Microscopic structure of the iliac artery in the rabbit.
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Animals↗
Biomedical subjects
S Wilk
Publications and source records attributed to S Wilk.
Aminoacylpyrrolidine-2-nitriles: potent and stable inhibitors of dipeptidyl-peptidase IV (CD 26).
Dipeptidyl-peptidase IV (EC 3.4.14.5) also known as CD26 is a membrane-bound serine peptidase which cleaves N-terminal dipeptides from a peptide chain containing a proline residue in the penultimate position. The enzyme is believed to play an important role in neuropeptide metabolism and T-cell activation. A series of aminoacylpyrrolidine-2-nitriles, in which the carboxyl group of proline is replaced by a nitrile group, was synthesized as inhibitors of dipeptidyl-peptidase IV. All compounds were found to competitively inhibit a homogeneous preparation of the rat kidney enzyme with Ki values in the low to submicromolar range. The nitriles presumably react with the active-site serine to form an imidate adduct. The compounds were stable following incubation either for 20 h at 37 degrees C or 72 h at room temperature. They proved to be poor inhibitors of dipeptidyl-peptidase II and prolyl oligopeptidase. These studies demonstrate that the generally held concept that nitriles are poor inhibitors of serine proteinases needs to be reconsidered. Amino-acylpyrrolidine-2-nitriles by virtue of their ease of synthesis, stability, specificity, and inhibitory potency appear to be superior to other described dipeptidyl-peptidase IV inhibitors.
Animals↗
Serine and cysteine proteinase inhibitors prevent nitric oxide production by activated macrophages by interfering with transcription of the inducible NO synthase gene.
The objective of this study was to ascertain the mechanism by which serine and cysteine proteinase inhibitors interfere with production of NO by LPS-activated rat alveolar macrophages. Macrophages were incubated in the presence of LPS+ test agent for 24 hr. Culture media were analyzed for NOX- accumulation, harvested cells were assayed for iNOS activity, and cellular RNA was extracted for determination of iNOS mRNA by Northern blot analysis. TPCK, TLCK, calpain inhibitor 1 (CPI-1) and calpain inhibitor 2 (CPI-2) each inhibited NOX- production and inducible iNOS expression in a concentration-dependent manner at 1-100 microM. TPCK and CPI-1 were about 10-fold more potent than TLCK and CPI-2, respectively. These data suggest that a chymotrypsin-like serine or cysteine proteinase is required for the LPS-inducible expression of the iNOS gene, perhaps by mechanisms involving activation of transcription factor NF-kappa B. Accordingly, a potent inhibitor of NF-kappa B activation whose action is attributed to inhibition of the chymotrypsin-like activity of the multicatalytic proteinase complex (MPC) was tested. Z-IE(O-t-Bu)A-Leucinal abolished NOX- production and inducible iNOS expression at 1 microM and showed over 50% inhibition at 10 nM. These observations indicate that inhibitors of MPC interfere with iNOS induction and provide strong evidence that MPC functions importantly in iNOS induction in macrophages.
Animals↗
Phosphorylation of human I kappa B-alpha on serines 32 and 36 controls I kappa B-alpha proteolysis and NF-kappa B activation in response to diverse stimuli.
Post-translational activation of the higher eukaryotic transcription factor NF-kappa B requires both phosphorylation and proteolytic degradation of the inhibitory subunit I kappa B-alpha. Inhibition of proteasome activity can stabilize an inducibly phosphorylated form of I kappa B-alpha in intact cells, suggesting that phosphorylation targets the protein for degradation. In this study, we have identified serines 32 and 36 in human I kappa B-alpha as essential for the control of I kappa B-alpha stability and the activation of NF-kappa B in HeLa cells. A point mutant substituting serines 32 and 36 by alanine residues was no longer phosphorylated in response to okadaic acid (OA) stimulation. This and various other Ser32 and Ser36 mutants behaved as potent dominant negative I kappa B proteins attenuating kappa B-dependent transactivation in response to OA, phorbol 12-myristate 13-acetate (PMA) and tumor necrosis factor-alpha (TNF). While both endogenous and transiently expressed wild-type I kappa B-alpha were proteolytically degraded in response to PMA and TNF stimulation of cells, the S32/36A mutant of I kappa B-alpha remained largely intact under these conditions. Our data suggest that such diverse stimuli as OA, TNF and PMA use the same kinase system to phosphorylate and thereby destabilize I kappa B-alpha, leading to NF-kappa B activation.
Base Sequence↗
A novel chymotrypsin-like component of the multicatalytic proteinase complex optimally active at acidic pH.
The multicatalytic proteinase complex (MPC) or proteasome is a multimeric, high-molecular-weight (700,000), extralysosomal proteolytic enzyme found in eukaryotes and in archaebacteria. Its multiple catalytic sites grant it a broad cleavage specificity toward short peptides and protein substrates. The pH optima of the catalytic activities of MPC are in the neutral or slightly alkaline range. We present here evidence for cryptic catalytic components of MPC optimally active at an acidic pH. Studies with a hydrophobic fluorescent probe provide direct evidence for conformational changes brought about by exposing the complex to an acidic environment. One of the newly described components, designated "acidic chymotrypsin-like activity," cleaves the Leu-2-naphthylamide bond in the substrate Boc-Val-Glu-Ala-Leu-2-naphythylamide. Compared with the classical "neutral" chymotrypsin-like activity defined by cleavage of the Leu-p-nitroanilide bond in Z-Gly-Gly-Leu-p-nitroanilide, the newly described component is not inhibited by monovalent cations and is less sensitive to the peptidyl aldehyde Z-Gly-Gly-leucinal, an inhibitor of the neutral chymotrypsin-like activity. In addition, we describe the properties of a novel potent peptidyl aldehyde, Z-Ile-Glu(OtBu)-Ala-leucinal, which is an inhibitor of both the acidic and neutral chymotrypsin-like activities of MPC, with IC50 values of 0.25 and 6.5 microM, respectively. In the presence of 65 microM of the newly synthesized peptidyl aldehyde, other MPC components such as the trypsin-like and peptidyl-glutamyl peptide hydrolyzing activities were decreased only by 14 and 9%, respectively. The hydrophobicity, potency, and specificity of Z-Ile-Glu(OtBu)-Ala-leucinal toward the chymotrypsin-like activities of the complex make it a valuable pharmacological tool with which to investigate the physiological roles of MPC.
Amino Acid Sequence↗
Pyroglutamyl peptidase-II ("thyroliberinase") activity in human serum: influence of weight and thyroid status.
The tripeptide hormone, TRH, is metabolized by three enzymes, the most specific of which is pyroglutamyl peptide hydrolase-II (also termed thyroliberinase), a metalloenzyme present in serum and brain. Because pyroglutamyl peptidase-II activity in rat serum is regulated by thyroid hormone levels, we tested the hypothesis that this activity is similarly altered in humans. We studied serum pyroglutamyl peptidase-II activity in 6 patients with hyperthyroidism, 18 patients with hypothyroidism, and 31 euthyroid, normal weight volunteers. Because TRH [or its metabolite cyclo(His-Pro)] is believed to be an important hormone regulating appetite and metabolism, we also evaluated pyroglutamyl peptidase-II activity in 27 euthyroid patients with obesity. Serum pyroglutamyl peptidase-II activity was elevated in patients with hypothyroidism (mean +/- SEM, 33.9 +/- 3.7 nmol/mL.h) compared to that in euthyroid, normal weight volunteers (24.5 +/- 2.8 nmol/mL.h; P < 0.05), but not that in patients with hyperthyroidism (28.3 +/- 4.1 nmol/mL.h; P = NS). Euthyroid obese patients had the highest pyroglutamyl peptidase-II activity (43.6 +/- 2.8 nmol/mL.h; P < 0.0001 vs. normal weight volunteers). Pyroglutamyl peptidase-II activity was positively correlated with body mass index (r2 = 0.30; P < 0.0001). After correction for body mass index, there were no difference in pyroglutamyl peptidase-II activity in hypothyroid, hyperthyroid, and euthyroid individuals. We conclude that serum pyroglutamyl peptidase-II activity is regulated by, or regulates, body weight.
Adult↗
A proteasome inhibitor prevents activation of NF-kappa B and stabilizes a newly phosphorylated form of I kappa B-alpha that is still bound to NF-kappa B.
Activation of the inducible transcription factor NF-kappa B involves removal of the inhibitory subunit I kappa B-alpha from a latent cytoplasmic complex. It has been reported that I kappa B-alpha is subject to both phosphorylation and proteolysis in the process of NF-kappa B activation. In this study, we present evidence that the multicatalytic cytosolic protease (proteasome) is involved in the degradation of I kappa B-alpha. Micromolar amounts of the peptide Cbz-Ile-Glu(O-t-Bu)-Ala-leucinal (PSI), a specific inhibitor of the chymotrypsin-like activity of the proteasome, prevented activation of NF-kappa B in response to tumor necrosis factor-alpha (TNF) and okadaic acid (OA) through inhibition of I kappa B-alpha degradation. The m-calpain inhibitor Cbz-Leu-leucinal was ineffective. In the presence of PSI, a newly phosphorylated form of I kappa B-alpha accumulated in TNF- and OA-stimulated cells. However, the covalent modification of I kappa B-alpha was not sufficient for activation of NF-kappa B: no substantial NF-kappa B DNA binding activity appeared in cells because the newly phosphorylated form of I kappa B-alpha was still tightly bound to p65 NF-kappa B. Pyrrolidinedithiocarbamate, an antioxidant inhibitor of NF-kappa B activation which did not interfere with proteasome activities, prevented de novo phosphorylation of I kappa B-alpha as well as its subsequent degradation. This suggests that phosphorylation of I kappa B-alpha is equally necessary for the activation of NF-kappa B.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Differential inhibition of aminopeptidase A and aminopeptidase N by new beta-amino thiols.
Aminopeptidase A (APA) is a highly selective peptidase, which cleaves the N-terminal Glu or Asp residues of biologically active peptides, and has therefore been proposed to be involved in angiotensin II and CCK8 metabolism. Highly potent and selective APA inhibitors are consequently required to study the physiological regulation of these two peptides. Using, as a model, Glu-thiol (4-amino-5-mercaptopentanoic acid), which was the first efficient APA inhibitor described but is however equipotent on APA (0.14 microM) and aminopeptidase N (APN) (0.12 microM), several beta-amino thiol inhibitors have been synthesized. In these molecules, the length of the side chain was varied and the carboxylate group of Glu-thiol was replaced by other negatively charged groups, such as phosphonate, sulfonate, hydroxamate, and thiol. The inhibitory potency of one of these compounds, 22h (S)-3-amino-4-mercaptobutanesulfonate, was found to be nearly 100-fold better for APA than for APN, with an affinity (0.29 microM) almost equivalent to that of Glu-thiol. Hence, this compound is the first selective APA inhibitor reported, and as such, it should be an interesting probe to explore the physiological involvement of APA in the metabolism of neuropeptides like angiotensin II and CCK8.
Aminopeptidases↗
Investigation of the active site of aminopeptidase A using a series of new thiol-containing inhibitors.
Aminopeptidase A (APA) and aminopeptidase N (APN) are two metallopeptidases which have been suggested to be involved in the enzymatic cascade of the renin-angiotensin system. APA liberates angotensin III from angiotensin II by releasing the N-terminal aspartate, and APN participates in the inactivation of angiotensin III. As the role of angiotensin III in the regulation of blood pressure in the central nervous system and at the periphery is controversial, it was of interest to develop selective and efficient inhibitors of APA. Starting from Glu-thiol(1), which was the first efficient APA inhibitor described, but however is equipotent on APA (Ki = 0.14 microM) and APN (Ki = 0.12 microM), beta-amino thiols bearing various carboxyalkyl chains have been synthesized and their inhibitory potencies measured on both purified enzymes. Compounds containing a carboxylated aromatic ring inhibited APA and APN with Ki values in the micromolar range but were slightly more active on APA. Conversely, inhibitors containing a cyclohexyl ring were more efficient on APN. Various modifications of the structure of Glu-thiol decreased inhibitory activity on both enzymes but increased the selectivity for APA, and compound 9d ((S)-4-amino-6-mercaptohexanoic acid) was 23 times more potent on APA (Ki = 2.0 microM) than on APN (Ki = 45 microM).
Acetylation↗
Dissociation and reassociation of the bovine pituitary multicatalytic proteinase complex.
The eukaryotic multicatalytic proteinase complex (proteasome) is a high molecular mass enzyme which contains 13-15 nonidentical subunits of similar size (molecular masses of 21-31 kDa), but differing widely in net charge (isoelectric points ranging from 3 to 10). At least four catalytic components termed chymotrypsin-like, trypsin-like, peptidylglutamyl peptide-hydrolyzing, and caseinolytic are associated with the proteinase. The catalytic nature of the components is unknown, since sequences of cloned subunits bear no homology to known proteinases and proteolytically active subunits have not been isolated. Analysis of the relationship between structure and catalytic function would be greatly facilitated if a means for reversibly dissociating and reassociating the proteinase were available. We provide the first evidence of reassembly of dissociated multicatalytic proteinase complex into a functional molecule. Incubation with the organic mercurial, p-chloromercuribenzoic acid disrupts in a concentration-dependent manner the quaternary structure of the enzyme, leading to formation of a heterogeneous population of subunits. Dissociation of the complex coincides with progressive loss of chymotrypsin-like, trypsin-like, and peptidylglutamyl peptide hydrolyzing activities. The caseinolytic activity of the residual undissociated enzyme is markedly activated. Exposure of the dissociated enzyme to dithiothreitol restores the catalytic profile and reassociates the enzyme. Evidence for catalytically active subcomplexes was not obtained indicating that structural integrity may be necessary for expression of all defined activities.
Amino Acid Sequence↗
Comparison of the effect of calpain inhibitors on two extralysosomal proteinases: the multicatalytic proteinase complex and m-calpain.
The potencies of three peptide aldehyde inhibitors of calpain (calpain inhibitors 1 and 2 and calpeptin) as inhibitors of four catalytic activities of the multicatalytic proteinase complex (MPC) were compared with their potencies as inhibitors of m-calpain. The chymotrypsinlike activity (cleavage after hydrophobic amino acids) and the caseinolytic activity (degradation of beta-casein) of MPC were strongly inhibited by calpain inhibitors 1 and 2 (IC50 values in the low micromolar range). Cleavage by MPC after acidic amino acids (peptidylglutamyl-peptide bond hydrolyzing activity) and basic amino acids (trypsinlike activity) was inhibited less effectively, declining moderately with increasing concentrations of calpain inhibitors 1 and 2. Calpeptin only weakly inhibited the four MPC activities, yet was the most potent inhibitor of m-calpain. These results indicate that caution must be exercised when calpain inhibitors 1 and 2 are used to infer calpain function. Calpeptin may be a better choice for such studies, although its effect on other cysteine or serine proteinases remains to be determined.
Amino Acid Sequence↗
A new inhibitor of the chymotrypsin-like activity of the multicatalytic proteinase complex (20S proteasome) induces accumulation of ubiquitin-protein conjugates in a neuronal cell.
Exposure of HT4 cells (a mouse neuronal cell line) to a new potent permeable peptidyl aldehyde inhibitor of the chymotrypsin-like activity of the multicatalytic proteinase complex (MPC) causes accumulation of ubiquitinylated proteins. In contrast, inhibition of calpain or treatment with a lysosomotropic agent failed to produce detectable ubiquitin-protein conjugates. The appearance of such conjugates is not a nonspecific phenomenon because incubation with the peptidyl alcohol analogue of the inhibitor does not produce accumulation of ubiquitinylated proteins. The MPC inhibitor may therefore be a useful tool for identification and study of physiological pathways involving MPC. Furthermore, the inhibitor may help develop a model for the study of neurodegeneration where accumulation of ubiquitin-protein conjugates is commonly detected in abnormal brain inclusions.
Animals↗
Rat kidney glutamyl aminopeptidase (aminopeptidase A): molecular identity and cellular localization.
Glutamyl aminopeptidase [aminopeptidase A (EAP), EC 3.4.11.7] is an ectoenzyme that selectively hydrolyzes acidic amino acid residues from the amino terminus of oligopeptides. EAP activity is highest within the kidney and small intestine. The murine pre-B cell BP-1/6C3 and the human kidney glycoprotein gp160 differentiation antigens have been reported to have biochemical properties indistinguishable from EAP. It is not known, however, if rat kidney EAP is a homologue of these antigens or molecularly distinct. Using the reverse transcription-polymerase chain reaction method with oligonucleotide primers based on the BP-1/6C3 nucleotide sequence, we isolated a 450-bp partial cDNA from rat kidney poly(A)+ RNA. The partial cDNA encoded a predicted protein that was 92% and 86% identical to the murine BP-1/6C3 and human gp160 antigens, respectively; the amino acid sequence within the zinc-binding domain was completely conserved. Purification of EAP from rat kidney and microsequence analysis of a tryptic digest peptide fragment (18-mer) indicated that the fragment was highly similar to a region within the BP-1/6C3 and gp160 proteins. Northern blot hybridization and immunoblot analyses were also consistent with labeling of products the same size as reported for the BP-1/6C3 and gp160 antigens. There was a good correlation between the cellular distribution of EAP mRNA and EAP immunoreactivity, with proximal tubules and glomerular mesangial cells having the highest densities. These results indicate that rat kidney EAP is a species homologue of the murine BP-1/6C3 and human gp160 antigens. Furthermore, on the basis of its cellular localization, rat kidney EAP is likely to be involved in degradation of oligopeptides within the glomerulus and the glomerular filtrate. Since cells that express EAP also express receptors for angiotensin II, an intrarenal vasoactive hormone that is a substrate for EAP, these results further suggest that EAP may play a role in modulating the activity of intrarenal angiotensin II.
Amino Acid Sequence↗
Localization of immunoreactive glutamyl aminopeptidase in rat brain. I. Association with cerebral microvessels.
Glutamyl aminopeptidase (aminopeptidase-A, EC 3.4.11.7) is an ectoenzyme that selectively hydrolyses N-terminal glutamyl and aspartyl residues from oligopeptides, including (Asp1) angiotensin II. Here we sought to determine the distribution of glutamyl aminopeptidase (EAP) in rat brain. EAP was purified to homogeneity from rat kidney and polyclonal antiserum raised in rabbits. Immune serum inhibited EAP enzyme activity in kidney homogenates and labeled two major protein bands of M(r) = 136,000 and M(r) = 101,000 in immunoblots of kidney protein. EAP-like immunoreactivity was concentrated on kidney proximal tubule brush borders. Immunocytochemical staining of rat brain indicated that EAP-like immunoreactivity was primarily associated with cerebral microvessels. Positive staining was detected in microvessels ranging in size from capillaries up to vessels approximately 50 microns in diameter. Isolated cerebral microvessels had a 23-fold enrichment in EAP enzyme activity (193.1 +/- 40.4 nmol/mg protein/h) compared to brain homogenates. Finally, immunoblots of isolated cerebral microvessels resulted in a pattern of labeling similar to that seen with kidney homogenates. These results indicate that EAP activity in brain is primarily associated with cerebral microvessels, and suggest that EAP may be involved in the metabolism of circulating or locally formed peptides.
Aminopeptidases↗
Localization of immunoreactive glutamyl aminopeptidase in rat brain. II. Distribution and correlation with angiotensin II.
Glutamyl aminopeptidase (EAP, EC 3.4.11.7) selectively hydrolyzes N-terminal glutamyl and aspartyl residues from oligopeptides and is present in the brain. (Asp1)Angiotensin II (Ang II) is a substrate for EAP, and increasing evidence suggests that des(Asp1)angiotensin II (Ang III) is an active angiotensin peptide in the brain. To determine whether a relationship exists between EAP and Ang II/III in rat brain, we compared their immunocytochemical distributions. EAP-like immunoreactivity was localized primarily to the adventitial surface of cerebral microvessels throughout the forebrain. Endothelial cells, neurons and glial cells were not labeled. The immunocytochemical staining of microvessel adventitium with EAP antiserum was suggestive of labeling of perivascular pericytes since intravenous horseradish peroxidase resulted in a similar adventitial pattern of staining, in addition to pericyte cell bodies. EAP immunoreactivity was highest within circumventricular organs, areas known to contain high levels of Ang II receptors. Positively stained EAP microvessels were also concentrated in areas containing Ang II/III immunoreactive neurons or nerve terminals, including the hypothalamic paraventricular nucleus and the median eminence. The immunocytochemical localization of EAP suggests that it may be involved in a wide variety of functions within the brain, including: (i) metabolism of circulating peptides in brain areas devoid of a blood-brain barrier, (ii) metabolism of circulating peptides as a component of the blood-brain barrier, (iii) metabolism of intravascularly synthesized peptides, (iv) metabolism of hypothalamic peptides released into the portal circulation, (v) metabolism/conversion of neuronally released Ang II to Ang III in the interstitial space, and (vi) metabolism of neuronally released neuropeptides with vasoactive properties.(ABSTRACT TRUNCATED AT 250 WORDS)
Aminopeptidases↗
Changes in the structure and catalytic activities of the bovine pituitary multicatalytic proteinase complex following dialysis.
The multicatalytic proteinase complex (proteasome) contains at least four distinct active sites catalyzing the degradation of selected chromogenic substrates (trypsin-like, chymotrypsin-like, and peptidylglutamyl peptide hydrolyzing activities) and proteins such as beta-casein. Oxidized insulin B chain was recently proposed as a model substrate for protein degradation by the multicatalytic proteinase complex (Dick, L. R., Moomaw, C. R., DeMartino, G. N., and Slaughter, C. A. (1991) Biochemistry 30, 2725-2734). We studied the dialysis-induced activation of the hydrolysis of oxidized insulin B chain by this enzyme. Removal of EDTA from purified preparations of bovine pituitary multicatalytic proteinase complex by dialysis against Tris-HCl buffers led to marked changes in the catalytic properties and structure of the enzyme. Dialysis produced a time-dependent activation of oxidized insulin B chain hydrolysis with predominant cleavage at the Glu13-Ala14 bond. A new chromogenic assay was developed for measurement of this activity. Activation was accompanied by a virtually total inactivation of the chymotrypsin-like, trypsin-like, and peptidylglutamyl peptide hydrolyzing activities. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a loss of the 24-kDa subunit and the appearance of a new band at 21 kDa. Amino-terminal amino acid analysis established that the 21-kDa band was autolytically derived from the 24-kDa subunit. Evidence for partial dissociation and/or aggregation indicated that autolysis destabilizes the complex. By altering the profile of catalytic activities of the multicatalytic proteinase complex, autolysis may serve as a mechanism for regulation of this macromolecule.
Amino Acid Sequence↗
Assessment of the role of TRH in the release of [3H]-dopamine from rat nucleus accumbens-lateral septum slices.
We have studied [3H]-dopamine ([3H]-DA) release from rat nucleus accumbens lateral septum slices in response to various paradigms aimed at increasing endogenous or exogenous thyrotropin releasing hormone (TRH) concentrations in the extracellular space. High KCl concentrations significantly enhanced [3H]-DA release by fourfold. TRH (10(-4) or 5 x 10(-4) M) did not affect [3H]-DA release. The release of [3H]-DA was not stimulated by TRH either in the presence of N-1-carboxy-2-phenylethyl (N(im)benzyl)-histidyl-beta naphthylamide, a specific pyroglutamyl peptidase II inhibitor, or that of specific inhibitors of prolyl endopeptidase and pyroglutamyl peptidase I. None of the peptidase inhibitors modified the [3H]-DA release by themselves. These results suggest that the TRH stimulation of [3H]-DA release in vitro observed in previous studies is not due to peptide inactivation but may be due to a nonspecific effect. TRH enhancement of DA release in nucleus accumbens in vivo may not be the result of a direct effect of TRH on DA terminals.
Animals↗
Synthetic inhibitors of the multicatalytic proteinase complex (proteasome).
Synthetic inhibitors of the multicatalytic proteinase complex (proteasome) can provide the means to uncover the functional significance and catalytic mechanism of this macromolecule. Although inhibitor development is still in its early stages, some useful compounds have already been prepared. Of the various types of inhibitors thus far studied, peptidyl aldehydes have been the most effective. Since peptidyl aldehydes inhibit both serine and cysteine proteinases, lack of specificity is their major limitation. The properties of one such compound N-benzyloxycarbonyl-IE(Ot-Bu)A-Leucinal, a potent inhibitor of suc-LLVY-MCA hydrolysis, are described in detail.
Amino Acid Sequence↗