Tissue-specific proteolysis of Huntingtin (htt) in human brain: evidence of enhanced levels of N- and C-terminal htt fragments in Huntington's disease striatum.

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 domain-specific 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.

Formation of the catecholamine release-inhibitory peptide catestatin from chromogranin A. Determination of proteolytic cleavage sites in hormone storage granules.

The catestatin fragment of chromogranin A is an inhibitor of catecholamine release, but its occurrence in vivo has not yet been verified, nor have its precise cleavage sites been established. Here we found extensive processing of catestatin in chromogranin A, as judged by catestatin radioimmunoassay of size-fractionated chromaffin granules. On mass spectrometry, a major catestatin form was bovine chromogranin A(332-364); identity of the peptide was confirmed by diagnostic Met(346) oxidation. Further analysis revealed two additional forms: bovine chromogranin A(333-364) and A(343-362). Synthetic longer (chromogranin A(332-364)) and shorter (chromogranin A(344-364)) versions of catestatin each inhibited catecholamine release from chromaffin cells, with superior potency for the shorter version (IC(50) approximately 2.01 versus approximately 0.35 microm). Radioimmunoassay demonstrated catestatin release from the regulated secretory pathway in chromaffin cells. Human catestatin was cleaved in pheochromocytoma chromaffin granules, with the major form, human chromogranin A(340-372), bounded by dibasic sites. We conclude that catestatin is cleaved extensively in vivo, and the peptide is released by exocytosis. In chromaffin granules, the major form of catestatin is cleaved at dibasic sites, while smaller carboxyl-terminal forms also occur. Knowledge of cleavage sites of catestatin from chromogranin A may provide a useful starting point in analysis of the relationship between structure and function for this peptide.

Molecular cloning of endopin 1, a novel serpin localized to neurosecretory vesicles of chromaffin cells. Inhibition of basic residue-cleaving proteases by endopin 1.

Serpins represent a diverse class of endogenous protease inhibitors that regulate important biological functions. In consideration of the importance of regulated proteolysis within secretory vesicles for the production of peptide hormones and neurotransmitters, this study revealed the molecular identity of a novel serpin, endopin 1, that is localized to neurosecretory vesicles of neuropeptide-containing chromaffin cells (chromaffin granules). Endopin 1 of 68-70 kDa was present within isolated chromaffin granules. Stimulated cosecretion of endopin 1 with chromaffin granule components, [Met]enkephalin and a cysteine protease known as "prohormone thiol protease," demonstrated localization of endopin 1 to functional secretory vesicles. Punctate, discrete immunofluorescence cellular localization of endopin 1 in chromaffin cells was consistent with its secretory vesicle localization. Endopin 1 contains a unique reactive site loop with Arg as the predicted P1 residue, suggesting inhibition of basic residue-cleaving proteases; indeed, trypsin was potently inhibited (K(i(app)) of 5 nM), and plasmin was moderately inhibited. Although endopin 1 possesses homology with alpha(1)-antichymotrypsin, chymotrypsin was not inhibited. Moreover, endopin 1 inhibited the chromaffin granule prohormone thiol protease (involved in proenkephalin processing). These results suggest a role for the novel serpin, endopin 1, in regulating basic residue-cleaving proteases within neurosecretory vesicles of chromaffin cells.

Evidence for functional localization of the proenkephalin-processing enzyme, prohormone thiol protease, to secretory vesicles of chromaffin cells.

The biosynthesis of enkephalin opioid neuropeptides as well as numerous peptide hormones and neurotransmitters requires proteolytic processing of the respective prohormone precursors. We previously identified a novel cysteine protease known as prohormone thiol protease (PTP) as the major proenkephalin-processing enzyme in chromaffin granules (secretory vesicles) of bovine adrenal medulla. In this study, colocalization of PTP with (Met)enkephalin in regulated secretory vesicles was assessed by immunochemical approaches. Western blots demonstrated the presence of PTP in chromaffin granules, with equivalent levels of PTP protein in the soluble and membrane components of the vesicle. The presence of PTP in pituitary was also demonstrated by immunoblots. Immunoelectron microscopy demonstrated immunogold-labeled PTP and (Met)enkephalin within isolated chromaffin granules. In primary cultures of chromaffin cells, the discrete pattern of PTP and (Met)enkephalin immunofluorescence staining in neuritic extensions and cytoplasmic (perinuclear) regions of chromaffin cells is consistent with localization to secretory vesicles. Moreover, cosecretion of PTP and (Met)enkephalin from chromaffin cells occurred upon KCl depolarization in a calcium-dependent manner, indicating the localization of PTP and (Met)enkephalin within regulated secretory vesicles. Calcium-dependent secretion is a well known property of regulated secretory vesicle exocytosis. Overall, these results are consistent with the localization of PTP to functional, regulated secretory vesicles that contain (Met)enkephalin.

Evidence for the proenkephalin processing enzyme prohormone thiol protease (PTP) as a multicatalytic cysteine protease complex: activation by glutathione localized to secretory vesicles.

The cysteine protease known as "prohormone thiol protease" (PTP) has been identified as a major proenkephalin processing enzyme in secretory vesicles of adrenal medulla (known as chromaffin granules). This study provides the first demonstration that PTP exists as a multicatalytic cysteine protease complex that can be activated by endogenous glutathione present in chromaffin granules. The high molecular mass nature of PTP, of approximately 185 kDa, was demonstrated by elution of a single peak of 35S-enkephalin precursor cleaving activity by Sephacryl S200 gel filtration chromatography and by a single band of 35S-enkephalin precursor cleaving activity detected on radiozymogram gels under native buffer conditions. Importantly, when 0.1% SDS was included in radiozymogram gels, PTP activity was resolved into three bands of proteolytic activity with apparent molecular masses of 88, 81, and 61 kDa. These activities were all cysteine proteases, since they were inhibited by the cysteine protease inhibitor E-64c but not by pepstatin A or EDTA that inhibit aspartyl protease and metalloprotease, respectively. Purification of native PTP by preparative gel electrophoresis indicated that PTP was composed of four polypeptides of 66, 60, 33, and 29 kDa detected on SDS-PAGE gels. These four protein subunits accounted for the three catalytic activities of PTP, as demonstrated on 35S-enkephalin precursor radiozymogram gels. Results also indicated that the electrophoretic mobilities of the four subunits differed under reducing compared to nonreducing conditions. The multicatalytic activities of the PTP complex all require reducing conditions for activity, which can be provided by endogenous reduced glutathione in chromaffin granules. These novel findings provide the first evidence for a role of a multicatalytic cysteine protease complex, PTP, in chromaffin granules that may be involved in the proteolytic processing of proenkephalin and perhaps other precursors into active neuropeptides.

The kunitz protease inhibitor form of the amyloid precursor protein (KPI/APP) inhibits the proneuropeptide processing enzyme prohormone thiol protease (PTP). Colocalization of KPI/APP and PTP in secretory vesicles.

Proteolytic processing of proenkephalin and proneuropeptides is required for the production of active neurotransmitters and peptide hormones. Variations in the extent of proenkephalin processing in vivo suggest involvement of endogenous protease inhibitors. This study demonstrates that "protease nexin 2 (PN2)," the secreted form of the kunitz protease inhibitor (KPI) of the amyloid precursor protein (APP), potently inhibited the proenkephalin processing enzyme known as prohormone thiol protease (PTP), with a Ki,app of 400 nM. Moreover, PTP and PN2 formed SDS-stable complexes that are typical of kunitz protease inhibitor interactions with target proteases. In vivo, KPI/APP (120 kDa), as well as a truncated form of KPI/APP that resembles PN2 in apparent molecular mass (110 kDa), were colocalized with PTP and (Met)enkephalin in secretory vesicles of adrenal medulla (chromaffin granules). KPI/APP (110-120 kDa) was also detected in pituitary secretory vesicles that contain PTP. In chromaffin cells, calcium-dependent secretion of KPI/APP with PTP and (Met)enkephalin demonstrated the colocalization of these components in functional secretory vesicles. These results suggest a role for KPI/APP inhibition of PTP in regulated secretory vesicles. In addition, these results are the first to identify an endogenous protease target of KPI/APP, which is developmentally regulated in aging and Alzheimer's disease.

Hemostatic factors in rabbit limb lymph: relationship to mechanisms regulating extravascular coagulation.

Mechanisms regulating extravascular coagulation in interstitial fluids of peripheral tissues are poorly understood, since measurements of hemostatic factors in these fluids are unavailable. Because lymph from a body region reflects the composition of its interstitial fluid, we measured hemostatic factors in limb lymph of rabbits both as activity and as antigen. Mean lymph-to-plasma activity ratios were the following: fibrinogen, 0.28; prothrombin, 0.26; factor X, 0.27; factor VII, 0.17; and factors V and VIII, 0.08. All lymph fibrinogen was clottable; fibrin degradation products were absent. Lymph von Willebrand factor antigen was < 10% of plasma antigen and consisted primarily of lower molecular weight multimers. Mean lymph-to-plasma activity ratio for antithrombin was 0.38 and for tissue factor pathway inhibitor the ratio was 0.40. Low levels of antithrombin-factor Xa were measurable in lymph. The data are compatible with a basal factor VIIa-tissue factor-catalyzed extravascular activation of factor X that is prevented from progressing to generation of fibrin in limb interstitial fluid and lymph by low levels of factor VIII and factor V and by the inhibitory activity of antithrombin and tissue factor pathway inhibitor.

Release of nontransmembrane full-length Alzheimer's amyloid precursor protein from the lumenar surface of chromaffin granule membranes.

We previously demonstrated the presence of a soluble form of full-length Alzheimer's amyloid precursor protein (APP) in the lumen of adrenal medullary chromaffin granules (CG). Furthermore, full-length APP is released from CG membranes in vitro at pH 9.0 by an enzymatic mechanism, sensitive to protease inhibitors [Vassilacopoulou et al. (1995) J. Neurochem. 64, 2140-2146]. In this study, we found that when intact CG were subjected to exogenous trypsin, a fraction of APP was not digested, consistent with an intragranular population of APP. To examine the substrate-product relationship between membrane and soluble full-length APP, we labeled CG transmembrane APP with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID), a lipophilic probe, specific for membrane-spanning domains of proteins. APP released from the membranes at pH 9.0 was not labeled with [125I]TID. In addition, this APP was not biotinylated in intact CG. Combined, the results indicate that APP released from CG membranes derives from a unique nontransmembrane population of membrane-associated APP, located in the lumenal side of CG membranes. Dithiobis(succinimidylpropionate) (DSP) cross-linking indicated that APP in CG is situated in close proximity with other proteins, possibly with APP itself. APP complexes were also detected under nonreducing conditions, without DSP cross-linking. These results, combined with our previous studies, indicate that full-length APP within CG exists as three different populations: (I) transmembrane, (II) membrane-associated/nontransmembrane, and (III) soluble. The existence of nontransmembrane populations suggests that putative gamma-secretase cleavage sites of APP, assumed to be buried within the lipid bilayer, could be accessible to proteolysis in a soluble intravesicular environment.

Arginine and lysine aminopeptidase activities in chromaffin granules of bovine adrenal medulla: relevance to prohormone processing.

Conversion of prohormones and neuropeptide precursors to smaller, biologically active peptides requires specific proteolytic processing at paired basic residues, which generates intermediate peptides with NH2 and COOH termini extended with Lys or Arg residues. These basic residues are then removed by aminopeptidase and carboxypeptidase activities, respectively. Among the proteases involved in prohormone processing, the basic residue aminopeptidase activity has not been well studied. This report demonstrates arginine and lysine aminopeptidase activities detected with Arg-methylcoumarinamide (Arg-MCA) and Lys-MCA substrates in neurosecretory vesicles of bovine adrenal medulla [chromaffin granules (CG)], which contain endoproteolytic processing enzymes co-localized with [Met]enkephalin and other neuropeptides. These arginine and lysine aminopeptidase activities showed many similarities and some differences. Both arginine and lysine aminopeptidase activities were stimulated by the reducing agent beta-mercaptoethanol (beta-ME) and inhibited by p-hydroxymercuribenzoate, suggesting involvement of reduced cysteinyl residues. The arginine aminopeptidase activity was stimulated by NaCl (150 mM), but the lysine aminopeptidase activity was minimally affected. Moreover, characteristic beta-ME/NaCl-stimulated Arg-MCA cleaving activity and beta-ME-stimulated Lys-MCA cleaving activity were detected only in CG and not in other subcellular fractions; these findings indicate the localization of these particular basic residue aminopeptidase activities to secretory vesicles. The arginine and lysine aminopeptidase activities showed pH optima at 6.7 and 7.0, respectively. Km(app) values for the arginine and lysine aminopeptidase activities were 104 and 160 microM, respectively. Inhibition by the aminopeptidase inhibitors bestatin, amastatin, and arphamenine was observed for Arg-MCA and Lys-MCA cleaving activities. Inhibition by the metal ion chelators indicated that metalloproteases were involved; Co2+ stimulated the arginine aminopeptidase activity but was less effective in stimulating lysine aminopeptidase activity. In addition, the lysine aminopeptidase activity was partially inhibited by Ni2+ and Zn2+ (1 mM), whereas the arginine aminopeptidase activity was minimally affected. These results demonstrate the presence of related arginine and lysine thiol metalloaminopeptidase activities in CG that may participate in prohormone processing.

The effect of immunodepletion of antithrombin III on the response of rabbits to Russell's viper venom-induced activation of factor X.

Many years ago it was shown that an infusion of tissue factor (TF) into rabbits causing only limited consumption of factor X and prothrombin resulted in extensive consumption of fibrinogen. More recently it was shown that an injection of a concentration of the factor X-activating fraction of Russell's viper venom (RVV-X) depleting rabbits of factor X resulted in only minimal consumption of both plasma prothrombin and fibrinogen. We report here experiments in which rabbits depleted of antithrombin III (ATIII) to different degrees were infused over 4 hours with a concentration of RVV-X, causing consumption of about 60% of plasma factor X. Similar minimal mean falls in plasma prothrombin and fibrinogen levels were observed in control rabbits given nonimmune goat IgG and in rabbits immunodepleted with goat anti-rabbit ATIII IgG to about 40% of normal plasma ATIII activity. However, if rabbits were immunodepleted to about 10% to 20% of normal plasma ATIII, then mean consumption of prothrombin was increased modestly and, more impressively, mean consumption of plasma fibrinogen was increased markedly. Whereas limited amounts of thrombin generated on the surface of phospholipid vesicles by factor VIIa/ TF can trigger extensive intravascular coagulation in rabbits with normal plasma ATIII levels, limited amounts of thrombin generated by reactions triggered by factor Xa formed in fluid phase did so only after plasma ATIII levels were markedly depleted. A possible reason for this difference is discussed.