Ubiquitination is one of the reversible protein post-translational modifications, in which ubiquitin molecules bind to the target protein in a cascade reaction of ubiquitin activating enzymes, ubiquitin conjugating enzymes, and ubiquitin ligases. The deubiquitinating enzymes (DUBs) remove ubiquitin residues from the substrates, which play key roles in the formation of mature ubiquitin, the removal and trimming of ubiquitin chains, as well as the recycling of free ubiquitin chains. Ubp14, a member of the ubiquitin specific proteases family in Saccharomyces cerevisiae, is mainly responsible for the recycling of intracellular free ubiquitin chains. To investigate its global biological function, a ubp14∆ mutant was constructed by homologous recombination technique. The growth rate of ubp14∆ mutant was lower than that of the wild-type (WT) strain. Using stable isotope labeling by amino acids in cell culture (SILAC) combined with deep coverage proteomics analysis, the differentially expressed proteins of ubp14∆ mutant relative to the wild-type strain were systematically analyzed. A total of 3 685 proteins were identified in this study, and 109 differentially expressed proteins were filtered out by statistical analysis. Gene ontology analysis found that differentially expressed proteins caused by Ubp14 loss were mainly involved in amino acid metabolism, REDOX, heat shock stress and etc, which shed light on the broad biological function of this DUB. This study provides highly reliable proteomic data for further exploring the biological functions of the deubiquitination enzyme Ubp14, and further understanding the relationship between the free ubiquitin homeostasis and biological process regulation.
Saccharomyces cerevisiae/metabolism* ; Proteomics ; Endopeptidases/metabolism* ; Ubiquitin/metabolism* ; Ubiquitination ; Proteins/metabolism* ; Deubiquitinating Enzymes/metabolism* ; Biological Phenomena

Proteolytic processing of viral polyproteins is indispensible for the lifecycle of coronaviruses. The main protease (M(pro)) of SARS-CoV is an attractive target for anti-SARS drug development as it is essential for the polyprotein processing. M(pro) is initially produced as part of viral polyproteins and it is matured by autocleavage. Here, we report that, with the addition of an N-terminal extension peptide, M(pro) can form a domain-swapped dimer. After complete removal of the extension peptide from the dimer, the mature M(pro) self-assembles into a novel super-active octamer (AO-M(pro)). The crystal structure of AO-M(pro) adopts a novel fold with four domain-swapped dimers packing into four active units with nearly identical conformation to that of the previously reported M(pro) active dimer, and 3D domain swapping serves as a mechanism to lock the active conformation due to entanglement of polypeptide chains. Compared with the previously well characterized form of M(pro), in equilibrium between inactive monomer and active dimer, the stable AO-M(pro) exhibits much higher proteolytic activity at low concentration. As all eight active sites are bound with inhibitors, the polyvalent nature of the interaction between AO-M(pro) and its polyprotein substrates with multiple cleavage sites, would make AO-M(pro) functionally much more superior than the M(pro) active dimer for polyprotein processing. Thus, during the initial period of SARS-CoV infection, this novel active form AOM(pro) should play a major role in cleaving polyproteins as the protein level is extremely low. The discovery of AOM(pro) provides new insights about the functional mechanism of M(pro) and its maturation process.
Coronavirus ; metabolism ; Cysteine Endopeptidases ; Endopeptidases ; metabolism ; Humans ; Peptides ; chemistry ; metabolism ; Polyproteins ; chemistry ; metabolism ; Protein Binding ; SARS Virus ; chemistry ; metabolism ; Viral Proteins

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

This study describes the characterization of 80 kDa protease showing gelationlytic property among three proteases in the excretory/secretory proteins (ESP) from Toxoplasma gondii. The protease activity was detected in the ESP but not in the somatic extract of RH tachyzoites. This protease was active only in the presence of calcium ion but not other divalent cationic ions such as Cu (2+), Zn (2+), Mg (2+), and Mn (2+), implying that Ca (2+) is critical factor for the activation of the protease. The 80 kDa protease was optimally active at pH 7.5. Its gelatinolytic activity was maximal at 37 degrees C, and significant level of enzyme activity of the protease remained after heat treatment at 56 degrees C for 30 min or 100 degrees C for 10 min. This thermostable enzyme was strongly inhibited by metal chelators, i.e., EDTA, EGTA, and 1, 10-phenanthroline. Thus, the 80 kDa protease in the ESP secreted by T. gondii was classified as a calcium dependent neutral metalloprotease.
Animals ; Calcium/metabolism ; Endopeptidases/*metabolism ; Hydrogen-Ion Concentration ; Molecular Weight ; Temperature ; Toxoplasma/*enzymology

OBJECTIVETo investigate the time course of changes in the expression of fibroblast activation protein (FAP) during healing of skin scald burns in rats.

METHODSAdult Wistar rats were randomized into two equal groups (n=42) and subject to superficial second degree and deep second degree scald burns on the dorsal skin groups, with 6 normal rats serving as the control group. At 6 h, 12 h, and 1, 3, 7, 14, and 21 days after burns, 6 rats from each group were sacrificed to detect FAP expression by immunohistochemistry and Western blotting.

RESULTSFAP was expressed on the cell membrane and in the cytoplasm of the fibroblasts, especially those around the neovessels. In both burn groups, FAP expression increased significantly at 6 h after burns. In superficial burn group, FAP expression was comparable between 6 and 12 h and between 1 and 3 days (P>0.05), but showed significant differences between the other time points (P<0.05). In deep burn group, FAP expression was comparable between 12 h, 1 day and 3 days (P>0.05) but differed significantly between the other time points (P<0.05). In both burn groups, FAP expression reached the peak level at 7 days followed by a gradual declination. At 21 days after the burns, FAP maintained a significantly higher expression level than the control level (P<0.05).

CONCLUSIONThe time course of the changes of FAP expression following scald burns suggests an important role of FAP in the healing process of scald burns.

Animals ; Burns ; metabolism ; rehabilitation ; Face ; Gelatinases ; metabolism ; Membrane Proteins ; metabolism ; Rats ; Rats, Wistar ; Serine Endopeptidases ; metabolism ; Skin ; metabolism ; Wound Healing

Helminthic cysteine proteases are well known to play critical roles in tissue invasion, nutrient uptake, and immune evasion of the parasites. In the same manner, the sparganum, the plerocercoid of Spirometra mansoni, is also known to secrete a large amount of cysteine proteases. However, cysteine protease inhibitors regulating the proteolytic activities of the cysteine protease are poorly illustrated. In this regard, we partially purified an endogenous cysteine protease inhibitor from spargana and characterized its biochemical properties. The cysteine protease inhibitor was purified by sequential chromatographies using Resource Q anion exchanger and Superdex 200 HR gel filtration from crude extracts of spargana. The molecular weight of the purified protein was estimated to be about 11 kD on SDS-PAGE. It was able to inhibit papain and 27 kDa cysteine protease of spargana with the ratio of 25.7% and 49.1%, respectively, while did not inhibit chymotrypsin. This finding suggests that the cysteine protease inhibitor of spargana may be involved in regulation of endogenous cysteine proteases of the parasite, rather than interact with cysteine proteases from their hosts.
Animals ; Cystatins/pharmacology ; Cysteine Endopeptidases/metabolism ; Cysteine Proteinase Inhibitors/chemistry/*metabolism/*pharmacology ; Helminth Proteins/*metabolism/*pharmacology ; Spirometra/*metabolism

OBJECTIVETo investigate the dynamic expression and role of SENP1 (SUMO-specific proteases-1) in the pulmonary vascular wall of rat during the development of hypoxic pulmonary hypertension (HPH).

METHODSForty adult male Wistar rats were randomly divided into 5 groups (n = 8), and exposed to normoxia (Control group) or exposed to hypoxia for 3, 7, 14 or 21 d, respectively. The HPH models were established by normobaric intermittent hypoxia. Mean pulmonary arterial pressure (mPAP), right ventricle hypertrophy index (RVHI), and vessel morphometry were measured. Reverse transcriptase-polymerase chain reaction(RT-PCR) and in situ hybridization were used to determine the mRNA expression of SENP1. Immunohistochemistry and Western blot were used to determine the protein expression of SENP1.

RESULTSThe hypoxic rats developed pulmonary vascular remodeling in pulmonary arterioles after 7 d of hypoxia exposure. Pulmonary vascular remodeling in pulmonary arterioles significantly increased after 14 d of hypoxia. The level of mPAP in hypoxic rats increased significantly after 7 d of hypoxia, reached its peak after 14 d of hypoxic exposure. RVHI was markedly increased after 14 d of hypoxia. In situ hybridization and immunohistochemical analysis showed that SENP1 mRNA and protein were positively stained in control. SENP1 mRNA expression had little changes after exposure to hypoxia compared with the control, however, SENP1 protein expression was declined gradually after 7 d of hypoxia. The results of RT-PCR and Western blot showed that the same dynamic expression of SENP1 mRNA and protein in lung tissues of rats. Linear correlation analysis showed that SENP1 protein were negatively correlated with mPAP, pulmonary vascular remodeling index and RVHI.

CONCLUSIONUnder chronic hypoxia, SENP1 protein can be degradated. The dynamic expression of SENP1 protein may play a role in implicating in the development of HPH.

Animals ; Endopeptidases ; metabolism ; Hypertension, Pulmonary ; etiology ; metabolism ; Hypoxia ; complications ; metabolism ; Male ; Pulmonary Artery ; metabolism ; Rats ; Rats, Wistar

Animals ; Hepatocyte Growth Factor ; metabolism ; Humans ; Proto-Oncogene Proteins c-met ; Serine Endopeptidases

OBJECTIVETo study the factors influencing the activity of fibrinolytic enzymes from earthworm and to obtain the better way to extract fibrinolytic enzymes as well as keep its optimum activity.

METHOD75% alcohol, 0.9% NaCl and 10% saccharose was used to extract the crude fibrinolytic enzymes from earthworm, the method of urokinase gelose-fibrin plate was used to measure the activity of fibrinolytic enzymes from earthworm. and the method of 3,3'-diaminobezidine tetrahydrochloride colorimetry to was used measure the content of selenium. The method use ts of measuring the content of arsenic was silver diethyldithiocarbamate colorimetry.

RESULTThe fibrinolytin of earthworms reared with cattle soils had higher activity than that reared with garbage. The arsenic in the earthworm's body could improve the activity of earthworm's fibrinolytin. However, the selenium had litter influence on it. Among the three methods of extraction, the 75% alcohol one was the most efficient, the 0.9% NaCl was next, and the 10% saccharose was the lowest. The influence of dialysis on the activity of fibrinolytin was less than that of ultrafiltration, when the earthworm's fibrinolytin enzyme was further sublimated.

CONCLUSIONThe activity of the earthworm's fibrinolytin will be increased earthworm is reared with the fitting baits and when appropriate methods, of extraction and purification are used.

Animal Feed ; Animals ; Endopeptidases ; isolation & purification ; metabolism ; Oligochaeta ; chemistry ; Selenium ; pharmacology

Detection of protein-protein interaction can provide valuable information for investigating the biological function of proteins. The current methods that applied in protein-protein interaction, such as co-immunoprecipitation and pull down etc., often cause plenty of working time due to the burdensome cloning and purification procedures. Here we established a system that characterization of protein-protein interaction was accomplished by co-expression and simply purification of target proteins from one expression cassette within E. coli system. We modified pET vector into co-expression vector pInvivo which encoded PPV NIa protease, two cleavage site F and two multiple cloning sites that flanking cleavage sites. The target proteins (for example: protein A and protein B) were inserted at multiple cloning sites and translated into polyprotein in the order of MBP tag-protein A-site F-PPV NIa protease-site F-protein B-His(6) tag. PPV NIa protease carried out intracellular cleavage along expression, then led to the separation of polyprotein components, therefore, the interaction between protein A-protein B can be detected through one-step purification and analysis. Negative control for protein B was brought into this system for monitoring interaction specificity. We successfully employed this system to prove two cases of reported protien-protein interaction: RHA2a/ANAC and FTA/FTB. In conclusion, a convenient and efficient system has been successfully developed for detecting protein-protein interaction.
Endopeptidases ; genetics ; metabolism ; Escherichia coli ; genetics ; Plum Pox Virus ; enzymology ; genetics ; Protein Interaction Mapping ; methods ; Proteolysis