Chemical Approaches for Functionally Probing the Proteome

Greenbaum, Doron C.; Baruch, Amos; Hayrapetian, Linda; Darula, Zsuzsanna; Burlingame, Alma L.; Medzihradszky, Katlin F.; Bogyo, Matthew

doi:10.1074/mcp.t100003-mcp200

Cited by 283 publications

(206 citation statements)

References 25 publications

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“…17 To identify the factors that upregulate cathepsin activity in macrophages, we developed a cell-based assay in which macrophages were differentiated from bone marrow cells in conditioned media, and cathepsin activity in the bone marrowderived macrophages (BMDMs) was assessed by flow cytometry using a cathepsin activity-based probe. 90 We found that tumor cell-conditioned media significantly induced cathepsin activity in BMDMs, and further identified IL-4 as a tumor-derived factor that triggers this induction. Treatment with IL-4 results in a significant increase in cathepsin activity, along with upregulation of several 'M2' marker genes including Ccl17, Ccl22, Arg1, Mrc1 and Cd36, and downregulation of 'M1' genes such as Tnf, Il12a and Nos2 in BMDMs.…”

Section: O N O T D I S T R I B U T Ementioning

confidence: 93%

Alternative activation of tumor-associated macrophages by IL-4

2010

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Section: O N O T D I S T R I B U T Ementioning

confidence: 93%

Alternative activation of tumor-associated macrophages by IL-4

2010

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“…The apparent molecular mass of the 31-kDa protein parallels that of the cysteine protease cathepsin B (21,22). Therefore, the effect of the selective cathepsin B inhibitor CA-074 (23, 24) on processing activity was tested.…”

Section: Resultsmentioning

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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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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“…These libraries were previously synthesized and successfully used for profiling cathepsins (34), to generate a cathepsin B-specific inhibitor (52) and also to find a falcipain 1-specific inhibitor in P. falciparum (53). In brief, each pool within a library contains a fixed amino acid (1 of 19 natural amino acids: no Cys, no Met, plus norleucine) at P2, P3, or P4 and a mixture of amino acids at the other positions (Fig.…”

Section: Specificity Profiling Using Positional Scanning Libraries-mentioning

confidence: 99%

Development of Calpain-specific Inactivators by Screening of Positional Scanning Epoxide Libraries

Cuerrier

Moldoveanu

Campbell

et al. 2007

Journal of Biological Chemistry

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Calpains are calcium-dependent proteases that are required for numerous intracellular processes but also play an important role in the development of pathologies such as ischemic injury and neurodegeneration. Many current small molecule calpain inhibitors also inhibit other cysteine proteases, including cathepsins, and need improved selectivity. The specificity of inhibition of several calpains and papain was profiled using synthetic positional scanning libraries of epoxide-based compounds that target the active-site cysteine. These peptidomimetic libraries probe the P4, P3, and P2 positions, display (S,S)-or (R,R)-epoxide stereochemistries, and incorporate both natural and nonnatural amino acids. To facilitate library screening, an SDS-PAGE assay that measures the extent of hydrolysis of an inactive recombinant m-calpain was developed. Individual epoxide inhibitors were synthesized guided by calpain-specific preferences observed from the profiles and tested for inhibition against calpain. The most potent compounds were assayed for specificity against cathepsins B, L, and K. Several compounds demonstrated high inhibition specificity for calpains over cathepsins. The best of these inhibitors, WRH(R,R), irreversibly inactivates m-and -calpain rapidly (k 2 /K i ‫؍‬ 131,000 and 16,500 M ؊1 s ؊1 , respectively) but behaves exclusively as a reversible and less potent inhibitor toward the cathepsins. X-ray crystallography of the proteolytic core of rat -calpain inactivated by the epoxide compounds WR ␥-cyano-␣-aminobutyric acid (S,S) and WR allylglycine (R,R) reveals that the stereochemistry of the epoxide influences positioning and orientation of the P2 residue, facilitating alternate interactions within the S2 pocket. Moreover, the WR ␥-cyano-␣-aminobutyric acid (S,S)-complexed structure defines a novel hydrogen-bonding site within the S2 pocket of calpains.

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Chemical Approaches for Functionally Probing the Proteome

Cited by 283 publications

References 25 publications

Alternative activation of tumor-associated macrophages by IL-4

Alternative activation of tumor-associated macrophages by IL-4

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

Development of Calpain-specific Inactivators by Screening of Positional Scanning Epoxide Libraries

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