碱性蛋白酶

碱性蛋白酶在洗涤剂、制革、丝绸、饲料、医药、食品、环保等领域被广泛应用,具有重要的工业和经济价值。在筛选新型蛋白酶产酶菌株方面,近年来已报道了具有较高pH适应性的碱性蛋白酶,碱性弹性蛋白酶,水解多种底物的碱性蛋白酶,具有耐热、耐表面活性剂、耐氧化剂等特     

Double-stranded DNA bacteriophage prohead protease is homologous to herpesvirus protease

Double-stranded DNA bacteriophages and herpesviruses assemble their heads in a similar fashion; a pre-formed precursor called a prohead or procapsid undergoes a conformational transition to give rise to a mature head or capsid. A virus-encoded prohead or procapsid protease is often required in this maturation process. Through computational analysis, we infer homology between bacteriophage prohead proteases (MEROPS families U9 and U35) and herpesvirus protease (MEROPS family S21), and unify them into a procapsid protease superfamily. We also extend this superfamily to include an uncharacterized cluster of orthologs (COG3566) and many other phage or bacteria-encoded hypothetical proteins. On the basis of this homology and the herpesvirus protease structure and catalytic mechanism, we predict that bacteriophage prohead proteases adopt the herpesvirus protease fold and exploit a conserved Ser and His residue pair in catalysis. Our study provides further support for the proposed evolutionary link between dsDNA bacteriophages and herpesviruses.     

Dengue virus protease activity modulated by dynamics of protease cofactor

The viral protease domain (NS3pro) of dengue virus is essential for virus replication, and its cofactor NS2B is indispensable for the proteolytic function. Although several NS3pro-NS2B complex structures have been obtained, the dynamic property of the complex remains poorly understood. Using NMR relaxation techniques, here we found that NS3pro-NS2B exists in both closed and open conformations that are in dynamic equilibrium on a submillisecond timescale in aqueous solution. Our structural information indicates that the C-terminal region of NS2B is disordered in the minor open conformation but folded in the major closed conformation. Using mutagenesis, we showed that the closed-open conformational equilibrium can be shifted by changing NS2B stability. Moreover, we revealed that the proteolytic activity of NS3pro-NS2B correlates well with the population of the closed conformation. Our results suggest that the closed-open conformational equilibrium can be used by both nature and humanity to control the replication of dengue virus.     

Proteolysis of the calcium-dependent protease inhibitor by myocardial calcium-dependent protease

Bovine heart peak II calcium-dependent protease was capable of hydrolyzing its specific inhibitor protein at high molar ratios of protease to inhibitor. The proteolysis was inhibited by leupeptin and required millimolar calcium. Thus, it appeared to be attributable to the calcium-dependent protease and not to possible contaminating proteases in the purified preparations of inhibitor or calcium-dependent protease. Incubation of the purified inhibitor with the calcium-dependent protease produced a discrete pattern of inhibitor fragments on Western blots developed with an inhibitor-specific monoclonal antibody. Traces of similar or identical lower molecular weight immunoreactive material could be observed in Western blots of bovine heart extracts, and the immunoreactivity present as these lower molecular weight forms could be increased by incubation of the extracts with calcium ion. These results suggest that the inhibitor can be proteolyzed to low molecular weight forms which can be detected in cardiac tissue extracts, and that calcium-dependent protease(s) may be responsible for this phenomenon.     

IgA1 protease

IgA1 proteases are proteolytic enzymes that cleave specific peptide bonds in the human immunoglobulin A1 (IgA1) hinge region sequence. Several species of pathogenic bacteria secrete IgA1 proteases at mucosal sites of infection to destroy the structure and function of human IgA1 thereby eliminating an important aspect of host defence. IgA1 proteases are known as autotransporter proteins as their gene structure encodes the information to direct their own secretion out of the bacterial cell. The iga gene structure is also thought to contribute to the antigenic heterogeneity demonstrated by the IgA1 proteases during infections and the cleavage specificity of the IgA1 proteases for human IgA1. The IgA1 proteases have therefore been implicated as important virulence factors that contribute to bacterial infection and colonisation. The development of strategies to inactivate these IgA1 proteases has become the subject of recent research, as this has the potential to reduce bacterial colonisation at mucosal surfaces.     

NS3 protease of Langat tick-borne flavivirus cleaves serine protease substrates

A DNA fragment of 163 bp containing 11 GGA repeats formed two-end positioned mononucleosomes as efficiently as that of CTG repeats. However, the rotational positioning of the GGA fragment was weak because clear DNase I cleavage patterns with 10-base periodicity were not seen near the center of the GGA fragment but were detected in the entire region of the CTG fragment. Incubation of the GGA mononucleosomes with the same fragment provided the DNA-DNA complex, which had been shown by using naked DNA fragments. DNase I digestion of the complex exhibited protection in the GGA repeats and in flanking sequences of about 30 bp at both sides, suggesting that both the repeat and flanking regions were involved in the association. Interestingly, histone H1, which enhanced DNA-DNA association on naked DNA, did not affect the complex formation on mononucleosomes. These results imply that GGA microsatellites in genomes could associate with one another at multiple sites and that the association may play a role in functional organization of higher order chromatin architecture.     

Enamino-oxindole HIV protease inhibitors

We have designed and synthesized a series of HIV protease inhibitors (PIs) with enamino-oxindole substituents optimized to interact with the S2' subsite of the HIV protease binding pocket. Several of these inhibitors have sub-nanomolar K(i) and antiviral IC(50) in the low nM range against WT HIV and against a panel of multi-drug resistant (MDR) strains.     

A mitochondrial rhomboid protease

Rhomboid proteases are integral membrane proteins, typically associated with cleavage of peptide hormones along the secretory pathway. Recent publications demonstrate that yeast mitochondria contain a rhomboid protease required for the cleavage of two mitochondrial intermembrane space proteins, suggesting that rhomboid proteases play a regulatory role in mitochondria.     

Inhibitor-assisted refolding of protease: a protease inhibitor as an intramolecular chaperone

Pleurotus ostrearus proteinase A inhibitor 1 (POIA1), which was discovered as a protease inhibitor, is unique in that it shows sequence homology to the propeptide of subtilisin, which functions as an intramolecular of a cognate protease. In this study, we demonstrate that POIA1 can function as an intramolecular chaperone of subtilisin by in vitro and in vivo experiments. The specific cleavage between POIA1 and the mature region of subtilisin BPN' occurred in a refolding process of a chimera protein, and Bacillus cells transformed with a chimera gene formed a halo on a skim milk plate. The mutational analyses of POIA1 in the chimera protein suggested that the tertiary structure of POIA1 is required for such a function, and that an increase in its ability to bind to subtilisin BPN' makes POIA1 a more effective intramolecular chaperone.     

Biosynthesis of protease nexin-I

Protease nexin-I (PN-I) is representative of a newly described class of serine protease inhibitors secreted by human fibroblasts, the protease nexins. Protease nexins form covalent complexes with their target proteases, subsequently binding to cells via specific receptors. PN-I preferentially binds thrombin, urokinase, trypsin, and plasmin, and its binding to thrombin is accelerated by heparin. We have previously described the production of a polyclonal antibody against PN-I which is able to block the binding of PN-I X proteinase complexes to cells and will immunoprecipitate metabolically labeled PN-I. Anti-PN-I was used to investigate the biosynthesis and regulation of PN-I in human fibroblasts. Unlabeled PN-I could compete for the binding of metabolically labeled PN-I to anti-PN-I, as shown by the elimination of the 43-kDa band representing PN-I on sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiographs. Excision of this 43-kDa band from gels, followed by amino-terminal sequencing, showed a homogeneous protein that is homologous with that described by Scott et al. (Scott, R. W., Bergman, B. L., Bajpai, A., Hersh, R. T., Rodriguez, H., Jones, B. N., Barreda, C., Watts, S., and Baker, J. B. (1985) J. Biol. Chem. 260, 7029-7034). An analysis of the biosynthesis of the PN-I revealed that a lower Mr precursor exists intracellularly. This apparent rough endoplasmic reticulum form appears as a doublet on sodium dodecyl sulfate gels, as does mature PN-I. The PN-I precursor was also sensitive to endoglycosidase H, suggesting that it contains N-linked carbohydrates of the high mannose form. Mature PN-I is not sensitive to endoglycosidase H, but does contain 3 kDa of N-linked carbohydrate. PN-I appears to be constitutively secreted by fibroblasts. PN-I levels in conditioned media reach a steady state within 48 h, although PN-I synthesis maintains a constant rate. This steady state is due to the continuous uptake of PN-I from medium, presumably through a specific receptor.     

Design of protease inhibitors

There is a general parallelism in the strategy followed in the design of hormonal peptide analogs and protease inhibitors. However, in the latter, one more dimension has been added with the development of mechanism-based inhibitors, a dimension that is not yet available for hormonal peptides because of the lack of knowledge about receptor mechanisms. The recently advanced concepts of transition state and bi-product analogs have made possible the development of highly potent active-site directed reversible protease inhibitors of great therapeutic potential.