The processing proteases prohormone thiol protease, PC1/3 and PC2, and 70-kDa aspartic proteinase show preferences among proenkephalin, proneuropeptide Y, and proopiomelanocortin substrates.

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Proteases of cysteine, aspartic, and subtilisin classes have been indicated as candidate prohormone processing enzymes. The chromaffin granule proenkephalin processing proteases have been characterized as the novel cysteine protease prohormone thiol protease (PTP), a 70-kDa aspartic proteinase, and the subtilisin-like PC1/3 and PC2 enzymes. The goal of this study was to assess whether these processing proteases possess preference(s) for prohormone substrates. The recombinant prohormones proenkephalin, proneuropeptide Y (pro-NPY), and proopiomelanocortin (POMC) were expressed in Escherichia coli using the T7 expression system and purified for in vitro processing studies. Results indicated that the chromaffin granule processing proteases possess selectivity for particular prohormones. PTP preferred proenkephalin, with good cleavage of pro-NPY and slow processing of POMC. In contrast, the 70-kDa aspartic proteinase cleaved POMC most readily, with cleavage of proenkephalin and some processing of pro-NPY. PC1/3 and PC2 preferred POMC among the prohormones tested. Importantly, these results indicate that prohormone selectivity of processing proteases may be an important factor in predicting the primary and rate-limiting protease(s) required for processing a particular prohormone.


Animals; Aspartic acid; Aspartic Acid Endopeptidases/metabolism; Base Sequence; Cattle; Chromaffin cells; Chromaffin Granules/enzymology; Cysteine Endopeptidases/metabolism; Endopeptidases; Enkephalins; Enkephalins/genetics; Enkephalins/metabolism; Enzymology; Genetics; Molecular Sequence Data; Nucleotide sequence; Neuropeptide Y; Neuropeptide Y/genetics; Neuropeptide Y/metabolism; Proopiomelanocortin; Pro-Opiomelanocortin/genetics; Pro-Opiomelanocortin/metabolism; Proprotein Convertase 2; Proprotein convertases; Protein precursors; Protein Precursors/genetics; Protein Precursors/metabolism; Protein Processing; Post-Translational; Proteolytic enzymes; Recombinant proteins; Recombinant Proteins/metabolism; Substrate Specificity; Subtilisins; Subtilisins/metabolism


Biochemistry | Biochemistry, Biophysics, and Structural Biology | Life Sciences | Medicine and Health Sciences

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