Leader peptidase is a novel serine protease in Escherichia coli, which catalyzes the cleavage of amino-terminal signal sequences from exported proteins. It is an integral membrane protein containing two transmembrane segments with its carboxy-terminal catalytic domain residing in the periplasmic space. Recently, the x-ray crystal structure of signal peptidase-inhibitor complex showed that Asp 280, a highly conserved consensus sequence of E. coli leader peptidase is the closest charged residue in the vicinity of two catalytic dyad, Ser 90 and Lys 145, and it is likely held in place by a salt bridge to Arg 282. Possible roles of Asp 280 and Arg 282 in the structure-catalytic function relationship were investigated by the site-directed mutagenesis of Asp 280 substituted with alanine, glutamic acid, glycine, or asparagine and of Arg 282 with methionine. All of mutants purified with nickel affinity chromatography were inactive using in vitro assay. It is surprising to find complete lose of activity by an extension of one carbon units in the mutant where Asp 280 is substituted with glutamic acid. These results suggest that Asp 280 and Arg 282 are in a sequence which constitutes catalytic crevice of leader peptidase and are essential for maintaining the conformation of catalytic pocket.
Aspartic Acid/chemistry* ; Bacterial Outer Membrane Proteins/metabolism ; Blotting, Western ; Escherichia coli/enzymology* ; Escherichia coli/chemistry ; Micrococcal Nuclease/metabolism ; Mutagenesis, Site-Directed ; Oligonucleotides ; Protein Precursors/metabolism ; Serine Endopeptidases/metabolism* ; Serine Endopeptidases/genetics ; Serine Endopeptidases/chemistry* ; Structure-Activity Relationship

Faldaprevir analogue molecule (FAM) has been reported to effectively inhibit the catalytic activity of HCV NS3/4A protease, making it a potential lead compound against HCV. A series of HCV NS3/4A protease crystal structures were analyzed by bioinformatics methods, and the FAM-HCV NS3/4A protease crystal structure was chosen for this study. A 20.4 ns molecular dynamics simulation of the complex consists of HCV NS3/4A protease and FAM was conducted. The key amino acid residues for interaction and the binding driving force for the molecular recognition between the protease and FAM were identified from the hydrogen bonds and binding free energy analyses. With the driving force of hydrogen bonds and van der Waals, FAM specifically bind to the active pocket of HCV NS3/4A protease, including V130-S137, F152-D166, D77-D79 and V55, which agreed with the experimental data. The effect of R155K, D168E/V and V170T site-directed mutagenesis on FAM molecular recognition was analyzed for their effect on drug resistance, which provided the possible molecular explanation of FAM resistance. Finally, the system conformational change was explored by using free energy landscape and conformational cluster. The result showed four kinds of dominant conformation, which provides theoretical basis for subsequent design of Faldaprevir analogue inhibitors based on the structure of HCV NS3/4A protease.
Antiviral Agents ; chemistry ; Carrier Proteins ; chemistry ; Drug Resistance, Viral ; Endopeptidases ; Hepacivirus ; Molecular Dynamics Simulation ; Mutagenesis, Site-Directed ; Oligopeptides ; chemistry ; Protease Inhibitors ; chemistry ; Serine Proteases ; Thiazoles ; chemistry ; Viral Nonstructural Proteins ; chemistry

New antibiotics with novel modes of action and structures are urgently needed to combat the emergence of multidrug-resistant bacteria. Bacterial signal peptidase I (SPase I) is an indispensable enzyme responsible for cleaving the signal peptide of preprotein to release the matured proteins. Increasing evidence suggests that SPase I plays a crucial role in bacterial pathogenesis by regulating the excretion of a variety of virulent factors, maturation of quorum sensing factor and the intrinsic resistance against beta-lactams. Recently, breakthrough has been achieved in the understanding of three-dimensional structure of SPase I as well as the mechanism of enzyme-inhibitors interaction. Three families of inhibitors are identified, i.e. signal peptide derivatives, beta-lactams and arylomycins. In this article, we summarize the recent advance in the study of structure, activity and structure-activity relationship of SPase I inhibitors.
Animals ; Anti-Bacterial Agents ; pharmacology ; Bacteria ; drug effects ; Escherichia coli ; drug effects ; Membrane Proteins ; antagonists & inhibitors ; metabolism ; Oligopeptides ; chemistry ; pharmacology ; Serine Endopeptidases ; metabolism ; Serine Proteinase Inhibitors ; chemistry ; pharmacology ; Structure-Activity Relationship ; beta-Lactams ; antagonists & inhibitors

In order to evaluate the possible roles of secretory proteases in the pathogenesis of amoebic keratitis, we purified and characterized a serine protease secreted by Acanthamoeba lugdunensis KA/E2, isolated from a Korean keratitis patient. The ammonium sulfate-precipitated culture supernatant of the isolate was purified by sequential chromatography on CM-Sepharose, Sephacryl S-200, and mono Q-anion exchange column. The purified 33 kDa protease had a pH optimum of 8.5 and a temperature optimum of 55 degrees C. Phenylmethylsulfonylfluoride and 4- (2- Aminoethyl) -benzenesulfonyl-fluoride, both serine protease specific inhibitors, inhibited almost completely the activity of the 33 kDa protease whereas other classes of inhibitors did not affect its activity. The 33 kDa enzyme degraded various extracellular matrix proteins and serum proteins. Our results strongly suggest that the 33 kDa serine protease secreted from this keratopathogenic Acanthamoeba play important roles in the pathogenesis of amoebic keratitis, such as in corneal tissue invasion, immune evasion and nutrient uptake.
Acanthamoeba/*enzymology/isolation & purification/pathogenicity ; Acanthamoeba Keratitis/*parasitology ; Animals ; Cornea/parasitology ; Humans ; Hydrogen-Ion Concentration ; Korea ; Serine Endopeptidases/chemistry/*isolation & purification/*metabolism ; Substrate Specificity ; Temperature ; Virulence Factors

This study sought to assess the effect of SspA on the formation of Staphylococcus aureus biofilm extending over the surfaces of Cardiovascular Biomaterial Dacron. SspA was extracted from the surface of staphylococcus aureus biofilm, purified, and then used to influence the adhesion of Staphylococcus aureus and the formation of Staphylococcus aureus biofilm on Dacron biomaterial surfaces. The formation of the Staphylococcus aureus biofilm on cardiovascular biomaterial Dacron surfaces under gradient SspA concentrations was evaluated by confocal laser microscopy. The result revealed that SspA inhibited the formation of Staphylococcus aureus biofilms on cardiovascular biomaterials surfaces effectively, and it was dose dependent. This study indicates that SspA is effective for preventing biomaterial centered infection and this method is conducive to clinical applications.
Bacterial Adhesion ; Biocompatible Materials ; chemistry ; Biofilms ; growth & development ; Polyethylene Terephthalates ; Prosthesis-Related Infections ; microbiology ; Serine Endopeptidases ; pharmacology ; Staphylococcus aureus ; pathogenicity ; physiology

Japanese encephalitis virus (JEV) is a single-stranded and positive-sense RNA, which has a single ORF (open reading frame), encoding a polyprotein precursor. Non-structural protein 3 (NS3) plays an important role in processing the polyprotein precursor and has become an important drug target of flavivirus. In this study, NS2BH-NS3 gene was amplified by PCR and subcloned to the prokaryotic expression plasmid, resulting pET30a-NS2BH-NS3. The fusion protein was expressed in Escherichia coli BL21 (DE3) in soluble form after induction by Isopropyl beta-D-1-Thiogalactopyranoside (IPTG). The recombinant protein was purified by Ni-NTA affinity column. Then a fluorescence resonance energy transfer (FRET) method was used to determine enzymatic activity and the assay conditions were optimized. After screening 113 compounds, we found two compounds inhibiting the activity of NS2BH-NS3. This study provides a convenient and cost-effective method for screening of JEV NS3 protease inhibitor.
Encephalitis Virus, Japanese ; enzymology ; Escherichia coli ; metabolism ; High-Throughput Screening Assays ; Protease Inhibitors ; chemistry ; RNA Helicases ; metabolism ; Recombinant Fusion Proteins ; metabolism ; Serine Endopeptidases ; metabolism ; Viral Nonstructural Proteins ; metabolism

A heterozygous GTG to ATG (Val297Met) mutation was detected in a patient with inherited protein C deficiency and deep vein thrombosis. Cosegregation of the mutation with protein C deficiency was observed through a family pedigree study. Molecular models of the serine protease domains of wild type and mutant protein C were constructed by standard comparative method. Val 297 was found to be located in the hydrophobic core of the protein. Although the substitution of Met for Val does not greatly alter the hydrophobicity of the protein, it introduces a bulkier side chain, which yields steric hindrance between this residue and adjacent residues, such as Met364, Tyr393, Ile321, Ile323, and Val378. It seems that the Met can not fit into the tight packing into which it is trapped, thereby probably inducing misfolding and/or greater instability of the protein. Such misfolding and/or instability thereby eventually disturbs the catalytic triad, in consistent with the observed type I deficiency state.
Adult ; Base Sequence ; Female ; Human ; Male ; Middle Age ; Models, Molecular* ; Molecular Sequence Data ; Pedigree ; Point Mutation* ; Polymerase Chain Reaction/methods ; Protein C/genetics* ; Protein C/chemistry* ; Protein C Deficiency/genetics* ; Protein Conformation ; Serine Endopeptidases/genetics ; Serine Endopeptidases/chemistry* ; Venous Thrombosis/genetics

OBJECTIVETo investigate the relationship between SNC19 protein and cancer metastasis.

METHODSExpression of SNC19 protein in cancer cell lines and tissues was examined by Western blot analysis using anti-SNC19 monoclonal antibody. In addition, Psectag2A-SNC19(ORF) eukaryotic expression vector was constructed and transfected into BCAP37 cells. After the target protein was expressed and purified, processing forms of SNC19 protein were further identified using anti-His mAb and each form was assayed for its gelatinase activity.

RESULTSDifferent expression and post-translational processing of the SNC19 proteins in the cancer cell lines and intestinal tissues were detected.BCAP37 cells transfected full-length SNC19 (ORF) generated two different sized proteins in cell lysates, 120 and 75 kD; 75 kD was detected to have proteolytic activity by gelatin zymography.

CONCLUSIONSNC19 protein presents different expression and post-translational processing in the cancer cells and tissues, of which 75 kD was identified to have gelatinase activity.

Breast Neoplasms ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Colonic Neoplasms ; genetics ; metabolism ; pathology ; DNA, Complementary ; chemistry ; Female ; Gelatinases ; metabolism ; Humans ; Neoplasm Metastasis ; Neoplasm Proteins ; chemistry ; genetics ; metabolism ; Protein Processing, Post-Translational ; Serine Endopeptidases ; chemistry ; genetics ; metabolism ; Transfection

OBJECTIVETo study the character of mutants originating from Japanese encephalitis viruses and the relationship between the characterization of mutant strains and E protein expression.

METHODSPersistent infection was established with standard strains of Japanese encephalitis viruse, known as parental viruse, in a human hepatoma cell line, KN73. Cells were subcultured weekly using trypsinization techniques. Cell-associated viruses of persistently infected cells were collected by a freeze and thaw method. Virus titers were examined by plaque method using baby hamster kidney (BHK) cells. Indirect immunofluorescence assays were used to examine E and NS3 protein antigens. Western blot analysis was used to test expression of E and NS3 proteins.

RESULTSIn the early phase (24 - 36 h) post-infection, virus titer in culture fluid from KN73 cells infected with parental viruses were 10(6) PFU/ml. They were 10(3 - 4) PFU/ml in the late phase (3 years) post-infection. The titer of cell-associated viruse was 10(2 - 3) PFU/ml. A virus super-infection assay found that virus titers in culture fluid from persistently infected KN73 cells acutely super- infected with parental viruses were much lower than that of culture fluids in acutely infected normal KN73 at the same phase. Indirect immunoflurescence assay revealed that the quantity of viral antigens in persistently infected KN73 cells was lower than that in acutely infected KN73 cells with parental viruses. Western blot analyses indicated that the molecular weights of E and NS3 proteins were 53 kD and 73 kD, respectively. Expression of NS3 protein in persistently infected KN73 cells was stable but expression of E protein was markedly suppressed.

CONCLUSIONSThe virulence and reproduction of viruses obtained from persistently infected KN73 cells, which have some features of DI viruses and were involved in persistent infection, was lower than that of parental viruses. These mutants may have be related to the decrease in E protein expression.

Carcinoma, Hepatocellular ; virology ; Defective Viruses ; physiology ; Encephalitis Virus, Japanese ; chemistry ; genetics ; physiology ; Humans ; Membrane Glycoproteins ; analysis ; Mutation ; RNA Helicases ; Serine Endopeptidases ; Tumor Cells, Cultured ; Viral Envelope Proteins ; analysis ; Viral Nonstructural Proteins ; analysis

OBJECTIVETo investigate the expression of serine protease Omi/HtrA2 in kidneys of postasphyxial neonatal rats, and to study the effects of Ucf-101 on apoptosis and the expression of Omi/HtrA2 in these rats.

METHODSSeventy-two neonatal Wistar rats of 7-10 days old were randomly divided into 3 groups: control, postasphyxial model, Ucf-101-treated postasphyxialThe postasphyxial model was established by normobaric asphyxiaExpression of Omi/HtrA2 was determined with streptavidin-peroxidase immunohistochemistry 2, 24 and 48 hrs after asphyxia. Terminal deoxynuleotidyl-mediated nick end labeling (TUNEL) was used to ascertain the apoptosis of renal cells.

RESULTSCompared with the control group, OmiHtrA2 expression in renal cells began to increase 2 hrs after asphyxia and peaked at 24 hrs. The expression of Omi/HtrA2 in the Ucf-101-treated postasphyxial group was significantly lower than that in the postasphyxial model group (P<0.01). TUNEL-positive cells began to increase 2 hrs after asphyxia and peaked at 24 hrs in the postasphyxial model group when compared with the control group. The number of TUNEL-positive cells in the Ucf-101-treated postasphyxial group was significantly lower than that in the postasphyxial model group at all time points (P<0.01).

CONCLUSIONSThe expression of Omi/HtrA2 in kidneys is increased in postasphyxial neonatal rats. The increased Omi/HtrA2 expression may play an important role in the development of postasphyxial renal injury. Treatment with Ucf-101 can reduce the expression of Omi/HtrA2 in kidneys of postasphyxial neonatal rats and thus reduce renal tububar epithelial cell apoptosis.

Animals ; Animals, Newborn ; Apoptosis ; drug effects ; Asphyxia Neonatorum ; drug therapy ; metabolism ; pathology ; Female ; High-Temperature Requirement A Serine Peptidase 2 ; Humans ; Immunohistochemistry ; In Situ Nick-End Labeling ; Infant, Newborn ; Kidney ; chemistry ; Male ; Mitochondrial Proteins ; analysis ; antagonists & inhibitors ; Pyrimidinones ; pharmacology ; therapeutic use ; Rats ; Rats, Wistar ; Serine Endopeptidases ; analysis ; Thiones ; pharmacology ; therapeutic use