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...

Alpha-melanocyte-stimulating hormone (a-MSH) is a neuropeptide expressed in pituitary and brain that is known to regulate energy balance, appetite control, and neuroimmune functions. The biosynthesis of a-MSH requires proteolytic processing of the proopiomelanocortin (POMC) precursor. Therefore, this study investigated the in vivo role of the prohormone convertase 2 (PC2) processing enzyme for production of a-MSH in PC2-deficient mice. Specific detection of a-MSH utilized radioimmunoassay (RIA) that does not crossreact with the POMC precursor, and which does not crossreact with other adrenocorticotropin hormone (ACTH) and b-endorphin peptide products derived from POMC. a-MSH in PC2-deficient mice was essentially obliterated in pituitary, hypothalamus, cortex, and other brain regions (collectively), compared to wild-type controls. These results demonstrate the critical requirement of PC2 for the production of a-MSH. The absence of a-MSH was accompanied by accumulation of ACTH, ACTH-containing imtermediates, and POMC precursor. ACTH was increased in pituitary and hypothalamus of PC2-deficient mice, evaluated by RIA and reversed-phase high pressure liquid chromatography (RP-HPLC). Accumulation of ACTH demonstrates its role as a PC2 substrate that can be converted for a-MSH production. Further analyses of POMC-derived intermediates in pituitary, conducted by denaturing western blot conditions, showed accumulation of ACTH-containing intermediates in pituitaries of PC2-deficient mice, which implicate participation of such intermediates as PC2 substrates. Moreover, accumulation of POMC was observed in PC2-deficient mice by western blots with anti-ACTH and anti-b-endorphin. In addition, increased b-endorphin 1)31 was observed in pituitary and hypothalamus of PC2-deficient mice, suggesting b-endorphin 1)31 as a substrate for PC2 in these tissues. Overall, these studies demonstrated that the PC2 processing enzyme is critical for the in vivo production of a-MSH in pituitary and brain. Keywords: alpha-melanocyte-stimulating hormone, brain, pituitary, prohormone convertase, proopiomelanocortin. The neuropeptide a-MSH (melanocyte-stimulating hormone) is derived from its POMC (proopiomelanocortin) precursor (Roberts et al. 1979;Chang et al. 1980) by proteolytic processing within neuroendocrine secretory vesicles of pituitary and brain (Steiner et al. 1992;Hook et al. 1994;Cawley et al. 1998;Seidah et al. 1999). Specific proteolytic processing of POMC at multibasic residues generates distinct neuropeptide products that consist of a-MSH, ACTH (adrenocorticotropin hormone), and b-endorphin. These POMC products are abundant in pituitary and brain where they are secreted for the regulation of pituitary and brain functions that include energy balance, appetite control, and neuroimmune functions (Ichiyama et al. 2000;Williams et al. 2001;Pritchard et al. 2002;Zimanyi and Pelleymounter 2003 These authors contributed equally to this study.Abbreviations used: ACTH, adrenocorticotropin hormone; a-MSH, amelanocyte-stimulating horm...

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