Anahit V. Azaryan

Proteases and the emerging role of protease inhibitors in prohormone processing.

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.—Hook, V. Y. H., Azaryan, A. V., Hwang, S.‐R., Tezapsidis, N. Proteases and the emerging role of protease inhibitors in prohormone processing. FASEB J. 8: 1269‐1278; 1994.

Effect of Chronic Cocaine Treatment on μ- and δ-Opioid Receptor mRNA Levels in Dopaminergically Innervated Brain Regions

Abstract: The regulation of μ‐(MOR) and δ‐opioid receptor (DOR) after chronic cocaine administration has been studied. Male Sprague‐Dawley rats were treated for 3 days with saline and cocaine (50 mg/kg/day) delivered by osmotic minipump. Expression of MOR and DOR mRNA in olfactory bulb, nucleus accumbens, and caudate‐putamen (caudal and rostral parts) was estimated using quantitative competitive PCR assays after reverse transcription. No changes in the levels of mRNA for DOR were detected after exposure to cocaine in the brain regions examined. A significant increase in the level of MOR mRNA was detected in nucleus accumbens after 3 days of cocaine treatment. In caudate‐putamen and olfactory bulb, no change in MOR mRNA was observed after cocaine administration. Both SCH 23390 and eticlopride, selective antagonists of D1‐ and D2‐dopamine receptors, respectively, blocked this cocaine‐induced up‐regulation of MOR mRNA in nucleus accumbens. We suggest that endogenous opioid systems in nucleus accumbens, the brain region specifically associated with the reinforcing properties of addictive drugs, are regulated by dopaminergic mechanisms and influenced by cocaine treatment.

Unique cleavage specificity of ‘prohormone thiol protease’ related to proenkephalin processing

‘Prohormone thiol protease’ (PTP) represents the major enkephalin precursor processing activity in chromaffin granules. In this study, cleavage specificity of PTP for paired basic and monobasic residues was examined with a series of model peptide‐MCA (‐methylcoumarinamide) substrates. Monobasic peptides were cleaved at the COOH‐ and NH2‐terminal sides of the single basic residue. Dibasic peptides, however, were preferentially cleaved at the NH2‐terminal side of the pair, or between the two basic residues, with low cleavage at the COOH‐terminal side of the pair. Inhibition by the peptide inhibitor (d‐Tyr)‐Glu‐Phe‐Lys‐Arg‐CH2Cl provided further evidence for ptps specificity for the dibasic Lys‐Arg site. Inhibition by Z‐Leu‐Val‐Gly‐CHN2; and Z‐Arg‐Leu‐Val‐Gly‐CHN2; suggests involvement of Val‐Gly in substrate binding to PTP; these two cystatin C‐related inhibitors also indicate PTP as a cysteine protease. These results demonstrate PTPs unique cleavage specificity that differs from other processing endopeptidases, including the subtilisin‐related proprotein convertases, PC1/PC3, and PC2, as well as the pituitary proopiomelanocortin‐converting enzyme, PCE. This study provides further evidence for PTP as a novel prohormone processing enzyme that belongs to the class of cysteine proteases.

Mu opioid receptor mRNA in nucleus accumbens is elevated following dopamine receptor activation

We have previously demonstrated that continuous cocaine treatment for three days induces a marked but transient increase in mu opioid receptor (MOR) mRNA in nucleus accumbens (n. acc.); SCH 23390 and eticlopride, selective antagonists of D1- and D2-like dopamine (DA) receptors, respectively, blocked this cocaine-induced upregulation of MOR mRNA in n. acc. suggesting involvement of both subfamilies of DA receptors in the effect of cocaine (1,2). In the present study the ability of the selective DA D3 receptor antagonist, nafadotride (3,4), to prevent the cocaine-induced upregulation of MOR mRNA in n. acc. has been examined. Also, regulation of MOR mRNA following chronic administration of the DA agonists, SKF 38393, R(+)-6-Bromo-APB hydrobromide, or bromocriptine, has been studied. Male Sprague-Dawley rats were treated for 3 days with saline, cocaine, the DA receptor agonists or antagonistsdelivered by osmotic minipump. Expression of MOR mRNA in n. acc. was estimated by quantitative competitive polymerase chain reaction (PCR) assays following reverse transcription. Nafadotride (1.0 mg/kg/day) prevented the cocaine-induced upregulation of MOR mRNA in n. acc. When administered alone, nafadotride did not change the expression of MOR mRNA. The levels of MOR mRNA were elevated in n. acc. after 3 days treatment with each of the DA agonists, SKF 38393 (4.0 mg/kg/day), R(+)-6-Bromo-APB hydrobromide (4.0 mg/kg/day), or bromocriptine (5.0 mg/kg/day). Thus, DA agonists mimick the effect of cocaine on the expression of MOR mRNA in n. acc. These data confirm the involvement of dopaminergic mechanisms in the mediation of cocaine effects, indicate the comparability of actions of indirect and direct DA agonists, and point to the usefulness of cocaine as a tool to expose interaction between dopaminergic and opioid systems. The results suggest that activation of more than one type of DA receptor is required for the increased expression of MOR mRNA.

Characteristics of the Chromaffin Granule Aspartic Proteinase Involved in Proenkephalin Processing

Abstract: Proteolytic processing of neuropeptide precursors is required for production of active neurotransmitters and hormones. In this study, a chromaffin granule (CG) aspartic proteinase of 70 kDa was found to contribute to enkephalin precursor cleaving activity, as assayed with recombinant ([35S]Met)preproenkephalin. The 70‐kDa CG aspartic proteinase was purified by concanavalin A‐Sepharose, Sephacryl S‐200, and pepstatin A agarose affinity chromatography. The proteinase showed optimal activity at pH 5.5. It was potently inhibited by pepstatin A, a selective aspartic proteinase inhibitor, but not by inhibitors of serine, cysteine, or metalloproteinases. Lack of inhibition by Val‐d‐Leu‐Pro‐Phe‐Val‐d‐Leu—an inhibitor of pepsin, cathepsin D, and cathepsin E—distinguishes the CG aspartic proteinase from classical members of the aspartic proteinase family. The CG aspartic proteinase cleaved recombinant proenkephalin between the Lys172‐Arg173 pair located at the COOH‐terminus of (Met)enkephalin‐Arg6‐Gly7‐Leu8, as assessed by peptide microsequencing. The importance of full‐length prohormone as substrate was demonstrated by the enzymes ability to hydrolyze 35S‐labeled proenkephalin and proopiomelanocortin and its inability to cleave tri‐ and tetrapeptide substrates containing dibasic or monobasic cleavage sites. In this study, results provide evidence for the role of an aspartic proteinase in proenkephalin and prohormone processing.

The presence of ATP + ubiquitin-dependent proteinase and multicatalytic proteinase complex in bovine brain

The presence of two distinct high-molecular-weight proteases with similar pH optima in the weakly alkaline region was shown in cytosol of the bovine brain cortex. They were separated by ammonium sulfate fractionation and each was further purified by DEAE-Sephacel Sephacryl S-300, DEAE-Cibacron Blue 3GA-agarose, heparin-agarose, and Sepharose 6B chromatography. The larger enzyme (Mr 1,400 kDa), which precipitates at 0–38% ammonium sulfate saturation, seems to be active in ATP+ubiquitin (Ub)-dependent proteolysis; it has low basal caseinolytic activity that is stimulated 3-fold by ATP, and when Ub is present ATP causes a 4.5-fold stimulation. A second proteinase was also found to be present (Mr 700 kDa) that precipitates at 38–80% ammonium sulfate saturation, is composed of multiple subunits ranging in Mr from 18 to 30 kDa, and degrades both protein and peptide substrates, demonstrating trypsin-, chymotrypsin- and cucumisin-like activities. Catalytic, biochemical, and immunological characteristics of this proteinase indicate that it is a multicatalytic proteinase complex (MPC), whose enzyme activity, in contrast to that of MPC from bovine pituitaries (1–3), is stimulated 1.7-fold by addition of ATP in the absence of ubiquitin at the early steps of purification; this property is lost during the course of further purification. Both proteinases are present in the nerve cells, since the primary chicken embryonic telencephalon neuronal cell culture extracts contain both ATP+Ub-dependent proteinase and MPC activities.

Action of brain cathepsin B, cathepsin D, and high-molecular-weight aspartic proteinase on angiotensins I and II.

The action of three previously isolated electrophoretically homogeneous brain proteinases—cathepsin B (EC 3.4.22.1), cathepsin D (EC 3.4.23.5), and high-molecular-weight aspartic proteinase (Mr=90K; EC 3.4.23.−)—on human angiotensins I and II has been investigated. The products of enzymatic hydrolysis have been identified by thin-layer chromatography on Silufol plates using authentic standards and by N-terminal amino acid residue analysis using a dansyl chloride method. Cathepsin D and high-molecular-weight aspartic proteinase did not split angiotensin I or angiotensin II. Cathepsin B hydrolyzed angiotensin I via a dipeptidyl carboxypeptidase mechanism removing His-Leu to form angiotensin II, and it degraded angiotensin II as an endopeptidase at the Val3-Tyr4 bond. Cathepsin B did not split off His-Leu from Z-Phe-His-Leu. Brain cathepsin B may have a role in the generation and degradation of angiotensin II in physiological conditions.

Proenkephalin-processing enzymes in chromaffin granules: model for neuropeptide biosynthesis.

Our discovery of precursor preference of processing enzymes indicates possible development of future drugs that target specific proteases uniquely associated associated with processing of a particular prohormone. For example, selective processing of PE by the PTP suggests that future evaluation of modulation of PTP through central nervous system drug reagents may modify the endogenous analgesic effects of the enkephalins. With respect to blood pressure, neuropeptide Y (NPY) that is released from sympathetic nerve terminals is a strong vasoconstrictor. Our finding that only PTP (not PC1/3, PC2, or the aspartic proteinase) possesses the ability to convert pro-NPY to NPY suggests that investigation of inhibitors of peripheral PTP in blood pressure regulation should be initiated. Overall, elucidation of the proteolytic components required in prohormone processing will provide insights into the molecular mechanisms of human disease.

Kex2-like proteolytic activity in adrenal medullary chromaffin granules

This study demonstrates the presence of boc-Gln-Arg-Arg-MCA cleaving activity in bovine chromaffin granule membranes that resembles yeast Kex2 proteolytic activity. The chromaffin granule boc-Gln-Arg-Arg-MCA cleaving activity, like Kex2 proteolytic activity, shows calcium dependence, optimum activity at pH 7.5-8.2, inhibition by serine protease inhibitors, and preference for cleavage at the COOH-terminal side of Arg-Arg and Lys-Arg, over Lys-Lys, paired basic residues. Potent inhibition by the active-site directed inhibitor [D-Tyr]-Glu-Phe-Lys-Arg-CK (20 microM) provided further evidence for dibasic residue cleavage site specificity. These results are the first report of endogenous mammalian Kex2-like proteolytic activity that may be related to PC1/PC3 and PC2 enzymes, the newly discovered mammalian homologues of Kex2 protease. It will be important to determine the role of this Kex2-like proteolytic activity in processing the precursors of adrenal medullary neuropeptides.

Some properties of human and bovine brain cathepsin B

Cathespin B has been purified 750-fold to apparent homogeneity from human and bovine brain cortex using ammonium sulfate fractionation (30–70%), chromatography on Sephadex G-100, CM-Sephadex C-50, and concanavalin A-Sepharose. Enzyme was assayed fluorometrically at pH 4.0 with pyridoxyl-hemoglobin in the presence of 1 mM DTT and 1 mM EDTA. Properties of the enzyme from the two sources proved to be similar. On disc PAGE the purified preparation produced two bands associated with proteinase activity that are due to existence of two multiple forms of brain cathepsin B with pI 6.1 and 6.8. The enzyme is completely inactivated by thiol-blocking reagents, leupeptin, E-64, and demands thiol compounds for its ultimate activity. Z-Phe-Ala-CHN2 is a potent inhibitor of the enzyme (K2nd=1280 M−1s−1) in contrast to Z-Phe-Phe-CHN2 (K2nd=264 M−1s−1). pH optimum in the reaction of hydrolysis of Pxy-Hb is 4.0–6.0,KM(app.) =10−5 M. Cathepsin B splits azocasein: pH optimum 5.0–6.0,KM(app.)=2.2·10−5 M, but inclusion of urea in the incubation medium depresses the azocaseinolytic activity of the enzyme 1.5-fold. It does not split Lys-NNap, Arg-NMec and is not inhibited by bestatin. The specific activity of brain cathepsin B with Z-Arg-Arg-NNapOMe at pH 6.0 is 10-fold higher than with Bz-Arg-NNap, Z-Gly-Gly-Arg-NNap is a poor substrate. With Z-Arg-Arg-NMec and Bz-Phe-Val-Arg-NMec the specific acitivity is 80 and 35%, respectively of that with Z-Phe-Arg-NMec.