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...

Peptide hormones and neurotransmitters constitute a large class of neurohumoral agents that mediate cell-cell communication in neuroendocrine systems. Their biosynthesis requires proteolytic processing of inactive protein precursors into active neuropeptides. Elucidation of the proteolytic components required for prohormone processing is important for identifying key proteases that may control the production of neuropeptides. This article compares the subtilisin-like PC1/3 and PC2 processing enzymes identified through molecular biological approaches, and several candidate processing enzymes identified biochemically, including the 'proopiomelanocortin converting enzyme' (PCE) and the 'prohormone thiol protease' (PTP), as well as others of different classes (aspartyl, cysteine, metallo, and serine proteases). A role for PTP in cellular proenkephalin processing is suggested by blockade of forskolin-stimulated (Met)enkephalin production by Ep453 that is converted intracellularly to E-64c, a selective cysteine protease inhibitor that potently inhibits PTP. A possible role for endogenous protease inhibitors in prohormone processing represents a new aspect of cellular mechanisms that may regulate neuropeptide biosynthesis. Future studies of the enzymology and molecular biology of processing enzymes and endogenous protease inhibitors will be necessary to elucidate mechanisms of prohormone processing.

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Proteolysis of mutant huntingtin (htt) has been hypothesized to occur in Huntington's disease (HD) brains. Therefore, this in vivo study examined htt fragments in cortex and striatum of adult HD and control human brains by Western blots, using domainspecific anti-htt antibodies that recognize N-and C-terminal domains of htt (residues 181-810 and 2146-2541, respectively), as well as the 17 residues at the N terminus of htt. On the basis of the patterns of htt fragments observed, different "protease-susceptible domains" were identified for proteolysis of htt in cortex compared with striatum, suggesting that htt undergoes tissue-specific proteolysis. In cortex, htt proteolysis occurs within two different N-terminal domains, termed protease-susceptible domains "A" and "B." However, in striatum, a different pattern of fragments indicated that proteolysis of striatal htt occurred within a C-terminal domain termed "C," as well as within the N-terminal domain region designated "A".Importantly, striatum from HD brains showed elevated levels of 40-50 kDa N-terminal and 30-50 kDa C-terminal fragments compared with that of controls. Increased levels of these htt fragments may occur from a combination of enhanced production or retarded degradation of fragments. Results also demonstrated tissue-specific ubiquitination of certain htt N-terminal fragments in striatum compared with cortex. Moreover, expansions of the triplet-repeat domain of the IT15 gene encoding htt was confirmed for the HD tissue samples studied. Thus, regulated tissue-specific proteolysis and ubiquitination of htt occur in human HD brains. These results suggest that the role of huntingtin proteolysis should be explored in the pathogenic mechanisms of HD. Key words: Huntington's disease; huntingtin; proteolytic fragments; brain; neurodegenerative disease; ubiquitinHuntington's disease (HD) is an inherited neurodegenerative disorder characterized by psychological, motor, and cognitive impairments (Vonsattel and DiFiglia, 1998;Petersen et al., 1999). The onset of HD generally occurs in adults in midlife, with a long-term duration of 15-20 years. The genetic mutation in HD has been identified as a CAG expansion near the 5Ј region of the IT15 gene that encodes the 350 kDa huntingtin (htt) protein, resulting in a greater number of polyglutamines near the N terminus of htt. Normal individuals contain Ͻ35 CAG repeats, whereas individuals with adult-onset HD possess an expansion of 38/39 -55 CAG repeats (MacDonald et al., 1993;Rubinsztein et al., 1997); expansions of 70 or more repeats occur in juvenileonset HD. HD brains display characteristic neuropathological alterations, graded from 0 to 4, with grade 4 representing severe brain atrophy. Advanced grades show a reduction in striatum, cerebral cortex, as well as hippocampus, amygdala, and thalamus brain tissues (de la Monte et al., 1988;Vonsattel and DiFiglia, 1998). Neuronal loss is especially severe in striatum.Studies of the role of the polyglutamine expansion within mutant huntingtin in HD pathogenesis in trans...

This study demonstrates that endopin 2 is a unique secretory vesicle serpin that displays cross-class inhibition of cysteine and serine proteases, indicated by effective inhibition of papain and elastase, respectively. Homology of the reactive site loop (RSL) domain of endopin 2, notably at P1-P1' residues, with other serpins that inhibit cysteine and serine proteases predicted that endopin 2 may inhibit similar proteases. Recombinant N-His-tagged endopin 2 inhibited papain and elastase with second-order rate constants (k(ass)) of 1.4 x 10(6) and 1.7 x 10(5) M(-1) s(-1), respectively. Endopin 2 formed SDS-stable complexes with papain and elastase, a characteristic property of serpins. Interactions of the RSL domain of endopin 2 with papain and elastase were indicated by cleavage of endopin 2 near the predicted P1-P1' residues by these proteases. Endopin 2 did not inhibit the cysteine protease cathepsin B, or the serine proteases chymotrypsin, trypsin, plasmin, and furin. Endopin 2 in neuroendocrine chromaffin cells was colocalized with the secretory vesicle component (Met)enkephalin by confocal immunonfluorescence microscopy, and was present in isolated secretory vesicles (chromaffin granules) from chromaffin cells as a glycoprotein of 72-73 kDa. Moreover, regulated secretion of endopin 2 from chromaffin cells was induced by nicotine and KCl depolarization. Overall, these results demonstrate that the serpin endopin 2 possesses dual specificity for inhibiting both papain-like cysteine and elastase-like serine proteases. These findings demonstrate that endopin 2 inhibitory functions may occur in the regulated secretory pathway.

The pituitary hormones adrenocorticotropic hormone (ACTH), ␤-endorphin, and ␣-melanocyte stimulating hormone (␣-MSH) are synthesized by proteolytic processing of their common proopiomelanocortin (POMC) precursor. Key findings from this study show that cathepsin L functions as a major proteolytic enzyme for the production of POMC-derived peptide hormones in secretory vesicles. Specifically, cathepsin L knockout mice showed major decreases in ACTH, ␤-endorphin, and ␣-MSH that were reduced to 23, 18, and 7% of wild-type controls (100%) in pituitary. These decreased peptide levels were accompanied by increased levels of POMC consistent with proteolysis of POMC by cathepsin L. The peptide hormones ACTH 2 , ␤-endorphin, and ␣-MSH are produced by proteolytic processing of their common proopiomelanocortin (POMC) prohormone precursor (1, 2). The mature peptide hormones are stored in pituitary secretory vesicles for regulated secretion and control of physiological functions in target organs. ACTH regulates the production of glucocorticoids in the adrenal cortex for control of metabolism (3, 4). ␣-MSH is implicated in the regulation of appetite and the production of melanin (5, 6). ␤-Endorphin is an endogenous opioid peptide involved in pain regulation (7,8). The proteolytic mechanisms that generate these functionally distinct peptide hormones from POMC are essential for these hormones to exert their biological functions.The role of cysteine protease activity for POMC processing has been implicated by several studies (9 -11), but the identity of the protease has not yet been achieved. In early studies of POMC processing activity in pituitary secretory vesicles, cysteine protease activity represented a significant portion of POMC cleaving activity, based on its inhibition by the thiol reagent p-chloromercuribenzoate (9). In anterior and intermediate pituitary cells in culture, treatment of cells with the cysteine protease inhibitor E64d reduced cell levels of POMC-derived ACTH, ␤-endorphin, and ␣-MSH (10, 11). Thus, it is likely that a cysteine protease participates in POMC processing. Participation of a cysteine protease in POMC processing would represent a new protease pathway, in addition to the subtilisinlike prohormone convertase enzymes (PC2 and PC1/3) that participate in processing POMC (12)(13)(14)(15). Therefore, the goal of this study was to identify the cysteine protease that produces ACTH, ␤-endorphin, and ␣-MSH peptide hormones derived from POMC.Herein we provide evidence indicating a key role for the cysteine protease cathepsin L in the biosynthesis of ACTH, ␤-endorphin, and ␣-MSH in secretory vesicles. Cathepsin L gene knock-out mice showed major reductions in pituitary tissue levels of ACTH, ␤-endorphin, and ␣-MSH. Furthermore, cathepsin L KO mouse pituitaries displayed accumulation of POMC, consistent with proteolysis of POMC by cathepsin L. In vivo cellular localization of cathepsin L in pituitary cells demonstrated a high degree of colocalization with ␤-endorphin, ␣-MSH, and ACTH in secretory vesicles. Ex...

Neuropeptide Y (NPY) functions as a peptide neurotransmitter and as a neuroendocrine hormone. The active NPY peptide is generated in secretory vesicles by proteolytic processing of proNPY. Novel findings from this study show that cathepsin L participates as a key proteolytic enzyme for NPY production in secretory vesicles. Notably, NPY levels in cathepsin L knockout (KO) mice were substantially reduced in brain and adrenal medulla by 80% and 90%, respectively. Participation of cathepsin L in producing NPY predicts their colocalization in secretory vesicles, a primary site of NPY production. Indeed, cathepsin L was colocalized with NPY in brain cortical neurons and in chromaffin cells of adrenal medulla, demonstrated by immunofluorescence confocal microscopy. Immunoelectron microscopy confirmed the localization of cathepsin L with NPY in regulated secretory vesicles of chromaffin cells. Functional studies showed that coexpression of proNPY with cathepsin L in neuroendocrine PC12 cells resulted in increased production of NPY. Furthermore, in vitro processing indicated cathepsin L processing of proNPY at paired basic residues. These findings demonstrate a role for cathepsin L in the production of NPY from its proNPY precursor. These studies illustrate the novel biological role of cathepsin L in the production of NPY, a peptide neurotransmitter, and neuroendocrine hormone.

Proteases within secretory vesicles are required for conversion of neuropeptide precursors into active peptide neurotransmitters and hormones. This study demonstrates the novel cellular role of the cysteine protease cathepsin L for producing the (Met)enkephalin peptide neurotransmitter from proenkephalin (PE) in the regulated secretory pathway of neuroendocrine PC12 cells. These findings were achieved by coexpression of PE and cathepsin L cDNAs in PC12 cells with analyses of PE-derived peptide products. Expression of cathepsin L resulted in highly increased cellular levels of (Met)enkephalin, resulting from the conversion of PE to enkephalin-containing intermediates of 23, 18 -19, 8 -9, and 4.5 kDa that were similar to those present in vivo. Furthermore, expression of cathepsin L with PE resulted in increased amounts of nicotine-induced secretion of (Met)enkephalin. These results indicate increased levels of (Met)enkephalin within secretory vesicles of the regulated secretory pathway. Importantly, cathespin L expression was directed to secretory vesicles, demonstrated by colocalization of cathepsin L-DsRed fusion protein with enkephalin and chromogranin A neuropeptides that are present in secretory vesicles. In vivo studies also showed that cathepsin L in vivo was colocalized with enkephalin. The newly defined secretory vesicle function of cathepsin L for biosynthesis of active enkephalin opioid peptide contrasts with its function in lysosomes for protein degradation. These findings demonstrate cathepsin L as a distinct cysteine protease pathway for producing the enkephalin member of neuropeptides.The biosynthesis of enkephalin opioid neuropeptides requires proteolytic processing of protein precursors within regulated secretory vesicles (1-3). Active enkephalin and related neuropeptides are stored in such secretory vesicles for regulated secretion that is induced by receptor-mediated mechanisms. Secreted enkephalin functions as an active peptide neurotransmitter in the control of analgesia for pain relief, behavioral responses, and related brain and physiological functions (4 -6).Secretory vesicles represent the primary subcellular site for proteolytic processing of proenkephalin and other proneuropeptides (1, 3). Secretory vesicles isolated from adrenal medullary chromaffin cells (also known as chromaffin granules) have provided a model system for identification of proteases for proneuropeptide processing, which includes PC1/3 and PC2 endopeptidases (7, 8) and the exopeptidase carboxypeptidase E/H (9, 10). These secretory vesicles contain (Met)enkephalin and its proenkephalin precursor (11, 12), and, therefore, contain the appropriate processing proteases.In vitro proneuropeptide processing assays have identified cathepsin L as a major proenkephalin-cleaving activity purified from neuropeptide-containing chromaffin granules (13,14). Cathepsin L generates (Met)enkephalin in vitro from enkephalin-containing peptide substrates, resulting from cleavage at paired basic residue processing sites, as well as monobasic ...