Cathepsin L participates in dynorphin production in brain cortex, illustrated by protease gene knockout and expression.

Dynorphin opioid neuropeptides mediate neurotransmission for analgesia and behavioral functions. Dynorphin A, dynorphin B, and alpha-neoendorphin are generated from prodynorphin by proteolytic processing. This study demonstrates the significant role of the cysteine protease cathepsin L for producing dynorphins. Cathepsin L knockout mouse brains showed extensive decreases in dynorphin A, dynorphin B, and alpha-neoendorphin that were reduced by 75%, 83%, and 90%, respectively, compared to controls. Moreover, cathepsin L in brain cortical neurons was colocalized with dynorphins in secretory vesicles, the primary site of neuropeptide production. Cellular coexpression of cathepsin L with prodynorphin in PC12 cells resulted in increased production of dynorphins A and B. Comparative studies of PC1/3 and PC2 convertases showed that PC1/3 knockout mouse brains had a modest decrease in dynorphin A, and PC2 knockout mice showed a minor decrease in alpha-neoendorphin. Overall, these results demonstrate a prominent role for cathepsin L, jointly with PC1/3 and PC2, for production of dynorphins in brain.

Human pituitary contains dual cathepsin L and prohormone convertase processing pathway components involved in converting POMC into the peptide hormones ACTH, alpha-MSH, and beta-endorphin.

The production of the peptide hormones ACTH, alpha-MSH, and beta-endorphin requires proteolytic processing of POMC which is hypothesized to utilize dual cysteine- and subtilisin-like protease pathways, consisting of the secretory vesicle cathepsin L pathway and the well-known subtilisin-like prohormone convertase (PC) pathway. To gain knowledge of these protease components in human pituitary where POMC-derived peptide hormones are produced, this study investigated the presence of these protease pathway components in human pituitary. With respect to the cathepsin L pathway, human pituitary contained cathepsin L of 27-29 kDa and aminopeptidase B of approximately 64 kDa, similar to those in secretory vesicles of related neuroendocrine tissues. The serpin inhibitor endopin 2, a selective inhibitor of cathepsin L, was also present. With respect to the PC pathway, human pituitary expresses PC1/3 and PC2 of approximately 60-65 kDa, which represent active PC1/3 and PC2; peptide hormone production then utilizes carboxypeptidase E (CPE) which is present as a protein of approximately 55 kDa. Analyses of POMC products in human pituitary showed that they resemble those in mouse pituitary which utilizes cathepsin L and PC2 for POMC processing. These findings suggest that human pituitary may utilize the cathepsin L and prohormone convertase pathways for producing POMC-derived peptide hormones.

Differential accessibilities of dibasic prohormone processing sites of proenkephalin to the aqueous environment revealed by H-D exchange mass spectrometry.

Proenkephalin (PE) is a prohormone containing dibasic sites that are cleaved by proteases to generate peptide neurotransmitters and hormones. Little is known about the conformational features of such protease cleavage sites within prohormone substrates. Therefore, the goal of this study was to investigate the relative accessibilities of multiple dibasic processing sites of PE by peptide amide hydrogen-deuterium exchange mass spectrometry (DXMS). DXMS demonstrated differences in the relative accessibilities of the KR, KK, and RR cleavage sites of PE to the aqueous environment. DXMS assesses relative rates of exchange of hydrogens of the polypeptide backbone of PE with deuterium atoms from D(2)O (heavy water) in solvent. Analyses of peptides spanning each of the 12 dibasic PE cleavage sites illustrated differences in H-D exchange rates that reflect relative solvent accessibility. The mid-domain cleavage sites (dibasic sites 4-8) exhibited greater accessibility to the aqueous solvent compared to regions of the NH(2) and COOH domains (dibasic sites 2, 3, and 9-11, respectively). The NH(2)- and COOH-terminal domains both exhibited relatively high H-D exchange rates. The hydrogen exchange rate profile of PE, as well as its circular dichroism (CD) features for secondary structure, was modified in trifluoroethanol, an organic solvent that represents a more hydrophobic environment. These findings suggest that the dibasic protease cleavage sites of the PE prohormone with differences in accessibility to the aqueous environment undergo proteolytic processing to generate active neuropeptides for cell-cell communication in neuroendocrine systems.

Endopin serpin protease inhibitors localize with neuropeptides in secretory vesicles and neuroendocrine tissues.

BACKGROUND/AIMS: The endopin serpin protease inhibitors have been identified by molecular studies as components of secretory vesicles that produce neuropeptides. Endopin 1 inhibits trypsin-like serine proteases, and endopin 2 inhibits cathepsin L that produces neuropeptides in secretory vesicles. To assess the secretory vesicle and neuroendocrine tissue distribution of these endopins, the goal of this study was to define specific antisera for each endopin isoform and to examine their localization with neuropeptides and in neuroendocrine tissues. METHODS: This study utilized methods consisting of Western blots, immunoelectron microscopy, and immunofluorescence microscopy for evaluation of the localization of endopin protease inhibitors in neuroendocrine tissues. RESULTS: Immunoelectron microscopy with these selective antisera demonstrated the localization of endopins 1 and 2 within secretory vesicles of adrenal medulla (bovine). Cellular immunofluorescence confocal microscopy illustrated the high level of colocalization of endopins 1 and 2 with enkephalin and NPY neuropeptides that are present in secretory vesicles of adrenal medullary chromaffin cells in primary culture. Tissue distribution studies (by Western blots) showed the expression of endopins 1 and 2 in bovine brain, pituitary, adrenal medulla, and other neuroendocrine tissues. CONCLUSIONS: These results implicate endopins 1 and 2 as endogenous protease inhibitors in neuropeptide-containing secretory vesicles and neuroendocrine tissues.

Major role of cathepsin L for producing the peptide hormones ACTH, beta-endorphin, and alpha-MSH, illustrated by protease gene knockout and expression.

The pituitary hormones adrenocorticotropic hormone (ACTH), beta-endorphin, and alpha-melanocyte stimulating hormone (alpha-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 knock-out mice showed major decreases in ACTH, beta-endorphin, and alpha-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. Immunofluorescence microscopy showed colocalization of cathepsin L with beta-endorphin and alpha-MSH in the intermediate pituitary and with ACTH in the anterior pituitary. In contrast, cathepsin L was only partially colocalized with the lysosomal marker Lamp-1 in pituitary, consistent with its extralysosomal function in secretory vesicles. Expression of cathepsin L in pituitary AtT-20 cells resulted in increased ACTH and beta-endorphin in the regulated secretory pathway. Furthermore, treatment of AtT-20 cells with CLIK-148, a specific inhibitor of cathepsin L, resulted in reduced production of ACTH and accumulation of POMC. These findings demonstrate a prominent role for cathepsin L in the production of ACTH, beta-endorphin, and alpha-MSH peptide hormones in the regulated secretory pathway.

Zinc regulation of aminopeptidase B involved in neuropeptide production.

Aminopeptidase B (AP-B) is a metallopeptidase that removes basic residues from the N-termini of neuropeptide substrates in secretory vesicles. This study assessed zinc regulation of AP-B activity, since secretory vesicles contain endogenous zinc. AP-B was inhibited by zinc at concentrations typically present in secretory vesicles. Zinc effects were dependent on concentration, incubation time, and the molar ratio of zinc to enzyme. AP-B activity was recovered upon removal of zinc. AP-B with zinc became susceptible to degradation by trypsin, suggesting that zinc alters enzyme conformation. Zinc regulation demonstrates the metallopeptidase property of AP-B.

Cathepsin L participates in the production of neuropeptide Y in secretory vesicles, demonstrated by protease gene knockout and expression.

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 for processing proneuropeptides into peptide neurotransmitters and hormones.

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, this review discusses important areas of current and future biomedical neuropeptide research with respect to biological regulation, inhibitors, structural features of proneuropeptide and protease interactions, and peptidomics combined with proteomics for systems biological approaches. Future studies that gain in-depth understanding of protease mechanisms for generating active neuropeptides will be instrumental for translational research to develop pharmacological strategies for regulation of neuropeptide functions. Pharmacological applications for neuropeptide research may provide valuable therapeutics in health and disease.

Multiple domains of endopin 2A for serpin cross-class inhibition of papain.

The serpin endopin 2A inhibits the cysteine protease papain in cross-class inhibition. This study demonstrates the novel finding that both the non-RSL NH(2)-domain and the RSL domain with P1-P1' residues participate in endopin 2A inhibition. Production of a chimeric mutant of endopin 2A with replacement of its NH(2)-domain with that of endopin 1 resulted in less effective inhibition of papain, indicated by its lower k(ass) association rate constant compared to wild-type endopin 2A. This chimeric mutant formed complexes with papain, but at lower levels compared to that with wild-type endopin 2A. Papain degradation of a portion of the chimeric mutant suggested a role for the NH(2)-domain in regulating relative amounts of endopin 2A that enter the substrate pathway compared to the serpin inhibitory pathway. Furthermore, site-directed mutagenesis demonstrated that the RSL domain with intact P1-P1' residues was necessary for inhibition. These findings indicate that the NH(2)-domain and the RSL region both participate in endopin 2A inhibition of papain.

Secretory vesicle aminopeptidase B related to neuropeptide processing: molecular identification and subcellular localization to enkephalin- and NPY-containing chromaffin granules.

Biosynthesis of peptide hormones and neurotransmittters involves proteolysis of proprotein precursors by secretory vesicle cathepsin L. Cathepsin L generates peptide intermediates with basic residues at their NH(2)-termini, indicating that Arg/Lys aminopeptidase is needed to generate the smaller biologically active peptide. Therefore, this study identified the Arg/Lys aminopeptidase that is present in secretory vesicles of adrenal medulla and neuroendocrine tissues, achieved by molecular cloning and localization in 'model' neuropeptide-containing secretory vesicles (bovine). Molecular cloning of the bovine aminopeptidase B (AP-B) cDNA defined its primary sequence that allowed selection of antisera for immunolocalization studies. AP-B was present in secretory vesicles that contain cathepsin L with the neuropeptides enkephalin and neuropeptide Y. The AP-B in several neuroendocrine tissues was detected by western blots. Recombinant bovine AP-B showed preference for Arg-methylcoumarinamide substrate. AP-B was inhibited by arphamenine, an inhibitor of aminopeptidases. Bovine AP-B showed similar activities for Arg-(Met)enkephalin (ME) and Lys-ME neuropeptide substrates to generate ME, while rat AP-B preferred Arg-ME. Furthermore, AP-B possesses an acidic pH optimum of 5.5-6.5 that is similar to the internal pH of secretory vesicles. The significant finding of the secretory vesicle localization of AP-B with neuropeptides and cathepsin L suggests a role for this exopeptidase in the biosynthesis of neuropeptides.

Cathepsin L expression is directed to secretory vesicles for enkephalin neuropeptide biosynthesis and secretion.

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.

Resistance of cathepsin L compared to elastase to proteolysis when complexed with the serpin endopin 2C, and recovery of cathepsin L activity.

This study demonstrates unique differences in the conformational nature of cathepsin L compared to elastase when complexed with the serpin endopin 2C, assessed by susceptibilities of protease/endopin 2C complexes to proteolysis by trypsin. Complexed and uncomplexed cathepsin L were resistant to degradation by trypsin, which indicated that trypsin cleavage sites within cathepsin L remain inaccessible when this cysteine protease is complexed with the endopin 2C serpin. In contrast, elastase in complexes with endopin 2C was degraded by trypsin, but uncomplexed elastase was not degraded. These results demonstrate a change in the conformational properties of trypsin cleavage sites within elastase when it is complexed with endopin 2C, compared to uncomplexed elastase. Cathepsin L complexes with endopin 2C were short-lived, but elastase complexes were stable. Furthermore, cathepsin L dissociated from complexes demonstrated recovery of cathepsin L activity, and reducing conditions provided optimum recovery of cathepsin L activity. These findings suggest that cathepsin L, when complexed with endopin 2C, maintains its general conformation in a manner that allows recovery of cathepsin L activity upon dissociation from endopin 2C. These results demonstrate differences in the relative conformational properties of the cysteine protease cathepsin L, compared to the serine protease elastase, in complexes with the serpin endopin 2C.

The novel bovine serpin endopin 2C demonstrates selective inhibition of the cysteine protease cathepsin L compared to the serine protease elastase, in cross-class inhibition.

Molecular cloning revealed the unique serpin endopin 2C that demonstrates selective inhibition of cathepsin L compared to papain or elastase. Endopin 2C, thus, functions as a serpin with the property of cross-class inhibition. Endopin 2C possesses homology in primary sequence to endopin 2A and other isoforms of endopins related to alpha1-antichymotrypsin, yet endopin 2C differs in its target protease specificity. Recombinant endopin 2C showed effective inhibition of cathepsin L with a stoichiometry of inhibition (SI) of 1/1 (molar ratio of inhibitor/protease), with the second-order rate constant, k(ass), of 7.2 x 10(5) M(-1) s(-1). Less effective endopin 2C inhibition of papain and elastase occurred with k(ass) association rate constants of approximately 1 x 10(4) M(-1) s(-1) with high SI values. Endopin 2C formed SDS-stable complexes with cathepsin L, papain, and elastase that are typical of serpins. These results are among the first to demonstrate stable serpin complexes with target cysteine proteases. Interactions of endopin 2C with cathepsin L and elastase were indicated by protease cleavage of the RSL region between P1-P1' residues of Thr-Ser. The hydrophobic Phe residue in the P2 position of the RSL region is consistent with the specificity of cathepsin L for hydrophobic residues in the P2 position of its substrate cleavage site. The NH2-terminal signal sequence of endopin 2C, like that of cathepsin L, predicts their colocalization to subcellular organelles. These findings demonstrate endopin 2C as a novel serpin that possesses cross-class inhibition with selectivity for inhibition of cathepsin L.

Demonstration of GTG as an alternative initiation codon for the serpin endopin 2B-2.

This study demonstrates GTG as a novel, alternative initiation codon for translation of bovine endopin 2B-2, a serpin protease inhibitor. Molecular cDNA cloning revealed the endopin 2B-1 and endopin 2B-2 isoforms that are predicted to inhibit papain and elastase. Notably, GTG was demonstrated as the initiation codon for endopin 2B-2, whereas endopin 2B-1 possesses ATG as its initiation codon. GTG mediated in vitro translation of 46kDa endopin 2B-2. GTG also mediated translation of EGFP by in vitro translation and by expression in mammalian cells. Notably, mutagenesis of GTG to GTC resulted in the absence of EGFP expression in cells. GTG produced a lower level of protein expression compared to ATG. The use of GTG as an initiation codon to direct translation of endopin 2B, as well as the heterologous protein EGFP, demonstrates the role of GTG in the regulation of mRNA translation in mammalian cells. Significantly, further analyses of mammalian genomes based on GTG as an alternative initiation codon may predict new candidate gene products expressed by mammalian and human genomes.

Demonstration of GTG as an endogenous initiation codon for a human mRNA transcript revealed by molecular cloning of the serpin endopin 2B.

This study demonstrates utilization of the novel GTG initiation codon for translation of a human mRNA transcript that encodes the serpin endopin 2B, a protease inhibitor. Molecular cloning revealed the nucleotide sequence of the human endopin 2B cDNA. Its deduced primary sequence shows high homology to bovine endopin 2A that possesses cross-class protease inhibition of elastase and papain. Notably, the human endopin 2B cDNA sequence revealed GTG as the predicted translation initiation codon; the predicted translation product of 46 kDa endopin 2B was produced by in vitro translation of 35S-endopin 2B with mammalian (rabbit) protein translation components. Importantly, bioinformatic studies demonstrated the presence of the entire human endopin 2B cDNA sequence with GTG as initiation codon within the human genome on chromosome 14. Further evidence for GTG as a functional initiation codon was illustrated by GTG-mediated in vitro translation of the heterologous protein EGFP, and by GTG-mediated expression of EGFP in mammalian PC12 cells. Mutagenesis of GTG to GTC resulted in the absence of EGFP expression in PC12 cells, indicating the function of GTG as an initiation codon. In addition, it was apparent that the GTG initiation codon produces lower levels of translated protein compared to ATG as initiation codon. Significantly, GTG-mediated translation of endopin 2B demonstrates a functional human gene product not previously predicted from initial analyses of the human genome. Further analyses based on GTG as an alternative initiation codon may predict new candidate genes of the human genome.

Novel chromaffin granule serpins, endopin 1 and endopin 2: endogenous protease inhibitors with distinct target protease specificities.

Endopin 1 and endopin 2 represent two novel serpin protease inhibitors localized within chromaffin granules, secretory vesicles of adrenomedullary chromaffin cells that represent a model neuroendocrine cell for synthesis and secretion of peptide neurotransmitters. This chapter describes the molecular features of the primary sequences of endopin 1 and endopin 2 that provided prediction of their distinct target protease specificities. Endopin 1 inhibits trypsin that cleaves at basic residues. In contrast, endopin 2 possesses cross-class inhibition of papain and elastase that represent cysteine and serine proteases, respectively. Cell biological studies indicate that endopin 1 and endopin 2 are localized within chromaffin granules. These results implicate endopin 1 inhibition in vivo of trypsin-like proteases in secretory vesicles, and endopin 2 inhibition of papain- or elastase-like proteases. Indeed, endopin 2 inhibits the endogenous cysteine protease PTP (prohormone thiol protease), present in chromaffin granules, that participates in the proteolytic processing of proenkephalin. These findings indicate the presence of endogenous endopin 1 and endopin 2 in secretory vesicle function.

Novel secretory vesicle serpins, endopin 1 and endopin 2: endogenous protease inhibitors with distinct target protease specificities.

Secretory vesicles of neuroendocrine cells possess multiple proteases for proteolytic processing of proteins into biologically active peptide components, such as peptide hormones and neurotransmitters. The importance of proteases within secretory vesicles predicts the presence of endogenous protease inhibitors in this subcellular compartment. Notably, serpins represent a diverse class of endogenous protease inhibitors that possess selective target protease specificities, defined by the reactive site loop domains (RSL). In the search for endogenous serpins in model secretory vesicles of neuroendocrine chromaffin cells, the presence of serpins related to alpha1-antichymotrypsin (ACT) was detected by Western blots with anti-ACT. Molecular cloning revealed the primary structures of two unique serpins, endopin 1 and endopin 2, that possess homology to ACT. Of particular interest was the observation that distinct RSL domains of these new serpins predicted that endopin 1 would inhibit trypsin-like serine proteases cleaving at basic residues, and endopin 2 would inhibit both elastase and papain that represent serine and cysteine proteases, respectively. Endopin 1 showed selective inhibition of trypsin, but did not inhibit chymotrypsin, elastase, or subtilisin. Endopin 2 demonstrated cross-class inhibition of the cysteine protease papain and the serine protease elastase. Endopin 2 did not inhibit chymotrypsin, trypsin, plasmin, thrombin, furin, or cathepsin B. Endopin 1 and endopin 2 each formed SDS-stable complexes with target proteases, a characteristic property of serpins. In neuroendocrine chromaffin cells from adrenal medulla, endopin 1 and endopin 2 were both localized to secretory vesicles. Moreover, the inhibitory activity of endopin 2 was optimized under reducing conditions, which required reduced Cys-374; this property is consistent with the presence of endogenous reducing agents in secretory vesicles in vivo. These new findings demonstrate the presence of unique secretory vesicle serpins, endopin 1 and endopin 2, which possess distinct target protease selectivities. Endopin 1 inhibits trypsin-like proteases; endopin 2 possesses cross-class inhibition for inhibition of papain-like cysteine proteases and elastase-like serine proteases. It will be of interest in future studies to define the endogenous protease targets of these two novel secretory vesicle serpins.

The novel serpin endopin 2 demonstrates cross-class inhibition of papain and elastase: localization of endopin 2 to regulated secretory vesicles of neuroendocrine chromaffin cells.

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.

Comparison of huntingtin proteolytic fragments in human lymphoblast cell lines and human brain.

Proteolytic fragments of huntingtin (htt) in human lymphoblast cell lines from HD and control cases were compared to those in human HD striatal and cortical brain regions, by western blots with epitope-specific antibodies. HD lymphoblast cell lines were heterozygous and homozygous for the expanded CAG triplet repeat mutations, which represented adult onset and juvenile HD. Lymphoblasts contained NH(2)- and COOH-terminal htt fragments of 20-100 kDa, with many similar htt fragments in HD compared to control lymphoblast cell lines. Detection of htt fragments in a homozygous HD lymphoblast cell line demonstrated proteolysis of mutant htt. It was of interest that adult HD lymphoblasts showed a 63-64 kDa htt fragment detected by the NH(2)-domain antibody, which was not found in controls. In addition, control and HD heterozygous cells showed a common 60-61 kDa band (detected by the NH(2)-domain antibody), which was absent in homozygous HD lymphoblast cells. These results suggest that the 63-64 kDa and 60-61 kDa NH(2)-domain htt fragments may be associated with mutant and normal htt, respectively. In juvenile HD lymphoblasts, the presence of a 66-kDa, instead of the 63-64 kDa N-domain htt fragment, may be consistent with the larger polyglutamine expansion of mutant htt in the juvenile case of HD. Lymphoblasts and striatal or cortical regions from HD brains showed similarities and differences in NH(2)- and COOH-terminal htt fragments. HD striatum showed elevated levels of 50 and 45 kDa NH(2)-terminal htt fragments [detected with anti(1-17) serum] compared to controls. Cortex from HD and control brains showed similar NH(2)-terminal htt fragments of 50, 43, 40, and 20 kDa; lymphoblasts also showed NH(2)-terminal htt fragments of 50, 43, 40, and 20 kDa. In addition, a 48-kDa COOH-terminal htt band was elevated in HD striatum, which was also detected in lymphoblasts. Overall, results demonstrate that mutant and normal htt undergo extensive proteolysis in lymphoblast cell lines, with similarities and differences compared to htt fragments observed in HD striatal and cortical brain regions. These data for in vivo proteolysis of htt are consistent with the observed neurotoxicity of recombinant NH(2)-terminal mutant htt fragments expressed in transgenic mice and in transfected cell lines that may be related to the pathogenesis of HD.