotransmitter

Cathepsin L in secretory vesicles functions as a prohormone-processing enzyme for production of the enkephalin peptide neurotransmitter

Cathepsin V, a Novel and Potent Elastolytic Activity Expressed in Activated Macrophages

Design of cell-permeable, fluorescent activity-based probes for the lysosomal cysteine protease asparaginyl endopeptidase (AEP)/legumain

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