The Cystatin-Related Epididymal Spermatogenic Protein Inhibits the Serine Protease Prohormone Convertase 2

Cornwall, Gail A.; Cameron, Angus; Lindberg, Iris; Hardy, Daniel M.; Cormier, Nathaly; Hsia, Nelson

doi:10.1210/en.2002-220997

Cited by 63 publications

(40 citation statements)

References 47 publications

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“…To date, the only nonpeptide PC1/3 inhibitors described are certain andrographolide derivatives, which only weakly inhibit PC1/3 (Basak et al, 1999). PC2 is known to have a potent natural inhibitor in the form of the 7B2 carboxyl-terminal peptide (Apletalina et al, 2000a); it is also inhibited by the cystatin-related epididymal spermatogenic protein (Cornwall et al, 2003). The only previous report of small molecule PC2 inhibitors are certain pyrrolidine bis-piperazines and bicyclic guanidines, which inhibit this enzyme with micromolar potency (Kowalska et al, 2009).…”

Section: Discussionmentioning

confidence: 99%

Inhibition of Prohormone Convertases PC1/3 and PC2 by 2,5-Dideoxystreptamine Derivatives

Vivoli¹,

Caulfield²,

Martínez‐Mayorga³

et al. 2011

Mol Pharmacol

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The prohormone convertases PC1/3 and PC2 are eukaryotic serine proteases involved in the proteolytic maturation of peptide hormone precursors and are implicated in a variety of pathological conditions, including obesity, diabetes, and neurodegenerative diseases. In this work, we screened 45 compounds obtained by derivatization of a 2,5-dideoxystreptamine scaffold with guanidinyl and aryl substitutions for convertase inhibition. We identified four promising PC1/3 competitive inhibitors and three PC2 inhibitors that exhibited various inhibition mechanisms (competitive, noncompetitive, and mixed), with sub-and low micromolar inhibitory potency against a fluorogenic substrate. Low micromolar concentrations of certain compounds blocked the processing of the physiological substrate proglucagon. The best PC2 inhibitor effectively inhibited glucagon synthesis, a known PC2-mediated process, in a pancreatic cell line; no cytotoxicity was observed. We also identified compounds that were able to stimulate both 87 kDa PC1/3 and PC2 activity, behavior related to the presence of aryl groups on the dideoxystreptamine scaffold. By contrast, inhibitory activity was associated with the presence of guanidinyl groups. Molecular modeling revealed interactions of the PC1/3 inhibitors with the active site that suggest structural modifications to further enhance potency. In support of kinetic data suggesting that PC2 inhibition probably occurs via an allosteric mechanism, we identified several possible allosteric binding sites using computational searches. It is noteworthy that one compound was found to both inhibit PC2 and stimulate PC1/3. Because glucagon acts in functional opposition to insulin in blood glucose homeostasis, blocking glucagon formation and enhancing proinsulin cleavage with a single compound could represent an attractive therapeutic approach in diabetes.

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Section: Discussionmentioning

confidence: 99%

Inhibition of Prohormone Convertases PC1/3 and PC2 by 2,5-Dideoxystreptamine Derivatives

Vivoli¹,

Caulfield²,

Martínez‐Mayorga³

et al. 2011

Mol Pharmacol

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“…Exogenous inhibitors as regulators of processing enzymes have been under much investigation (86)(87)(88)(89)(90)(91)(92)(93)(94)(95)(96), but few selective inhibitors exist. Specific active-site directed inhibitors of processing enzymes are desirable for mechanistic studies, but such inhibitors are unlikely to provide selective regulation for processing specific proneuropeptide or prohormones, since each protease appears to process multiple proneuropeptide substrates.…”

Section: Inhibitors and Modulators Of Processing Enzymesmentioning

confidence: 99%

Proteases for Processing Proneuropeptides into Peptide Neurotransmitters and Hormones

Hook

Funkelstein

et al. 2008

Annu. Rev. Pharmacol. Toxicol.

220

304

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Summary Points List:1. Proteases are essential for proteolytic processing of proneuropeptide precursors into active peptide neurotransmitters and hormones.2. Secretory vesicles represent the primary subcellular site of neuropeptide biosynthesis, which are produced, stored, and secreted to mediate cell-cell communication. 3.Protease pathways for proneuropeptide processing have been elucidated consisting of (a) the newly identified cysteine protease cathepsin L with aminopeptidase B in secretory vesicles, and (b) the well-established, proprotein convertase family that include the neuroendocrine-specific prohormone convertases 1 and 2 (PC1/3 and PC2) with carboxypeptidase E. 4.Protease gene knockout experiments have validated the roles of PC1/3, PC2, as well as cathepsin L for the production of neuropeptides in nervous and endocrine tissues. 5.Endogenous regulators consisting of inhibitors and activators participate in the in vivo control of processing enzyme functions.6. Structural biology of protease and proneuropeptides will be important to understand interacting mechanisms for proneuropeptide processing. 7.Neuropeptidomics has recently been applied to investigations of neuropeptide systems for their primary sequence and structural identification, as well as quantitation by LC-MS/MS tandem mass spectrometry. 8.Proteomic studies have revealed functional protein families that participate in secretory vesicle functions for the production, storage, and secretion of neuropeptides.9. Pharmacological evaluation of unique specificities among neuropeptide processing systems will be valuable for design of future strategies to develop selective small molecule modulators of processing enzymes for therapeutic applications in health and disease.Future Issues: Areas of Neuropeptide Research for Exploration. 1.How are cathepsin L and prohormone convertase protease pathways coordinately regulated? 2.What is the proteolytic basis for tissue-specific processing of proneuropeptides, such as that for POMC?3. Selective and potent inhibitors of protease components for processing prohormones should be developed to facilitate basic and pharmacological research. 4.What are the structural features of prohormone and protease interactions for functional processing? Peptide neurotransmitters and peptide hormones, collectively known as neuropeptides, are required for cell-cell communication in neurotransmission and for regulation of endocrine functions. Neuropeptides are synthesized from protein precursors (termed proneuropeptides or prohormones) that require proteolytic processing primarily within secretory vesicles that store and secrete the mature neuropeptides to control target cellular and organ systems. This review describes interdisciplinary strategies that have elucidated two primary protease pathways for prohormone processing consisting of the cysteine protease pathway mediated by secretory vesicle cathepsin L and the well-known subtilisin-like proprotein convertase pathway that together support neuropeptide biosynthesis. Importantly...

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“…CRES is synthesized and secreted into the lumen by the epithelial cells in the most proximal part of the epididymis and then abruptly disappears from the lumen a short time later (5). In vitro CRES does not inhibit cysteine proteases but rather inhibited the serine protease prohormone convertase 2, suggesting an intracellular rather than an extracellular role for CRES (6). Although a function of CRES within the secretory pathway of the epididymal epithelial cells would make it dispensable once it was secreted into the luminal fluid, it is not clear why or how CRES is so rapidly removed from the lumen.…”

mentioning

confidence: 97%

Oligomerization and Transglutaminase Cross-linking of the Cystatin CRES in the Mouse Epididymal Lumen

Horsten

Johnson

SanFrancisco³

et al. 2007

Journal of Biological Chemistry

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CRES (cystatin-related epididymal spermatogenic), a member of the cystatin superfamily of cysteine protease inhibitors, is expressed in the epididymis and spermatozoa, suggesting specialized roles in reproduction. Several cystatin family members oligomerize, including cystatin C that forms amyloid deposits associated with cerebral amyloid angiopathy. Our studies demonstrate that CRES also forms oligomers. Size exclusion chromatography revealed the presence of multiple forms of CRES in the epididymal luminal fluid, including SDS-sensitive and SDSresistant high molecular mass complexes. In vitro experiments demonstrated that CRES is a substrate for transglutaminase and that an endogenous transglutaminase activity in the epididymal lumen catalyzed the formation of SDS-resistant CRES complexes. The use of a conformation-dependent antibody that recognizes only the oligomeric precursors to amyloid, negative stain electron microscopy, and Congo Red staining showed that CRES adopted similar oligomeric and fibrillar structures during its aggregation as other amyloidogenic proteins, suggesting that CRES has the potential to form amyloid in the epididymal lumen. The addition of transglutaminase, however, prevented the formation of CRES oligomers recognized by the conformation antibody by cross-linking CRES into an amorphous structure. We propose that transglutaminase activity in the epididymal lumen may function as a mechanism of extracellular quality control by diverting proteins such as CRES from the amyloidogenic pathway.As spermatozoa migrate through the long convoluted tubule known as the epididymis, they undergo maturation and acquire motility and fertility. Since sperm are synthetically inactive, the maturation process requires the interaction of sperm with proteins that are synthesized and secreted in a region-dependent manner by the epididymal epithelium. Following secretion, the fate of proteins in the epididymal lumen is varied. Some proteins bind to sperm and presumably affect sperm function directly, whereas others remain in the lumen throughout the length of the tubule (1, 2). Other proteins are present in the epididymal lumen for only a short time, suggesting that their continued presence may be detrimental to sperm maturation and/or epididymal cell functions, and thus selective mechanisms are in place for their removal.CRES is the defining member of a reproductive subgroup of family 2 cystatins within the cystatin superfamily of cysteine protease inhibitors (MEROPS classification subfamily I25B) (3, 4). CRES is synthesized and secreted into the lumen by the epithelial cells in the most proximal part of the epididymis and then abruptly disappears from the lumen a short time later (5). In vitro CRES does not inhibit cysteine proteases but rather inhibited the serine protease prohormone convertase 2, suggesting an intracellular rather than an extracellular role for CRES (6). Although a function of CRES within the secretory pathway of the epididymal epithelial cells would make it dispensable once it was secrete...

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The Cystatin-Related Epididymal Spermatogenic Protein Inhibits the Serine Protease Prohormone Convertase 2

Cited by 63 publications

References 47 publications

Inhibition of Prohormone Convertases PC1/3 and PC2 by 2,5-Dideoxystreptamine Derivatives

Inhibition of Prohormone Convertases PC1/3 and PC2 by 2,5-Dideoxystreptamine Derivatives

Proteases for Processing Proneuropeptides into Peptide Neurotransmitters and Hormones

Oligomerization and Transglutaminase Cross-linking of the Cystatin CRES in the Mouse Epididymal Lumen

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