The general secretory (Sec) pathway represents a common mechanism by which bacteria secrete proteins, including virulence factors, into the extracytoplasmic milieu. However, there is little information about this system, as well as its associated secretory proteins, in relation to the fire blight pathogen Erwinia amylovora. In this study, data mining revealed that E. amylovora harbors all of the essential components of the Sec system. Based on this information, we identified putative Sec-dependent secretory proteases in E. amylovora on a genome-wide scale. Using the programs SignalP, LipoP, and Phobius, a total of 15 putative proteases were predicted to contain the N-terminal signal peptides (SPs) that might link them to the Sec-dependent pathway. The activities of the predicted SPs were further validated using an Escherichia coli-based alkaline phosphatase (PhoA) gene fusion system that confirmed their extracytoplasmic property. Transcriptional analyses showed that the expression of 11 of the 15 extracytoplasmic protease genes increased significantly when E. amylovora was used to inoculate immature pears, suggesting their potential roles in plant infection. The results of this study support the suggestion that E. amylovora might employ the Sec system to secrete a suite of proteases to enable successful infection of plants, and shed new light on the interaction of E. amylovora with host plants.
Erwinia amylovora/metabolism* ; Escherichia coli/genetics* ; Peptide Hydrolases/genetics* ; Plant Diseases/microbiology* ; Pyrus/microbiology*

We assessed the effects of trxS gene on changes of proteinase activity, contents of different protein fractions and SDS-PAGE profiles in germinating seeds of contrasting transgenic and nontransgenic barley variety. Proteinase activity was enhanced by 70.28% in transgenic than nontransgenic barley seeds, whereas contents of albumin, globulin, hordein and glutelin in transgenic seeds were 3.68%, 23.52%, 31.37%, and 21.04%, lower than those in nontransgenic seeds. Degradation rates of hordein and glutelin in transgenic seeds were faster than those in nontransgenic seedlings as indicated by the SDS-PAGE profiles. Our data imply that the transformation of trxS gene could promote the degradation of protein, providing theoretic basis for the use of trxS gene and barley quality breeding.
Germination ; Hordeum ; genetics ; growth & development ; metabolism ; Peptide Hydrolases ; metabolism ; Plant Proteins ; metabolism ; Plants, Genetically Modified ; genetics ; growth & development ; metabolism ; Seeds ; growth & development ; Thioredoxins ; genetics ; metabolism ; Transformation, Genetic

A new feather-degrading bacterium was isolated from a local feather waste site and identified as Bacillus subtilis based on morphological, physiochemical, and phylogenetic characteristics. Screening for mutants with elevated keratinolytic activity using N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis resulted in a mutant strain KD-N2 producing keratinolytic activity about 2.5 times that of the wild-type strain. The mutant strain produced inducible keratinase in different substrates of feathers, hair, wool and silk under submerged cultivation. Scanning electron microscopy studies showed the degradation of feathers, hair and silk by the keratinase. The optimal conditions for keratinase production include initial pH of 7.5, inoculum size of 2% (v/v), age of inoculum of 16 h, and cultivation at 23 degrees C. The maximum keratinolytic activity of KD-N2 was achieved after 30 h. Essential amino acids like threonine, valine, methionine as well as ammonia were produced when feathers were used as substrates. Strain KD-N2, therefore, shows great promise of finding potential applications in keratin hydrolysis and keratinase production.
Amino Acids ; biosynthesis ; Bacillus subtilis ; genetics ; metabolism ; Culture Media ; Hydrogen-Ion Concentration ; Keratins ; metabolism ; Mutation ; Peptide Hydrolases ; biosynthesis

Enzymes can degrade the anti-nutrient factors in feedstuff, increase nutrient digestibility, and reduce pollution to environment, and have been widely supplemented in animal feedstuff. However, the use of enzymes is limited because of their undesirable properties, such as thermoliability and susceptibility against protease digestions. And its commercialization is also limited by low production efficiency and high cost. Therefore, the focuses for future enzyme development will be: (1) to obtain novel enzymes with better properties by high-throughput screening of enzyme encoding genes, especially those from extreme and special environments; (2) to improve enzyme properties using directed mutagenesis and protein engineering methods; (3) to achieve high-level fermentation of enzymes by heterogonous expression and optimization of codons, vectors and fermentation conditions; (4) to determine the effect of enzymes to animals and utilize enzymes efficiently.
6-Phytase ; genetics ; metabolism ; pharmacology ; Animal Feed ; Animals ; Dietary Supplements ; Lipase ; genetics ; metabolism ; pharmacology ; Peptide Hydrolases ; genetics ; metabolism ; pharmacology ; Protein Engineering

Monoclonal antibody (mAb) Tg786 against Toxoplasma gondii has been found to detect a 42-kDa rhoptry protein (ROP6) which showed protease activity and host cell binding characteristics after secretion. Using the mAb, a colony containing a 3'-UTR was probed in a T. gondii cDNA expression library. A full length cDNA sequence of the rhoptry protein was completed after 5'-RACE, which consisted of 1,908 bp with a 1,443 bp ORF. The deduced amino acid sequence of ROP6 consisted of a polypeptide of 480 amino acids without significant homology to any other known proteins. This sequence contains an amino terminal stop transfer sequence downstream of a short neutral sequence, hydrophilic middle sequence, and hydrophobic carboxy terminus. It is suggested that the ROP6 is inserted into the rhoptry membrane with both N- and C-termini.
Toxoplasma/enzymology/*genetics ; Protozoan Proteins/*genetics/metabolism ; Peptide Hydrolases/genetics/metabolism ; Molecular Sequence Data ; Gene Library ; Cloning, Molecular ; Animals ; Amino Acid Sequence ; 3' Untranslated Regions

AX15 is a mutein of naturally occurring human ciliary neurophic factor (hCNTF), with improved biological activity, stability and solubility. AX15 is susceptible to protease degradation when expressed in Pichia pastoris. Amino acid sequencing revealed the degradation was occurred behind position 12 and 13 amino acid residues, which constitute a dibasic site, RR. Based on the substrate specificity of KEX2, a KEX2 resistant mutein of AX15-AX15 (R13K) was constructed, in which RR was replaced by RK. It was demonstrated that the stability of AX15 (R13K) improved significantly, as no degradation was detected even after 120 hours of induction. AX15 (R13K) was purified to homogeneity by ultrafiltration and gel filtration. TF-1 cell survival bioassay showed AX15 (R13K) had equivalent specific activity to AX15. The protease resistant mutein of AX15 may have greater in vivo stability and thus have superior therapeutic potential.
Ciliary Neurotrophic Factor ; biosynthesis ; genetics ; Genetic Vectors ; Humans ; Mutant Proteins ; biosynthesis ; genetics ; Mutation ; Peptide Hydrolases ; chemistry ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification

Huntington's disease is caused by CAG trinucleotide expansions in the gene encoding huntingtin. N- terminal fragments of huntingtin with polyglutamine produce aggregates in the endosome-lysosomal system, where proteolytic fragments of huntingtin is generated. Heat shock protein 70 (HSP70) prevents the formation of protein aggregates, but the effect of HSP70 on the huntingtin in the endosome-lysosomal system is unknown. This study was to determine whether HSP70 alters the distribution of huntingtin in endosome-lysosomal system. HSP70 expressing stable cells (NIH/3T3 or cerebral hybrid cell line A1) were generated, and mutant [(CAG)100] huntingtin was transiently overexpressed. Analysis of subcellular distribution by immnuocytochemistry or proteolysis cleavage by Western blotting was performed. 18 CAG repeat wild type [WT; (CAG)18] huntingtin was used as a control. Cells with huntingtin showed patterns of endosome- lysosomal accumulation, from a 'dispersed vacuole (DV)' type into a coalescent 'perinuclear vacuole (PV)' type over time. In WT huntingtin, HSP70 increased the cells with the PV types that enhanced the proteolytic cleavage of huntingtin. However, HSP70 reduced cells of the DV and PV types expressing mutant huntingtin, that result in less proteolysis than that of control. In addition, intranuclear inclusions were formed only in mutant cells, which was not affected by HSP70. These results suggest that HSP70 alters the distribution of huntingtin in the endosome-lysosomal system, and that this contributes to huntingtin proteolysis.
Peptide Hydrolases/metabolism ; Nuclear Proteins/genetics/*metabolism ; Nerve Tissue Proteins/genetics/*metabolism ; NIH 3T3 Cells ; Mice ; Lysosomes/*metabolism ; HSP70 Heat-Shock Proteins/genetics/*metabolism ; Endosomes/*metabolism ; Cytoplasm/metabolism ; Cell Survival ; Animals

COP9 Signalosome Complex ; Carcinoma, Hepatocellular ; genetics ; metabolism ; Cyclin-Dependent Kinase Inhibitor p27 ; chemistry ; genetics ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Gene Expression Regulation, Neoplastic ; Humans ; Intracellular Signaling Peptides and Proteins ; chemistry ; genetics ; metabolism ; Peptide Hydrolases ; chemistry ; genetics ; metabolism ; Phosphorylation ; Phosphoserine ; metabolism

Pseudomonas aeruginosa is an important opportunistic human pathogen. It encodes many virulence factors and one of them is type III secretion system (TTSS). Effectors proteins can be delivered into host cells directly by this system, causing necrosis or apoptosis. popN gene is the first gene in the popN operon of TTSS gene cluster. To investigate its function, popN gene deletion mutant was generated in this study, and we found this mutant can secrete effectors proteins constitutively under non-inducting condition in DMEM medium containing serum. The results indicated that PopN is a negative regulator of the TTSS expression. However, no secreted effector proteins were detectable when the popN- mutant was grown in LB medium under non-inducting condition. To investigate the possible reasons, effects of growth status and protease (s) inhibitors on the TTSS were investigated. We present evidences that indicate protease mediated degradation of secreted effector proteins played a key role in the phenotypic inconsistency of popN- mutant.
ADP Ribose Transferases ; metabolism ; secretion ; Bacterial Proteins ; genetics ; metabolism ; secretion ; Bacterial Toxins ; metabolism ; Gene Expression Regulation, Bacterial ; Mutation ; Peptide Hydrolases ; genetics ; metabolism ; Pore Forming Cytotoxic Proteins ; genetics ; secretion ; Protease Inhibitors ; pharmacology ; Pseudomonas aeruginosa ; genetics ; metabolism ; pathogenicity

OBJECTIVETo report a protocol using biotin-labelled PrP protein in cell free conversion assay instead of isotope.

METHODSA hamster PrP protein (HaPrP) was expressed in E. coli and purified with HIS-tag affinity chromatograph. After being labelled with biotin, HaPrP was mixed with PrPSc preparation from scrapie strain 263K.

RESULTSProtease-resistant bands were detected after four-day incubation.

CONCLUSIONThe new conversion model provides a reliable, easily handling, and environment-friendly method for studies of prion and transmissible spongiform encephalopathies.

Animals ; Biotin ; Blotting, Western ; Cell-Free System ; Chromatography, Affinity ; methods ; Cricetinae ; Escherichia coli ; genetics ; Gene Expression Regulation ; Molecular Weight ; Peptide Hydrolases ; analysis ; metabolism ; PrPSc Proteins ; genetics ; metabolism ; Prion Diseases ; genetics ; pathology ; Recombinant Proteins ; genetics ; metabolism ; Staining and Labeling