Identification of a novel cleavage activity of the first papain-like proteinase domain encoded by open reading frame 1a of the coronavirus Avian infectious bronchitis virus and characterization of the cleavage products.
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The coronavirus Avian infectious bronchitis virus (IBV) employs polyprotein processing as a strategy to express its gene products. Previously we identified the first cleavage event as proteolysis at the Gly(673)-Gly(674) dipeptide bond mediated by the first papain-like proteinase domain (PLPD-1) to release an 87-kDa mature protein. In this report, we demonstrate a novel cleavage activity of PLPD-1. Expression, deletion, and mutagenesis studies showed that the product encoded between nucleotides 2548 and 8865 was further cleaved by PLPD-1 at the Gly(2265)-Gly(2266) dipeptide bond to release an N-terminal 195-kDa and a C-terminal 41-kDa cleavage product. Characterization of the cleavage activity revealed that the proteinase is active on this scissile bond when expressed in vitro in rabbit reticulocyte lysates and can act on the same substrate in trans when expressed in intact cells. Both the N- and C-terminal cleavage products were detected in virus-infected cells and were found to be physically associated. Glycosidase digestion and site-directed mutagenesis studies of the 41-kDa protein demonstrated that it is modified by N-linked glycosylation at the Asn(2313) residue encoded by nucleotides 7465 to 7467. By using a region-specific antiserum raised against the IBV sequence encoded by nucleotides 8865 to 9786, we also demonstrated that a 33-kDa protein, representing the 3C-like proteinase (3CLP), was specifically immunoprecipitated from the virus-infected cells. Site-directed mutagenesis and expression studies showed that a previously predicted cleavage site (Q(2583)-G(2584)) located within the 41-kDa protein-encoding region was not utilized by 3CLP, supporting the conclusion that the 41-kDa protein is a mature viral product. (+info)
Inactivation of cysteine proteases by peptidyl epoxides: characterization of the alkylation sites on the enzyme and the inactivator.
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Erythro peptidyl epoxides are selective inactivators of cysteine proteases. The alkylation site, both on the enzyme papain and on the epoxide itself, was characterized. The inactivation of papain with the peptidyl epoxide erythro benzyloxycarbonyl-Phe-Ala-epoxide was followed by total hydrolysis by acid. Mass spectral analysis of the hydrolysate revealed, in addition to the expected amino acids, a unique signal of m/z 209 (MH(+)). Its high-resolution mass spectrum and daughter peak analysis correspond to the product of alkylation on cysteine and the expected fragmentation. A similar MS pattern was obtained for a synthetic model compound corresponding to the expected hydrolysis product. A (13)C NMR analysis of papain inactivated by a specifically (13)C-labelled peptidyl epoxide indicated that the alkylation of the enzyme's cysteine residue occurs on the primary carbon of the epoxide moiety. (+info)
Selective targeting of lysosomal cysteine proteases with radiolabeled electrophilic substrate analogs.
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BACKGROUND: The lysosomal cysteine proteases of the papain family are some of the best studied proteolytic enzymes. Small-molecule inhibitors and fluorogenic substrate mimics have been used to probe the physiological roles of these proteases. A high degree of homology between family members and overlap in substrate specificity have made elucidating individual protease function, expression and activity difficult. RESULTS: Using peptide vinyl sulfones and epoxide as templates, we have generated probes that can be tagged with radioactive iodine. The resulting compounds covalently label various cathepsins and several unidentified polypeptides likely to be proteases. MB-074 was found to be a highly selective probe of cathepsin B activity. Probes that labeled several cathepsins were used to examine the specificity and cell permeability of the CA-074 family of inhibitors. Although CA-074 reportedly acts in vivo, we find it is unable to penetrate cells. Esterifying CA-074 resulted in a cell-permeable inhibitor with dramatically reduced activity and specificity for cathepsin B. The probes were also used to monitor protease activity in primary human tumor tissue and cells derived from human placenta. CONCLUSIONS: We have generated a highly selective cathepsin B probe and several less specific reagents for the study of cathepsin biology. The reagents have several advantages over commonly used fluorogenic substrates, allowing inhibitor targets to be identified in a pool of total cellular enzymes. We have used the probes to show that cathepsin activity is regulated in tumor tissues and during differentiation of placental-derived cytotrophoblasts to invasive cells required for establishing blood circulation in a developing embryo. (+info)
Angiotensin converting enzyme in human synovium: increased stromal [(125)I]351A binding in rheumatoid arthritis.
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OBJECTIVE: To determine whether tissue angiotensin converting enzyme (ACE) is increased in synovia from patients with rheumatoid arthritis, osteoarthritis or chondromalacia patellae. METHODS: Sections of synovia from patients with rheumatoid arthritis (n = 7), osteoarthritis (n = 7) or chondromalacia patellae (n = 6) were tested for immunoreactivity for ACE, and for binding of the ACE inhibitor [(125)I]351A. The amount of ACE was measured with computer assisted image analysis as the proportion of synovial section area occupied by ACE-immunoreactive cells, and the density of [(125)I]351A binding. RESULTS: [(125)I]351A binding sites had characteristics of ACE and colocalised with ACE-like immunoreactivity to microvascular endothelium and fibroblast-like stromal cells in inflamed and non-inflamed human synovium. Stromal [(125)I]351A binding densities (B(eq)) and the fraction of synovial section area occupied by ACE-immunoreactivity (fractional area) were higher in synovia from patients with rheumatoid arthritis (B(eq) 28 amol/mm(2), fractional area 0.21) than from those with osteoarthritis (B(eq) 9 amol/mm(2), fractional area 0.10) or chondromalacia patellae (B(eq) 9 amol/mm(2), fractional area 0.09)(p < 0.05). Density of [(125)I]351A binding to stroma was similar to that to blood vessels in rheumatoid arthritis, but less dense than vascular binding in chondromalacia patellae and osteoarthritis. Increases in [(125)I]351A binding densities were attributable to increases in the numbers of binding sites, and were consistent with an increase in the density of ACE bearing stromal cells. CONCLUSION: ACE is upregulated in synovial stroma in rheumatoid arthritis. Increased tissue ACE may result in increased local generation of the vasoconstrictor and mitogenic peptide angiotensin II and thereby potentiate synovial hypoxia and proliferation in rheumatoid arthritis. (+info)
Activity and stability of a neutral protease from Vibrio sp. (vimelysin) in a pressure-temperature gradient.
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The apparent second-order rate constant of hydrolysis of Fua-Gly-LeuNH2 by vimelysin, a neutral protease from Vibrio sp. T1800, was measured in a variable pressure-temperature gradient (0. 1-400 MPa and 5-40 degrees C). The apparent maximum rate was observed at approximately 15 degrees C and 150-200 MPa; the pressure-activation ratio (kcat/Km(max)/kcat/Km(0.1 MPa)) was reached about sevenfold. The pressure dependence of the kcat and Km parameters at constant temperature (25 degrees C) revealed that the pressure-activation below 200 MPa was mainly caused by a change in the kcat parameter. The change in the intrinsic fluorescence intensity of vimelysin was also measured in a pressure-temperature plane (0.1-400 MPa and -20 to +60 degrees C). The fluorescence intensity was found to decrease by increasing pressure and temperature, and the isointensity contours were more or less circular. The tangential lines to the contours at high temperatures and low to medium pressures seem to have slightly positive slopes, which was reflected by the higher residual activities left after incubations at higher temperatures and medium pressure (200 MPa and 50 degrees C) and by the almost intact secondary structure left after 1 h of incubation at 200 MPa and 40 degrees C, as studied by circular dichroism. These results were compared with the corresponding results for thermolysin, a moderately thermostable protease from Bacillus thermoproteolyticus. Apparent differences that might be related to the temperature adaptations of the respective source microbes are also discussed. (+info)
Specificity of the wound-induced leucine aminopeptidase (LAP-A) of tomato activity on dipeptide and tripeptide substrates.
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Wounding of tomato leaves results in the accumulation of an exoprotease called leucine aminopeptidase (LAP-A) that preferentially hydrolyzes amino acid-p-nitroanilide and -beta-naphthylamide substrates with N-terminal Leu, Met and Arg residues. To determine the substrate specificity of LAP-A on more natural substrates, the rates of hydrolysis of 60 dipeptide and seven tripeptide substrates were determined. For comparison, the specificities of the porcine and Escherichia coli LAPs were evaluated in parallel. Several marked differences in substrate specificities for the animal, plant and prokaryotic LAP enzymes were observed. Substrates with variable N-terminal (P1) residues (Xaa) were evaluated; these substrates had Leu or Gly in the penultimate (P1') position. The plant, animal, and prokaryotic LAPs hydrolyzed dipeptides with N-terminal nonpolar aliphatic (Leu, Val, Ile, and Ala), basic (Arg), and sulfur-containing (Met) residues rapidly, while P1 Asp or Gly were cleaved inefficiently from peptides. Significant differences in the cleavage of dipeptides with P1 aromatic residues (Phe, Tyr, and Trp) were noted. To systematically evaluate the impact of the P1' residue on cleavage of dipeptides, three series of dipeptides (Leu-Xaa, Gly-Xaa, and Arg-Xaa) were evaluated. The P1' residue strongly influenced hydrolysis of dipeptides and the magnitude of its effect was dependent on the P1 residue. P1' Pro, Asp, Lys and Gly slowed the hydrolysis rates of the tomato LAP-A, porcine LAP, and E. coli PepA markedly. Analysis six Arg-Gly-Xaa tripeptides showed that more diversity was tolerated in the P2' position. P2' Arg inhibited tripeptide cleavage by all three enzymes, while P2' Asp enhanced hydrolysis rates for the porcine and prokaryotic LAPs. (+info)
Results of a randomized study of IM862 nasal solution in the treatment of AIDS-related Kaposi's sarcoma.
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PURPOSE: Although advances have been made in the treatment of AIDS-related Kaposi's sarcoma (AIDS-KS) with systemic chemotherapy, less toxic therapies are needed. IM862 is a naturally occurring peptide with antiangiogenic properties and was thus studied in patients with AIDS-KS. PATIENTS AND METHODS: IM862 was given as intranasal drops at a dose of 5 mg. Patients were randomized to two dosing schedules given in repeated cycles until disease progression or unacceptable toxicity: 5 days of therapy followed by 5 days off (n = 18) and every other day dosing (n = 26). RESULTS: Forty-two male patients and two female patients with a median age of 38 years (range, 22 to 53 years) were accrued. Twenty-one patients (47%) had more than 50 mucocutaneous lesions, 14 (32%) had lymphedema, and none had visceral involvement. Thirty-three patients (75%) had received prior systemic chemotherapy. Twenty-four patients (55%) had CD4(+) lymphocyte count +info)
A novel stable inhibitor of endopeptidases EC 3.4.24.15 and 3.4.24.16 potentiates bradykinin-induced hypotension.
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We have developed a novel inhibitor of the metalloendopeptidases EC 3.4.24.15 (EP24.15) and EC 3.4.24.16 (EP24.16), N-[1-(R, S)-carboxy-3-phenylpropyl]-Ala-Aib-Tyr-p-aminobenzoate (JA2), in which alpha-aminoisobutyric acid (Aib) is substituted for an alanine in a well-described but unstable inhibitor, cFP-AAY-pAB. This substitution increases the resistance of the inhibitor to degradation without altering potency. In the present study, we investigated the effects of JA2 (5 mg/kg) on the responses of mean arterial pressure to bradykinin, angiotensin I, and angiotensin II in conscious rabbits. The depressor responses to both low (10 ng/kg) and high (100 ng/kg) doses of bradykinin were increased 7.0+/-2. 7-fold and 1.5+/-0.3-fold, respectively, during the 30 minutes after JA2 administration (mean+/-SEM, n=8). Bradykinin potentiation was undiminished 4 hours after JA2 injection. In contrast, the hypertensive effects of angiotensins I and II were unaltered, indicating that the bradykinin-potentiating effects were not due to angiotensin-converting enzyme inhibition. These data suggest that JA2 is not only a potent and specific inhibitor of EP24.15 and EP24. 16 but is also stable in vivo. Furthermore, the potentiation of bradykinin-induced hypotension by JA2 suggests for the first time a role for one or both of these peptidases in the metabolism of bradykinin in the circulation. (+info)