(1/976) Expression of murine coronavirus recombinant papain-like proteinase: efficient cleavage is dependent on the lengths of both the substrate and the proteinase polypeptides.
Proteolytic processing of the replicase gene product of mouse hepatitis virus (MHV) is essential for viral replication. In MHV strain A59 (MHV-A59), the replicase gene encodes two predicted papain-like proteinase (PLP) domains, PLP-1 and PLP-2. Previous work using viral polypeptide substrates synthesized by in vitro transcription and translation from the replicase gene demonstrated both cis and trans cleavage activities for PLP-1. We have cloned and overexpressed the PLP-1 domain in Escherichia coli by using a T7 RNA polymerase promoter system or as a maltose-binding protein (MBP) fusion protein. With both overexpression systems, the recombinant PLP-1 exhibited trans cleavage activity when incubated with in vitro-synthesized viral polypeptide substrates. Subsequent characterization of the recombinant PLP-1 revealed that in vitro trans cleavage is more efficient at 22 degrees C than at higher temperatures. Using substrates of increasing lengths, we observed efficient cleavage by PLP-1 requires a substrate greater than 69 kDa. In addition, when PLP-1 was expressed as a polypeptide that included additional viral sequences at the carboxyl terminus of the predicted PLP-1 domain, a fivefold increase in proteolytic activity was observed. The data presented here support previous data suggesting that in vitro and in vivo cleavage of the ORF 1a polyprotein by PLP-1 can occur in both in cis and in trans. In contrast to the cleavage activity demonstrated for PLP-1, no in vitro cleavage in cis or in trans could be detected with PLP-2 expressed either as a polypeptide, including flanking viral sequences, or as an MBP fusion enzyme. (+info)
(2/976) Expression and characterization of a DNase I-Fc fusion enzyme.
Recombinant human deoxyribonuclease I (DNase I) is an important clinical agent that is inhaled into the airways where it degrades DNA to lower molecular weight fragments, thus reducing the viscoelasticity of sputum and improving the lung function of cystic fibrosis patients. To investigate DNases with potentially improved properties, we constructed a molecular fusion of human DNase I with the hinge and Fc region of human IgG1 heavy chain, creating a DNase I-Fc fusion protein. Infection of Sf9 insect cells with recombinant baculovirus resulted in the expression and secretion of the DNase I-Fc fusion protein. The fusion protein was purified from the culture medium using protein A affinity chromatography followed by desalting by gel filtration and was characterized by amino-terminal sequence, amino acid composition, and a variety of enzyme-linked immunosorbent assays (ELISA) and activity assays. The purified fusion contains DNase I, as determined by a DNase I ELISA and an actin-binding ELISA, and an intact antibody Fc region, which was quantified by an Fc ELISA, in a 2:1 stoichiometric ratio, respectively. The dimeric DNase I-Fc fusion was functionally active in enzymatic DNA digestion assays, albeit about 10-fold less than monomeric DNase I. Cleavage of the DNase I-Fc fusion by papain resulted in a specific activity comparable to the monomeric enzyme. Salt was inhibitory for wild type monomeric DNase I but actually enhanced the activity of the dimeric DNase I-Fc fusion. The DNase I-Fc fusion protein was also less Ca2+-dependent than DNase I itself. These results are consistent with a higher affinity of the dimeric fusion protein to DNA than monomeric DNase I. The engineered DNase I-Fc fusion protein described herein has properties that may have clinical benefits. (+info)
(3/976) Revisiting the S2 specificity of papain by structural analogs of Phe.
Papain characteristically has a strong preference for encoded L-aromatic amino acids (Phe > Tyr) at P2 position. We re-examined papain S2 specificity using structural analogs of Phe, in fluorogenic substrates of the series: dansyl-Xaa-Arg-Ala-Pro-Trp (Xaa = P2 residue). Kinetic analyses showed that the S2 pocket accommodates a broad spectrum of Phe derivatives. Papain is poorly stereoselective towards Dns-(D/L)-Phe-Arg-Ala-Pro-Trp and binding is not critically affected by replacement of the benzyl ring by the non-aromatic lateral chain of cyclohexylalanine. The Km was significantly improved by mono- and di-chlorination of Phe, or by its substitution by an electronegative group-like NO2, but the specificity constant was unchanged. Shortening or lengthening the side chain by adding or removing a methylene group impairs the P2/S2 interactions significantly, as do constrained structural analogs of Phe. Incorporation of benzyl-substituted phenylalanyl amino acid could help to design peptide-derived inhibitors with greater affinity and bioavailability. (+info)
(4/976) A human RNA viral cysteine proteinase that depends upon a unique Zn2+-binding finger connecting the two domains of a papain-like fold .
A cysteine proteinase, papain-like proteinase (PL1pro), of the human coronavirus 229E (HCoV) regulates the expression of the replicase polyproteins, pp1a and ppa1ab, by cleavage between Gly111 and Asn112, far upstream of its own catalytic residue Cys1054. In this report, using bioinformatics tools, we predict that, unlike its distant cellular homologues, HCoV PL1pro and its coronaviral relatives have a poorly conserved Zn2+ finger connecting the left and right hand domains of a papain-like fold. Optical emission spectrometry has been used to confirm the presence of Zn2+ in a purified and proteolytically active form of the HCoV PL1pro fused with the Escherichia coli maltose-binding protein. In denaturation/renaturation experiments using the recombinant protein, its activity was shown to be strongly dependent upon Zn2+, which could be partly substituted by Co2+ during renaturation. The reconstituted, Zn2+-containing PL1pro was not sensitive to 1,10-phenanthroline, and the Zn2+-depleted protein was not reactivated by adding Zn2+ after renaturation. Consistent with the proposed essential structural role of Zn2+, PL1pro was selectively inactivated by mutations in the Zn2+ finger, including replacements of any of four conserved Cys residues predicted to co-ordinate Zn2+. The unique domain organization of HCoV PL1pro provides a potential framework for regulatory processes and may be indicative of a nonproteolytic activity of this enzyme. (+info)
(5/976) Neutrophil elastase increases MUC5AC mRNA and protein expression in respiratory epithelial cells.
Chronic neutrophil-predominant inflammation and hypersecretion of mucus are common pathophysiological features of cystic fibrosis, chronic bronchitis, and viral- or pollution-triggered asthma. Neutrophils release elastase, a serine protease, that causes increased mucin production and secretion. The molecular mechanisms of elastase-induced mucin production are unknown. We hypothesized that as part of this mechanism, elastase upregulates expression of a major respiratory mucin gene, MUC5AC. A549, a human lung carcinoma cell line that expresses MUC5AC mRNA and protein, and normal human bronchial epithelial cells in an air-liquid interface culture were stimulated with neutrophil elastase. Neutrophil elastase increased MUC5AC mRNA levels in a time-dependent manner in both cell culture systems. Neutrophil elastase treatment also increased MUC5AC protein levels in A549 cells. The mechanism of MUC5AC gene regulation by elastase was determined in A549 cells. The induction of MUC5AC gene expression required serine protease activity; other classes of proteases had no effect on MUC5AC gene expression. Neutrophil elastase increased MUC5AC mRNA levels by enhancing mRNA stability. This is the first report of mucin gene regulation by this mechanism. (+info)
(6/976) Collagen type I antisense and collagen type IIA messenger RNA is expressed in adult murine articular cartilage.
OBJECTIVE: Articular cartilage has only limited capacities for repair and it is not known what is the exact mechanism of matrix restoration. It was investigated whether the reparative process in murine articular cartilage after moderate proteoglycan depletion is accompanied by a change in the chondrocyte phenotype either to hypertrophy or to a less differentiated phenotype as assayed by the expression of specific collagen subtypes. DESIGN: Moderate proteoglycan depletion was induced by injection of papain whereafter the expression of collagen type I mRNA, collagen IIA and IIB mRNA and type X collagen mRNA in patellar cartilage, as markers for chondrocyte phenotype, was investigated by RT-PCR during normal cartilage physiology and matrix restoration. In addition, in-situ expression of collagen subtypes was assayed by immunolocalisation. RESULTS: In normal articular cartilage collagen I, collagen IIB and collagen type X transcripts were easily detected. Surprisingly, collagen type I sense as well as antisense mRNA was detected and in addition to IIB transcripts collagen IIA transcripts were detected in a number of samples. During cartilage matrix restoration no change in the expression of collagen I, collagen IIA or IIB or collagen type X mRNA transcripts could be detected. Immunolocalization demonstrated the presence of type I (pericellular) and type II collagen in the extracellular matrix. The pericellular matrix of hypertrophic chondrocytes showed collagen type X staining in the calcified cartilage in normal and papain-injected knee joints. Increased staining for collagen type X was found in the upper cartilage layer in the interterritorial matrix from day 7 after papain injection. CONCLUSION: The absence of changes in collagen mRNA expression indicates that alteration of chondrocyte phenotype does not occur during the successful repair process after moderate proteoglycan depletion. Collagen type X appears to be deposited in the upper cartilage layer during this process. (+info)
(7/976) Binding modes of a new epoxysuccinyl-peptide inhibitor of cysteine proteases. Where and how do cysteine proteases express their selectivity?
Papain from Carica papaya, an easily available cysteine protease, is the best-studied representative of this family of enzymes. The three dimensional structure of papain is very similar to that of other cysteine proteases of either plant (actinidin, caricain, papaya protease IV) or animal (cathepsins B, K, L, H) origin. As abnormalities in the activities of mammalian cysteine proteases accompany a variety of diseases, there has been a long-lasting interest in the development of potent and selective inhibitors for these enzymes. A covalent inhibitor of cysteine proteases, designed as a combination of epoxysuccinyl and peptide moieties, has been modeled in the catalytic pocket of papain. A number of its configurations have been generated and relaxed by constrained simulated annealing-molecular dynamics in water. A clear conformational variability of this inhibitor is discussed in the context of a conspicuous conformational diversity observed earlier in several solid-state structures of other complexes between cysteine proteases and covalent inhibitors. The catalytic pockets S2 and even more so S3, as defined by the pioneering studies on the papain-ZPACK, papain-E64c and papain-leupeptin complexes, appear elusive in view of the evident flexibility of the present inhibitor and in confrontation with the obvious conformational scatter seen in other examples. This predicts limited chances for the development of selective structure-based inhibitors of thiol proteases, designed to exploit the minute differences in the catalytic pockets of various members of this family. A simultaneous comparison of the three published proenzyme structures suggests the enzyme's prosegment binding loop-prosegment interface as a new potential target for selective inhibitors of papain-related thiol proteases. (+info)
(8/976) The affinity and kinetics of inhibition of cysteine proteinases by intact recombinant bovine cystatin C.
Recent studies have shown that the bovine cysteine proteinase inhibitor, cystatin C, is synthesized as a preprotein containing a 118-residue mature protein. However, the forms of the inhibitor isolated previously from bovine tissues had shorter N-terminal regions than expected from these results, and also lower affinity for proteinases than human cystatin C. In this work, we report the properties of recombinant, full-length bovine cystatin C having a complete N-terminal region. The general characteristics of this form of the inhibitor, as reflected by the isoelectric point, the far-ultraviolet circular dichroism spectrum, the thermal stability and the changes of tryptophan fluorescence on interaction with papain, resembled those of human cystatin C. The affinity and kinetics of inhibition of papain and cathepsins B, H and L by the bovine inhibitor were also comparable with those of the human inhibitor, although certain differences were apparent. Notably, the affinity of bovine cystatin C for cathepsin H was somewhat weaker than that of human cystatin C, and bovine cystatin C bound to cathepsin L with about a four-fold higher association rate constant than the human inhibitor. This rate constant is comparable with the highest values reported previously for cystatin-cysteine proteinase reactions. The full-length, recombinant bovine cystatin C bound appreciably more tightly to proteinases than the shorter form characterized previously. Digestion of the recombinant inhibitor with neutrophil elastase resulted in forms with truncated N-terminal regions and appreciably decreased affinity for papain, consistent with the forms of bovine cystatin C isolated previously having arisen by proteolytic cleavage of a mature, full-length inhibitor. (+info)