Sample records for a1 protease gene

  1. Transcriptional activation of a 37 kDa ethylene responsive cysteine protease gene, RbCP1, is associated with protein degradation during petal abscission in rose

    PubMed Central

    Tripathi, Siddharth Kaushal; Singh, Amar Pal; Sane, Aniruddha P.; Nath, Pravendra

    2009-01-01

    Cysteine proteases play an important role in several developmental processes in plants, particularly those related to senescence and cell death. A cysteine protease gene, RbCP1, has been identified that encodes a putative protein of 357 amino acids and is expressed in the abscission zone (AZ) of petals in rose. The gene was responsive to ethylene in petals, petal abscission zones, leaves, and thalamus. The expression of RbCP1 increased during both ethylene-induced as well as natural abscission and was inhibited by 1-MCP. Transcript accumulation of RbCP1 was accompanied by the appearance of a 37 kDa cysteine protease, a concomitant increase in protease activity and a substantial decrease in total protein content in the AZ of petals. Agro-injection of rose petals with a 2.0 kb region upstream of the RbCP1 gene could drive GUS expression in an abscission zone-specific manner and was blocked by 1-MCP. It is concluded that petal abscission is associated with a decrease in total protein content resulting from rapid transcription of RbCP1 and the expression of a 37 kDa protease. PMID:19346241

  2. Transcriptional Activation by Heat and Cold of a Thiol Protease Gene in Tomato

    PubMed Central

    Schaffer, Mark A.; Fischer, Robert L.

    1990-01-01

    We previously determined that low temperature induces the accumulation in tomato (Lycopersicon esculentum) fruit of a cloned mRNA, designated C14, encoding a polypeptide related to thiol proteases (MA Schaffer, RL Fischer [1988] Plant Physiol 87: 431-436). We now demonstrate that C14 mRNA accumulation is a response common to both high (40°C) and low (4°C) temperature stresses. Exposure of tomato fruit to 40°C results in the accumulation of C14 mRNA, by 8 hours. This response is more rapid than that to 4°C, but slower than the induction of many heat shock messages by 40°C, and therefore unique. We have also studied the mechanism by which heat and cold exposure activate C14 gene expression. Both high and low temperature regulate protease gene expression through transcriptional induction of a single C14 gene. A hypothesis for the function of C14 thiol protease gene expression in response to heat and cold is discussed. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:16667644

  3. Detergent alkaline proteases: enzymatic properties, genes, and crystal structures.

    PubMed

    Saeki, Katsuhisa; Ozaki, Katsuya; Kobayashi, Tohru; Ito, Susumu

    2007-06-01

    Subtilisin-like serine proteases from bacilli have been used in various industrial fields worldwide, particularly in the production of laundry and automatic dishwashing detergents. They belong to family A of the subtilase superfamily, which is composed of three clans, namely, true subtilisins, high-alkaline proteases, and intracellular proteases. We succeeded in the large-scale production of a high-alkaline protease (M-protease) from alkaliphilic Bacillus clausii KSM-K16, and the enzyme has been introduced into compact heavy-duty laundry detergents. We have also succeeded in the industrial-scale production of a new alkaline protease, KP-43, which was originally resistant to chemical oxidants and to surfactants, produced by alkaliphilic Bacillus sp. strain KSM-KP43 and have incorporated it into laundry detergents. KP-43 and related proteases form a new clan, oxidatively stable proteases, in subtilase family A. In this review, we describe the enzymatic properties, gene sequences, and crystal structures of M-protease, KP-43, and related enzymes.

  4. Polymorphisms and resistance mutations in the protease and reverse transcriptase genes of HIV-1 F subtype Romanian strains.

    PubMed

    Paraschiv, Simona; Otelea, Dan; Dinu, Magdalena; Maxim, Daniela; Tinischi, Mihaela

    2007-03-01

    To evaluate the prevalence of resistance mutations in the genome of HIV-1 F subtype strains isolated from Romanian antiretroviral (ARV) treatment-naïve patients and to assess the phylogenetic relatedness of these strains with other HIV-1 strains. Twenty-nine HIV-1 strains isolated from treatment-naïve adolescents (n=15) and adults (n=14) were included in this study. Resistance genotyping was performed by using Big Dye Terminator chemistry provided by the ViroSeq Genotyping System. The sequences of the protease and reverse transcriptase genes were aligned (ClustalW) and a phylogenetic tree was built (MEGA 3 software). For subtyping purposes, all the nucleotide sequences were submitted to the Stanford database. All the studied strains were found to harbor accessory mutations in the protease gene. The most frequent mutation was M36I (29 of 29 strains), followed by L63T, K20R, and L10V. The number of polymorphisms associated with protease inhibitor resistance was different for the two age groups. Intraphylogenetic divergence was greater for adults than for adolescents infected in childhood. All the strains were found to belong to the F1 subtype. The phylogenetic analysis revealed that Romanian strains clustered together, but distinctly from F1 HIV-1 strains isolated in other parts of the world (Brazil, Finland, and Belgium). Protease secondary mutations are present with high frequency in the HIV-1 F subtype strains isolated from Romanian ARV treatment-naïve patients, but no major resistance mutations were found.

  5. A Comprehensive Genetic Study of Streptococcal Immunoglobulin A1 Proteases: Evidence for Recombination within and between Species

    PubMed Central

    Poulsen, Knud; Reinholdt, Jesper; Jespersgaard, Christina; Boye, Kit; Brown, Thomas A.; Hauge, Majbritt; Kilian, Mogens

    1998-01-01

    An analysis of 13 immunoglobulin A1 (IgA1) protease genes (iga) of strains of Streptococcus pneumoniae, Streptococcus oralis, Streptococcus mitis, and Streptococcus sanguis was carried out to obtain information on the structure, polymorphism, and phylogeny of this specific protease, which enables bacteria to evade functions of the predominant Ig isotype on mucosal surfaces. The analysis included cloning and sequencing of iga genes from S. oralis and S. mitis biovar 1, sequencing of an additional seven iga genes from S. sanguis biovars 1 through 4, and restriction fragment length polymorphism (RFLP) analyses of iga genes of another 10 strains of S. mitis biovar 1 and 6 strains of S. oralis. All 13 genes sequenced had the potential of encoding proteins with molecular masses of approximately 200 kDa containing the sequence motif HEMTH and an E residue 20 amino acids downstream, which are characteristic of Zn metalloproteinases. In addition, all had a typical gram-positive cell wall anchor motif, LPNTG, which, in contrast to such motifs in other known streptococcal and staphylococcal proteins, was located in their N-terminal parts. Repeat structures showing variation in number and sequence were present in all strains and may be of relevance to the immunogenicities of the enzymes. Protease activities in cultures of the streptococcal strains were associated with species of different molecular masses ranging from 130 to 200 kDa, suggesting posttranslational processing possibly as a result of autoproteolysis at post-proline peptide bonds in the N-terminal parts of the molecules. Comparison of deduced amino acid sequences revealed a 94% similarity between S. oralis and S. mitis IgA1 proteases and a 75 to 79% similarity between IgA1 proteases of these species and those of S. pneumoniae and S. sanguis, respectively. Combined with the results of RFLP analyses using different iga gene fragments as probes, the results of nucleotide sequence comparisons provide evidence of

  6. Identification and Partial Characterization of Extracellular Aspartic Protease Genes from Metschnikowia pulcherrima IWBT Y1123 and Candida apicola IWBT Y1384

    PubMed Central

    Reid, Vernita J.; Theron, Louwrens W.; du Toit, Maret

    2012-01-01

    The extracellular acid proteases of non-Saccharomyces wine yeasts may fulfill a number of roles in winemaking, which include increasing the available nitrogen sources for the growth of fermentative microbes, affecting the aroma profile of the wine, and potentially reducing protein haze formation. These proteases, however, remain poorly characterized, especially at genetic level. In this study, two extracellular aspartic protease-encoding genes were identified and sequenced, from two yeast species of enological origin: one gene from Metschnikowia pulcherrima IWBT Y1123, named MpAPr1, and the other gene from Candida apicola IWBT Y1384, named CaAPr1. In silico analysis of these two genes revealed a number of features peculiar to aspartic protease genes, and both the MpAPr1 and CaAPr1 putative proteins showed homology to proteases of yeast genera. Heterologous expression of MpAPr1 in Saccharomyces cerevisiae YHUM272 confirmed that it encodes an aspartic protease. MpAPr1 production, which was shown to be constitutive, and secretion were confirmed in the presence of bovine serum albumin (BSA), casein, and grape juice proteins. The MpAPr1 gene was found to be present in 12 other M. pulcherrima strains; however, plate assays revealed that the intensity of protease activity was strain dependent and unrelated to the gene sequence. PMID:22820332

  7. Physical characterization of the cloned protease III gene from Escherichia coli K-12.

    PubMed

    Dykstra, C C; Kushner, S R

    1985-09-01

    Analysis of the cloned protease III gene (ptr) from Escherichia coli K-12 has demonstrated that in addition to the previously characterized 110,000-Mr protease III protein, a second 50,000-Mr polypeptide (p50) is derived from the amino-terminal end of the coding sequence. The p50 polypeptide is found predominantly in the periplasmic space along with protease III, but does not proteolytically degrade insulin, a substrate for protease III. p50 does not appear to originate from autolysis of the larger protein. Protease III is not essential for normal cell growth since deletion of the structural gene causes no observed alterations in the phenotypic properties of the bacteria. A 30-fold overproduction of protease III does not affect cell viability. A simple new purification method for protease III is described.

  8. Disruption of ten protease genes in the filamentous fungus Aspergillus oryzae highly improves production of heterologous proteins.

    PubMed

    Yoon, Jaewoo; Maruyama, Jun-ichi; Kitamoto, Katsuhiko

    2011-02-01

    Proteolytic degradation by secreted proteases into the culture medium is one of the significant problems to be solved in heterologous protein production by filamentous fungi including Aspergillus oryzae. Double (tppA, and pepE) and quintuple (tppA, pepE, nptB, dppIV, and dppV) disruption of protease genes enhanced human lysozyme (HLY) and bovine chymosin (CHY) production by A. oryzae. In this study, we used a quintuple protease gene disruptant and performed successive rounds of disruption for five additional protease genes (alpA, pepA, AopepAa, AopepAd, and cpI), which were previously investigated by DNA microarray analyses for their expression. Gene disruption was performed by pyrG marker recycling with a highly efficient gene-targeting background (∆ligD) as previously reported. As a result, the maximum yields of recombinant CHY and HLY produced by a decuple protease gene disruptant were approximately 30% and 35%, respectively, higher than those produced by a quintuple protease gene disruptant. Thus, we successfully constructed a decuple protease gene disruptant possessing highly improved capability of heterologous protein production. This is the first report on decuple protease gene disruption that improved the levels of heterologous protein production by the filamentous fungus A. oryzae.

  9. Loss of hippocampal serine protease BSP1/neuropsin predisposes to global seizure activity.

    PubMed

    Davies, B; Kearns, I R; Ure, J; Davies, C H; Lathe, R

    2001-09-15

    Serine proteases in the adult CNS contribute both to activity-dependent structural changes accompanying learning and to the regulation of excitotoxic cell death. Brain serine protease 1 (BSP1)/neuropsin is a trypsin-like serine protease exclusively expressed, within the CNS, in the hippocampus and associated limbic structures. To explore the role of this enzyme, we have used gene targeting to disrupt this gene in mice. Mutant mice were viable and overtly normal; they displayed normal hippocampal long-term synaptic potentiation (LTP) and exhibited no deficits in spatial navigation (water maze). Nevertheless, electrophysiological studies revealed that the hippocampus of mice lacking this specifically expressed protease possessed an increased susceptibility for hyperexcitability (polyspiking) in response to repetitive afferent stimulation. Furthermore, seizure activity on kainic acid administration was markedly increased in mutant mice and was accompanied by heightened immediate early gene (c-fos) expression throughout the brain. In view of the regional selectivity of BSP1/neuropsin brain expression, the observed phenotype may selectively reflect limbic function, further implicating the hippocampus and amygdala in controlling cortical activation. Within the hippocampus, our data suggest that BSP1/neuropsin, unlike other serine proteases, has little effect on physiological synaptic remodeling and instead plays a role in limiting neuronal hyperexcitability induced by epileptogenic insult.

  10. Transcriptional activation by heat and cold of a thiol protease gene in tomato. [Lycopersicon esculentum

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Schaffer, M.A.; Fischer, R.L.

    We previously determined that low temperature induces the accumulation in tomato (Lycopersicon esculentum) fruit of a cloned mRNA, designated C14, encoding a polypeptide related to thiol proteases. We now demonstrate that C14 mRNA accumulation is a response common to both high (40{degree}C) and low (4{degree}C) temperature stresses. Exposure of tomato fruit to 40{degree}C results in the accumulation of C14 mRNA, by 8 hours. This response is more rapid than that to 4{degree}C, but slower than the induction of many heat shock messages by 40{degree}C, and therefore unique. We have also studied the mechanism by which heat and cold exposure activatemore » C14 gene expression. Both high and low temperature regulate protease gene expression through transcriptional induction of a single C14 gene. A hypothesis for the function of C14 thiol protease gene expression in response to heat and cold is discussed.« less

  11. A complex of serine protease genes expressed preferentially in cytotoxic T-lymphocytes is closely linked to the T-cell receptor alpha- and delta-chain genes on mouse chromosome 14.

    PubMed

    Crosby, J L; Bleackley, R C; Nadeau, J H

    1990-02-01

    A complex of genes encoding serine proteases that are preferentially expressed in cytotoxic T-cells was shown to be closely linked to the T-cell receptor alpha- and delta-chain genes on mouse chromosome 14. A striking difference in recombination frequencies among linkage crosses was reported. Two genes, Np-1 and Tcra, which fail to recombine in crosses involving conventional strains of mice, were shown to recombine readily in interspecific crosses involving Mus spretus. This difference in recombination frequency suggests chromosomal rearrangements that suppress recombination in conventional crosses, recombination hot spots in interspecific crosses, or selection against recombinant haplotypes during development of recombinant inbred strains. Finally, a mutation called disorganization, which is located near the serine protease complex, is of considerable interest because it causes an extraordinarily wide variety of congenital defects. Because of the involvement of serine protease loci in several homeotic mutations in Drosophila, disorganization must be considered a candidate for a mutation in a serine protease-encoding gene.

  12. Cloning and characterization of the gene for an additional extracellular serine protease of Bacillus subtilis.

    PubMed Central

    Sloma, A; Rufo, G A; Theriault, K A; Dwyer, M; Wilson, S W; Pero, J

    1991-01-01

    We have purified a minor extracellular serine protease from a strain of Bacillus subtilis bearing null mutations in five extracellular protease genes: apr, npr, epr, bpr, and mpr (A. Sloma, C. Rudolph, G. Rufo, Jr., B. Sullivan, K. Theriault, D. Ally, and J. Pero, J. Bacteriol. 172:1024-1029, 1990). During purification, this novel protease (Vpr) was found bound in a complex in the void volume after gel filtration chromatography. The amino-terminal sequence of the purified protein was determined, and an oligonucleotide probe was constructed on the basis of the amino acid sequence. This probe was used to clone the structural gene (vpr) for this protease. The gene encodes a primary product of 806 amino acids. The amino acid sequence of the mature protein was preceded by a signal sequence of approximately 28 amino acids and a prosequence of approximately 132 amino acids. The mature protein has a predicted molecular weight of 68,197; however, the isolated protein has an apparent molecular weight of 28,500, suggesting that Vpr undergoes C-terminal processing or proteolysis. The vpr gene maps in the ctrA-sacA-epr region of the chromosome and is not required for growth or sporulation. Images FIG. 1 PMID:1938892

  13. Erwinia carotovora subsp. carotovora extracellular protease: characterization and nucleotide sequence of the gene.

    PubMed Central

    Kyöstiö, S R; Cramer, C L; Lacy, G H

    1991-01-01

    The prt1 gene encoding extracellular protease from Erwinia carotovora subsp. carotovora EC14 in cosmid pCA7 was subcloned to create plasmid pSK1. The partial nucleotide sequence of the insert in pSK1 (1,878 bp) revealed a 1,041-bp open reading frame (ORF1) that correlated with protease activity in deletion mutants. ORF1 encodes a polypeptide of 347 amino acids with a calculated molecular mass of 38,826 Da. Escherichia coli transformed with pSK1 or pSK23, a subclone of pSK1, produces a protease (Prt1) intracellularly with a molecular mass of 38 kDa and a pI of 4.8. Prt1 activity was inhibited by phenanthroline, suggesting that it is a metalloprotease. The prt1 promoter was localized between 173 and 1,173 bp upstream of ORF1 by constructing transcriptional lacZ fusions. Primer extension identified the prt1 transcription start site 205 bp upstream of ORF1. The deduced amino acid sequence of ORF1 showed significant sequence identity to metalloproteases from Bacillus thermoproteolyticus (thermolysin), B. subtilis (neutral protease), Legionella pneumophila (metalloprotease), and Pseudomonas aeruginosa (elastase). It has less sequence similarity to metalloproteases from Serratia marcescens and Erwinia chrysanthemi. Locations for three zinc ligands and the active site for E. carotovora subsp. carotovora protease were predicted from thermolysin. Images FIG. 2 FIG. 5 FIG. 6 FIG. 8 FIG. 9 PMID:1917878

  14. Nematicidal protease genes screened from a soil metagenomic library to control Radopholus similis mediated by Pseudomonas fluorescens pf36.

    PubMed

    Chen, Deqiang; Wang, Dongwei; Xu, Chunling; Chen, Chun; Li, Junyi; Wu, Wenjia; Huang, Xin; Xie, Hui

    2018-04-01

    Controlling Radopholus similis, an important phytopathogenic nematode, is a challenge worldwide. Herein, we constructed a metagenomic fosmid library from the rhizosphere soil of banana plants, and six clones with protease activity were obtained by functionally screening the library. Furthermore, subclones were constructed using the six clones, and three protease genes with nematicidal activity were identified: pase1, pase4, and pase6. The pase4 gene was successfully cloned and expressed, demonstrating that the protease PASE4 could effectively degrade R. similis tissues and result in nematode death. Additionally, we isolated a predominant R. similis-associated bacterium, Pseudomonas fluorescens (pf36), from 10 R. similis populations with different hosts. The pase4 gene was successfully introduced into the pf36 strain by vector transformation and conjugative transposition, and two genetically modified strains were obtained: p4MCS-pf36 and p4Tn5-pf36. p4MCS-pf36 had significantly higher protease expression and nematicidal activity (p < 0.05) than p4Tn5-pf36 in a microtiter plate assay, whereas p4Tn5-pf36 was superior to p4MCS-pf36 in terms of genetic stability and controlling R. similis in growth pot tests. This study confirmed that R. similis is inhibited by the associated bacterium pf36-mediated expression of nematicidal proteases. Herein, a novel approach is provided for the study and development of efficient, environmentally friendly, and sustainable biocontrol techniques against phytonematodes.

  15. Molecular characterization of alkaline protease of Bacillus amyloliquefaciens SP1 involved in biocontrol of Fusarium oxysporum.

    PubMed

    Guleria, Shiwani; Walia, Abhishek; Chauhan, Anjali; Shirkot, C K

    2016-09-02

    An alkaline protease gene was amplified from genomic DNA of Bacillus amyloliquefaciens SP1 which was involved in effective biocontrol of Fusarium oxysporum. We investigated the antagonistic capacity of protease of B. amyloliquifaciens SP1, under in vitro conditions. The 5.62 fold purified enzyme with specific activity of 607.69U/mg reported 24.14% growth inhibition of F. oxysporum. However, no antagonistic activity was found after addition of protease inhibitor i.e. PMSF (15mM) to purified enzyme. An 1149bp nucleotide sequence of protease gene encoded 382 amino acids of 43kDa and calculated isoelectric point of 9.29. Analysis of deduced amino acid sequence revealed high homology (86%) with subtilisin E of Bacillus subtilis. The B. amyloliquefaciens SP1 protease gene was expressed in Escherichiax coli BL21. The expressed protease was secreted into culture medium by E. coli and exhibited optimum activity at pH8.0 and 60°C. The most reliable three dimensional structure of alkaline protease was determined using Phyre 2 server which was validated on the basis of Ramachandran plot and ERRAT value. The expression and structure prediction of the enzyme offers potential value for commercial application in agriculture and industry. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Cloning and sequencing of an alkaline protease gene from Bacillus lentus and amplification of the gene on the B. lentus chromosome by an improved technique.

    PubMed

    Jørgensen, P L; Tangney, M; Pedersen, P E; Hastrup, S; Diderichsen, B; Jørgensen, S T

    2000-02-01

    A gene encoding an alkaline protease was cloned from an alkalophilic bacillus, and its nucleotide sequence was determined. The cloned gene was used to increase the copy number of the protease gene on the chromosome by an improved gene amplification technique.

  17. Cloning, characterization, expression analysis and inhibition studies of a novel gene encoding Bowman-Birk type protease inhibitor from rice bean

    USDA-ARS?s Scientific Manuscript database

    This paper presents the first study describing the isolation, cloning and characterization of a full length gene encoding Bowman-Birk protease inhibitor (RbTI) from rice bean (Vigna umbellata). A full-length protease inhibitor gene with complete open reading frame of 327bp encoding 109 amino acids w...

  18. Plasmodium vivax rhomboid-like protease 1 gene diversity in Thailand.

    PubMed

    Mataradchakul, Touchchapol; Uthaipibull, Chairat; Nosten, Francois; Vega-Rodriguez, Joel; Jacobs-Lorena, Marcelo; Lek-Uthai, Usa

    2017-10-01

    Plasmodium vivax infection remains a major public health problem, especially along the Thailand border regions. We examined the genetic diversity of this parasite by analyzing single-nucleotide polymorphisms (SNPs) of the P. vivax rhomboid-like protease 1 gene (Pvrom1) in parasites collected from western (Tak province, Thai-Myanmar border) and eastern (Chanthaburi province, Thai-Cambodia border) regions. Data were collected by a cross-sectional survey, consisting of 47 and 45 P. vivax-infected filter paper-spotted blood samples from the western and eastern regions of Thailand, respectively during September 2013 to May 2014. Extracted DNA was examined for presence of P. vivax using Plasmodium species-specific nested PCR. Pvrom1 gene was PCR amplified, sequenced and the SNP diversity was analyzed using F-STAT, DnaSP, MEGA and LIAN programs. Comparison of sequences of the 92 Pvrom1 831-base open reading frames with that of a reference sequence (GenBank acc. no. XM001615211) revealed 17 samples with a total of 8 polymorphic sites, consisting of singleton (exon 3, nt 645) and parsimony informative (exon 1, nt 22 and 39; exon 3, nt 336, 537 and 656; and exon 4, nt 719 and 748) sites, which resulted in six different deduced Pvrom1 variants. Non-synonymous to synonymous substitutions ratio estimated by the DnaSP program was 1.65 indicating positive selection, but the Z-tests of selection showed no significant deviations from neutrality for Pvrom1 samples from western region of Thailand. In addition McDonald Kreitman test (MK) showed not significant, and Fst values are not different between the two regions and the regions combined. Interestingly, only Pvrom1 exon 2 was the most conserved sequences among the four exons. The relatively high degree of Pvrom1 polymorphism suggests that the protein is important for parasite survival in face of changes in both insect vector and human populations. These polymorphisms could serve as a sensitive marker for studying plasmodial

  19. A novel aspartic acid protease gene from pineapple fruit (Ananas comosus): cloning, characterization and relation to postharvest chilling stress resistance.

    PubMed

    Raimbault, Astrid-Kim; Zuily-Fodil, Yasmine; Soler, Alain; Cruz de Carvalho, Maria H

    2013-11-15

    A full-length cDNA encoding a putative aspartic acid protease (AcAP1) was isolated for the first time from the flesh of pineapple (Ananas comosus) fruit. The deduced sequence of AcAP1 showed all the common features of a typical plant aspartic protease phytepsin precursor. Analysis of AcAP1 gene expression under postharvest chilling treatment in two pineapple varieties differing in their resistance to blackheart development revealed opposite trends. The resistant variety showed an up-regulation of AcAP1 precursor gene expression whereas the susceptible showed a down-regulation in response to postharvest chilling treatment. The same trend was observed regarding specific AP enzyme activity in both varieties. Taken together our results support the involvement of AcAP1 in postharvest chilling stress resistance in pineapple fruits. Copyright © 2013 Elsevier GmbH. All rights reserved.

  20. Dysregulation of Protease and Protease Inhibitors in a Mouse Model of Human Pelvic Organ Prolapse

    PubMed Central

    Budatha, Madhusudhan; Silva, Simone; Montoya, Teodoro Ignacio; Suzuki, Ayako; Shah-Simpson, Sheena; Wieslander, Cecilia Karin; Yanagisawa, Masashi; Word, Ruth Ann; Yanagisawa, Hiromi

    2013-01-01

    Mice deficient for the fibulin-5 gene (Fbln5−/−) develop pelvic organ prolapse (POP) due to compromised elastic fibers and upregulation of matrix metalloprotease (MMP)-9. Here, we used casein zymography, inhibitor profiling, affinity pull-down, and mass spectrometry to discover additional protease upregulated in the vaginal wall of Fbln5−/− mice, herein named V1 (25 kDa). V1 was a serine protease with trypsin-like activity similar to protease, serine (PRSS) 3, a major extrapancreatic trypsinogen, was optimum at pH 8.0, and predominantly detected in estrogenized vaginal epithelium of Fbln5−/− mice. PRSS3 was (a) localized in epithelial secretions, (b) detected in media of vaginal organ culture from both Fbln5−/− and wild type mice, and (c) cleaved fibulin-5 in vitro. Expression of two serine protease inhibitors [Serpina1a1-antitrypsin) and Elafin] was dysregulated in Fbln5−/− epithelium. Finally, we confirmed that PRSS3 was expressed in human vaginal epithelium and that SERPINA1 and Elafin were downregulated in vaginal tissues from women with POP. These data collectively suggest that the balance between proteases and their inhibitors contributes to support of the pelvic organs in humans and mice. PMID:23437119

  1. Cloning and characterization of the gene for an additional extracellular serine protease of Bacillus subtilis.

    PubMed

    Sloma, A; Rufo, G A; Theriault, K A; Dwyer, M; Wilson, S W; Pero, J

    1991-11-01

    We have purified a minor extracellular serine protease from a strain of Bacillus subtilis bearing null mutations in five extracellular protease genes: apr, npr, epr, bpr, and mpr (A. Sloma, C. Rudolph, G. Rufo, Jr., B. Sullivan, K. Theriault, D. Ally, and J. Pero, J. Bacteriol. 172:1024-1029, 1990). During purification, this novel protease (Vpr) was found bound in a complex in the void volume after gel filtration chromatography. The amino-terminal sequence of the purified protein was determined, and an oligonucleotide probe was constructed on the basis of the amino acid sequence. This probe was used to clone the structural gene (vpr) for this protease. The gene encodes a primary product of 806 amino acids. The amino acid sequence of the mature protein was preceded by a signal sequence of approximately 28 amino acids and a prosequence of approximately 132 amino acids. The mature protein has a predicted molecular weight of 68,197; however, the isolated protein has an apparent molecular weight of 28,500, suggesting that Vpr undergoes C-terminal processing or proteolysis. The vpr gene maps in the ctrA-sacA-epr region of the chromosome and is not required for growth or sporulation.

  2. Curcumin derivatives as HIV-1 protease inhibitors

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sui, Z.; Li, J.; Craik, C.S.

    1993-12-31

    Curcumin, a non-toxic natural compound from Curcuma longa, has been found to be an HIV-1 protease inhibitor. Some of its derivatives were synthesized and their inhibitory activity against the HIV-1 protease was tested. Curcumin analogues containing boron enhanced the inhibitory activity. At least of the the synthesized compounds irreversibly inhibits the HIV-1 protease.

  3. Secretory leukocyte protease inhibitor gene deletion alters bleomycin-induced lung injury, but not development of pulmonary fibrosis.

    PubMed

    Habgood, Anthony N; Tatler, Amanda L; Porte, Joanne; Wahl, Sharon M; Laurent, Geoffrey J; John, Alison E; Johnson, Simon R; Jenkins, Gisli

    2016-06-01

    Idiopathic pulmonary fibrosis is a progressive, fatal disease with limited treatment options. Protease-mediated transforming growth factor-β (TGF-β) activation has been proposed as a pathogenic mechanism of lung fibrosis. Protease activity in the lung is tightly regulated by protease inhibitors, particularly secretory leukocyte protease inhibitor (SLPI). The bleomycin model of lung fibrosis was used to determine the effect of increased protease activity in the lungs of Slpi(-/-) mice following injury. Slpi(-/-), and wild-type, mice received oropharyngeal administration of bleomycin (30 IU) and the development of pulmonary fibrosis was assessed. Pro and active forms of matrix metalloproteinase (MMP)-2 and MMP-9 were measured. Lung fibrosis was determined by collagen subtype-specific gene expression, hydroxyproline concentration, and histological assessment. Alveolar TGF-β activation was measured using bronchoalveolar lavage cell pSmad2 levels and global TGF-β activity was assessed by pSmad2 immunohistochemistry. The active-MMP-9 to pro-MMP-9 ratio was significantly increased in Slpi(-/-) animals compared with wild-type animals, demonstrating enhanced metalloproteinase activity. Wild-type animals showed an increase in TGF-β activation following bleomycin, with a progressive and sustained increase in collagen type I, alpha 1 (Col1α1), III, alpha 1(Col3α1), IV, alpha 1(Col4α1) mRNA expression, and a significant increase in total lung collagen 28 days post bleomycin. In contrast Slpi(-/-) mice showed no significant increase of alveolar TGF-β activity following bleomycin, above their already elevated levels, although global TGF-β activity did increase. Slpi(-/-) mice had impaired collagen gene expression but animals demonstrated minimal reduction in lung fibrosis compared with wild-type animals. These data suggest that enhanced proteolysis does not further enhance TGF-β activation, and inhibits sustained Col1α1, Col3α1, and Col4α1 gene expression

  4. Comprehensive mutagenesis of HIV-1 protease: a computational geometry approach.

    PubMed

    Masso, Majid; Vaisman, Iosif I

    2003-05-30

    A computational geometry technique based on Delaunay tessellation of protein structure, represented by C(alpha) atoms, is used to study effects of single residue mutations on sequence-structure compatibility in HIV-1 protease. Profiles of residue scores derived from the four-body statistical potential are constructed for all 1881 mutants of the HIV-1 protease monomer and compared with the profile of the wild-type protein. The profiles for an isolated monomer of HIV-1 protease and the identical monomer in a dimeric state with an inhibitor are analyzed to elucidate changes to structural stability. Protease residues shown to undergo the greatest impact are those forming the dimer interface and flap region, as well as those known to be involved in inhibitor binding.

  5. HIV-1 Protease in the Fission Yeast Schizosaccharomyces pombe.

    PubMed

    Benko, Zsigmond; Elder, Robert T; Li, Ge; Liang, Dong; Zhao, Richard Y

    2016-01-01

    HIV-1 protease (PR) is an essential viral enzyme. Its primary function is to proteolyze the viral Gag-Pol polyprotein for production of viral enzymes and structural proteins and for maturation of infectious viral particles. Increasing evidence suggests that PR cleaves host cellular proteins. However, the nature of PR-host cellular protein interactions is elusive. This study aimed to develop a fission yeast (Schizosaccharomyces pombe) model system and to examine the possible interaction of HIV-1 PR with cellular proteins and its potential impact on cell proliferation and viability. A fission yeast strain RE294 was created that carried a single integrated copy of the PR gene in its chromosome. The PR gene was expressed using an inducible nmt1 promoter so that PR-specific effects could be measured. HIV-1 PR from this system cleaved the same indigenous viral p6/MA protein substrate as it does in natural HIV-1 infections. HIV-1 PR expression in fission yeast cells prevented cell proliferation and induced cellular oxidative stress and changes in mitochondrial morphology that led to cell death. Both these PR activities can be prevented by a PR-specific enzymatic inhibitor, indinavir, suggesting that PR-mediated proteolytic activities and cytotoxic effects resulted from enzymatic activities of HIV-1 PR. Through genome-wide screening, a serine/threonine kinase, Hhp2, was identified that suppresses HIV-1 PR-induced protease cleavage and cell death in fission yeast and in mammalian cells, where it prevented PR-induced apoptosis and cleavage of caspase-3 and caspase-8. This is the first report to show that HIV-1 protease is functional as an enzyme in fission yeast, and that it behaves in a similar manner as it does in HIV-1 infection. HIV-1 PR-induced cell death in fission yeast could potentially be used as an endpoint for mechanistic studies, and this system could be used for developing a high-throughput system for drug screenings.

  6. Inhibition of Prevotella and Capnocytophaga immunoglobulin A1 proteases by human serum.

    PubMed

    Frandsen, E V; Kjeldsen, M; Kilian, M

    1997-07-01

    Oral Prevotella and Capnocytophaga species, regularly isolated from periodontal pockets and associated with extraoral infections, secret specific immunoglobulin A1 (IgA1) proteases cleaving human IgA1 in the hinge region into intact Fab and Fc fragments. To investigate whether these enzymes are subject to inhibition in vivo in humans, we tested 34 sera from periodontally diseased and healthy individuals in an enzyme-linked immunosorbent assay for the presence and titers of inhibition of seven Prevotella and Capnocytophaga proteases. All or nearly all of the sera inhibited the IgA1 protease activity of Prevotella buccae, Prevotella oris, and Prevotella loescheii. A minor proportion of the sera inhibited Prevotella buccalis, Prevotella denticola, and Prevotella melaninogenica IgA1 proteases, while no sera inhibited Capnocytophaga ochracea IgA1 protease. All inhibition titers were low, ranging from 5 to 55, with titer being defined as the reciprocal of the dilution of serum causing 50% inhibition of one defined unit of protease activity. No correlation between periodontal disease status and the presence, absence, or titer of inhibition was observed. The nature of the low titers of inhibition in all sera of the IgA1 proteases of P. buccae, P. oris, and P. loescheii was further examined. In size exclusion chromatography, inhibitory activity corresponded to the peak volume of IgA. Additional inhibition of the P. oris IgA1 protease was found in fractions containing both IgA and IgG. Purification of the IgG fractions of five sera by passage of the sera on a protein G column resulted in recovery of inhibitory IgG antibodies against all three IgA1 proteases, with the highest titer being for the P. oris enzyme. These finding indicate that inhibitory activity is associated with enzyme-neutralizing antibodies.

  7. Inhibition of Prevotella and Capnocytophaga immunoglobulin A1 proteases by human serum.

    PubMed Central

    Frandsen, E V; Kjeldsen, M; Kilian, M

    1997-01-01

    Oral Prevotella and Capnocytophaga species, regularly isolated from periodontal pockets and associated with extraoral infections, secret specific immunoglobulin A1 (IgA1) proteases cleaving human IgA1 in the hinge region into intact Fab and Fc fragments. To investigate whether these enzymes are subject to inhibition in vivo in humans, we tested 34 sera from periodontally diseased and healthy individuals in an enzyme-linked immunosorbent assay for the presence and titers of inhibition of seven Prevotella and Capnocytophaga proteases. All or nearly all of the sera inhibited the IgA1 protease activity of Prevotella buccae, Prevotella oris, and Prevotella loescheii. A minor proportion of the sera inhibited Prevotella buccalis, Prevotella denticola, and Prevotella melaninogenica IgA1 proteases, while no sera inhibited Capnocytophaga ochracea IgA1 protease. All inhibition titers were low, ranging from 5 to 55, with titer being defined as the reciprocal of the dilution of serum causing 50% inhibition of one defined unit of protease activity. No correlation between periodontal disease status and the presence, absence, or titer of inhibition was observed. The nature of the low titers of inhibition in all sera of the IgA1 proteases of P. buccae, P. oris, and P. loescheii was further examined. In size exclusion chromatography, inhibitory activity corresponded to the peak volume of IgA. Additional inhibition of the P. oris IgA1 protease was found in fractions containing both IgA and IgG. Purification of the IgG fractions of five sera by passage of the sera on a protein G column resulted in recovery of inhibitory IgG antibodies against all three IgA1 proteases, with the highest titer being for the P. oris enzyme. These finding indicate that inhibitory activity is associated with enzyme-neutralizing antibodies. PMID:9220164

  8. Extracellular proteases of Trichoderma species. A review.

    PubMed

    Kredics, L; Antal, Zsuzsanna; Szekeres, A; Hatvani, L; Manczinger, L; Vágvölgyi, Cs; Nagy, Erzsébet

    2005-01-01

    Cellulolytic, xylanolytic, chitinolytic and beta-1,3-glucanolytic enzyme systems of species belonging to the filamentous fungal genus Trichoderma have been investigated in details and are well characterised. The ability of Trichoderma strains to produce extracellular proteases has also been known for a long time, however, the proteolytic enzyme system is relatively unknown in this genus. Fortunately, in the recent years more and more attention is focused on the research in this field. The role of Trichoderma proteases in the biological control of plant pathogenic fungi and nematodes has been demonstrated, and it is also suspected that they may be important for the competitive saprophytic ability of green mould isolates and may represent potential virulence factors of Trichoderma strains as emerging fungal pathogens of clinical importance. The aim of this review is to summarize the information available about the extracellular proteases of Trichoderma. Numerous studies are available about the extracellular proteolytic enzyme profiles of Trichoderma strains and about the effect of abiotic environmental factors on protease activities. A number of protease enzymes have been purified to homogeneity and some protease encoding genes have been cloned and characterized. These results will be reviewed and the role of Trichoderma proteases in biological control as well as their advantages and disadvantages in biotechnology will be discussed.

  9. Lon Protease of Azorhizobium caulinodans ORS571 Is Required for Suppression of reb Gene Expression

    PubMed Central

    Nakajima, Azusa; Tsukada, Shuhei; Siarot, Lowela; Ogawa, Tetsuhiro; Oyaizu, Hiroshi

    2012-01-01

    Bacterial Lon proteases play important roles in a variety of biological processes in addition to housekeeping functions. In this study, we focused on the Lon protease of Azorhizobium caulinodans, which can fix nitrogen both during free-living growth and in stem nodules of the legume Sesbania rostrata. The nitrogen fixation activity of an A. caulinodans lon mutant in the free-living state was not significantly different from that of the wild-type strain. However, the stem nodules formed by the lon mutant showed little or no nitrogen fixation activity. By microscopic analyses, two kinds of host cells were observed in the stem nodules formed by the lon mutant. One type has shrunken host cells containing a high density of bacteria, and the other type has oval or elongated host cells containing a low density or no bacteria. This phenotype is similar to a praR mutant highly expressing the reb genes. Quantitative reverse transcription-PCR analyses revealed that reb genes were also highly expressed in the lon mutant. Furthermore, a lon reb double mutant formed stem nodules showing higher nitrogen fixation activity than the lon mutant, and shrunken host cells were not observed in these stem nodules. These results suggest that Lon protease is required to suppress the expression of the reb genes and that high expression of reb genes in part causes aberrance in the A. caulinodans-S. rostrata symbiosis. In addition to the suppression of reb genes, it was found that Lon protease was involved in the regulation of exopolysaccharide production and autoagglutination of bacterial cells. PMID:22752172

  10. HIV-1 protease-substrate coevolution in nelfinavir resistance.

    PubMed

    Kolli, Madhavi; Ozen, Ayşegül; Kurt-Yilmaz, Nese; Schiffer, Celia A

    2014-07-01

    Resistance to various human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) challenges the effectiveness of therapies in treating HIV-1-infected individuals and AIDS patients. The virus accumulates mutations within the protease (PR) that render the PIs less potent. Occasionally, Gag sequences also coevolve with mutations at PR cleavage sites contributing to drug resistance. In this study, we investigated the structural basis of coevolution of the p1-p6 cleavage site with the nelfinavir (NFV) resistance D30N/N88D protease mutations by determining crystal structures of wild-type and NFV-resistant HIV-1 protease in complex with p1-p6 substrate peptide variants with L449F and/or S451N. Alterations of residue 30's interaction with the substrate are compensated by the coevolving L449F and S451N cleavage site mutations. This interdependency in the PR-p1-p6 interactions enhances intermolecular contacts and reinforces the overall fit of the substrate within the substrate envelope, likely enabling coevolution to sustain substrate recognition and cleavage in the presence of PR resistance mutations. Resistance to human immunodeficiency virus type 1 (HIV-1) protease inhibitors challenges the effectiveness of therapies in treating HIV-1-infected individuals and AIDS patients. Mutations in HIV-1 protease selected under the pressure of protease inhibitors render the inhibitors less potent. Occasionally, Gag sequences also mutate and coevolve with protease, contributing to maintenance of viral fitness and to drug resistance. In this study, we investigated the structural basis of coevolution at the Gag p1-p6 cleavage site with the nelfinavir (NFV) resistance D30N/N88D protease mutations. Our structural analysis reveals the interdependency of protease-substrate interactions and how coevolution may restore substrate recognition and cleavage in the presence of protease drug resistance mutations. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  11. Sweet potato SPAP1 is a typical aspartic protease and participates in ethephon-mediated leaf senescence.

    PubMed

    Chen, Hsien-Jung; Huang, Yu-Hsuan; Huang, Guan-Jhong; Huang, Shyh-Shyun; Chow, Te-Jin; Lin, Yaw-Huei

    2015-05-15

    Plant aspartic proteases are generally divided into three categories: typical, nucellin-like, and atypical aspartic proteases based on their gene and protein structures. In this report, a full-length cDNA SPAP1 was cloned from sweet potato leaves, which contained 1515 nucleotides (504 amino acids) and exhibited high amino acid sequence identity (ca. 51-72%) with plant typical aspartic proteases, including tomato LeAspP, potato StAsp, and wheat WAP2. SPAP1 also contained conserved DTG and DSG amino acid residues within its catalytic domain and plant specific insert (PSI) at the C-terminus. The cDNA corresponding to the mature protein (starting from the 66th to 311th amino acid residues) without PSI domain was constructed with pET30a expression vector for fusion protein and antibody production. RT-PCR and protein blot hybridization showed that SPAP1 expression level was the highest in L3 mature leaves, then gradually declined until L5 completely yellow leaves. Ethephon, an ethylene-releasing compound, also enhanced SPAP1 expression at the time much earlier than the onset of leaf senescence. Exogenous application of SPAP1 fusion protein promoted ethephon-induced leaf senescence, which could be abolished by pre-treatment of SPAP1 fusion protein with (a) 95 °C for 5 min, (b) aspartic protease inhibitor pepstatin A, and (c) anti-SPAP1 antibody, respectively. Exogenous SPAP1 fusion protein, whereas, did not significantly affect leaf senescence under dark. These data conclude that sweet potato SPAP1 is a functional typical aspartic protease and participates in ethephon-mediated leaf senescence. The SPAP1-promoted leaf senescence and its activity are likely not associated with the PSI domain. Interaction of ethephon-inducible components for effective SPAP1 promotion on leaf senescence is also suggested. Copyright © 2015 Elsevier GmbH. All rights reserved.

  12. Cloning and characterization of TPE4A, a thiol-protease gene induced during ovary senescence and seed germination in pea.

    PubMed

    Cercós, M; Santamaría, S; Carbonell, J

    1999-04-01

    A cDNA clone encoding a thiol-protease (TPE4A) was isolated from senescent ovaries of pea (Pisum sativum) by reverse transcriptase-polymerase chain reaction. The deduced amino acid sequence of TPE4A has the conserved catalytic amino acids of papain. It is very similar to VSCYSPROA, a thiol-protease induced during seed germination in common vetch. TPE4A mRNA levels increase during the senescence of unpollinated pea ovaries and are totally suppressed by treatment with gibberellic acid. In situ hybridization indicated that TPE4A mRNA distribution in senescent pea ovaries is different from that of previously reported thiol-proteases induced during senescence, suggesting the involvement of different proteases in the mobilization of proteins from senescent pea ovaries. TPE4A is also induced during the germination of pea seeds, indicating that a single protease gene can be induced during two different physiological processes, senescence and germination, both of which require protein mobilization.

  13. A Maize Cystatin Suppresses Host Immunity by Inhibiting Apoplastic Cysteine Proteases[C][W

    PubMed Central

    van der Linde, Karina; Hemetsberger, Christoph; Kastner, Christine; Kaschani, Farnusch; van der Hoorn, Renier A.L.; Kumlehn, Jochen; Doehlemann, Gunther

    2012-01-01

    Ustilago maydis is a biotrophic pathogen causing maize (Zea mays) smut disease. Transcriptome profiling of infected maize plants indicated that a gene encoding a putative cystatin (CC9) is induced upon penetration by U. maydis wild type. By contrast, cc9 is not induced after infection with the U. maydis effector mutant Δpep1, which elicits massive plant defenses. Silencing of cc9 resulted in a strongly induced maize defense gene expression and a hypersensitive response to U. maydis wild-type infection. Consequently, fungal colonization was strongly reduced in cc9-silenced plants, while recombinant CC9 prevented salicylic acid (SA)–induced defenses. Protease activity profiling revealed a strong induction of maize Cys proteases in SA-treated leaves, which could be inhibited by addition of CC9. Transgenic maize plants overexpressing cc9-mCherry showed an apoplastic localization of CC9. The transgenic plants showed a block in Cys protease activity and SA-dependent gene expression. Moreover, activated apoplastic Cys proteases induced SA-associated defense gene expression in naïve plants, which could be suppressed by CC9. We show that apoplastic Cys proteases play a pivotal role in maize defense signaling. Moreover, we identified cystatin CC9 as a novel compatibility factor that suppresses Cys protease activity to allow biotrophic interaction of maize with the fungal pathogen U. maydis. PMID:22454455

  14. Human High Temperature Requirement Serine Protease A1 (HTRA1) Degrades Tau Protein Aggregates*

    PubMed Central

    Tennstaedt, Annette; Pöpsel, Simon; Truebestein, Linda; Hauske, Patrick; Brockmann, Anke; Schmidt, Nina; Irle, Inga; Sacca, Barbara; Niemeyer, Christof M.; Brandt, Roland; Ksiezak-Reding, Hanna; Tirniceriu, Anca Laura; Egensperger, Rupert; Baldi, Alfonso; Dehmelt, Leif; Kaiser, Markus; Huber, Robert; Clausen, Tim; Ehrmann, Michael

    2012-01-01

    Protective proteases are key elements of protein quality control pathways that are up-regulated, for example, under various protein folding stresses. These proteases are employed to prevent the accumulation and aggregation of misfolded proteins that can impose severe damage to cells. The high temperature requirement A (HtrA) family of serine proteases has evolved to perform important aspects of ATP-independent protein quality control. So far, however, no HtrA protease is known that degrades protein aggregates. We show here that human HTRA1 degrades aggregated and fibrillar tau, a protein that is critically involved in various neurological disorders. Neuronal cells and patient brains accumulate less tau, neurofibrillary tangles, and neuritic plaques, respectively, when HTRA1 is expressed at elevated levels. Furthermore, HTRA1 mRNA and HTRA1 activity are up-regulated in response to elevated tau concentrations. These data suggest that HTRA1 is performing regulated proteolysis during protein quality control, the implications of which are discussed. PMID:22535953

  15. Papain-like cysteine proteases in Carica papaya: lineage-specific gene duplication and expansion.

    PubMed

    Liu, Juan; Sharma, Anupma; Niewiara, Marie Jamille; Singh, Ratnesh; Ming, Ray; Yu, Qingyi

    2018-01-06

    Papain-like cysteine proteases (PLCPs), a large group of cysteine proteases structurally related to papain, play important roles in plant development, senescence, and defense responses. Papain, the first cysteine protease whose structure was determined by X-ray crystallography, plays a crucial role in protecting papaya from herbivorous insects. Except the four major PLCPs purified and characterized in papaya latex, the rest of the PLCPs in papaya genome are largely unknown. We identified 33 PLCP genes in papaya genome. Phylogenetic analysis clearly separated plant PLCP genes into nine subfamilies. PLCP genes are not equally distributed among the nine subfamilies and the number of PLCPs in each subfamily does not increase or decrease proportionally among the seven selected plant species. Papaya showed clear lineage-specific gene expansion in the subfamily III. Interestingly, all four major PLCPs purified from papaya latex, including papain, chymopapain, glycyl endopeptidase and caricain, were grouped into the lineage-specific expansion branch in the subfamily III. Mapping PLCP genes on chromosomes of five plant species revealed that lineage-specific expansions of PLCP genes were mostly derived from tandem duplications. We estimated divergence time of papaya PLCP genes of subfamily III. The major duplication events leading to lineage-specific expansion of papaya PLCP genes in subfamily III were estimated at 48 MYA, 34 MYA, and 16 MYA. The gene expression patterns of the papaya PLCP genes in different tissues were assessed by transcriptome sequencing and qRT-PCR. Most of the papaya PLCP genes of subfamily III expressed at high levels in leaf and green fruit tissues. Tandem duplications played the dominant role in affecting copy number of PLCPs in plants. Significant variations in size of the PLCP subfamilies among species may reflect genetic adaptation of plant species to different environments. The lineage-specific expansion of papaya PLCPs of subfamily III might

  16. In vivo cleavage of immunoglobulin A1 by immunoglobulin A1 proteases from Prevotella and Capnocytophaga species.

    PubMed

    Frandsen, E V; Reinholdt, J; Kjeldsen, M; Kilian, M

    1995-10-01

    Immunoglobulin A1 (IgA1) proteases secreted by oral Prevotella and Capnocytophaga species specifically cleave IgA1 at the same peptide bond in the hinge region, leaving intact monomeric Fab and Fc fragments. Assuming that Prevotella- and Capnocytophaga-induced Fab fragments of IgA1 expose a specific immunogenic neoepitope at the cleavage site, we established an enzyme-linked immunosorbent assay to measure human serum antibodies to this neoepitope as indirect evidence of in vivo activity of Prevotella and Capnocytophaga IgA1 proteases. The assay used a monoclonal antibody with specificity for the neoepitope, and the ability to block binding of the monoclonal antibody to the neoepitope was investigated. Absorption of sera with Prevotella melaninogenica-induced Fab fragments of IgA1 resulted in removal of antibodies blocking binding of the monoclonal antibody, whereas absorption with Fab fragments induced by bacterial IgA1 proteases of other cleavage specificities did not remove blocking antibodies. Consequently, we assume that the antibodies detected had been induced by a neoepitope an the Fab fragment of IgA1 exposed exclusively after cleavage with IgA1 proteases from Prevotella and Capnocytophaga, indicating in vivo activity of these IgA1 proteases. Evidence, though indirect, of in vivo activity of Prevotella and Capnocytophaga IgA1 proteases was present in 42 of 92 sera examined and in a significantly higher proportion of sera from adults with periodontal disease compared with control individuals. No correlation with disease was observed for the juvenile periodontitis groups.

  17. A cathepsin L-like protease from Strongylus vulgaris: an orthologue of Caenorhabditis elegans CPL-1.

    PubMed

    Ultaigh, Sinéad Nic An; Carolan, James C; Britton, Collette; Murray, Linda; Ryan, Michael F

    2009-04-01

    Cathespin L-like proteases (CPLs), characterized from a wide range of helminths, are significant in helminth biology. For example, in Caenorhabditis elegans CPL is essential for embryogenesis. Here, we report a cathepsin L-like gene from three species of strongyles that parasitize the horse, and describe the isolation of a cpl gene (Sv-cpl-1) from Strongylus vulgaris, the first such from equine strongyles. It encodes a protein of 354 amino acids with high similarity to other parasitic Strongylida (90-91%), and C.elegans CPL-1 (87%), a member of the same Clade. As S.vulgaris cpl-1 rescued the embryonic lethal phenotype of the C.elegans cpl-1 mutant, these genes may be orthologues, sharing the same function in each species. Targeting Sv-CPL-1 might enable novel control strategies by decreasing parasite development and transmission.

  18. Heterologous expression of an aspartic protease gene from biocontrol fungus Trichoderma asperellum in Pichia pastoris.

    PubMed

    Yang, Xiaoxue; Cong, Hua; Song, Jinzhu; Zhang, Junzheng

    2013-11-01

    Trichoderma asperellum parasitizes a large variety of phytopathogenic fungi. The mycoparasitic activity of T. asperellum depends on the secretion of complex mixtures of hydrolytic enzymes able to degrade the host cell wall and proteases which are a group of enzymes capable of degrading proteins from host. In this study, a full-length cDNA clone of aspartic protease gene, TaAsp, from T. asperellum was obtained and sequenced. The 1,185 bp long cDNA sequence was predicted to encode a 395 amino acid polypeptide with molecular mass of 42.3 kDa. The cDNA of TaAsp was inserted into the pPIC9K vector and transformed into yeast Pichia pastoris GS115 for heterologous expression. A clearly visible band with molecular mass about 42 kDa in the SDS-PAGE gel indicated that the transformant harboring the gene TaAsp had been successfully translated in P. pastoris and produced a recombinant protein. Enzyme characterization test showed that the optimum fermentation time for P. pastoris GS115 transformant was 72 h. Enzyme activity of the recombinant aspartic proteinase remained relatively stable at 25-60 °C and pH 3.0-9.0, which indicated its good prospect of application in biocontrol. The optimal pH value and temperature of the enzyme activity were pH 4.0 and 40 °C, and under this condition, with casein as the substrate, the recombinant protease activity was 18.5 U mL(-1). In order to evaluate antagonistic activity of the recombinant protease against pathogenic fungi, five pathogenic fungi, Fusarium oxysporum, Alternaria alternata, Cytospora chrysosperma, Sclerotinia sclerotiorum and Rhizoctonia solani, were applied to the test of in vitro inhibition of their mycelial growth by culture supernatant of P. pastoris GS115 transformant.

  19. Serological Analysis of Immunogenic Properties of Recombinant Meningococcus IgA1 Protease-Based Proteins.

    PubMed

    Kotelnikova, O V; Zinchenko, A A; Vikhrov, A A; Alliluev, A P; Serova, O V; Gordeeva, E A; Zhigis, L S; Zueva, V S; Razgulyaeva, O A; Melikhova, T D; Nokel, E A; Drozhzhina, E Yu; Rumsh, L D

    2016-07-01

    Using the genome sequence of IgA1 protease of N. meningitidis of serogroup B, four recombinant proteins of different structure and molecular weight were constructed. These proteins were equal in inducing the formation of specific antibodies to IgA1 protease and had protective properties against meningococci. In the sera of immunized mice, anti-IgA1 protease antibodies were detected by whole-cell ELISA, which indicated the presence of IgA1 protease on the surface of these bacteria. We hypothesized that the protective properties of IgA1 protease-based antigens and IgA1 protease analogs could be realized not only via impairment of bacterium adhesion to the mucosa, but also via suppression of this pathogen in the organism. The presented findings seem promising for using these proteins as the basis for anti-meningococcus vaccine.

  20. MOLECULAR IDENTIFICATION OF CYSTEINE AND TRYPSIN PROTEASE, EFFECT OF DIFFERENT HOSTS ON PROTEASE EXPRESSION, AND RNAI MEDIATED SILENCING OF CYSTEINE PROTEASE GENE IN THE SUNN PEST.

    PubMed

    Amiri, Azam; Bandani, Ali Reza; Alizadeh, Houshang

    2016-04-01

    Sunn pest, Eurygaster integriceps, is a serious pest of cereals in the wide area of the globe from Near and Middle East to East and South Europe and North Africa. This study described for the first time, identification of E. integriceps trypsin serine protease and cathepsin-L cysteine, transcripts involved in digestion, which might serve as targets for pest control management. A total of 478 and 500 base pair long putative trypsin and cysteine gene sequences were characterized and named Tryp and Cys, respectively. In addition, the tissue-specific relative gene expression levels of these genes as well as gluten hydrolase (Gl) were determined under different host kernels feeding conditions. Result showed that mRNA expression of Cys, Tryp, and Gl was significantly affected after feeding on various host plant species. Transcript levels of these genes were most abundant in the wheat-fed E. integriceps larvae compared to other hosts. The Cys transcript was detected exclusively in the gut, whereas the Gl and Tryp transcripts were detectable in both salivary glands and gut. Also possibility of Sunn pest gene silencing was studied by topical application of cysteine double-stranded RNA (dsRNA). The results indicated that topically applied dsRNA on fifth nymphal stage can penetrate the cuticle of the insect and induce RNA interference. The Cys gene mRNA transcript in the gut was reduced to 83.8% 2 days posttreatment. Also, it was found that dsRNA of Cys gene affected fifth nymphal stage development suggesting the involvement of this protease in the insect growth, development, and molting. © 2015 Wiley Periodicals, Inc.

  1. RpA, an extracellular protease similar to the metalloprotease of serralysin family, is required for pathogenicity of Ralstonia pickettii.

    PubMed

    Chen, C-M; Liu, J-J; Chou, C-W; Lai, C-H; Wu, L-T

    2015-10-01

    To investigate the biochemical and functional properties of an extracellular protease, RpA, in Ralstonia pickettii WP1 isolated from water supply systems. An extracellular protease was identified and characterized from R. pickettii WP1. A mutant strain WP1M2 was created from strain WP1 by mini-Tn5 transposition. The culture filtrates from WP1M2 had a lower cytotoxic effect than the parental WP1 on several mammalian cell lines. Cloning and sequence analysis revealed the Tn5 transposon inserted at a protease gene (rpA) which is 81% homologous to prtA and aprX genes of Pseudomonas fluorescens. The rpA gene encodes a 482-residue protein showing sequence similarity to metalloproteases of the serralysin family. The RpA protein was expressed in Escherichia coli using a pET expression vector and purified as a 55 kDa molecular weight protein. Furthermore, the protease activity of RpA was inhibited by protease inhibitor and heat treatment. The in vitro cytotoxic activity of R. pickettii culture filtrates was attributed to RpA protease. An extracellular protease, RpA, was identified from R. pickettii WP1 isolated from water supply system. The RpA metalloproteases is required for the pathogenicity of R. pickettii to mammalian cell lines. © 2015 The Society for Applied Microbiology.

  2. Characterization of thermostable alkaline proteases from Bacillus infantis SKS1 isolated from garden soil.

    PubMed

    Saggu, Sandeep Kaur; Mishra, Prakash Chandra

    2017-01-01

    Proteases are one of the largest groups of hydrolytic enzymes constituting about 60% of total worldwide sales of industrial enzymes due to their wide applications in detergent, leather, textile, food and pharmaceutical industry. Microbial proteases have been preferred over animal and plant proteases because of their fundamental features and ease in production. Bacillus infantis SKS1, an alkaline protease producing bacteria has been isolated from garden soil of north India and identified using morphological, biochemical and molecular methods. 16S rDNA sequence amplified using universal primers has 99% sequence identity with corresponding gene sequence of Bacillus infantis strain FM 34 and Bacillus sp. Beige. The bacterial culture and its 16S rDNA gene sequence have been deposited to Microbial Culture Collection (Pune, India) with accession number MCC 3035 and GenBank with accession number KR092197 respectively. The partially purified extract of Bacillus infantis SKS1 was thermostable and active in presence of Mg2+, acetyl acetone and laundry detergents implicating its application in industry. Production of these enzymes using this strain was maximized by optimization of various parameters including temperature, pH, media components and other growth conditions. Our results show that fructose and dextrose serve as the best carbon sources for production of these enzymes, highlighting the use of this strain for enzyme production utilizing relatively inexpensive substrates like beet molasses and corn steep liquor. Additionally, this strain showed maximum production of enzymes at 40°C similar to bacterial species used for commercial production of alkaline proteases. Characterization of alkaline proteases from this strain of Bacillus infantis and optimization of parameters for its production would help in understanding its industrial application and large-scale production.

  3. Characterization of thermostable alkaline proteases from Bacillus infantis SKS1 isolated from garden soil

    PubMed Central

    Saggu, Sandeep Kaur

    2017-01-01

    Proteases are one of the largest groups of hydrolytic enzymes constituting about 60% of total worldwide sales of industrial enzymes due to their wide applications in detergent, leather, textile, food and pharmaceutical industry. Microbial proteases have been preferred over animal and plant proteases because of their fundamental features and ease in production. Bacillus infantis SKS1, an alkaline protease producing bacteria has been isolated from garden soil of north India and identified using morphological, biochemical and molecular methods. 16S rDNA sequence amplified using universal primers has 99% sequence identity with corresponding gene sequence of Bacillus infantis strain FM 34 and Bacillus sp. Beige. The bacterial culture and its 16S rDNA gene sequence have been deposited to Microbial Culture Collection (Pune, India) with accession number MCC 3035 and GenBank with accession number KR092197 respectively. The partially purified extract of Bacillus infantis SKS1 was thermostable and active in presence of Mg2+, acetyl acetone and laundry detergents implicating its application in industry. Production of these enzymes using this strain was maximized by optimization of various parameters including temperature, pH, media components and other growth conditions. Our results show that fructose and dextrose serve as the best carbon sources for production of these enzymes, highlighting the use of this strain for enzyme production utilizing relatively inexpensive substrates like beet molasses and corn steep liquor. Additionally, this strain showed maximum production of enzymes at 40°C similar to bacterial species used for commercial production of alkaline proteases. Characterization of alkaline proteases from this strain of Bacillus infantis and optimization of parameters for its production would help in understanding its industrial application and large-scale production. PMID:29190780

  4. Protease and Protease-Activated Receptor-2 Signaling in the Pathogenesis of Atopic Dermatitis

    PubMed Central

    Lee, Sang Eun; Jeong, Se Kyoo

    2010-01-01

    Proteases in the skin are essential to epidermal permeability barrier homeostasis. In addition to their direct proteolytic effects, certain proteases signal to cells by activating protease-activated receptors (PARs), the G-protein-coupled receptors. The expression of functional PAR-2 on human skin and its role in inflammation, pruritus, and skin barrier homeostasis have been demonstrated. Atopic dermatitis (AD) is a multifactorial inflammatory skin disease characterized by genetic barrier defects and allergic inflammation, which is sustained by gene-environmental interactions. Recent studies have revealed aberrant expression and activation of serine proteases and PAR-2 in the lesional skin of AD patients. The imbalance between proteases and protease inhibitors associated with genetic defects in the protease/protease inhibitor encoding genes, increase in skin surface pH, and exposure to proteolytically active allergens contribute to this aberrant protease/PAR-2 signaling in AD. The increased protease activity in AD leads to abnormal desquamation, degradation of lipid-processing enzymes and antimicrobial peptides, and activation of primary cytokines, thereby leading to permeability barrier dysfunction, inflammation, and defects in the antimicrobial barrier. Moreover, up-regulated proteases stimulate PAR-2 in lesional skin of AD and lead to the production of cytokines and chemokines involved in inflammation and immune responses, itching sensation, and sustained epidermal barrier perturbation with easier allergen penetration. In addition, PAR-2 is an important sensor for exogenous danger molecules, such as exogenous proteases from various allergens, and plays an important role in AD pathogenesis. Together, these findings suggest that protease activity or PAR-2 may be a future target for therapeutic intervention for the treatment of AD. PMID:20879045

  5. Fusion of the SUMO/Sentrin-specific protease 1 gene SENP1 and the embryonic polarity-related mesoderm development gene MESDC2 in a patient with an infantile teratoma and a constitutional t(12;15)(q13;q25).

    PubMed

    Veltman, Imke M; Vreede, Lilian A; Cheng, Jinke; Looijenga, Leendert H J; Janssen, Bert; Schoenmakers, Eric F P M; Yeh, Edward T H; van Kessel, Ad Geurts

    2005-07-15

    Recently, we identified a patient with an infantile sacrococcygeal teratoma and a constitutional t(12;15)(q13;q25). Here, we show that, as a result of this chromosomal translocation, the SUMO/Sentrin-specific protease 1 gene (SENP1) on chromosome 12 and the embryonic polarity-related mesoderm development gene (MESDC2) on chromosome 15 are disrupted and fused. Both reciprocal SENP1-MESDC2 (SEME) and MESDC2-SENP1 (MESE) fusion genes are transcribed in tumor-derived cells and their open reading frames encode aberrant proteins. As a consequence of this, and in contrast to wild-type (WT) MESDC2, the translocation-associated SEME protein is no longer targeted to the endoplasmatic reticulum, leading to a presumed loss-of-function as a chaperone for the WNT co-receptors LRP5 and/or LRP6. Ultimately, this might lead to abnormal development and/or routing of germ cell tumor precursor cells. SUMO, a post-translational modifier, plays an important role in several cellular key processes and is cleaved from its substrates by WT SENP1. Using a PML desumoylation assay, we found that translocation-associated MESE proteins exhibit desumoylation capacities similar to those observed for WT SENP1. We speculate that spatio-temporal disturbances in desumoylating activities during critical stages of embryonic development might have predisposed the patient. Together, the constitutional t(12;15)(q13;q25) translocation revealed two novel candidate genes for neonatal/infantile GCT development: MESDC2 and SENP1.

  6. Proteases in Fas-mediated apoptosis.

    PubMed

    Zhivotovsky, B; Burgess, D H; Schlegel, J; Pörn, M I; Vanags, D; Orrenius, S

    1997-01-01

    Involvement of a unique family of cysteine proteases in the multistep apoptotic process has been documented. Cloning of several mammalian genes identifies some components of this cellular response. However, it is currently unclear which protease plays a role as a signal and/or effector of apoptosis. We summarize contributions to the data concerning proteases in Fas-mediated apoptosis.

  7. Insecticide resistance and intracellular proteases.

    PubMed

    Wilkins, Richard M

    2017-12-01

    Pesticide resistance is an example of evolution in action with mechanisms of resistance arising from mutations or increased expression of intrinsic genes. Intracellular proteases have a key role in maintaining healthy cells and in responding to stressors such as pesticides. Insecticide-resistant insects have constitutively elevated intracellular protease activity compared to corresponding susceptible strains. This increase was shown for some cases originally through biochemical enzyme studies and subsequently putatively by transcriptomics and proteomics methods. Upregulation and expression of proteases have been characterised in resistant strains of some insect species, including mosquitoes. This increase in proteolysis results in more degradation products (amino acids) of intracellular proteins. These may be utilised in the resistant strain to better protect the cell from stress. There are changes in insect intracellular proteases shortly after insecticide exposure, suggesting a role in stress response. The use of protease and proteasome inhibitors or peptide mimetics as synergists with improved application techniques and through protease gene knockdown using RNA interference (possibly expressed in crop plants) may be potential pest management strategies, in situations where elevated intracellular proteases are relevant. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  8. Cloning, expression and activity analysis of a novel fibrinolytic serine protease from Arenicola cristata

    NASA Astrophysics Data System (ADS)

    Zhao, Chunling; Ju, Jiyu

    2015-06-01

    The full-length cDNA of a protease gene from a marine annelid Arenicola cristata was amplified through rapid amplification of cDNA ends technique and sequenced. The size of the cDNA was 936 bp in length, including an open reading frame encoding a polypeptide of 270 amino acid residues. The deduced amino acid sequnce consisted of pro- and mature sequences. The protease belonged to the serine protease family because it contained the highly conserved sequence GDSGGP. This protease was novel as it showed a low amino acid sequence similarity (< 40%) to other serine proteases. The gene encoding the active form of A. cristata serine protease was cloned and expressed in E. coli. Purified recombinant protease in a supernatant could dissolve an artificial fibrin plate with plasminogen-rich fibrin, whereas the plasminogen-free fibrin showed no clear zone caused by hydrolysis. This result suggested that the recombinant protease showed an indirect fibrinolytic activity of dissolving fibrin, and was probably a plasminogen activator. A rat model with venous thrombosis was established to demonstrate that the recombinant protease could also hydrolyze blood clot in vivo. Therefore, this recombinant protease may be used as a thrombolytic agent for thrombosis treatment. To our knowledge, this study is the first of reporting the fibrinolytic serine protease gene in A. cristata.

  9. MicroRNA Regulation of Human Protease Genes Essential for Influenza Virus Replication

    PubMed Central

    Meliopoulos, Victoria A.; Andersen, Lauren E.; Brooks, Paula; Yan, Xiuzhen; Bakre, Abhijeet; Coleman, J. Keegan; Tompkins, S. Mark; Tripp, Ralph A.

    2012-01-01

    Influenza A virus causes seasonal epidemics and periodic pandemics threatening the health of millions of people each year. Vaccination is an effective strategy for reducing morbidity and mortality, and in the absence of drug resistance, the efficacy of chemoprophylaxis is comparable to that of vaccines. However, the rapid emergence of drug resistance has emphasized the need for new drug targets. Knowledge of the host cell components required for influenza replication has been an area targeted for disease intervention. In this study, the human protease genes required for influenza virus replication were determined and validated using RNA interference approaches. The genes validated as critical for influenza virus replication were ADAMTS7, CPE, DPP3, MST1, and PRSS12, and pathway analysis showed these genes were in global host cell pathways governing inflammation (NF-κB), cAMP/calcium signaling (CRE/CREB), and apoptosis. Analyses of host microRNAs predicted to govern expression of these genes showed that eight miRNAs regulated gene expression during virus replication. These findings identify unique host genes and microRNAs important for influenza replication providing potential new targets for disease intervention strategies. PMID:22606348

  10. MicroRNA regulation of human protease genes essential for influenza virus replication.

    PubMed

    Meliopoulos, Victoria A; Andersen, Lauren E; Brooks, Paula; Yan, Xiuzhen; Bakre, Abhijeet; Coleman, J Keegan; Tompkins, S Mark; Tripp, Ralph A

    2012-01-01

    Influenza A virus causes seasonal epidemics and periodic pandemics threatening the health of millions of people each year. Vaccination is an effective strategy for reducing morbidity and mortality, and in the absence of drug resistance, the efficacy of chemoprophylaxis is comparable to that of vaccines. However, the rapid emergence of drug resistance has emphasized the need for new drug targets. Knowledge of the host cell components required for influenza replication has been an area targeted for disease intervention. In this study, the human protease genes required for influenza virus replication were determined and validated using RNA interference approaches. The genes validated as critical for influenza virus replication were ADAMTS7, CPE, DPP3, MST1, and PRSS12, and pathway analysis showed these genes were in global host cell pathways governing inflammation (NF-κB), cAMP/calcium signaling (CRE/CREB), and apoptosis. Analyses of host microRNAs predicted to govern expression of these genes showed that eight miRNAs regulated gene expression during virus replication. These findings identify unique host genes and microRNAs important for influenza replication providing potential new targets for disease intervention strategies.

  11. Analysis of the regulatory region of the protease III (ptr) gene of Escherichia coli K-12.

    PubMed

    Claverie-Martin, F; Diaz-Torres, M R; Kushner, S R

    1987-01-01

    The ptr gene of Escherichia coli encodes protease III (Mr 110,000) and a 50-kDa polypeptide, both of which are found in the periplasmic space. The gene is physically located between the recC and recB loci on the E. coli chromosome. The nucleotide sequence of a 1167-bp EcoRV-ClaI fragment of chromosomal DNA containing the promoter region and 885 bp of the ptr coding sequence has been determined. S1 nuclease mapping analysis showed that the major 5' end of the ptr mRNA was localized 127 bp upstream from the ATG start codon. The open reading frame (ORF), preceded by a Shine-Dalgarno sequence, extends to the end of the sequenced DNA. Downstream from the -35 and -10 regions is a sequence that strongly fits the consensus sequence of known nitrogen-regulated promoters. A signal peptide of 23 amino acids residues is present at the N terminus of the derived amino acid sequence. The cleavage site as well as the ORF were confirmed by sequencing the N terminus of mature protease III.

  12. StAR Enhances Transcription of Genes Encoding the Mitochondrial Proteases Involved in Its Own Degradation

    PubMed Central

    Bahat, Assaf; Perlberg, Shira; Melamed-Book, Naomi; Lauria, Ines; Langer, Thomas

    2014-01-01

    Steroidogenic acute regulatory protein (StAR) is essential for steroid hormone synthesis in the adrenal cortex and the gonads. StAR activity facilitates the supply of cholesterol substrate into the inner mitochondrial membranes where conversion of the sterol to a steroid is catalyzed. Mitochondrial import terminates the cholesterol mobilization activity of StAR and leads to mounting accumulation of StAR in the mitochondrial matrix. Our studies suggest that to prevent mitochondrial impairment, StAR proteolysis is executed by at least 2 mitochondrial proteases, ie, the matrix LON protease and the inner membrane complexes of the metalloproteases AFG3L2 and AFG3L2:SPG7/paraplegin. Gonadotropin administration to prepubertal rats stimulated ovarian follicular development associated with increased expression of the mitochondrial protein quality control system. In addition, enrichment of LON and AFG3L2 is evident in StAR-expressing ovarian cells examined by confocal microscopy. Furthermore, reporter studies of the protease promoters examined in the heterologous cell model suggest that StAR expression stimulates up to a 3.5-fold increase in the protease gene transcription. Such effects are StAR-specific, are independent of StAR activity, and failed to occur upon expression of StAR mutants that do not enter the matrix. Taken together, the results of this study suggest the presence of a novel regulatory loop, whereby acute accumulation of an apparent nuisance protein in the matrix provokes a mitochondria to nucleus signaling that, in turn, activates selected transcription of genes encoding the enrichment of mitochondrial proteases relevant for enhanced clearance of StAR. PMID:24422629

  13. A cysteine protease encoded by the baculovirus Bombyx mori nuclear polyhedrosis virus.

    PubMed Central

    Ohkawa, T; Majima, K; Maeda, S

    1994-01-01

    Sequence analysis of the BamHI F fragment of the genome of Bombyx mori nuclear polyhedrosis virus (BmNPV) revealed an open reading frame whose deduced amino acid sequence had homology to those of cysteine proteases of the papain superfamily. The putative cysteine protease sequence (BmNPV-CP) was 323 amino acids long and showed 35% identity to a cysteine proteinase precursor from Trypanosoma brucei. Of 36 residues conserved among cathepsins B, H, L, and S and papain, 31 were identical in BmNPV-CP. In order to determine the activity and function of the putative cysteine protease, a BmNPV mutant (BmCysPD) was constructed by homologous recombination of the protease gene with a beta-galactosidase gene cassette. BmCysPD-infected BmN cell extracts were significantly reduced in acid protease activity compared with wild-type virus-infected cell extracts. The cysteine protease inhibitor E-64 [trans-epoxysuccinylleucylamido-(4-guanidino)butane] inhibited wild-type virus-expressed protease activity. Deletion of the cysteine protease gene had no significant effect on viral growth or polyhedron production in BmN cells, indicating that the cysteine protease was not essential for viral replication in vitro. However, B. mori larvae infected with BmCysPD showed symptoms different from those of wild-type BmNPV-infected larvae, e.g., less degradation of the body, including fat body cells, white body surface color due presumably to undegraded epidermal cells, and an increase in the number of polyhedra released into the hemolymph. This is the first report of (i) a virus-encoded protease with activity on general substrates and (ii) evidence that a virus-encoded protease may play a role in degradation of infected larvae to facilitate horizontal transmission of the virus. Images PMID:8083997

  14. Lack of cleavage of immunoglobulin A (IgA) from rhesus monkeys by bacterial IgA1 proteases.

    PubMed Central

    Reinholdt, J; Kilian, M

    1991-01-01

    Bacterial immunoglobulin A1 (IgA1) proteases cleaving IgA1 and secretory IgA1 molecules in the hinge region are believed to be important virulence factors. Previous studies have indicated that IgA of humans, gorillas, and chimpanzees are the exclusive substrates of these enzymes. In a recent study, IgA from the rhesus monkey was found to be susceptible to the IgA1 protease activity of Streptococcus pneumoniae. In an attempt to reproduce this observation, we found that neither five isolates of S. pneumoniae nor other IgA1 protease-producing bacteria representing different cleavage specificities caused cleavage of rhesus monkey IgA. Hence, the rhesus monkey does not appear to be a suitable animal model for studies of IgA1 proteases as virulence factors. Images PMID:2037384

  15. Serine protease activity in m-1 cortical collecting duct cells.

    PubMed

    Liu, Lian; Hering-Smith, Kathleen S; Schiro, Faith R; Hamm, L Lee

    2002-04-01

    An apical serine protease, channel-activating protease 1 (CAP1), augments sodium transport in A6 cells. Prostasin, a novel serine protease originally purified from seminal fluid, has been proposed to be the mammalian ortholog of CAP1. We have recently found functional evidence for a similar protease activity in the M-1 cortical collecting duct cell line. The purposes of the present studies were to determine whether prostasin (or CAP1) is present in collecting duct cells by use of mouse M-1 cells, to sequence mouse prostasin, and to further characterize the identity of the serine protease activity and additional functional features in M-1 cells. Using mouse expressed sequence tag sequences that are highly homologous to the published human prostasin sequence as templates, reverse transcription-polymerase chain reaction and RACE (rapid amplification of cDNA ends) were used to sequence mouse prostasin mRNA, which shows 99% identical to published mouse CAP1 sequence. A single 1800-bp transcript was found by Northern analysis, and this was not altered by aldosterone. Equivalent short-circuit current (I(eq)), which represents sodium transport in these cells, dropped to 59+/-3% of control value within 1 hour of incubation with aprotinin, a serine protease inhibitor. Trypsin increased the I(eq) in aprotinin-treated cells to the value of the control group within 5 minutes. Application of aprotinin not only inhibited amiloride sensitive I(eq) but also reduced transepithelial resistance (R(te)) to 43+/-2%, an effect not expected with simple inhibition of sodium channels. Trypsin partially reversed the effect of aprotinin on R(te). Another serine protease inhibitor, soybean trypsin inhibitor (STI), decreased I(eq) in M-1 cells. STI inhibited I(eq) gradually over 6 hours, and the inhibition of I(eq) by 2 inhibitors was additive. STI decreased transepithelial resistance much less than did aprotinin. Neither aldosterone nor dexamethasone significantly augmented protease activity

  16. Identification of SlpB, a Cytotoxic Protease from Serratia marcescens

    PubMed Central

    Stella, Nicholas A.; Hunt, Kristin M.; Brothers, Kimberly M.; Zhang, Liang; Thibodeau, Patrick H.

    2015-01-01

    The Gram-negative bacterium and opportunistic pathogen Serratia marcescens causes ocular infections in healthy individuals. Secreted protease activity was characterized from 44 ocular clinical isolates, and a higher frequency of protease-positive strains was observed among keratitis isolates than among conjunctivitis isolates. A positive correlation between protease activity and cytotoxicity to human corneal epithelial cells in vitro was determined. Deletion of prtS in clinical keratitis isolate K904 reduced, but did not eliminate, cytotoxicity and secreted protease production. This indicated that PrtS is necessary for full cytotoxicity to ocular cells and implied the existence of another secreted protease(s) and cytotoxic factors. Bioinformatic analysis of the S. marcescens Db11 genome revealed three additional open reading frames predicted to code for serralysin-like proteases noted here as slpB, slpC, and slpD. Induced expression of prtS and slpB, but not slpC and slpD, in strain PIC3611 rendered the strain cytotoxic to a lung carcinoma cell line; however, only prtS induction was sufficient for cytotoxicity to a corneal cell line. Strain K904 with deletion of both prtS and slpB genes was defective in secreted protease activity and cytotoxicity to human cell lines. PAGE analysis suggests that SlpB is produced at lower levels than PrtS. Purified SlpB demonstrated calcium-dependent and AprI-inhibited protease activity and cytotoxicity to airway and ocular cell lines in vitro. Lastly, genetic analysis indicated that the type I secretion system gene, lipD, is required for SlpB secretion. These genetic data introduce SlpB as a new cytotoxic protease from S. marcescens. PMID:25939509

  17. A Comparative Study: Taxonomic Grouping of Alkaline Protease Producing Bacilli.

    PubMed

    Tekin, Nilgun; Cihan, Arzu Coleri; Karaca, Basar; Cokmus, Cumhur

    2017-03-30

    Alkaline proteases have biotechnological importance due to their activity and stability at alkaline pH. 56 bacteria, capable of growing under alkaline conditions were isolated and their alkaline protease activities were carried out at different parameters to determine their optimum alkaline protease production conditions. Seven isolates were showed higher alkaline protease production capacity than the reference strains. The highest alkaline protease producing isolates (103125 U/g), E114 and C265, were identified as Bacillus licheniformis with 99.4% and Bacillus mojavensis 99.8% based on 16S rRNA gene sequence similarities, respectively. Interestingly, the isolates identified as Bacillus safensis were also found to be high alkaline protease producing strains. Genotypic characterizations of the isolates were also determined by using a wide range of molecular techniques (ARDRA, ITS-PCR, (GTG)5-PCR, BOX-PCR). These different techniques allowed us to differentiate the alkaliphilic isolates and the results were in concurrence with phylogenetic analyses of the 16S rRNA genes. While ITS-PCR provided the highest correlation with 16S rRNA groups, (GTG)5-PCR showed the highest differentiation at species and intra-species level. In this study, each of the biotechnologically valuable alkaline protease producing isolates was grouped into their taxonomic positions with multi-genotypic analyses.

  18. A Rhomboid Protease Gene Deletion Affects a Novel Oligosaccharide N-Linked to the S-layer Glycoprotein of Haloferax volcanii*

    PubMed Central

    Parente, Juliana; Casabuono, Adriana; Ferrari, María Celeste; Paggi, Roberto Alejandro; De Castro, Rosana Esther; Couto, Alicia Susana; Giménez, María Inés

    2014-01-01

    Rhomboid proteases occur in all domains of life; however, their physiological role is not completely understood, and nothing is known of the biology of these enzymes in Archaea. One of the two rhomboid homologs of Haloferax volcanii (RhoII) is fused to a zinc finger domain. Chromosomal deletion of rhoII was successful, indicating that this gene is not essential for this organism; however, the mutant strain (MIG1) showed reduced motility and increased sensitivity to novobiocin. Membrane preparations of MIG1 were enriched in two glycoproteins, identified as the S-layer glycoprotein and an ABC transporter component. The H. volcanii S-layer glycoprotein has been extensively used as a model to study haloarchaeal protein N-glycosylation. HPLC analysis of oligosaccharides released from the S-layer glycoprotein after PNGase treatment revealed that MIG1 was enriched in species with lower retention times than those derived from the parent strain. Mass spectrometry analysis showed that the wild type glycoprotein released a novel oligosaccharide species corresponding to GlcNAc-GlcNAc(Hex)2-(SQ-Hex)6 in contrast to the mutant protein, which contained the shorter form GlcNAc2(Hex)2-SQ-Hex-SQ. A glycoproteomics approach of the wild type glycopeptide fraction revealed Asn-732 peptide fragments linked to the sulfoquinovose-containing oligosaccharide. This work describes a novel N-linked oligosaccharide containing a repeating SQ-Hex unit bound to Asn-732 of the H. volcanii S-layer glycoprotein, a position that had not been reported as glycosylated. Furthermore, this study provides the first insight on the biological role of rhomboid proteases in Archaea, suggesting a link between protein glycosylation and this protease family. PMID:24596091

  19. A rhomboid protease gene deletion affects a novel oligosaccharide N-linked to the S-layer glycoprotein of Haloferax volcanii.

    PubMed

    Parente, Juliana; Casabuono, Adriana; Ferrari, María Celeste; Paggi, Roberto Alejandro; De Castro, Rosana Esther; Couto, Alicia Susana; Giménez, María Inés

    2014-04-18

    Rhomboid proteases occur in all domains of life; however, their physiological role is not completely understood, and nothing is known of the biology of these enzymes in Archaea. One of the two rhomboid homologs of Haloferax volcanii (RhoII) is fused to a zinc finger domain. Chromosomal deletion of rhoII was successful, indicating that this gene is not essential for this organism; however, the mutant strain (MIG1) showed reduced motility and increased sensitivity to novobiocin. Membrane preparations of MIG1 were enriched in two glycoproteins, identified as the S-layer glycoprotein and an ABC transporter component. The H. volcanii S-layer glycoprotein has been extensively used as a model to study haloarchaeal protein N-glycosylation. HPLC analysis of oligosaccharides released from the S-layer glycoprotein after PNGase treatment revealed that MIG1 was enriched in species with lower retention times than those derived from the parent strain. Mass spectrometry analysis showed that the wild type glycoprotein released a novel oligosaccharide species corresponding to GlcNAc-GlcNAc(Hex)2-(SQ-Hex)6 in contrast to the mutant protein, which contained the shorter form GlcNAc2(Hex)2-SQ-Hex-SQ. A glycoproteomics approach of the wild type glycopeptide fraction revealed Asn-732 peptide fragments linked to the sulfoquinovose-containing oligosaccharide. This work describes a novel N-linked oligosaccharide containing a repeating SQ-Hex unit bound to Asn-732 of the H. volcanii S-layer glycoprotein, a position that had not been reported as glycosylated. Furthermore, this study provides the first insight on the biological role of rhomboid proteases in Archaea, suggesting a link between protein glycosylation and this protease family.

  20. Analysis of 16S rRNA gene lactic acid bacteria (LAB) isolate from Markisa fruit (Passiflora sp.) as a producer of protease enzyme and probiotics

    NASA Astrophysics Data System (ADS)

    Hidayat, Habibi

    2017-03-01

    16S rRNA gene analysis of bacteria lactic acid (LAB) isolate from Markisa Kuning Fruit (Passiflora edulis var. flavicarpa) as a producer of protease enzyme and probiotics has been done. The aim of the study is to determine the protease enzyme activity and 16S rRNA gene amplification using PCR. The calculation procedure was done to M4 isolate bacteria lactic acid (LAB) Isolate which has been resistant to acids with pH 2.0 in the manner of screening protease enzyme activity test result 6.5 to clear zone is 13 mm againts colony diametre is 2 mm. The results of study enzyme activity used spectrophotometer UV-Vis obtainable the regression equation Y=0.02983+0.001312X, with levels of protein M4 isolate is 0.6594 mg/mL and enzyme activity of obtainable is 0.8626 unit/ml while the spesific enzyme activity produced is 1.308 unit/mg. Then, 16S rRNA gene amplificatiom and DNA sequencing has been done. The results of study showed that the bacteria species contained from M4 bacteria lactic acid (LAB) isolate is Weisella cibiria strain II-I-59. Weisella cibiria strain II-I-59 is one of bacteria could be utilized in the digestive tract.

  1. Purification and molecular characterization of subtilisin-like alkaline protease BPP-A from Bacillus pumilus strain MS-1.

    PubMed

    Miyaji, T; Otta, Y; Nakagawa, T; Watanabe, T; Niimura, Y; Tomizuka, N

    2006-03-01

    The present study was conducted by screening zein-degrading bacteria in an attempt to obtain zein-degrading protease. Soil bacteria were screened by formation of a clear zone on zein plates. Characterization of a zein-degrading bacterium indicated a taxonomic affiliation to Bacillus pumilus, and was named MS-1 strain. The strain produced two different types of extracellular proteases, BPP-A and BPP-B. In this study, we purified and characterized BPP-A because it exhibited a higher ability to hydrolyze zein than BPP-B. When casein was used as the substrate, the optimal pH for BPP-A was 11.0. In BPP-A, zein was better substrate than casein at pH 13.0, whereas casein was better one than zein at pH 11.0. The bppA gene encoded a 383-amino acid pre-pro form of BPP-A, and mature BPP-A contained 275 amino acid residues. It was concluded that BPP-A belonged to the subtilisin family. A zein-degrading bacterium assigned to B. pumilus produced two different types of extracellular proteases, BPP-A and BPP-B. BPP-A exhibited an ability to hydrolyze zein in an extreme alkaline condition. This is a first report on screening for zein-degrading micro-organisms. The subtilisin-like protease BPP-A is possible to utilize as an industrial enzyme for the production of zein hydrolysates.

  2. Interdependence of Inhibitor Recognition in HIV-1 Protease.

    PubMed

    Paulsen, Janet L; Leidner, Florian; Ragland, Debra A; Kurt Yilmaz, Nese; Schiffer, Celia A

    2017-05-09

    Molecular recognition is a highly interdependent process. Subsite couplings within the active site of proteases are most often revealed through conditional amino acid preferences in substrate recognition. However, the potential effect of these couplings on inhibition and thus inhibitor design is largely unexplored. The present study examines the interdependency of subsites in HIV-1 protease using a focused library of protease inhibitors, to aid in future inhibitor design. Previously a series of darunavir (DRV) analogs was designed to systematically probe the S1' and S2' subsites. Co-crystal structures of these analogs with HIV-1 protease provide the ideal opportunity to probe subsite interdependency. All-atom molecular dynamics simulations starting from these structures were performed and systematically analyzed in terms of atomic fluctuations, intermolecular interactions, and water structure. These analyses reveal that the S1' subsite highly influences other subsites: the extension of the hydrophobic P1' moiety results in 1) reduced van der Waals contacts in the P2' subsite, 2) more variability in the hydrogen bond frequencies with catalytic residues and the flap water, and 3) changes in the occupancy of conserved water sites both proximal and distal to the active site. In addition, one of the monomers in this homodimeric enzyme has atomic fluctuations more highly correlated with DRV than the other monomer. These relationships intricately link the HIV-1 protease subsites and are critical to understanding molecular recognition and inhibitor binding. More broadly, the interdependency of subsite recognition within an active site requires consideration in the selection of chemical moieties in drug design; this strategy is in contrast to what is traditionally done with independent optimization of chemical moieties of an inhibitor.

  3. The spectrum of low molecular weight alpha-amylase/protease inhibitor genes expressed in the US bread wheat cultivar Butte 86

    PubMed Central

    2011-01-01

    Background Wheat grains accumulate a variety of low molecular weight proteins that are inhibitors of alpha-amylases and proteases and play an important protective role in the grain. These proteins have more balanced amino acid compositions than the major wheat gluten proteins and contribute important reserves for both seedling growth and human nutrition. The alpha-amylase/protease inhibitors also are of interest because they cause IgE-mediated occupational and food allergies and thereby impact human health. Results The complement of genes encoding alpha-amylase/protease inhibitors expressed in the US bread wheat Butte 86 was characterized by analysis of expressed sequence tags (ESTs). Coding sequences for 19 distinct proteins were identified. These included two monomeric (WMAI), four dimeric (WDAI), and six tetrameric (WTAI) inhibitors of exogenous alpha-amylases, two inhibitors of endogenous alpha-amylases (WASI), four putative trypsin inhibitors (CMx and WTI), and one putative chymotrypsin inhibitor (WCI). A number of the encoded proteins were identical or very similar to proteins in the NCBI database. Sequences not reported previously included variants of WTAI-CM3, three CMx inhibitors and WTI. Within the WDAI group, two different genes encoded the same mature protein. Based on numbers of ESTs, transcripts for WTAI-CM3 Bu-1, WMAI Bu-1 and WTAI-CM16 Bu-1 were most abundant in Butte 86 developing grain. Coding sequences for 16 of the inhibitors were unequivocally associated with specific proteins identified by tandem mass spectrometry (MS/MS) in a previous proteomic analysis of milled white flour from Butte 86. Proteins corresponding to WDAI Bu-1/Bu-2, WMAI Bu-1 and the WTAI subunits CM2 Bu-1, CM3 Bu-1 and CM16 Bu-1 were accumulated to the highest levels in flour. Conclusions Information on the spectrum of alpha-amylase/protease inhibitor genes and proteins expressed in a single wheat cultivar is central to understanding the importance of these proteins in both

  4. A novel mutation of the high-temperature requirement A serine peptidase 1 (HTRA1) gene in a Chinese family with cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL).

    PubMed

    Chen, Yan; He, Zhiyi; Meng, Su; Li, Lei; Yang, Hua; Zhang, Xiaotang

    2013-10-01

    Mutations in the high-temperature requirement A serine peptidase 1 (HTRA1) gene were studied in a Chinese family with cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL). Exons 1-9 of the HTRA1 gene were amplified and bidirectionally sequenced in a Chinese family with CARASIL. Mutation effects were analysed by three-dimensional modelling of the serine protease HTRA1 protein. The proband was found to be homozygous for a novel missense mutation (c.854 C > T) identified in exon 4 of the HTRA1 gene; the parents of the proband were heterozygous for the same missense mutation. This c.854 C > T mutation resulted in a change from proline to leucine (p.P285L) in serine protease HTRA1, and was absent in 260 control chromosomes. Three-dimensional models showed that the change from proline to leucine (p.P285L) could attenuate the hydrogen bond between S284 and S287 residues, which might affect function of serine protease HTRA1. Discovery of a novel missense mutation (c.854C>T) associated with CARASIL expands the known CARASIL-related mutations in HTRA1.

  5. Production and characterization of a novel protease from Bacillus sp. RRM1 under solid state fermentation.

    PubMed

    Rajkumar, Renganathan; Kothilmozhian, Jayappriyan; Ramasamy, Rengasamy

    2011-06-01

    A commercially important alkaline protease, produced by Bacillus sp. RRM1 isolated from the red seaweed Kappaphycus alvarezii (Doty) Doty ex Silva, was first recognized and characterized in the present study. Identification of the isolated bacterium was done using both biochemical characterization as well as 16S rRNA gene sequencing. The bacterial strain, Bacillus sp. RRM1, produced a high level of protease using easily available, inexpensive agricultural residues solid-state fermentation (SSF). Among them, wheat bran was found to be the best substrate. Influences of process parameters such as moistening agents, moisture level, temperature, inoculum concentration, and co-carbon and co-nitrogen sources on the fermentation were also evaluated. Under optimized conditions, maximum protease production (i.e., 2081 U/g) was obtained from wheat bran, which is about 2-fold greater than the initial conditions. The protease enzyme was stable over a temperature range of 30-60 degrees C and pH 6-12, with maximum activity at 50 degrees C and pH 9.0. Whereas the metal ions Na+, Ca2+, and K+ enhanced the activity of the enzyme, others such as Hg2+, Cu2+, Fe2+, Co2+, and Zn2+ had rendered negative effects. The activity of the enzyme was inhibited by EDTA and enhanced by Cu2+ ions, thus indicating the nature of the enzyme as a metalloprotease. The enzyme showed extreme stability and activity even in the presence of detergents, surfactants, and organic solvents. Moreover, the present findings opened new vistas in the utilization of wheat bran, a cheap, abundantly available, and effective waste as a substrate for SSF.

  6. Identification of SlpB, a Cytotoxic Protease from Serratia marcescens.

    PubMed

    Shanks, Robert M Q; Stella, Nicholas A; Hunt, Kristin M; Brothers, Kimberly M; Zhang, Liang; Thibodeau, Patrick H

    2015-07-01

    The Gram-negative bacterium and opportunistic pathogen Serratia marcescens causes ocular infections in healthy individuals. Secreted protease activity was characterized from 44 ocular clinical isolates, and a higher frequency of protease-positive strains was observed among keratitis isolates than among conjunctivitis isolates. A positive correlation between protease activity and cytotoxicity to human corneal epithelial cells in vitro was determined. Deletion of prtS in clinical keratitis isolate K904 reduced, but did not eliminate, cytotoxicity and secreted protease production. This indicated that PrtS is necessary for full cytotoxicity to ocular cells and implied the existence of another secreted protease(s) and cytotoxic factors. Bioinformatic analysis of the S. marcescens Db11 genome revealed three additional open reading frames predicted to code for serralysin-like proteases noted here as slpB, slpC, and slpD. Induced expression of prtS and slpB, but not slpC and slpD, in strain PIC3611 rendered the strain cytotoxic to a lung carcinoma cell line; however, only prtS induction was sufficient for cytotoxicity to a corneal cell line. Strain K904 with deletion of both prtS and slpB genes was defective in secreted protease activity and cytotoxicity to human cell lines. PAGE analysis suggests that SlpB is produced at lower levels than PrtS. Purified SlpB demonstrated calcium-dependent and AprI-inhibited protease activity and cytotoxicity to airway and ocular cell lines in vitro. Lastly, genetic analysis indicated that the type I secretion system gene, lipD, is required for SlpB secretion. These genetic data introduce SlpB as a new cytotoxic protease from S. marcescens. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  7. Evaluation of cysteine proteases of Plasmodium vivax as antimalarial drug targets: sequence analysis and sensitivity to cysteine protease inhibitors.

    PubMed

    Na, Byoung-Kuk; Kim, Tong-Soo; Rosenthal, Philip J; Lee, Jong-Koo; Kong, Yoon

    2004-10-01

    Cysteine proteases perform critical roles in the life cycles of malaria parasites. In Plasmodium falciparum, treatment of cysteine protease inhibitors inhibits hemoglobin hydrolysis and blocks the parasite development in vitro and in vivo, suggesting that plasmodial cysteine proteases may be interesting targets for new chemotherapeutics. To determine whether sequence diversity may limit chemotherapy against Plasmodium vivax, we analyzed sequence variations in the genes encoding three cysteine proteases, vivapain-1, -2 and -3, in 22 wild isolates of P. vivax. The sequences were highly conserved among wild isolates. A small number of substitutions leading to amino acid changes were found, while they did not modify essential residues for the function or structure of the enzymes. The substrate specificities and sensitivities to synthetic cysteine protease inhibitors of vivapain-2 and -3 from wild isolates were also very similar. These results support the suggestion that cysteine proteases of P. vivax are promising antimalarial chemotherapeutic targets.

  8. Interdependence of Inhibitor Recognition in HIV-1 Protease

    PubMed Central

    2017-01-01

    Molecular recognition is a highly interdependent process. Subsite couplings within the active site of proteases are most often revealed through conditional amino acid preferences in substrate recognition. However, the potential effect of these couplings on inhibition and thus inhibitor design is largely unexplored. The present study examines the interdependency of subsites in HIV-1 protease using a focused library of protease inhibitors, to aid in future inhibitor design. Previously a series of darunavir (DRV) analogs was designed to systematically probe the S1′ and S2′ subsites. Co-crystal structures of these analogs with HIV-1 protease provide the ideal opportunity to probe subsite interdependency. All-atom molecular dynamics simulations starting from these structures were performed and systematically analyzed in terms of atomic fluctuations, intermolecular interactions, and water structure. These analyses reveal that the S1′ subsite highly influences other subsites: the extension of the hydrophobic P1′ moiety results in 1) reduced van der Waals contacts in the P2′ subsite, 2) more variability in the hydrogen bond frequencies with catalytic residues and the flap water, and 3) changes in the occupancy of conserved water sites both proximal and distal to the active site. In addition, one of the monomers in this homodimeric enzyme has atomic fluctuations more highly correlated with DRV than the other monomer. These relationships intricately link the HIV-1 protease subsites and are critical to understanding molecular recognition and inhibitor binding. More broadly, the interdependency of subsite recognition within an active site requires consideration in the selection of chemical moieties in drug design; this strategy is in contrast to what is traditionally done with independent optimization of chemical moieties of an inhibitor. PMID:28358514

  9. The cysteine protease CEP1, a key executor involved in tapetal programmed cell death, regulates pollen development in Arabidopsis.

    PubMed

    Zhang, Dandan; Liu, Di; Lv, Xiaomeng; Wang, Ying; Xun, Zhili; Liu, Zhixiong; Li, Fenglan; Lu, Hai

    2014-07-01

    Tapetal programmed cell death (PCD) is a prerequisite for pollen grain development in angiosperms, and cysteine proteases are the most ubiquitous hydrolases involved in plant PCD. We identified a papain-like cysteine protease, CEP1, which is involved in tapetal PCD and pollen development in Arabidopsis thaliana. CEP1 is expressed specifically in the tapetum from stages 5 to 11 of anther development. The CEP1 protein first appears as a proenzyme in precursor protease vesicles and is then transported to the vacuole and transformed into the mature enzyme before rupture of the vacuole. cep1 mutants exhibited aborted tapetal PCD and decreased pollen fertility with abnormal pollen exine. A transcriptomic analysis revealed that 872 genes showed significantly altered expression in the cep1 mutants, and most of them are important for tapetal cell wall organization, tapetal secretory structure formation, and pollen development. CEP1 overexpression caused premature tapetal PCD and pollen infertility. ELISA and quantitative RT-PCR analyses confirmed that the CEP1 expression level showed a strong relationship to the degree of tapetal PCD and pollen fertility. Our results reveal that CEP1 is a crucial executor during tapetal PCD and that proper CEP1 expression is necessary for timely degeneration of tapetal cells and functional pollen formation. © 2014 American Society of Plant Biologists. All rights reserved.

  10. A novel serine protease predominately expressed in macrophages.

    PubMed Central

    Chen, Cailin; Darrow, Andrew L; Qi, Jian-Shen; D'Andrea, Michael R; Andrade-Gordon, Patricia

    2003-01-01

    We have identified a novel serine protease designated EOS by sequence identity searches. The deduced protein contains 284 amino acids with an active form containing 248 amino acids starting from an Ile-Val-Gly-Gly motif. The active form comprises a catalytic triad of conserved amino acids: His77, Asp126 and Ser231. It shares 44% identity with beta-tryptase and belongs to the S1 trypsin-like serine-protease family. Interestingly, this gene also maps to human chromosome 16p13.3. The purified protease showed amidolytic activity, cleaving its substrates before arginine residues. Tissue distribution by immunohistochemistry analysis demonstrated that EOS is highly expressed in spleen and moderately expressed in intestine, colon, lung and brain. We confirmed this expression pattern at the mRNA level by performing in situ hybridization. The results from both immunohistochemistry and in situ hybridization indicate that EOS is associated with macrophages. We corroborated this observation by double immunofluorescence using the anti-EOS antibody and an anti-CD68 antibody, a macrophage specific marker. Furthermore, we have detected a dramatic increase in immune staining of EOS in cultured U937 cells treated with PMA, which represent activated macrophages. This up-regulation is also reflected by elevated EOS mRNA in the PMA-treated U937 cells detected by Northern blotting. Since macrophages have important roles in various pathological conditions, such as wound healing, atherosclerosis and numerous inflammatory diseases, the localization of this novel serine protease to active macrophages may help to further the elucidation of the roles of this gene product in modulating these disorders. PMID:12795636

  11. The proteases HtrA2/Omi and UCH-L1 regulate TNF-induced necroptosis

    PubMed Central

    2013-01-01

    Background In apoptosis, proteolysis by caspases is the primary mechanism for both initiation and execution of programmed cell death (PCD). In contrast, the impact of proteolysis on the regulation and execution of caspase-independent forms of PCD (programmed necrosis, necroptosis) is only marginally understood. Likewise, the identity of the involved proteases has remained largely obscure. Here, we have investigated the impact of proteases in TNF-induced necroptosis. Results The serine protease inhibitor TPKC protected from TNF-induced necroptosis in multiple murine and human cells systems whereas inhibitors of metalloproteinases or calpain/cysteine and cathepsin proteases had no effect. A screen for proteins labeled by a fluorescent TPCK derivative in necroptotic cells identified HtrA2/Omi (a serine protease previously implicated in PCD) as a promising candidate. Demonstrating its functional impact, pharmacological inhibition or genetic deletion of HtrA2/Omi protected from TNF-induced necroptosis. Unlike in apoptosis, HtrA2/Omi did not cleave another protease, ubiquitin C-terminal hydrolase (UCH-L1) during TNF-induced necroptosis, but rather induced monoubiquitination indicative for UCH-L1 activation. Correspondingly, pharmacologic or RNA interference-mediated inhibition of UCH-L1 protected from TNF-induced necroptosis. We found that UCH-L1 is a mediator of caspase-independent, non-apoptotic cell death also in diseased kidney podocytes by measuring cleavage of the protein PARP-1, caspase activity, cell death and cell morphology. Indicating a role of TNF in this process, podocytes with stably downregulated UCH-L1 proved resistant to TNF-induced necroptosis. Conclusions The proteases HtrA2/Omi and UCH-L1 represent two key components of TNF-induced necroptosis, validating the relevance of proteolysis not only for apoptosis, but also for caspase-independent PCD. Since UCH-L1 clearly contributes to the non-apoptotic death of podocytes, interference with the necroptotic

  12. Hinnuliquinone, a C2-symmetric dimeric non-peptide fungal metabolite inhibitor of HIV-1 protease.

    PubMed

    Singh, Sheo B; Ondeyka, John G; Tsipouras, Nasios; Ruby, Carolyn; Sardana, Vinod; Schulman, Marvin; Sanchez, Manuel; Pelaez, Fernando; Stahlhut, Mark W; Munshi, Sanjeev; Olsen, David B; Lingham, Russell B

    2004-11-05

    HIV-1 protease is one of several key enzymes required for the replication and maturation of HIV-1 virus. An almost two-decade research effort by academic and pharmaceutical institutions resulted in the successful commercialization of seven drugs that are potent inhibitors of HIV-1 protease activity and which, if used correctly, are highly effective in managing viral load. However, identification of clinical viral isolates that are resistant to these drugs indicates that this is a significant problem and that new classes of inhibitors are continually needed. Screening of microbial extracts followed by bioassay-guided isolation led to the discovery of a natural hinnuliquinone, a C(2)-symmetric bis-indolyl quinone natural product that inhibited the wild-type and a clinically resistant (A44) strain of HIV-1 protease with K(i) values of 0.97 and 1.25microM, respectively. Crystallographic analysis of the inhibitor-bound HIV-1 protease helped explain the importance of the C(2)-symmetry of hinnuliquinone for activity. Details of the isolation, biological activity, and crystallographic analysis of the inhibitor-bound protease are herein described.

  13. A de novo variant in the ASPRV1 gene in a dog with ichthyosis.

    PubMed

    Bauer, Anina; Waluk, Dominik P; Galichet, Arnaud; Timm, Katrin; Jagannathan, Vidhya; Sayar, Beyza S; Wiener, Dominique J; Dietschi, Elisabeth; Müller, Eliane J; Roosje, Petra; Welle, Monika M; Leeb, Tosso

    2017-03-01

    Ichthyoses are a heterogeneous group of inherited cornification disorders characterized by generalized dry skin, scaling and/or hyperkeratosis. Ichthyosis vulgaris is the most common form of ichthyosis in humans and caused by genetic variants in the FLG gene encoding filaggrin. Filaggrin is a key player in the formation of the stratum corneum, the uppermost layer of the epidermis and therefore crucial for barrier function. During terminal differentiation of keratinocytes, the precursor profilaggrin is cleaved by several proteases into filaggrin monomers and eventually processed into free amino acids contributing to the hydration of the cornified layer. We studied a German Shepherd dog with a novel form of ichthyosis. Comparing the genome sequence of the affected dog with 288 genomes from genetically diverse non-affected dogs we identified a private heterozygous variant in the ASPRV1 gene encoding "aspartic peptidase, retroviral-like 1", which is also known as skin aspartic protease (SASPase). The variant was absent in both parents and therefore due to a de novo mutation event. It was a missense variant, c.1052T>C, affecting a conserved residue close to an autoprocessing cleavage site, p.(Leu351Pro). ASPRV1 encodes a retroviral-like protease involved in profilaggrin-to-filaggrin processing. By immunofluorescence staining we showed that the filaggrin expression pattern was altered in the affected dog. Thus, our findings provide strong evidence that the identified de novo variant is causative for the ichthyosis in the affected dog and that ASPRV1 plays an essential role in skin barrier formation. ASPRV1 is thus a novel candidate gene for unexplained human forms of ichthyoses.

  14. A New Subtilase-Like Protease Deriving from Fusarium equiseti with High Potential for Industrial Applications.

    PubMed

    Juntunen, Kari; Mäkinen, Susanna; Isoniemi, Sari; Valtakari, Leena; Pelzer, Alexander; Jänis, Janne; Paloheimo, Marja

    2015-09-01

    A gene encoding a novel extracellular subtilisin-like protease was cloned from the ascomycete Fusarium equiseti and expressed in Trichoderma reesei. The F. equiseti protease (Fe protease) showed excellent performance in stain removal and good compatibility with several commercial laundry detergent formulations, suggesting that it has high potential for use in various industrial applications. The recombinant enzyme was purified and characterized. The temperature optimum of the Fe protease was 60 °C and it showed high activity in the pH range of 6-10, with a sharp decline in activity at pH above 10. The amino acid specificity of the Fe protease was studied using casein, cytochrome c, and ubiquitin as substrates. The Fe protease had broad substrate specificity: almost all amino acid residues were accepted at position P1, even though it showed some preference for cleavage at the C-terminal side of asparagine and histidine residues. The S4 subsite of Fe protease favors aspartic acid and threonine. The other well-characterized proteases from filamentous fungi, Proteinase K from Engyodontium album, Thermomycolin from Malbranchea sulfurea, and alkaline subtilisins from Bacillus species prefer hydrophobic amino acids in both the S1 and S4 subsites. Due to its different specificity compared to the members of the S8 family of clan SB of proteases, we consider that the Fe protease is a new protease. It does not belong to any previously defined IUBMB groups of proteases.

  15. The Cysteine Protease CEP1, a Key Executor Involved in Tapetal Programmed Cell Death, Regulates Pollen Development in Arabidopsis[W][OPEN

    PubMed Central

    Zhang, Dandan; Liu, Di; Lv, Xiaomeng; Wang, Ying; Xun, Zhili; Liu, Zhixiong; Li, Fenglan; Lu, Hai

    2014-01-01

    Tapetal programmed cell death (PCD) is a prerequisite for pollen grain development in angiosperms, and cysteine proteases are the most ubiquitous hydrolases involved in plant PCD. We identified a papain-like cysteine protease, CEP1, which is involved in tapetal PCD and pollen development in Arabidopsis thaliana. CEP1 is expressed specifically in the tapetum from stages 5 to 11 of anther development. The CEP1 protein first appears as a proenzyme in precursor protease vesicles and is then transported to the vacuole and transformed into the mature enzyme before rupture of the vacuole. cep1 mutants exhibited aborted tapetal PCD and decreased pollen fertility with abnormal pollen exine. A transcriptomic analysis revealed that 872 genes showed significantly altered expression in the cep1 mutants, and most of them are important for tapetal cell wall organization, tapetal secretory structure formation, and pollen development. CEP1 overexpression caused premature tapetal PCD and pollen infertility. ELISA and quantitative RT-PCR analyses confirmed that the CEP1 expression level showed a strong relationship to the degree of tapetal PCD and pollen fertility. Our results reveal that CEP1 is a crucial executor during tapetal PCD and that proper CEP1 expression is necessary for timely degeneration of tapetal cells and functional pollen formation. PMID:25035401

  16. The higher barrier of darunavir and tipranavir resistance for HIV-1 protease

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang, Yong; Liu, Zhigang; Brunzelle, Joseph S.

    2011-11-17

    Darunavir and tipranavir are two inhibitors that are active against multi-drug resistant (MDR) HIV-1 protease variants. In this study, the invitro inhibitory efficacy was tested against a MDR HIV-1 protease variant, MDR 769 82T, containing the drug resistance mutations of 46L/54V/82T/84V/90M. Crystallographic and enzymatic studies were performed to examine the mechanism of resistance and the relative maintenance of potency. The key findings are as follows: (i) The MDR protease exhibits decreased susceptibility to all nine HIV-1 protease inhibitors approved by the US Food and Drug Administration (FDA), among which darunavir and tipranavir are the most potent; (ii) the threonine 82more » mutation on the protease greatly enhances drug resistance by altering the hydrophobicity of the binding pocket; (iii) darunavir or tipranavir binding facilitates closure of the wide-open flaps of the MDR protease; and (iv) the remaining potency of tipranavir may be preserved by stabilizing the flaps in the inhibitor-protease complex while darunavir maintains its potency by preserving protein main chain hydrogen bonds with the flexible P2 group. These results could provide new insights into drug design strategies to overcome multi-drug resistance of HIV-1 protease variants.« less

  17. Cloning a Chymotrypsin-Like 1 (CTRL-1) Protease cDNA from the Jellyfish Nemopilema nomurai

    PubMed Central

    Heo, Yunwi; Kwon, Young Chul; Bae, Seong Kyeong; Hwang, Duhyeon; Yang, Hye Ryeon; Choudhary, Indu; Lee, Hyunkyoung; Yum, Seungshic; Shin, Kyoungsoon; Yoon, Won Duk; Kang, Changkeun; Kim, Euikyung

    2016-01-01

    An enzyme in a nematocyst extract of the Nemopilema nomurai jellyfish, caught off the coast of the Republic of Korea, catalyzed the cleavage of chymotrypsin substrate in an amidolytic kinetic assay, and this activity was inhibited by the serine protease inhibitor, phenylmethanesulfonyl fluoride. We isolated the full-length cDNA sequence of this enzyme, which contains 850 nucleotides, with an open reading frame of 801 encoding 266 amino acids. A blast analysis of the deduced amino acid sequence showed 41% identity with human chymotrypsin-like (CTRL) and the CTRL-1 precursor. Therefore, we designated this enzyme N. nomurai CTRL-1. The primary structure of N. nomurai CTRL-1 includes a leader peptide and a highly conserved catalytic triad of His69, Asp117, and Ser216. The disulfide bonds of chymotrypsin and the substrate-binding sites are highly conserved compared with the CTRLs of other species, including mammalian species. Nemopilema nomurai CTRL-1 is evolutionarily more closely related to Actinopterygii than to Scyphozoan (Aurelia aurita) or Hydrozoan (Hydra vulgaris). The N. nomurai CTRL1 was amplified from the genomic DNA with PCR using specific primers designed based on the full-length cDNA, and then sequenced. The N. nomurai CTRL1 gene contains 2434 nucleotides and four distinct exons. The 5′ donor splice (GT) and 3′ acceptor splice sequences (AG) are wholly conserved. This is the first report of the CTRL1 gene and cDNA structures in the jellyfish N. nomurai. PMID:27399771

  18. Cloning a Chymotrypsin-Like 1 (CTRL-1) Protease cDNA from the Jellyfish Nemopilema nomurai.

    PubMed

    Heo, Yunwi; Kwon, Young Chul; Bae, Seong Kyeong; Hwang, Duhyeon; Yang, Hye Ryeon; Choudhary, Indu; Lee, Hyunkyoung; Yum, Seungshic; Shin, Kyoungsoon; Yoon, Won Duk; Kang, Changkeun; Kim, Euikyung

    2016-07-05

    An enzyme in a nematocyst extract of the Nemopilema nomurai jellyfish, caught off the coast of the Republic of Korea, catalyzed the cleavage of chymotrypsin substrate in an amidolytic kinetic assay, and this activity was inhibited by the serine protease inhibitor, phenylmethanesulfonyl fluoride. We isolated the full-length cDNA sequence of this enzyme, which contains 850 nucleotides, with an open reading frame of 801 encoding 266 amino acids. A blast analysis of the deduced amino acid sequence showed 41% identity with human chymotrypsin-like (CTRL) and the CTRL-1 precursor. Therefore, we designated this enzyme N. nomurai CTRL-1. The primary structure of N. nomurai CTRL-1 includes a leader peptide and a highly conserved catalytic triad of His(69), Asp(117), and Ser(216). The disulfide bonds of chymotrypsin and the substrate-binding sites are highly conserved compared with the CTRLs of other species, including mammalian species. Nemopilema nomurai CTRL-1 is evolutionarily more closely related to Actinopterygii than to Scyphozoan (Aurelia aurita) or Hydrozoan (Hydra vulgaris). The N. nomurai CTRL1 was amplified from the genomic DNA with PCR using specific primers designed based on the full-length cDNA, and then sequenced. The N. nomurai CTRL1 gene contains 2434 nucleotides and four distinct exons. The 5' donor splice (GT) and 3' acceptor splice sequences (AG) are wholly conserved. This is the first report of the CTRL1 gene and cDNA structures in the jellyfish N. nomurai.

  19. Extreme Entropy-Enthalpy Compensation in a Drug Resistant Variant of HIV-1 Protease

    PubMed Central

    King, Nancy M.; Prabu-Jeyabalan, Moses; Bandaranayake, Rajintha M.; Nalam, Madhavi N. L.; Nalivaika, Ellen A.; Özen, Ayşegül; Haliloglu, Türkan; Yılmaz, Neşe Kurt; Schiffer, Celia A.

    2012-01-01

    The development of HIV-1 protease inhibitors has been the historic paradigm of rational structure-based drug design, where structural and thermodynamic analyses have assisted in the discovery of novel inhibitors. While the total enthalpy and entropy change upon binding determine the affinity, often the thermodynamics are considered in terms of inhibitor properties only. In the current study, profound changes are observed in the binding thermodynamics of a drug resistant variant compared to wild-type HIV-1 protease, irrespective of the inhibitor bound. This variant (Flap+) has a combination of flap and active site mutations and exhibits extremely large entropy-enthalpy compensation compared to wild-type protease, 5–15 kcal/mol, while losing only 1–3 kcal/mol in total binding free energy for any of six FDA approved inhibitors. Although entropy-enthalpy compensation has been previously observed for a variety of systems, never have changes of this magnitude been reported. The co-crystal structures of Flap+ protease with four of the inhibitors were determined and compared with complexes of both the wildtype protease and another drug resistant variant that does not exhibit this energetic compensation. Structural changes conserved across the Flap+ complexes, which are more pronounced for the flaps covering the active site, likely contribute to the thermodynamic compensation. The finding that drug resistant mutations can profoundly modulate the relative thermodynamic properties of a therapeutic target independent of the inhibitor presents a new challenge for rational drug design. PMID:22712830

  20. Development and evaluation of a phenotypic assay monitoring resistance formation to protease inhibitors in HIV-1-infected patients.

    PubMed

    Gehringer, Heike; Von der Helm, Klaus; Seelmeir, Sigrid; Weissbrich, Benedikt; Eberle, Josef; Nitschko, Hans

    2003-05-01

    A novel phenotypic assay, based on recombinant expression of the HIV-1-protease was developed and evaluated; it monitors the formation of resistance to protease inhibitors. The HIV-1 protease-encoding region from the blood sample of patients was amplified, ligated into the expression vector pBD2, and recombinantly expressed in Escherichia coli TG1 cells. The resulting recombinant enzyme was purified by a newly developed one-step acid extraction protocol. The protease activity was determined in presence of five selected HIV protease inhibitors and the 50% inhibitory concentration (IC(50)) to the respective protease inhibitors determined. The degree of resistance was expressed in terms of x-fold increase in IC(50) compared to the IC(50) value of an HIV-1 wild type protease preparation. The established test system showed a reproducible recombinant expression of each individual patients' HIV-1 protease population. Samples of nine clinically well characterised HIV-1-infected patients with varying degrees of resistance were analysed. There was a good correlation between clinical parameters and the results obtained by this phenotypic assay. For the majority of patients a blind genotypic analysis of the patients' protease domain revealed a fair correlation to the results of the phenotypic assay. In a minority of patients our phenotypic results diverged from the genotypic ones. This novel phenotypic assay can be carried out within 8-10 days, and offers a significant advantage in time to the current employed phenotypic tests.

  1. A Molecular Approach to Nested RT-PCR Using a New Set of Primers for the Detection of the Human Immunodeficiency Virus Protease Gene.

    PubMed

    Zarei, Mohammad; Ravanshad, Mehrdad; Bagban, Ashraf; Fallahi, Shahab

    2016-07-01

    The human immunodeficiency virus (HIV-1) is the etiologic agent of AIDS. The disease can be transmitted via blood in the window period prior to the development of antibodies to the disease. Thus, an appropriate method for the detection of HIV-1 during this window period is very important. This descriptive study proposes a sensitive, efficient, inexpensive, and easy method to detect HIV-1. In this study 25 serum samples of patients under treatment and also 10 positive and 10 negative control samples were studied. Twenty-five blood samples were obtained from HIV-1-infected individuals who were receiving treatment at the acquired immune deficiency syndrome (AIDS) research center of Imam Khomeini hospital in Tehran. The identification of HIV-1-positive samples was done by using reverse transcription to produce copy deoxyribonucleic acid (cDNA) and then optimizing the nested polymerase chain reaction (PCR) method. Two pairs of primers were then designed specifically for the protease gene fragment of the nested real time-PCR (RT-PCR) samples. Electrophoresis was used to examine the PCR products. The results were analyzed using statistical tests, including Fisher's exact test, and SPSS17 software. The 325 bp band of the protease gene was observed in all the positive control samples and in none of the negative control samples. The proposed method correctly identified HIV-1 in 23 of the 25 samples. These results suggest that, in comparison with viral cultures, antibody detection by enzyme linked immunosorbent assay (ELISAs), and conventional PCR methods, the proposed method has high sensitivity and specificity for the detection of HIV-1.

  2. Conformations of the HIV-1 protease: A crystal structure data set analysis.

    PubMed

    Palese, Luigi Leonardo

    2017-11-01

    The HIV protease is an important drug target for HIV/AIDS therapy, and its structure and function have been extensively investigated. This enzyme performs an essential role in viral maturation by processing specific cleavage sites in the Gag and Gag-Pol precursor polyproteins so as to release their mature forms. This 99 amino acid aspartic protease works as a homodimer, with the active site localized in a central cavity capped by two flexible flap regions. The dimer presents closed or open conformations, which are involved in the substrate binding and release. Here the results of the analysis of a HIV-1 protease data set containing 552 dimer structures are reported. Different dimensionality reduction methods have been used in order to get information from this multidimensional database. Most of the structures in the data set belong to two conformational clusters. An interesting observation that comes from the analysis of these data is that some protease sequences are localized preferentially in specific areas of the conformational landscape of this protein. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Structural, kinetic, and thermodynamic studies of specificity designed HIV-1 protease.

    PubMed

    Alvizo, Oscar; Mittal, Seema; Mayo, Stephen L; Schiffer, Celia A

    2012-07-01

    HIV-1 protease recognizes and cleaves more than 12 different substrates leading to viral maturation. While these substrates share no conserved motif, they are specifically selected for and cleaved by protease during viral life cycle. Drug resistant mutations evolve within the protease that compromise inhibitor binding but allow the continued recognition of all these substrates. While the substrate envelope defines a general shape for substrate recognition, successfully predicting the determinants of substrate binding specificity would provide additional insights into the mechanism of altered molecular recognition in resistant proteases. We designed a variant of HIV protease with altered specificity using positive computational design methods and validated the design using X-ray crystallography and enzyme biochemistry. The engineered variant, Pr3 (A28S/D30F/G48R), was designed to preferentially bind to one out of three of HIV protease's natural substrates; RT-RH over p2-NC and CA-p2. In kinetic assays, RT-RH binding specificity for Pr3 increased threefold compared to the wild-type (WT), which was further confirmed by isothermal titration calorimetry. Crystal structures of WT protease and the designed variant in complex with RT-RH, CA-p2, and p2-NC were determined. Structural analysis of the designed complexes revealed that one of the engineered substitutions (G48R) potentially stabilized heterogeneous flap conformations, thereby facilitating alternate modes of substrate binding. Our results demonstrate that while substrate specificity could be engineered in HIV protease, the structural pliability of protease restricted the propagation of interactions as predicted. These results offer new insights into the plasticity and structural determinants of substrate binding specificity of the HIV-1 protease. Copyright © 2012 The Protein Society.

  4. Degradation of the HilC and HilD regulator proteins by ATP-dependent Lon protease leads to downregulation of Salmonella pathogenicity island 1 gene expression.

    PubMed

    Takaya, Akiko; Kubota, Yohsuke; Isogai, Emiko; Yamamoto, Tomoko

    2005-02-01

    Salmonella pathogenicity island 1 (SPI1) enables infecting Salmonella to cross the small intestinal barrier and to escape phagocytosis by inducing apoptosis. Several environmental signals and transcriptional regulators modulate the expression of hilA, which encodes a protein playing a central role in the regulatory hierarchy of SPI1 gene expression. We have previously shown that Lon, a stress-induced ATP-dependent protease, is a negative regulator of hilA, suggesting that it targets factors required for activating hilA expression. To elucidate the mechanisms by which Lon protease negatively regulates SPI1 transcription, we looked for its substrate proteins. We found that HilC and HilD, which are positive regulators of hilA expression, accumulate in Lon-depleted cells, and that the enhancement of SPI1 expression that occurs in a lon-disrupted mutant is not observed in the lon hilC hilD triple null mutant. Furthermore, we demonstrated that the half-lives of HilC and HilD are, respectively, about 12 times and three times longer in the Lon-depleted mutant, than in the Lon+ cells, suggesting that Lon targets both of HilC and HilD. In view of these findings, we suggest that the regulation of SPI1 expression is negatively controlled through degradation of the HilC and HilD transcriptional regulators by Lon.

  5. HIV-1 protease inhibitor mutations affect the development of HIV-1 resistance to the maturation inhibitor bevirimat.

    PubMed

    Fun, Axel; van Maarseveen, Noortje M; Pokorná, Jana; Maas, Renée Em; Schipper, Pauline J; Konvalinka, Jan; Nijhuis, Monique

    2011-08-24

    Maturation inhibitors are an experimental class of antiretrovirals that inhibit Human Immunodeficiency Virus (HIV) particle maturation, the structural rearrangement required to form infectious virus particles. This rearrangement is triggered by the ordered cleavage of the precursor Gag polyproteins into their functional counterparts by the viral enzyme protease. In contrast to protease inhibitors, maturation inhibitors impede particle maturation by targeting the substrate of protease (Gag) instead of the protease enzyme itself. Direct cross-resistance between protease and maturation inhibitors may seem unlikely, but the co-evolution of protease and its substrate, Gag, during protease inhibitor therapy, could potentially affect future maturation inhibitor therapy. Previous studies showed that there might also be an effect of protease inhibitor resistance mutations on the development of maturation inhibitor resistance, but the exact mechanism remains unclear. We used wild-type and protease inhibitor resistant viruses to determine the impact of protease inhibitor resistance mutations on the development of maturation inhibitor resistance. Our resistance selection studies demonstrated that the resistance profiles for the maturation inhibitor bevirimat are more diverse for viruses with a mutated protease compared to viruses with a wild-type protease. Viral replication did not appear to be a major factor during emergence of bevirimat resistance. In all in vitro selections, one of four mutations was selected: Gag V362I, A364V, S368N or V370A. The impact of these mutations on maturation inhibitor resistance and viral replication was analyzed in different protease backgrounds. The data suggest that the protease background affects development of HIV-1 resistance to bevirimat and the replication profiles of bevirimat-selected HIV-1. The protease-dependent bevirimat resistance and replication levels can be explained by differences in CA/p2 cleavage processing by the different

  6. Intracellular serine protease 1 of Bacillus subtilis is formed in vivo as an unprocessed, active protease in stationary cells.

    PubMed Central

    Sheehan, S M; Switzer, R L

    1990-01-01

    Western immunoblots and assays of Bacillus subtilis extracts showed that intracellular serine protease 1 is produced in a form larger than previously reported, appears not to have undergone N-terminal processing, and is active in the presence or absence of calcium. No evidence for an inactive precursor form of the protease was found. Images FIG. 1 PMID:2104610

  7. Characterization of the Mamestra configurata (Lepidoptera: Noctuidae) larval midgut protease complement and adaptation to feeding on artificial diet, Brassica species, and protease inhibitor.

    PubMed

    Erlandson, Martin A; Hegedus, Dwayne D; Baldwin, Douglas; Noakes, Amy; Toprak, Umut

    2010-10-01

    The midgut protease profiles from 5th instar Mamestra configurata larvae fed various diets (standard artificial diet, low protein diet, low protein diet with soybean trypsin inhibitor [SBTI], or Brassica napus) were characterized by one-dimensional enzymography in gelatin gels. The gut protease profile of larvae fed B. napus possessed protease activities of molecular masses of approximately 33 and 55 kDa, which were not present in the guts of larvae fed artificial diet. Similarly, larvae fed artificial diet had protease activities of molecular masses of approximately 21, 30, and 100 kDa that were absent in larvae fed B. napus. Protease profiles changed within 12 to 24 h after switching larvae from artificial diet to plant diet and vice versa. The gut protease profiles from larvae fed various other brassicaceous species and lines having different secondary metabolite profiles did not differ despite significant differences in larval growth rates on the different host plants. Genes encoding putative digestive proteolytic enzymes, including four carboxypeptidases, five aminopeptidases, and 48 serine proteases, were identified in cDNA libraries from 4th instar M. configurata midgut tissue. Many of the protease-encoding genes were expressed at similar levels on all diets; however, three chymoptrypsin-like genes (McSP23, McSP27, and McSP37) were expressed at much higher levels on standard artificial diet and diet containing SBTI as was the trypsin-like gene McSP34. The expression of the trypsin-like gene McSP50 was highest on B. napus. The adaptation of M. configurata digestive biochemistry to different diets is discussed in the context of the flexibility of polyphagous insects to changing diet sources.

  8. Revealing interaction mode between HIV-1 protease and mannitol analog inhibitor.

    PubMed

    Yan, Guan-Wen; Chen, Yue; Li, Yixue; Chen, Hai-Feng

    2012-06-01

    HIV protease is a key enzyme to play a key role in the HIV-1 replication cycle and control the maturation from HIV viruses to an infectious virion. HIV-1 protease has become an important target for anti-HIV-1 drug development. Here, we used molecular dynamics simulation to study the binding mode between mannitol derivatives and HIV-1 protease. The results suggest that the most active compound (M35) has more stable hydrogen bonds and stable native contacts than the less active one (M17). These mannitol derivatives might have similar interaction mode with HIV-1 protease. Then, 3D-QSAR was used to construct quantitative structure-activity models. The cross-validated q(2) values are found as 0.728 and 0.611 for CoMFA and CoMSIA, respectively. And the non-cross-validated r(2) values are 0.973 and 0.950. Nine test set compounds validate the model. The results show that this model possesses better prediction ability than the previous work. This model can be used to design new chemical entities and make quantitative prediction of the bioactivities for HIV-1 protease inhibitors before resorting to in vitro and in vivo experiment. © 2012 John Wiley & Sons A/S.

  9. Frequency and patterns of protease gene resistance mutations in HIV-infected patients treated with lopinavir/ritonavir as their first protease inhibitor.

    PubMed

    Barber, Tristan J; Harrison, Linda; Asboe, David; Williams, Ian; Kirk, Stuart; Gilson, Richard; Bansi, Loveleen; Pillay, Deenan; Dunn, David

    2012-04-01

    Selection of protease mutations on antiretroviral therapy (ART) including a ritonavir-boosted protease inhibitor (PI) has been reported infrequently. Scarce data exist from long-term cohorts on resistance incidence or mutational patterns emerging to different PIs. We studied UK patients receiving lopinavir/ritonavir as their first PI, either while naive to ART or having previously received non-PI-based ART. Virological failure was defined as viral load ≥ 400 copies/mL after previous suppression <400 copies/mL, or failure to achieve <400 copies/mL during the first 6 months. pol sequences whilst failing lopinavir or within 30 days after stopping were analysed. Major and minor mutations (IAS-USA 2008-after exclusion of polymorphisms) were considered. Predicted susceptibility was determined using the Stanford HIVdb algorithm. Three thousand and fifty-six patients were followed for a median (IQR) of 14 (6-30) months, of whom 811 (27%) experienced virological failure. Of these, resistance test results were available on 291 (36%). One or more protease mutations were detected in 32 (11%) patients; the most frequent were I54V (n = 12), M46I (n = 11), V82A (n = 7) and L76V (n = 3). No association with viral subtype was evident. Many patients retained virus predicted to be susceptible to lopinavir (14, 44%), tipranavir (26, 81%) and darunavir (27, 84%). This study reflects the experience of patients in routine care. Selection of protease gene mutations by lopinavir/ritonavir occurred at a much higher rate than in clinical trials. The mutations observed showed only partial overlap with those previously identified by structural chemistry models, serial cell culture passage and genotype-phenotype analyses. There remained a low degree of predicted cross-resistance to other widely used PIs.

  10. Proteolytic Activation of the Protease-activated Receptor (PAR)-2 by the Glycosylphosphatidylinositol-anchored Serine Protease Testisin*

    PubMed Central

    Driesbaugh, Kathryn H.; Buzza, Marguerite S.; Martin, Erik W.; Conway, Gregory D.; Kao, Joseph P. Y.; Antalis, Toni M.

    2015-01-01

    Protease-activated receptors (PARs) are a family of seven-transmembrane, G-protein-coupled receptors that are activated by multiple serine proteases through specific N-terminal proteolytic cleavage and the unmasking of a tethered ligand. The majority of PAR-activating proteases described to date are soluble proteases that are active during injury, coagulation, and inflammation. Less investigation, however, has focused on the potential for membrane-anchored serine proteases to regulate PAR activation. Testisin is a unique trypsin-like serine protease that is tethered to the extracellular membrane of cells through a glycophosphatidylinositol (GPI) anchor. Here, we show that the N-terminal domain of PAR-2 is a substrate for testisin and that proteolytic cleavage of PAR-2 by recombinant testisin activates downstream signaling pathways, including intracellular Ca2+ mobilization and ERK1/2 phosphorylation. When testisin and PAR-2 are co-expressed in HeLa cells, GPI-anchored testisin specifically releases the PAR-2 tethered ligand. Conversely, knockdown of endogenous testisin in NCI/ADR-Res ovarian tumor cells reduces PAR-2 N-terminal proteolytic cleavage. The cleavage of PAR-2 by testisin induces activation of the intracellular serum-response element and NFκB signaling pathways and the induction of IL-8 and IL-6 cytokine gene expression. Furthermore, the activation of PAR-2 by testisin results in the loss and internalization of PAR-2 from the cell surface. This study reveals a new biological substrate for testisin and is the first demonstration of the activation of a PAR by a serine protease GPI-linked to the cell surface. PMID:25519908

  11. Protease signaling through protease activated receptor 1 mediate nerve activation by mucosal supernatants from irritable bowel syndrome but not from ulcerative colitis patients

    PubMed Central

    Buhner, Sabine; Hahne, Hannes; Hartwig, Kerstin; Li, Qin; Vignali, Sheila; Ostertag, Daniela; Meng, Chen; Hörmannsperger, Gabriele; Braak, Breg; Pehl, Christian; Frieling, Thomas; Barbara, Giovanni; De Giorgio, Roberto; Demir, Ihsan Ekin; Ceyhan, Güralp Onur; Zeller, Florian; Boeckxstaens, Guy; Haller, Dirk; Kuster, Bernhard

    2018-01-01

    Background & aims The causes of gastrointestinal complaints in irritable bowel syndrome (IBS) remain poorly understood. Altered nerve function has emerged as an important pathogenic factor as IBS mucosal biopsy supernatants consistently activate enteric and sensory neurons. We investigated the neurally active molecular components of such supernatants from patients with IBS and quiescent ulcerative colitis (UC). Method Effects of supernatants from 7 healthy controls (HC), 20 IBS and 12 UC patients on human and guinea pig submucous neurons were studied with neuroimaging techniques. We identify differentially expressed proteins with proteome analysis. Results Nerve activation by IBS supernatants was prevented by the protease activated receptor 1 (PAR1) antagonist SCHE79797. UC supernatants also activated enteric neurons through protease dependent mechanisms but without PAR1 involvement. Proteome analysis of the supernatants identified 204 proteins, among them 17 proteases as differentially expressed between IBS, UC and HC. Of those the four proteases elastase 3a, chymotrypsin C, proteasome subunit type beta-2 and an unspecified isoform of complement C3 were significantly more abundant in IBS compared to HC and UC supernatants. Of eight proteases, which were upregulated in IBS, the combination of elastase 3a, cathepsin L and proteasome alpha subunit-4 showed the highest prediction accuracy of 98% to discriminate between IBS and HC groups. Elastase synergistically potentiated the effects of histamine and serotonin–the two other main neuroactive substances in the IBS supernatants. A serine protease inhibitor isolated from the probiotic Bifidobacterium longum NCC2705 (SERPINBL), known to inhibit elastase-like proteases, prevented nerve activation by IBS supernatants. Conclusion Proteases in IBS and UC supernatants were responsible for nerve activation. Our data demonstrate that proteases, particularly those signalling through neuronal PAR1, are biomarker candidates for

  12. Design of novel HIV-1 protease inhibitors incorporating isophthalamide-derived P2-P3 ligands: Synthesis, biological evaluation and X-ray structural studies of inhibitor-HIV-1 protease complex

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ghosh, Arun K.; Brindisi, Margherita; Nyalapatla, Prasanth R.

    Based upon molecular insights from the X-ray structures of inhibitor-bound HIV-1 protease complexes, we have designed a series of isophthalamide-derived inhibitors incorporating substituted pyrrolidines, piperidines and thiazolidines as P2-P3 ligands for specific interactions in the S2-S3 extended site. Compound 4b has shown an enzyme Ki of 0.025 nM and antiviral IC50 of 69 nM. An X-ray crystal structure of inhibitor 4b-HIV-1 protease complex was determined at 1.33 Å resolution. We have also determined X-ray structure of 3b-bound HIV-1 protease at 1.27 Å resolution. These structures revealed important molecular insight into the inhibitor–HIV-1 protease interactions in the active site.

  13. Purification, Characterization, and Cloning of a Cold-Adapted Protease from Antarctic Janthinobacterium lividum.

    PubMed

    Kim, Hyun-Do; Kim, Su-Mi; Choi, Jong-Il

    2018-03-28

    In this study, a 107 kDa protease from psychrophilic Janthinobacterium lividum PAMC 26541 was purified by anion-exchange chromatography. The specific activity of the purified protease was 264 U/mg, and the overall yield was 12.5%. The J. lividum PAMC 25641 protease showed optimal activity at pH 7.0-7.5 and 40°C. Protease activity was inhibited by PMSF, but not by DTT. On the basis of the N-terminal sequence of the purified protease, the gene encoding the cold-adapted protease from J. lividum PAMC 25641 was cloned into the pET-28a(+) vector and heterologously expressed in Escherichia coli BL21(DE3) as an intracellular soluble protein.

  14. PhAP protease from Pseudoalteromonas haloplanktis TAC125: Gene cloning, recombinant production in E. coli and enzyme characterization

    NASA Astrophysics Data System (ADS)

    de Pascale, D.; Giuliani, M.; De Santi, C.; Bergamasco, N.; Amoresano, A.; Carpentieri, A.; Parrilli, E.; Tutino, M. L.

    2010-08-01

    Cold-adapted proteases have been found to be the dominant activity throughout the cold marine environment, indicating their importance in bacterial acquisition of nitrogen-rich complex organic compounds. However, few extracellular proteases from marine organisms have been characterized so far, and the mechanisms that enable their activity in situ are still largely unknown. Aside from their ecological importance and use as model enzyme for structure/function investigations, cold-active proteolytic enzymes offer great potential for biotechnological applications. Our studies on cold adapted proteases were performed on exo-enzyme produced by the Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125. By applying a proteomic approach, we identified several proteolytic activities from its culture supernatant. PhAP protease was selected for further investigations. The encoding gene was cloned and the protein was recombinantly produced in E. coli cells. The homogeneous product was biochemically characterised and it turned out that the enzyme is a Zn-dependent aminopeptidase, with an activity dependence from assay temperature typical of psychrophilic enzymes.

  15. The dimer interfaces of protease and extra-protease domains influence the activation of protease and the specificity of GagPol cleavage.

    PubMed

    Pettit, Steven C; Gulnik, Sergei; Everitt, Lori; Kaplan, Andrew H

    2003-01-01

    Activation of the human immunodeficiency virus type 1 (HIV-1) protease is an essential step in viral replication. As is the case for all retroviral proteases, enzyme activation requires the formation of protease homodimers. However, little is known about the mechanisms by which retroviral proteases become active within their precursors. Using an in vitro expression system, we have examined the determinants of activation efficiency and the order of cleavage site processing for the protease of HIV-1 within the full-length GagPol precursor. Following activation, initial cleavage occurs between the viral p2 and nucleocapsid proteins. This is followed by cleavage of a novel site located in the transframe domain. Mutational analysis of the dimer interface of the protease produced differential effects on activation and specificity. A subset of mutations produced enhanced cleavage at the amino terminus of the protease, suggesting that, in the wild-type precursor, cleavages that liberate the protease are a relatively late event. Replacement of the proline residue at position 1 of the protease dimer interface resulted in altered cleavage of distal sites and suggests that this residue functions as a cis-directed specificity determinant. In summary, our studies indicate that interactions within the protease dimer interface help determine the order of precursor cleavage and contribute to the formation of extended-protease intermediates. Assembly domains within GagPol outside the protease domain also influence enzyme activation.

  16. Serine protease-related proteins in the malaria mosquito, Anopheles gambiae.

    PubMed

    Cao, Xiaolong; Gulati, Mansi; Jiang, Haobo

    2017-09-01

    Insect serine proteases (SPs) and serine protease homologs (SPHs) participate in digestion, defense, development, and other physiological processes. In mosquitoes, some clip-domain SPs and SPHs (i.e. CLIPs) have been investigated for possible roles in antiparasitic responses. In a recent test aimed at improving quality of gene models in the Anopheles gambiae genome using RNA-seq data, we observed various discrepancies between gene models in AgamP4.5 and corresponding sequences selected from those modeled by Cufflinks, Trinity and Bridger. Here we report a comparative analysis of the 337 SP-related proteins in A. gambiae by examining their domain structures, sequence diversity, chromosomal locations, and expression patterns. One hundred and ten CLIPs contain 1 to 5 clip domains in addition to their protease domains (PDs) or non-catalytic, protease-like domains (PLDs). They are divided into five subgroups: CLIPAs (22) are clip 1-5 -PLD; CLIPBs (29), CLIPCs (12) and CLIPDs (14) are mainly clip-PD; most CLIPEs (33) have a domain structure of PD/PLD-PLD-clip-PLD 0-1 . While expression of the CLIP genes in group-1 is generally low and detected in various tissue- and stage-specific RNA-seq libraries, some putative GPs/GPHs (i.e. single domain gut SPs/SPHs) in group-2 are highly expressed in midgut, whole larva or whole adult libraries. In comparison, 46 SPs, 26 SPHs, and 37 multi-domain SPs/SPHs (i.e. PD/PLD-PLD ≥1 ) in group-3 do not seem to be specifically expressed in digestive tract. There are 16 SPs and 2 SPH containing other types of putative regulatory domains (e.g. LDLa, CUB, Gd). Of the 337 SP and SPH genes, 159 were sorted into 46 groups (2-8 members/group) based on similar phylogenetic tree position, chromosomal location, and expression profile. This information and analysis, including improved gene models and protein sequences, constitute a solid foundation for functional analysis of the SP-related proteins in A. gambiae. Copyright © 2017 Elsevier Ltd

  17. Serine Proteases-Like Genes in the Asian Rice Gall Midge Show Differential Expression in Compatible and Incompatible Interactions with Rice

    PubMed Central

    Sinha, Deepak Kumar; Lakshmi, Mulagondla; Anuradha, Ghanta; Rahman, Shaik J.; Siddiq, Ebrahimali A.; Bentur, Jagadish S.; Nair, Suresh

    2011-01-01

    The Asian rice gall midge, Orseolia oryzae (Wood-Mason), is a serious pest of rice. Investigations into the gall midge-rice interaction will unveil the underlying molecular mechanisms which, in turn, can be used as a tool to assist in developing suitable integrated pest management strategies. The insect gut is known to be involved in various physiological and biological processes including digestion, detoxification and interaction with the host. We have cloned and identified two genes, OoprotI and OoprotII, homologous to serine proteases with the conserved His87, Asp136 and Ser241 residues. OoProtI shared 52.26% identity with mosquito-type trypsin from Hessian fly whereas OoProtII showed 52.49% identity to complement component activated C1s from the Hessian fly. Quantitative real time PCR analysis revealed that both the genes were significantly upregulated in larvae feeding on resistant cultivar than in those feeding on susceptible cultivar. These results provide an opportunity to understand the gut physiology of the insect under compatible or incompatible interactions with the host. Phylogenetic analysis grouped these genes in the clade containing proteases of phytophagous insects away from hematophagous insects. PMID:21686154

  18. N-Terminal Protease Gene Phylogeny Reveals the Potential for Novel Cyanobactin Diversity in Cyanobacteria

    PubMed Central

    Martins, Joana; Leão, Pedro N.; Ramos, Vitor; Vasconcelos, Vitor

    2013-01-01

    Cyanobactins are a recently recognized group of ribosomal cyclic peptides produced by cyanobacteria, which have been studied because of their interesting biological activities. Here, we have used a PCR-based approach to detect the N-terminal protease (A) gene from cyanobactin synthetase gene clusters, in a set of diverse cyanobacteria from our culture collection (Laboratory of Ecotoxicology, Genomics and Evolution (LEGE) CC). Homologues of this gene were found in Microcystis and Rivularia strains, and for the first time in Cuspidothrix, Phormidium and Sphaerospermopsis strains. Phylogenetic relationships inferred from available A-gene sequences, including those obtained in this work, revealed two new groups of phylotypes, harboring Phormidium, Sphaerospermopsis and Rivularia LEGE isolates. Thus, this study shows that, using underexplored cyanobacterial strains, it is still possible to expand the known genetic diversity of genes involved in cyanobactin biosynthesis. PMID:24351973

  19. The Dimer Interfaces of Protease and Extra-Protease Domains Influence the Activation of Protease and the Specificity of GagPol Cleavage

    PubMed Central

    Pettit, Steven C.; Gulnik, Sergei; Everitt, Lori; Kaplan, Andrew H.

    2003-01-01

    Activation of the human immunodeficiency virus type 1 (HIV-1) protease is an essential step in viral replication. As is the case for all retroviral proteases, enzyme activation requires the formation of protease homodimers. However, little is known about the mechanisms by which retroviral proteases become active within their precursors. Using an in vitro expression system, we have examined the determinants of activation efficiency and the order of cleavage site processing for the protease of HIV-1 within the full-length GagPol precursor. Following activation, initial cleavage occurs between the viral p2 and nucleocapsid proteins. This is followed by cleavage of a novel site located in the transframe domain. Mutational analysis of the dimer interface of the protease produced differential effects on activation and specificity. A subset of mutations produced enhanced cleavage at the amino terminus of the protease, suggesting that, in the wild-type precursor, cleavages that liberate the protease are a relatively late event. Replacement of the proline residue at position 1 of the protease dimer interface resulted in altered cleavage of distal sites and suggests that this residue functions as a cis-directed specificity determinant. In summary, our studies indicate that interactions within the protease dimer interface help determine the order of precursor cleavage and contribute to the formation of extended-protease intermediates. Assembly domains within GagPol outside the protease domain also influence enzyme activation. PMID:12477841

  20. Tunable protease-activatable virus nanonodes.

    PubMed

    Judd, Justin; Ho, Michelle L; Tiwari, Abhinav; Gomez, Eric J; Dempsey, Christopher; Van Vliet, Kim; Igoshin, Oleg A; Silberg, Jonathan J; Agbandje-McKenna, Mavis; Suh, Junghae

    2014-05-27

    We explored the unique signal integration properties of the self-assembling 60-mer protein capsid of adeno-associated virus (AAV), a clinically proven human gene therapy vector, by engineering proteolytic regulation of virus-receptor interactions such that processing of the capsid by proteases is required for infection. We find the transfer function of our engineered protease-activatable viruses (PAVs), relating the degree of proteolysis (input) to PAV activity (output), is highly nonlinear, likely due to increased polyvalency. By exploiting this dynamic polyvalency, in combination with the self-assembly properties of the virus capsid, we show that mosaic PAVs can be constructed that operate under a digital AND gate regime, where two different protease inputs are required for virus activation. These results show viruses can be engineered as signal-integrating nanoscale nodes whose functional properties are regulated by multiple proteolytic signals with easily tunable and predictable response surfaces, a promising development toward advanced control of gene delivery.

  1. Tunable Protease-Activatable Virus Nanonodes

    PubMed Central

    2015-01-01

    We explored the unique signal integration properties of the self-assembling 60-mer protein capsid of adeno-associated virus (AAV), a clinically proven human gene therapy vector, by engineering proteolytic regulation of virus–receptor interactions such that processing of the capsid by proteases is required for infection. We find the transfer function of our engineered protease-activatable viruses (PAVs), relating the degree of proteolysis (input) to PAV activity (output), is highly nonlinear, likely due to increased polyvalency. By exploiting this dynamic polyvalency, in combination with the self-assembly properties of the virus capsid, we show that mosaic PAVs can be constructed that operate under a digital AND gate regime, where two different protease inputs are required for virus activation. These results show viruses can be engineered as signal-integrating nanoscale nodes whose functional properties are regulated by multiple proteolytic signals with easily tunable and predictable response surfaces, a promising development toward advanced control of gene delivery. PMID:24796495

  2. Biochemical and molecular characterization of a detergent-stable serine alkaline protease from Bacillus pumilus CBS with high catalytic efficiency.

    PubMed

    Jaouadi, Bassem; Ellouz-Chaabouni, Semia; Rhimi, Moez; Bejar, Samir

    2008-09-01

    We have described previously the potential use of an alkaline protease from Bacillus pumilus CBS as an effective additive in laundry detergent formulations [B. Jaouadi, S. Ellouz-Chaabouni, M. Ben Ali, E. Ben Messaoud, B. Naili, A. Dhouib, S. Bejar, A novel alkaline protease from Bacillus pumilus CBS having a high compatibility with laundry detergent and a high feather-degrading activity, Process Biochem, submitted for publication]. Here, we purified this enzyme (named SAPB) and we cloned, sequenced and over-expressed the corresponding gene. The enzyme was purified to homogeneity using salt precipitation and gel filtration HPLC. The pure protease was found to be monomeric protein with a molecular mass of 34598.19Da as determined by MALDI-TOF mass spectrometry. The NH2-terminal sequence of first 21 amino acids (aa) of the purified SAPB was AQTVPYGIPQIKAPAVHAQGY and was completely identical to proteases from other Bacillus pumilus species. This protease is strongly inhibited by PMSF and DFP, showing that it belongs to the serine proteases superfamily. Interestingly, the optimum pH is 10.6 while the optimum temperature was determined to be 65 degrees C. The enzyme was completely stable within a wide range of pH (7.0-10.6) and temperature (30-55 degrees C). One of the distinguishing properties is its catalytic efficiency (kcat/Km) calculated to be 45,265min(-1)mM(-1) and 147,000min(-1)mM(-1) using casein and AAPF as substrates, respectively, which is higher than that of Subtilisin Carlsberg, Subtilisin BPN' and Subtilisin 309 determined under the same conditions. In addition, SAPB showed remarkable stability, for 24h at 40 degrees C, in the presence of 5% Tween-80, 1% SDS, 15% urea and 10% H2O2, which comprise the common bleach-based detergent formulation. The sapB gene encoding SAPB was cloned, sequenced and over-expressed in Escherichia coli. The purified recombinant enzyme (rSAPB) has the same physicochemical and kinetic properties as the native one. SapB gene had

  3. Proteolytic activation of the protease-activated receptor (PAR)-2 by the glycosylphosphatidylinositol-anchored serine protease testisin.

    PubMed

    Driesbaugh, Kathryn H; Buzza, Marguerite S; Martin, Erik W; Conway, Gregory D; Kao, Joseph P Y; Antalis, Toni M

    2015-02-06

    Protease-activated receptors (PARs) are a family of seven-transmembrane, G-protein-coupled receptors that are activated by multiple serine proteases through specific N-terminal proteolytic cleavage and the unmasking of a tethered ligand. The majority of PAR-activating proteases described to date are soluble proteases that are active during injury, coagulation, and inflammation. Less investigation, however, has focused on the potential for membrane-anchored serine proteases to regulate PAR activation. Testisin is a unique trypsin-like serine protease that is tethered to the extracellular membrane of cells through a glycophosphatidylinositol (GPI) anchor. Here, we show that the N-terminal domain of PAR-2 is a substrate for testisin and that proteolytic cleavage of PAR-2 by recombinant testisin activates downstream signaling pathways, including intracellular Ca(2+) mobilization and ERK1/2 phosphorylation. When testisin and PAR-2 are co-expressed in HeLa cells, GPI-anchored testisin specifically releases the PAR-2 tethered ligand. Conversely, knockdown of endogenous testisin in NCI/ADR-Res ovarian tumor cells reduces PAR-2 N-terminal proteolytic cleavage. The cleavage of PAR-2 by testisin induces activation of the intracellular serum-response element and NFκB signaling pathways and the induction of IL-8 and IL-6 cytokine gene expression. Furthermore, the activation of PAR-2 by testisin results in the loss and internalization of PAR-2 from the cell surface. This study reveals a new biological substrate for testisin and is the first demonstration of the activation of a PAR by a serine protease GPI-linked to the cell surface. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Identification of unannotated exons of low abundance transcripts in Drosophila melanogaster and cloning of a new serine protease gene upregulated upon injury.

    PubMed

    Maia, Rafaela M; Valente, Valeria; Cunha, Marco A V; Sousa, Josane F; Araujo, Daniela D; Silva, Wilson A; Zago, Marco A; Dias-Neto, Emmanuel; Souza, Sandro J; Simpson, Andrew J G; Monesi, Nadia; Ramos, Ricardo G P; Espreafico, Enilza M; Paçó-Larson, Maria L

    2007-07-24

    The sequencing of the D.melanogaster genome revealed an unexpected small number of genes (~ 14,000) indicating that mechanisms acting on generation of transcript diversity must have played a major role in the evolution of complex metazoans. Among the most extensively used mechanisms that accounts for this diversity is alternative splicing. It is estimated that over 40% of Drosophila protein-coding genes contain one or more alternative exons. A recent transcription map of the Drosophila embryogenesis indicates that 30% of the transcribed regions are unannotated, and that 1/3 of this is estimated as missed or alternative exons of previously characterized protein-coding genes. Therefore, the identification of the variety of expressed transcripts depends on experimental data for its final validation and is continuously being performed using different approaches. We applied the Open Reading Frame Expressed Sequence Tags (ORESTES) methodology, which is capable of generating cDNA data from the central portion of rare transcripts, in order to investigate the presence of hitherto unnanotated regions of Drosophila transcriptome. Bioinformatic analysis of 1,303 Drosophila ORESTES clusters identified 68 sequences derived from unannotated regions in the current Drosophila genome version (4.3). Of these, a set of 38 was analysed by polyA+ northern blot hybridization, validating 17 (50%) new exons of low abundance transcripts. For one of these ESTs, we obtained the cDNA encompassing the complete coding sequence of a new serine protease, named SP212. The SP212 gene is part of a serine protease gene cluster located in the chromosome region 88A12-B1. This cluster includes the predicted genes CG9631, CG9649 and CG31326, which were previously identified as up-regulated after immune challenges in genomic-scale microarray analysis. In agreement with the proposal that this locus is co-regulated in response to microorganisms infection, we show here that SP212 is also up-regulated upon

  5. Identification of unannotated exons of low abundance transcripts in Drosophila melanogaster and cloning of a new serine protease gene upregulated upon injury

    PubMed Central

    Maia, Rafaela M; Valente, Valeria; Cunha, Marco AV; Sousa, Josane F; Araujo, Daniela D; Silva, Wilson A; Zago, Marco A; Dias-Neto, Emmanuel; Souza, Sandro J; Simpson, Andrew JG; Monesi, Nadia; Ramos, Ricardo GP; Espreafico, Enilza M; Paçó-Larson, Maria L

    2007-01-01

    Background The sequencing of the D.melanogaster genome revealed an unexpected small number of genes (~ 14,000) indicating that mechanisms acting on generation of transcript diversity must have played a major role in the evolution of complex metazoans. Among the most extensively used mechanisms that accounts for this diversity is alternative splicing. It is estimated that over 40% of Drosophila protein-coding genes contain one or more alternative exons. A recent transcription map of the Drosophila embryogenesis indicates that 30% of the transcribed regions are unannotated, and that 1/3 of this is estimated as missed or alternative exons of previously characterized protein-coding genes. Therefore, the identification of the variety of expressed transcripts depends on experimental data for its final validation and is continuously being performed using different approaches. We applied the Open Reading Frame Expressed Sequence Tags (ORESTES) methodology, which is capable of generating cDNA data from the central portion of rare transcripts, in order to investigate the presence of hitherto unnanotated regions of Drosophila transcriptome. Results Bioinformatic analysis of 1,303 Drosophila ORESTES clusters identified 68 sequences derived from unannotated regions in the current Drosophila genome version (4.3). Of these, a set of 38 was analysed by polyA+ northern blot hybridization, validating 17 (50%) new exons of low abundance transcripts. For one of these ESTs, we obtained the cDNA encompassing the complete coding sequence of a new serine protease, named SP212. The SP212 gene is part of a serine protease gene cluster located in the chromosome region 88A12-B1. This cluster includes the predicted genes CG9631, CG9649 and CG31326, which were previously identified as up-regulated after immune challenges in genomic-scale microarray analysis. In agreement with the proposal that this locus is co-regulated in response to microorganisms infection, we show here that SP212 is also up

  6. Docking analysis of gallic acid derivatives as HIV-1 protease inhibitors.

    PubMed

    Singh, Anjali; Pal, Tapan Kumar

    2015-01-01

    HIV-1 Protease (HIV-1 PR) enzymes are essential for accurate assembly and maturation of infectious HIV retroviruses. The significant role of HIV-1 protease in viral replication has made it a potential drug target. In the recent past, phytochemical Gallic Acid (GA) derivatives have been screened for protease inhibitor activity. The present work aims to design and evaluate potential GA-based HIV-1 PR phytoinhibitors by docking approach. The ligands were prepared by ChemDraw and docking was performed in HEX software. In this present study, one of the GA analogues (GA4) emerged as a potent drug candidate for HIV-1 PR inhibition, and docking results showed it to be comparable with anti-HIV drugs, darunavir and amprenavir. The GA4 derivative provided a lead for designing more effective HIV-1 PR inhibitors.

  7. Identification of Broad-Based HIV-1 Protease Inhibitors From Combinatorial Libraries

    PubMed Central

    Chang, Max W.; Giffin, Michael J.; Muller, Rolf; Savage, Jeremiah; Lin, Ying C.; Hong, Sukwon; Jin, Wei; Whitby, Landon R.; Elder, John H.; Boger, Dale L.; Torbett, Bruce E.

    2011-01-01

    Clinically approved inhibitors of HIV-1 protease function via a competitive mechanism. A particular vulnerability of competitive inhibitors is their sensitivity to increases in substrate concentration, as may occur during virion assembly, budding and processing into a mature, infectious viral particle. Advances in chemical synthesis have led to the development of new chemical libraries with high diversity using rapid in-solution syntheses. These libraries have been previously shown to be effective at disrupting protein-protein and protein-nucleic acid interfaces. We have screened 44,000 compounds from such a library to identify inhibitors of HIV-1 protease. One compound was identified that inhibits wild type protease, as well as a drug-resistant protease with 6 mutations. Moreover, analysis of this compound suggests an allosteric, non-competitive mechanism of inhibition and may represent a starting point for an additional strategy for anti-retroviral therapy. PMID:20507280

  8. Splicing Variants of SERPINA1 Gene in Ovine Milk: Characterization of cDNA and Identification of Polymorphisms

    PubMed Central

    Marchitelli, Cinzia; Crisà, Alessandra; Mostarda, Elisa; Napolitano, Francesco; Moioli, Bianca

    2013-01-01

    The serine protease inhibitor, clade A, member 1 (SERPINA1) is the gene for a protein called alpha-1-antitrypsin (AAT), which is a member of the serine protease inhibitor (serpin) superfamily of proteins. By conformational change, serpins control several chemical reactions inhibiting the activity of proteases. AAT is the most abundant endogenous serpin in blood circulation and it is present in relatively high concentration in human milk as well as in bovine and porcine colostrum. Here we report for the first time the molecular characterization and sequence variability of the ovine SERPINA1 cDNA and gene. cDNAs from mammary gland and from milk were PCR amplified, and three different transcripts (1437, 1166 and 521bp) of the SERPINA1 gene were identified. We amplified and sequenced different regions of the gene (5’ UTR, from exon 2 to exon 5 and 3’ UTR), and we found that the exon-intron structure of the gene is similar to that of human and bovine. We detected a total of 97 SNPs in cDNAs and gene sequences from 10 sheep of three different breeds. In adult sheep tissues a SERPINA1 gene expression analysis indicated a differential expression of the three different transcripts. The finding reported in this paper will aid further studies on possible involvement of the SERPINA1 gene in different physiological states and its possible association with production traits. PMID:24009725

  9. Learning and memory deficits in mice lacking protease activated receptor-1

    PubMed Central

    Almonte, Antoine G.; Hamill, Cecily E.; Chhatwal, Jasmeer P.; Wingo, Thomas S.; Barber, Jeremy A.; Lyuboslavsky, Polina N.; Sweatt, J. David; Ressler, Kerry J.; White, David A.; Traynelis, Stephen F.

    2007-01-01

    The roles of serine proteases and protease activated receptors have been extensively studied in coagulation, wound healing, inflammation, and neurodegeneration. More recently, serine proteases have been suggested to influence synaptic plasticity. In this context, we examined the role of protease activated receptor 1 (PAR1), which is activated following proteolytic cleavage by thrombin and plasmin, in emotionally-motivated learning. We were particularly interested in PAR1 because its activation enhances the function of NMDA receptors, which are required for some forms of synaptic plasticity. We examined several baseline behavioral measures, including locomotor activity, expression of anxiety-like behavior, motor task acquisition, nociceptive responses, and startle responses in C57Bl/6 mice in which the PAR1 receptor has been genetically deleted. In addition, we evaluated learning and memory in these mice using two memory tasks, passive avoidance and cued fear-conditioning. Whereas locomotion, pain response, startle, and measures of baseline anxiety were largely unaffected by PAR1 removal, PAR1−/− animals showed significant deficits in a passive avoidance task and in cued fear conditioning. These data suggest that PAR1 may play an important role in emotionally-motivated learning. PMID:17544303

  10. Potential elucidation of a novel CTL epitope in HIV-1 protease by the protease inhibitor resistance mutation L90M.

    PubMed

    Smidt, Werner

    2013-01-01

    The combination of host immune responses and use of antiretrovirals facilitate partial control of human immunodeficiency virus type 1 (HIV-1) infection and result in delayed progression to Acquired Immunodeficiency Syndrome (AIDS). Both treatment and host immunity impose selection pressures on the highly mutable HIV-1 genome resulting in antiretroviral resistance and immune escape. Researchers have shown that antiretroviral resistance mutations can shape cytotoxic T-lymphocyte immunity by altering the epitope repertoire of HIV infected cells. Here it was discovered that an important antiretroviral resistance mutation, L90M in HIV protease, occurs at lower frequencies in hosts that harbor the B*15, B*48 or A*32 human leukocyte antigen subtypes. A likely reason is the elucidation of novel epitopes by L90M. NetMHCPan predictions reveal increased affinity of the peptide spanning the HIV protease region, PR 89-97 and PR 90-99 to HLA-B*15/B*48 and HLA-A*32 respectively due to the L90M substitution. The higher affinity could increase the chance of the epitope being presented and recognized by Cytotoxic T-lymphocytes and perhaps provide additional immunological pressures in the presence of antiretroviral attenuating mutations. This evidence supports the notion that knowledge of HLA allotypes in HIV infected individuals could augment antiretroviral treatment by the elucidation of epitopes due to antiretroviral resistance mutations in HIV protease.

  11. Molecular characterization of protease activity in Serratia sp. strain SCBI and its importance in cytotoxicity and virulence.

    PubMed

    Petersen, Lauren M; Tisa, Louis S

    2014-11-01

    A newly recognized Serratia species, termed South African Caenorhabditis briggsae isolate (SCBI), is both a mutualist of the nematode Caenorhabditis briggsae KT0001 and a pathogen of lepidopteran insects. Serratia sp. strain SCBI displays high proteolytic activity, and because secreted proteases are known virulence factors for many pathogens, the purpose of this study was to identify genes essential for extracellular protease activity in Serratia sp. strain SCBI and to determine what role proteases play in insect pathogenesis and cytotoxicity. A bank of 2,100 transposon mutants was generated, and six SCBI mutants with defective proteolytic activity were identified. These mutants were also defective in cytotoxicity. The mutants were found defective in genes encoding the following proteins: alkaline metalloprotease secretion protein AprE, a BglB family transcriptional antiterminator, an inosine/xanthosine triphosphatase, GidA, a methyl-accepting chemotaxis protein, and a PIN domain protein. Gene expression analysis on these six mutants showed significant downregulation in mRNA levels of several different types of predicted protease genes. In addition, transcriptome sequencing (RNA-seq) analysis provided insight into how inactivation of AprE, GidA, and a PIN domain protein influences motility and virulence, as well as protease activity. Using quantitative reverse transcription-PCR (qRT-PCR) to further characterize expression of predicted protease genes in wild-type Serratia sp. SCBI, the highest mRNA levels for the alkaline metalloprotease genes (termed prtA1 to prtA4) occurred following the death of an insect host, while two serine protease and two metalloprotease genes had their highest mRNA levels during active infection. Overall, these results indicate that proteolytic activity is essential for cytotoxicity in Serratia sp. SCBI and that its regulation appears to be highly complex. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  12. Molecular Characterization of Protease Activity in Serratia sp. Strain SCBI and Its Importance in Cytotoxicity and Virulence

    PubMed Central

    Petersen, Lauren M.

    2014-01-01

    A newly recognized Serratia species, termed South African Caenorhabditis briggsae isolate (SCBI), is both a mutualist of the nematode Caenorhabditis briggsae KT0001 and a pathogen of lepidopteran insects. Serratia sp. strain SCBI displays high proteolytic activity, and because secreted proteases are known virulence factors for many pathogens, the purpose of this study was to identify genes essential for extracellular protease activity in Serratia sp. strain SCBI and to determine what role proteases play in insect pathogenesis and cytotoxicity. A bank of 2,100 transposon mutants was generated, and six SCBI mutants with defective proteolytic activity were identified. These mutants were also defective in cytotoxicity. The mutants were found defective in genes encoding the following proteins: alkaline metalloprotease secretion protein AprE, a BglB family transcriptional antiterminator, an inosine/xanthosine triphosphatase, GidA, a methyl-accepting chemotaxis protein, and a PIN domain protein. Gene expression analysis on these six mutants showed significant downregulation in mRNA levels of several different types of predicted protease genes. In addition, transcriptome sequencing (RNA-seq) analysis provided insight into how inactivation of AprE, GidA, and a PIN domain protein influences motility and virulence, as well as protease activity. Using quantitative reverse transcription-PCR (qRT-PCR) to further characterize expression of predicted protease genes in wild-type Serratia sp. SCBI, the highest mRNA levels for the alkaline metalloprotease genes (termed prtA1 to prtA4) occurred following the death of an insect host, while two serine protease and two metalloprotease genes had their highest mRNA levels during active infection. Overall, these results indicate that proteolytic activity is essential for cytotoxicity in Serratia sp. SCBI and that its regulation appears to be highly complex. PMID:25182493

  13. Differential Response of Extracellular Proteases of Trichoderma Harzianum Against Fungal Phytopathogens.

    PubMed

    Sharma, Vivek; Salwan, Richa; Sharma, Prem N

    2016-09-01

    In the present study, production of extracellular proteases by Trichoderma harzianum was evaluated based on the relative gene expression and spectrophotometric assay. The fungal isolates were grown in Czapek Dox Broth medium supplemented with deactivated mycelium of plant fungal pathogens such as Fusarium oxysporum, Colletotrichum capsici, Gloeocercospora sorghi, and Colletotrichum truncatum. The maximum protease activity was detected after 48 h of incubation against Colletotrichum spp. Similarly in qRT-PCR, the relative gene expression of four proteases varied from 48 to 96 h against host pathogens in a time-independent manner. Among proteases, statistically significant upregulation of asp, asp, and srp was observed against Colletotrichum spp., followed by F. oxysporum. But in the case of pepM22, maximum upregulation was observed against F. oxysporum. The variation in enzyme assay and qRT-PCR of proteases at different time intervals against various fungal phytopathogens could be due to the limitation of using casein as a substrate for all types of proteases or protease-encoding transcripts selected for qRT-PCR, which may not be true representative of total protease activity.

  14. Evaluation of retro-inverso modifications of HTLV-1 protease inhibitors containing a hydroxyethylamine isoster.

    PubMed

    Tatsumi, Tadashi; Awahara, Chiyuki; Naka, Hiromi; Aimoto, Saburo; Konno, Hiroyuki; Nosaka, Kazuto; Akaji, Kenichi

    2010-04-01

    Effects of retro-inverso (RI) modifications of HTLV-1 protease inhibitors containing a hydroxyethylamine isoster backbone were clarified. Construction of the isoster backbone was achieved by a stereoselective aldol reaction. Four diastereomers with different configurations at the isoster hydroxyl site and the scissile site substituent were synthesized. Inhibitory activities of the new inhibitors suggest that partially modified RI inhibitors would interact with HTLV-1 protease in the same manner as the parent hydroxyethylamine inhibitor. Copyright 2010 Elsevier Ltd. All rights reserved.

  15. Shear stress reduces protease activated receptor-1 expression in human endothelial cells

    NASA Technical Reports Server (NTRS)

    Nguyen, K. T.; Eskin, S. G.; Patterson, C.; Runge, M. S.; McIntire, L. V.

    2001-01-01

    Shear stress has been shown to regulate several genes involved in the thrombotic and proliferative functions of endothelial cells. Thrombin receptor (protease-activated receptor-1: PAR-1) increases at sites of vascular injury, which suggests an important role for PAR-1 in vascular diseases. However, the effect of shear stress on PAR-1 expression has not been previously studied. This work investigates effects of shear stress on PAR-1 gene expression in both human umbilical vein endothelial cells (HUVECs) and microvascular endothelial cells (HMECs). Cells were exposed to different shear stresses using a parallel plate flow system. Northern blot and flow cytometry analysis showed that shear stress down-regulated PAR-1 messenger RNA (mRNA) and protein levels in both HUVECs and HMECs but with different thresholds. Furthermore, shear-reduced PAR-1 mRNA was due to a decrease of transcription rate, not increased mRNA degradation. Postshear stress release of endothelin-1 in response to thrombin was reduced in HUVECs and HMECs. Moreover, inhibitors of potential signaling pathways applied during shear stress indicated mediation of the shear-decreased PAR-1 expression by protein kinases. In conclusion, shear stress exposure reduces PAR-1 gene expression in HMECs and HUVECs through a mechanism dependent in part on protein kinases, leading to altered endothelial cell functional responses to thrombin.

  16. Molecular characterization of two serine proteases expressed in gut tissue of the African trypanosome vector, Glossina morsitans morsitans.

    PubMed

    Yan, J; Cheng, Q; Li, C B; Aksoy, S

    2001-02-01

    Serine proteases are major insect gut enzymes involved in digestion of dietary proteins, and in addition they have been implicated in the process of pathogen establishment in several vector insects. The medically important vector, tsetse fly (Diptera:Glossinidiae), is involved in the transmission of African trypanosomes, which cause devastating diseases in animals and humans. Both the male and female tsetse can transmit trypanosomes and both are strict bloodfeeders throughout all stages of their development. Here, we describe the characterization of two putative serine protease-encoding genes, Glossina serine protease-1 (Gsp1) and Glossina serine protease-2 (Gsp2) from gut tissue. Both putative cDNA products represent prepro peptides with hydrophobic signal peptide sequences associated with their 5'-end terminus. The Gsp1 cDNA encodes a putative mature protein of 245 amino acids with a molecular mass of 26 428 Da, while the predicted size of the 228 amino acid mature peptide encoded by Gsp2 cDNA is 24 573 Da. Both deduced peptides contain the Asp/His/Ser catalytic triad and the conserved residues surrounding it which are characteristic of serine proteases. In addition, both proteins have the six-conserved cysteine residues to form the three-cysteine bonds typically present in invertebrate serine proteases. Based on the presence of substrate specific residues, the Gsp1 gene encodes a chymotrypsin-like protease while Gsp2 gene encodes for a protein with trypsin-like activity. Both proteins are encoded by few loci in tsetse genome, being present in one or two copies only. The mRNA expression levels for the genes do not vary extensively throughout the digestive cycle, and high levels of mRNAs can be readily detected in the gut tissue of newly emerged flies. The levels of trypsin and chymotrypsin activities in the gut lumen increase following blood feeding and change significantly in the gut cells throughout the digestion cycle. Hence, the regulation of expression for

  17. Expression and characterization of Coprothermobacter proteolyticus alkaline serine protease

    USDA-ARS?s Scientific Manuscript database

    TECHNICAL ABSTRACT A putative protease gene (aprE) from the thermophilic bacterium Coprothermobacter proteolyticus was cloned and expressed in Bacillus subtilis. The enzyme was determined to be a serine protease based on inhibition by PMSF. Biochemical characterization demonstrated the enzyme had...

  18. Protease-deficient herpes simplex virus protects mice from lethal herpesvirus infection.

    PubMed Central

    Hippenmeyer, P J; Rankin, A M; Luckow, V A; Neises, G R

    1997-01-01

    Null mutants and attenuated mutants of herpes simplex virus (HSV) have been shown to induce immunity against challenge from wild-type virus. Null viruses with a defect in late gene products would be expected to express more viral genes than viruses with defects in essential early gene products and thus induce a better immune response. Herpesviruses encode a late gene product (serine protease) that is autocatalytic and cleaves the capsid assembly protein during viral replication. To determine whether a virus with a mutation in this gene could induce immunity, we constructed a recombinant virus containing the gusA reporter gene in the protease domain of the HSV type 1 UL26 open reading frame (ORF). Consistent with previous results (M. Gao, L. Matusick-Kumar, W. Hurlburt, S. F. DiTusa, W. W. Newcomb, J. C. Brown, P. J. McCann, I. Deckman, and R. J. Colonno, J. Virol. 68:3702-3712, 1994), recombinant virus could be isolated only from helper cell lines expressing the product of the UL26 ORF. Mice inoculated with the recombinant virus were unaffected by doses of virus that were lethal to mice infected with wild-type virus. Mice which were previously inoculated with the recombinant virus were also protected by a subsequent challenge with wild-type virus in a dose-dependent manner. These results indicate that recombinant viruses lacking the protease gene are avirulent but render protection from subsequent challenge. PMID:8995617

  19. The Pochonia chlamydosporia serine protease gene vcp1 is subject to regulation by carbon, nitrogen and pH: implications for nematode biocontrol.

    PubMed

    Ward, Elaine; Kerry, Brian R; Manzanilla-López, Rosa H; Mutua, Gerald; Devonshire, Jean; Kimenju, John; Hirsch, Penny R

    2012-01-01

    The alkaline serine protease VCP1 of the fungus Pochonia chlamydosporia belongs to a family of subtilisin-like enzymes that are involved in infection of nematode and insect hosts. It is involved early in the infection process, removing the outer proteinaceous vitelline membrane of nematode eggs. Little is known about the regulation of this gene, even though an understanding of how nutrients and other factors affect its expression is critical for ensuring its efficacy as a biocontrol agent. This paper provides new information on the regulation of vcp1 expression. Sequence analysis of the upstream regulatory region of this gene in 30 isolates revealed that it was highly conserved and contained sequence motifs characteristic of genes that are subject to carbon, nitrogen and pH-regulation. Expression studies, monitoring enzyme activity and mRNA, confirmed that these factors affect VCP1 production. As expected, glucose reduced VCP1 expression and for a few hours so did ammonium chloride. Surprisingly, however, by 24 h VCP1 levels were increased in the presence of ammonium chloride for most isolates. Ambient pH also regulated VCP1 expression, with most isolates producing more VCP1 under alkaline conditions. There were some differences in the response of one isolate with a distinctive upstream sequence including a variant regulatory-motif profile. Cryo-scanning electron microscopy studies indicated that the presence of nematode eggs stimulates VCP1 production by P. chlamydosporia, but only where the two are in close contact. Overall, the results indicate that readily-metabolisable carbon sources and unfavourable pH in the rhizosphere/egg-mass environment may compromise nematode parasitism by P. chlamydosporia. However, contrary to previous indications using other nematophagous and entomopathogenic fungi, ammonium nitrate (e.g. from fertilizers) may enhance biocontrol potential in some circumstances.

  20. The Degradome database: expanding roles of mammalian proteases in life and disease

    PubMed Central

    Pérez-Silva, José G.; Español, Yaiza; Velasco, Gloria; Quesada, Víctor

    2016-01-01

    Since the definition of the degradome as the complete repertoire of proteases in a given organism, the combined effort of numerous laboratories has greatly expanded our knowledge of its roles in biology and pathology. Once the genomic sequences of several important model organisms were made available, we presented the Degradome database containing the curated sets of known protease genes in human, chimpanzee, mouse and rat. Here, we describe the updated Degradome database, featuring 81 new protease genes and 7 new protease families. Notably, in this short time span, the number of known hereditary diseases caused by mutations in protease genes has increased from 77 to 119. This increase reflects the growing interest on the roles of the degradome in multiple diseases, including cancer and ageing. Finally, we have leveraged the widespread adoption of new webtools to provide interactive graphic views that show information about proteases in the global context of the degradome. The Degradome database can be accessed through its web interface at http://degradome.uniovi.es. PMID:26553809

  1. Immunoglobulins in Nasal Secretions of Healthy Humans: Structural Integrity of Secretory Immunoglobulin A1 (IgA1) and Occurrence of Neutralizing Antibodies to IgA1 Proteases of Nasal Bacteria

    PubMed Central

    Kirkeby, Line; Rasmussen, Trine Tang; Reinholdt, Jesper; Kilian, Mogens

    2000-01-01

    Certain bacteria, including overt pathogens as well as commensals, produce immunoglobulin A1 (IgA1) proteases. By cleaving IgA1, including secretory IgA1, in the hinge region, these enzymes may interfere with the barrier functions of mucosal IgA antibodies, as indicated by experiments in vitro. Previous studies have suggested that cleavage of IgA1 in nasal secretions may be associated with the development and perpetuation of atopic disease. To clarify the potential effect of IgA1 protease-producing bacteria in the nasal cavity, we have analyzed immunoglobulin isotypes in nasal secretions of 11 healthy humans, with a focus on IgA, and at the same time have characterized and quantified IgA1 protease-producing bacteria in the nasal flora of the subjects. Samples in the form of nasal wash were collected by using a washing liquid that contained lithium as an internal reference. Dilution factors and, subsequently, concentrations in undiluted secretions could thereby be calculated. IgA, mainly in the secretory form, was found by enzyme-linked immunosorbent assay to be the dominant isotype in all subjects, and the vast majority of IgA (median, 91%) was of the A1 subclass, corroborating results of previous analyses at the level of immunoglobulin-producing cells. Levels of serum-type immunoglobulins were low, except for four subjects in whom levels of IgG corresponded to 20 to 66% of total IgA. Cumulative levels of IgA, IgG, and IgM in undiluted secretions ranged from 260 to 2,494 (median, 777) μg ml−1. IgA1 protease-producing bacteria (Haemophilus influenzae, Streptococcus pneumoniae, or Streptococcus mitis biovar 1) were isolated from the nasal cavities of seven subjects at 2.1 × 103 to 7.2 × 106 CFU per ml of undiluted secretion, corresponding to 0.2 to 99.6% of the flora. Nevertheless, α-chain fragments characteristic of IgA1 protease activity were not detected in secretions from any subject by immunoblotting. Neutralizing antibodies to IgA1 proteases of autologous

  2. Effectiveness of commercial inhibitors against subtype F HIV-1 protease.

    PubMed

    Krauchenco, Sandra; Martins, Nadia H; Sanches, Mario; Polikarpov, Igor

    2009-06-01

    Subtype F wild type HIV protease has been kinetically characterized using six commercial inhibitors (amprenavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir) commonly used for HIV/AIDS treatment, as well as inhibitor TL-3 and acetyl-pepstatin. We also obtained kinetic parameters for two multi-resistant proteases (one of subtype B and one of subtype F) harboring primary and secondary mutations selected by intensive treatment with ritonavir/nelfinavir. This newly obtained biochemical data shows that all six studied commercially available protease inhibitors are significantly less effective against subtype F HIV proteases than against HIV proteases of subtype B, as judged by increased K(i) and biochemical fitness (vitality) values. Comparison with previously reported kinetic values for subtype A and C HIV proteases show that subtype F wild type proteases are significantly less susceptible to inhibition. These results demonstrate that the accumulation of natural polymorphisms in subtype F proteases yields catalytically more active enzymes with a large degree of cross-resistance, which thus results in strong virus viability.

  3. The Three Streptomyces lividans HtrA-Like Proteases Involved in the Secretion Stress Response Act in a Cooperative Manner

    PubMed Central

    Vicente, Rebeca L.; Gullón, Sonia; Marín, Silvia; Mellado, Rafael P.

    2016-01-01

    Overproduction of Sec-proteins in S. lividans accumulates misfolded proteins outside of the cytoplasmic membrane where the accumulated proteins interfere with the correct functioning of the secretion machinery and with the correct cell functionality, triggering the expression in S. lividans of a CssRS two-component system which regulates the degradation of the accumulated protein, the so-called secretion stress response. Optimization of secretory protein production via the Sec route requires the identification and characterisation of quality factors involved in this process. The phosphorylated regulator (CssR) interacts with the regulatory regions of three genes encoding three different HtrA-like proteases. Individual mutations in each of these genes render degradation of the misfolded protein inoperative, and propagation in high copy number of any of the three proteases encoding genes results on indiscriminate alpha-amylase degradation. None of the proteases could complement the other two deficiencies and only propagation of each single copy protease gene can restore its own deficiency. The obtained results strongly suggest that the synthesis of the three HtrA-like proteases needs to be properly balanced to ensure the effective degradation of misfolded overproduced secretory proteins and, at the same time, avoid negative effects in the secreted proteins and the secretion machinery. This is particularly relevant when considering the optimisation of Streptomyces strains for the overproduction of homologous or heterologous secretory proteins of industrial application. PMID:27977736

  4. Multiple Classes of Immune-Related Proteases Associated with the Cell Death Response in Pepper Plants

    PubMed Central

    Bae, Chungyun; Kim, Su-min; Lee, Dong Ju; Choi, Doil

    2013-01-01

    Proteases regulate a large number of biological processes in plants, such as metabolism, physiology, growth, and defense. In this study, we carried out virus-induced gene silencing assays with pepper cDNA clones to elucidate the biological roles of protease superfamilies. A total of 153 representative protease genes from pepper cDNA were selected and cloned into a Tobacco rattle virus-ligation independent cloning vector in a loss-of-function study. Silencing of 61 proteases resulted in altered phenotypes, such as the inhibition of shoot growth, abnormal leaf shape, leaf color change, and lethality. Furthermore, the silencing experiments revealed that multiple proteases play a role in cell death and immune response against avirulent and virulent pathogens. Among these 153 proteases, 34 modulated the hypersensitive cell death response caused by infection with an avirulent pathogen, and 16 proteases affected disease symptom development caused by a virulent pathogen. Specifically, we provide experimental evidence for the roles of multiple protease genes in plant development and immune defense following pathogen infection. With these results, we created a broad sketch of each protease function. This information will provide basic information for further understanding the roles of the protease superfamily in plant growth, development, and defense. PMID:23696830

  5. Molecular and Biochemical Characterization of a Cathepsin B-Like Protease Family Unique to Trypanosoma congolense▿ †

    PubMed Central

    Mendoza-Palomares, Carlos; Biteau, Nicolas; Giroud, Christiane; Coustou, Virginie; Coetzer, Theresa; Authié, Edith; Boulangé, Alain; Baltz, Théo

    2008-01-01

    Cysteine proteases have been shown to be essential virulence factors and drug targets in trypanosomatids and an attractive antidisease vaccine candidate for Trypanosoma congolense. Here, we describe an important amplification of genes encoding cathepsin B-like proteases unique to T. congolense. More than 13 different genes were identified, whereas only one or two highly homologous genes have been identified in other trypanosomatids. These proteases grouped into three evolutionary clusters: TcoCBc1 to TcoCBc5 and TcoCBc6, which possess the classical catalytic triad (Cys, His, and Asn), and TcoCBs7 to TcoCBs13, which contains an unusual catalytic site (Ser, Xaa, and Asn). Expression profiles showed that members of the TcoCBc1 to TcoCBc5 and the TcoCBs7 to TcoCBs13 groups are expressed mainly in bloodstream forms and localize in the lysosomal compartment. The expression of recombinant representatives of each group (TcoCB1, TcoCB6, and TcoCB12) as proenzymes showed that TcoCBc1 and TcoCBc6 are able to autocatalyze their maturation 21 and 31 residues, respectively, upstream of the predicted start of the catalytic domain. Both displayed a carboxydipeptidase function, while only TcoCBc1 behaved as an endopeptidase. TcoCBc1 exhibited biochemical differences regarding inhibitor sensitivity compared to that of other cathepsin B-like proteases. Recombinant pro-TcoCBs12 did not automature in vitro, and the pepsin-matured enzyme was inactive in tests with cathepsin B fluorogenic substrates. In vivo inhibition studies using CA074Me (a cell-permeable cathepsin B-specific inhibitor) demonstrated that TcoCB are involved in lysosomal protein degradation essential for survival in bloodstream form. Furthermore, TcoCBc1 elicited an important immune response in experimentally infected cattle. We propose this family of proteins as a potential therapeutic target and as a plausible antigen for T. congolense diagnosis. PMID:18281598

  6. Evolution of a mass spectrometry-grade protease with PTM-directed specificity.

    PubMed

    Tran, Duc T; Cavett, Valerie J; Dang, Vuong Q; Torres, Héctor L; Paegel, Brian M

    2016-12-20

    Mapping posttranslational modifications (PTMs), which diversely modulate biological functions, represents a significant analytical challenge. The centerpiece technology for PTM site identification, mass spectrometry (MS), requires proteolytic cleavage in the vicinity of a PTM to yield peptides for sequencing. This requirement catalyzed our efforts to evolve MS-grade mutant PTM-directed proteases. Citrulline, a PTM implicated in epigenetic and immunological function, made an ideal first target, because citrullination eliminates arginyl tryptic sites. Bead-displayed trypsin mutant genes were translated in droplets, the mutant proteases were challenged to cleave bead-bound fluorogenic probes of citrulline-dependent proteolysis, and the resultant beads (1.3 million) were screened. The most promising mutant efficiently catalyzed citrulline-dependent peptide bond cleavage (k cat /K M = 6.9 × 10 5 M -1 ⋅s -1 ). The resulting C-terminally citrullinated peptides generated characteristic isotopic patterns in MALDI-TOF MS, and both a fragmentation product y 1 ion corresponding to citrulline (176.1030 m/z) and diagnostic peak pairs in the extracted ion chromatograms of LC-MS/MS analysis. Using these signatures, we identified citrullination sites in protein arginine deiminase 4 (12 sites) and in fibrinogen (25 sites, two previously unknown). The unique mass spectral features of PTM-dependent proteolytic digest products promise a generalized PTM site-mapping strategy based on a toolbox of such mutant proteases, which are now accessible by laboratory evolution.

  7. Kinetic characterization of the critical step in HIV-1 protease maturation.

    PubMed

    Sadiq, S Kashif; Noé, Frank; De Fabritiis, Gianni

    2012-12-11

    HIV maturation requires multiple cleavage of long polyprotein chains into functional proteins that include the viral protease itself. Initial cleavage by the protease dimer occurs from within these precursors, and yet only a single protease monomer is embedded in each polyprotein chain. Self-activation has been proposed to start from a partially dimerized protease formed from monomers of different chains binding its own N termini by self-association to the active site, but a complete structural understanding of this critical step in HIV maturation is missing. Here, we captured the critical self-association of immature HIV-1 protease to its extended amino-terminal recognition motif using large-scale molecular dynamics simulations, thus confirming the postulated intramolecular mechanism in atomic detail. We show that self-association to a catalytically viable state requires structural cooperativity of the flexible β-hairpin "flap" regions of the enzyme and that the major transition pathway is first via self-association in the semiopen/open enzyme states, followed by enzyme conformational transition into a catalytically viable closed state. Furthermore, partial N-terminal threading can play a role in self-association, whereas wide opening of the flaps in concert with self-association is not observed. We estimate the association rate constant (k(on)) to be on the order of ∼1 × 10(4) s(-1), suggesting that N-terminal self-association is not the rate-limiting step in the process. The shown mechanism also provides an interesting example of molecular conformational transitions along the association pathway.

  8. HvPap-1 C1A Protease and HvCPI-2 Cystatin Contribute to Barley Grain Filling and Germination1

    PubMed Central

    Velasco-Arroyo, Blanca; Cambra, Ines; Gonzalez-Melendi, Pablo; Lopez-Gonzalvez, Angeles; Garcia, Antonia

    2016-01-01

    Proteolysis is an essential process throughout the mobilization of storage proteins in barley (Hordeum vulgare) grains during germination. It involves numerous types of enzymes, with C1A Cys proteases the most abundant key players. Manipulation of the proteolytic machinery is a potential way to enhance grain yield and quality, and it could influence the mobilization of storage compounds along germination. Transgenic barley plants silencing or over-expressing the cathepsin F-like HvPap-1 Cys protease show differential accumulation of storage molecules such as starch, proteins, and free amino acids in the grain. It is particularly striking that the HvPap-1 artificial microRNA lines phenotype show a drastic delay in the grain germination process. Alterations to the proteolytic activities in the over-expressing and knock-down grains associated with changes in the level of expression of several C1A peptidases were also detected. Similarly, down-regulating cystatin Icy-2, one of the proteinaceous inhibitors of the cathepsin F-like protease, also has important effects on grain filling. However, the ultimate physiological influence of manipulating a peptidase or an inhibitor cannot be always predicted, since the plant tries to compensate the modified proteolytic effects by modulating the expression of some other peptidases or their inhibitors. PMID:26912343

  9. A study of proteases and protease-inhibitor complexes in biological fluids

    PubMed Central

    Granelli-Piperno, A; Reich, E

    1978-01-01

    We have (a) screened a variety of cell lines and body fluids for plasminogen activators and (b) studied the activity of proteases bound to α2- macroglobulin after exposing the complexes to partial degradation and/or denaturing procedures to unmask proteolytic activity. The respective results show (a) that the plasminogen activators in urine and cell culture media are generally of lower molecular weight than those in plasma; and (b) that proteases bound to α2-macroglobulin recover the ability to attack macromolecular substrates after exposure to sodium dodecyl sulfate while retaining the electrophoretic mobility of the protease inhibitor complex. This indicates that the protease and inhibitor are probably linked by covalent bonds. In contrast, other complexes formed between proteases and inhibitors of lower molecular weight (such as soybean or Kunitz inhibitors) are fully dissociated by sodium dodecyl sulfate (SDS). The experiments described were based on a new procedure for detecting proteolytic enzyme activity in SDS-polyacrylamide gels. The method relies on solutions of nonionic detergents for extracting SDS, after which the electrophoretic gel is applied to an indicator gel consisting of a fibrin- agar mixture. The method is sensitive, permitting the detection of proteinases in less than 1 μl of fresh plasma, and it is effective for resolving small differences in molecular weight. The procedure can be quantitated and, with minor modifications appropriate to each particular system, it has been applied to a broad spectrum of serine enzymes and proenzymes, including some that function in the pathways of fibrinolysis, coagulation and kinin-generation. Other potential applications appear likely. PMID:78958

  10. Altered Substrate Specificity of Drug-Resistant Human Immunodeficiency Virus Type 1 Protease

    PubMed Central

    Dauber, Deborah S.; Ziermann, Rainer; Parkin, Neil; Maly, Dustin J.; Mahrus, Sami; Harris, Jennifer L.; Ellman, Jon A.; Petropoulos, Christos; Craik, Charles S.

    2002-01-01

    Resistance to human immunodeficiency virus type 1 protease (HIV PR) inhibitors results primarily from the selection of multiple mutations in the protease region. Because many of these mutations are selected for the ability to decrease inhibitor binding in the active site, they also affect substrate binding and potentially substrate specificity. This work investigates the substrate specificity of a panel of clinically derived protease inhibitor-resistant HIV PR variants. To compare protease specificity, we have used positional-scanning, synthetic combinatorial peptide libraries as well as a select number of individual substrates. The subsite preferences of wild-type HIV PR determined by using the substrate libraries are consistent with prior reports, validating the use of these libraries to compare specificity among a panel of HIV PR variants. Five out of seven protease variants demonstrated subtle differences in specificity that may have significant impacts on their abilities to function in viral maturation. Of these, four variants demonstrated up to fourfold changes in the preference for valine relative to alanine at position P2 when tested on individual peptide substrates. This change correlated with a common mutation in the viral NC/p1 cleavage site. These mutations may represent a mechanism by which severely compromised, drug-resistant viral strains can increase fitness levels. Understanding the altered substrate specificity of drug-resistant HIV PR should be valuable in the design of future generations of protease inhibitors as well as in elucidating the molecular basis of regulation of proteolysis in HIV. PMID:11773410

  11. Two Membrane-Anchored Aspartic Proteases Contribute to Pollen and Ovule Development1[OPEN

    PubMed Central

    Gao, Hui; Zhang, Yinghui; Wang, Wanlei; Zhao, Keke; Liu, Chunmei; Bai, Lin; Li, Rui

    2017-01-01

    Aspartic proteases are a class of proteolytic enzymes with conserved aspartate residues, which are implicated in protein processing, maturation, and degradation. Compared with yeast and animals, plants possess a larger aspartic protease family. However, little is known about most of these enzymes. Here, we characterized two Arabidopsis (Arabidopsis thaliana) putative glycosylphosphatidylinositol (GPI)-anchored aspartic protease genes, A36 and A39, which are highly expressed in pollen and pollen tubes. a36 and a36 a39 mutants display significantly reduced pollen activity. Transmission electron microscopy and terminal-deoxynucleotidyl transferase-mediated nick end labeling assays further revealed that the unviable pollen in a36 a39 may undergo unanticipated apoptosis-like programmed cell death. The degeneration of female gametes also occurred in a36 a39. Aniline Blue staining, scanning electron microscopy, and semi in vitro guidance assays indicated that the micropylar guidance of pollen tubes is significantly compromised in a36 a39. A36 and A39 that were fused with green fluorescent protein are localized to the plasma membrane and display punctate cytosolic localization and colocalize with the GPI-anchored protein COBRA-LIKE10. Furthermore, in a36 a39, the abundance of highly methylesterified homogalacturonans and xyloglucans was increased significantly in the apical pollen tube wall. These results indicate that A36 and A39, two putative GPI-anchored aspartic proteases, play important roles in plant reproduction in Arabidopsis. PMID:27872247

  12. Transcriptional and proteomic analysis of the Aspergillus fumigatus ΔprtT protease-deficient mutant.

    PubMed

    Hagag, Shelly; Kubitschek-Barreira, Paula; Neves, Gabriela W P; Amar, David; Nierman, William; Shalit, Itamar; Shamir, Ron; Lopes-Bezerra, Leila; Osherov, Nir

    2012-01-01

    Aspergillus fumigatus is the most common opportunistic mold pathogen of humans, infecting immunocompromised patients. The fungus invades the lungs and other organs, causing severe damage. Penetration of the pulmonary epithelium is a key step in the infectious process. A. fumigatus produces extracellular proteases to degrade the host structural barriers. The A. fumigatus transcription factor PrtT controls the expression of multiple secreted proteases. PrtT shows similarity to the fungal Gal4-type Zn(2)-Cys(6) DNA-binding domain of several transcription factors. In this work, we further investigate the function of this transcription factor by performing a transcriptional and a proteomic analysis of the ΔprtT mutant. Unexpectedly, microarray analysis revealed that in addition to the expected decrease in protease expression, expression of genes involved in iron uptake and ergosterol synthesis was dramatically decreased in the ΔprtT mutant. A second finding of interest is that deletion of prtT resulted in the upregulation of four secondary metabolite clusters, including genes for the biosynthesis of toxic pseurotin A. Proteomic analysis identified reduced levels of three secreted proteases (ALP1 protease, TppA, AFUA_2G01250) and increased levels of three secreted polysaccharide-degrading enzymes in the ΔprtT mutant possibly in response to its inability to derive sufficient nourishment from protein breakdown. This report highlights the complexity of gene regulation by PrtT, and suggests a potential novel link between the regulation of protease secretion and the control of iron uptake, ergosterol biosynthesis and secondary metabolite production in A. fumigatus.

  13. Functional Proteomic Profiling of Secreted Serine Proteases in Health and Inflammatory Bowel Disease.

    PubMed

    Denadai-Souza, Alexandre; Bonnart, Chrystelle; Tapias, Núria Solà; Marcellin, Marlène; Gilmore, Brendan; Alric, Laurent; Bonnet, Delphine; Burlet-Schiltz, Odile; Hollenberg, Morley D; Vergnolle, Nathalie; Deraison, Céline

    2018-05-18

    While proteases are essential in gastrointestinal physiology, accumulating evidence indicates that dysregulated proteolysis plays a pivotal role in the pathophysiology of inflammatory bowel disease (IBD). Nonetheless, the identity of overactive proteases released by human colonic mucosa remains largely unknown. Studies of protease abundance have primarily investigated expression profiles, not taking into account their enzymatic activity. Herein we have used serine protease-targeted activity-based probes (ABPs) coupled with mass spectral analysis to identify active forms of proteases secreted by the colonic mucosa of healthy controls and IBD patients. Profiling of (Pro-Lys)-ABP bound proteases revealed that most of hyperactive proteases from IBD secretome are clustered at 28-kDa. We identified seven active proteases: the serine proteases cathepsin G, plasma kallikrein, plasmin, tryptase, chymotrypsin-like elastase 3 A, and thrombin and the aminopeptidase B. Only cathepsin G and thrombin were overactive in supernatants from IBD patient tissues compared to healthy controls. Gene expression analysis highlighted the transcription of genes encoding these proteases into intestinal mucosae. The functional ABP-targeted proteomic approach that we have used to identify active proteases in human colonic samples bears directly on the understanding of the role these enzymes may play in the pathophysiology of IBD.

  14. Kunitz-type protease inhibitors group B from Solanum palustre.

    PubMed

    Speransky, Anna S; Cimaglia, Fabio; Krinitsina, Anastasya A; Poltronieri, Palmiro; Fasano, Pasqua; Bogacheva, Anna M; Valueva, Tatiana A; Halterman, Dennis; Shevelev, Alexei B; Santino, Angelo

    2007-11-01

    Five Kunitz protease inhibitor group B genes were isolated from the genome of the diploid non-tuber-forming potato species Solanum palustre. Three of five new genes share 99% identity to the published KPI-B genes from various cultivated potato accessions, while others exhibit 96% identity. Spls-KPI-B2 and Spls-KPI-B4 proteins contain unique substitutions of the most conserved residues usually involved to trypsin and chymotrypsin-specific binding sites of Kunitz-type protease inhibitor (KPI)-B, respectively. To test the inhibition of trypsin and chymotrypsin by Spls-KPI proteins, five of them were produced in E. coli purified using a Ni-sepharose resin and ion-exchange chromatography. All recombinant Spls-KPI-B inhibited trypsin; K(i) values ranged from 84.8 (Spls-KPI-B4), 345.5 (Spls-KPI-B1), and 1310.6 nM (Spls-KPI-B2) to 3883.5 (Spls-KPI-B5) and 8370 nM (Spls-KPI-B3). In addition, Spls-KPI-B1 and Spls-KPI-B4 inhibited chymotrypsin. These data suggest that regardless of substitutions of key active-center residues both Spls-KPI-B4 and Spls-KPI-B1 are functional trypsin-chymotrypsin inhibitors.

  15. Effects of different dietary conditions on the expression of trypsin- and chymotrypsin-like protease genes in the digestive system of the migratory locust, Locusta migratoria.

    PubMed

    Spit, Jornt; Zels, Sven; Dillen, Senne; Holtof, Michiel; Wynant, Niels; Vanden Broeck, Jozef

    2014-05-01

    While technological advancements have recently led to a steep increase in genomic and transcriptomic data, and large numbers of protease sequences are being discovered in diverse insect species, little information is available about the expression of digestive enzymes in Orthoptera. Here we describe the identification of Locusta migratoria serine protease transcripts (cDNAs) involved in digestion, which might serve as possible targets for pest control management. A total of 5 putative trypsin and 15 putative chymotrypsin gene sequences were characterized. Phylogenetic analysis revealed that these are distributed among 3 evolutionary conserved clusters. In addition, we have determined the relative gene expression levels of representative members in the gut under different feeding conditions. This study demonstrated that the transcript levels for all measured serine proteases were strongly reduced after starvation. On the other hand, larvae of L. migratoria displayed compensatory effects to the presence of Soybean Bowman Birk (SBBI) and Soybean Trypsin (SBTI) inhibitors in their diet by differential upregulation of multiple proteases. A rapid initial upregulation was observed for all tested serine protease transcripts, while only for members belonging to class I, the transcript levels remained elevated after prolonged exposure. In full agreement with these results, we also observed an increase in proteolytic activity in midgut secretions of locusts that were accustomed to the presence of protease inhibitors in their diet, while no change in sensitivity to these inhibitors was observed. Taken together, this paper is the first comprehensive study on dietary dependent transcript levels of proteolytic enzymes in Orthoptera. Our data suggest that compensatory response mechanisms to protease inhibitor ingestion may have appeared early in insect evolution. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. A New Method for the Characterization of Strain-Specific Conformational Stability of Protease-Sensitive and Protease-Resistant PrPSc

    PubMed Central

    Pirisinu, Laura; Di Bari, Michele; Marcon, Stefano; Vaccari, Gabriele; D'Agostino, Claudia; Fazzi, Paola; Esposito, Elena; Galeno, Roberta; Langeveld, Jan; Agrimi, Umberto; Nonno, Romolo

    2010-01-01

    Although proteinacious in nature, prions exist as strains with specific self-perpetuating biological properties. Prion strains are thought to be associated with different conformers of PrPSc, a disease-associated isoform of the host-encoded cellular protein (PrPC). Molecular strain typing approaches have been developed which rely on the characterization of protease-resistant PrPSc. However, PrPSc is composed not only of protease-resistant but also of protease-sensitive isoforms. The aim of this work was to develop a protocol for the molecular characterization of both, protease-resistant and protease-sensitive PrPSc aggregates. We first set up experimental conditions which allowed the most advantageous separation of PrPC and PrPSc by means of differential centrifugation. The conformational solubility and stability assay (CSSA) was then developed by measuring PrPSc solubility as a function of increased exposure to GdnHCl. Brain homogenates from voles infected with human and sheep prion isolates were analysed by CSSA and showed strain-specific conformational stabilities, with mean [GdnHCl]1/2 values ranging from 1.6 M for MM2 sCJD to 2.1 for scrapie and to 2.8 M for MM1/MV1 sCJD and E200K gCJD. Interestingly, the rank order of [GdnHCl]1/2 values observed in the human and sheep isolates used as inocula closely matched those found following transmission in voles, being MM1 sCJD the most resistant (3.3 M), followed by sheep scrapie (2.2 M) and by MM2 sCJD (1.6 M). In order to test the ability of CSSA to characterise protease-sensitive PrPSc, we analysed sheep isolates of Nor98 and compared them to classical scrapie isolates. In Nor98, insoluble PrPSc aggregates were mainly protease-sensitive and showed a conformational stability much lower than in classical scrapie. Our results show that CSSA is able to reveal strain-specified PrPSc conformational stabilities of protease-resistant and protease-sensitive PrPSc and that it is a valuable tool for strain typing in natural

  17. Structural insights into the mechanisms of drug resistance in HIV-1 protease NL4-3.

    PubMed

    Heaslet, Holly; Kutilek, Victoria; Morris, Garrett M; Lin, Ying-Chuan; Elder, John H; Torbett, Bruce E; Stout, C David

    2006-03-03

    The development of resistance to anti-retroviral drugs targeted against HIV is an increasing clinical problem in the treatment of HIV-1-infected individuals. Many patients develop drug-resistant strains of the virus after treatment with inhibitor cocktails (HAART therapy), which include multiple protease inhibitors. Therefore, it is imperative that we understand the mechanisms by which the viral proteins, in particular HIV-1 protease, develop resistance. We have determined the three-dimensional structure of HIV-1 protease NL4-3 in complex with the potent protease inhibitor TL-3 at 2.0 A resolution. We have also obtained the crystal structures of three mutant forms of NL4-3 protease containing one (V82A), three (V82A, M46I, F53L) and six (V82A, M46I, F53L, V77I, L24I, L63P) point mutations in complex with TL-3. The three protease mutants arose sequentially under ex vivo selective pressure in the presence of TL-3, and exhibit fourfold, 11-fold, and 30-fold resistance to TL-3, respectively. This series of protease crystal structures offers insights into the biochemical and structural mechanisms by which the enzyme can overcome inhibition by TL-3 while recovering some of its native catalytic activity.

  18. Expression of Bacillus protease (Protease BYA) from Bacillus sp. Y in Bacillus subtilis and enhancement of its specific activity by site-directed mutagenesis-improvement in productivity of detergent enzyme-.

    PubMed

    Tobe, Seiichi; Shimogaki, Hisao; Ohdera, Motoyasu; Asai, Yoshio; Oba, Kenkichi; Iwama, Masanori; Irie, Masachika

    2006-01-01

    An attempt was made to express protease BYA produced by an alkalophilic Bacillus sp. Y in Bacillus subtilis by gene engineering methods. The gene encoding protease BYA was cloned from Bacillus sp. Y, and expression vector pTA71 was constructed from the amylase promoter of Bacillus licheniformis, DNA fragments encoding the open reading frame of protease BYA, and pUB110. Protease BYA was secreted at an activity level of 5100 APU/ml in the common industrial culture medium of Bacillus subtilis transformed with pTA71. We then attempted to increase the specific activity of protease BYA by site-directed mutagenesis. Amino acid residue Ala29 next to catalytic Asp30 was replaced by one of three uncharged amino acid residues (Val29, Leu29, Ile29), and each mutant enzyme was expressed and isolated from the culture medium. Val29 mutant enzyme was secreted at an activity level of greater than 7000 APU/ml in culture medium, and its specific activity was 1.5-fold higher than that of the wild-type enzyme. Other mutant enzymes had specific activity similar to that of the original one and were less stabile than the wild-type enzyme. It can be thought that the substitution at amino acid residue 29 affects the level of activity and stability of protease BYA.

  19. Occurrence and Evolution of the Paralogous Zinc Metalloproteases IgA1 Protease, ZmpB, ZmpC, and ZmpD in Streptococcus pneumoniae and Related Commensal Species

    PubMed Central

    Bek-Thomsen, Malene; Poulsen, Knud; Kilian, Mogens

    2012-01-01

    ABSTRACT The distribution, genome location, and evolution of the four paralogous zinc metalloproteases, IgA1 protease, ZmpB, ZmpC, and ZmpD, in Streptococcus pneumoniae and related commensal species were studied by in silico analysis of whole genomes and by activity screening of 154 representatives of 20 species. ZmpB was ubiquitous in the Mitis and Salivarius groups of the genus Streptococcus and in the genera Gemella and Granulicatella, with the exception of a fragmented gene in Streptococcus thermophilus, the only species with a nonhuman habitat. IgA1 protease activity was observed in all members of S. pneumoniae, S. pseudopneumoniae, S. oralis, S. sanguinis, and Gemella haemolysans, was variably present in S. mitis and S. infantis, and absent in S. gordonii, S. parasanguinis, S. cristatus, S. oligofermentans, S. australis, S. peroris, and S. suis. Phylogenetic analysis of 297 zmp sequences and representative housekeeping genes provided evidence for an unprecedented selection for genetic diversification of the iga, zmpB, and zmpD genes in S. pneumoniae and evidence of very frequent intraspecies transfer of entire genes and combination of genes. Presumably due to their adaptation to a commensal lifestyle, largely unaffected by adaptive mucosal immune factors, the corresponding genes in commensal streptococci have remained conserved. The widespread distribution and significant sequence diversity indicate an ancient origin of the zinc metalloproteases predating the emergence of the humanoid species. zmpB, which appears to be the ancestral gene, subsequently duplicated and successfully diversified into distinct functions, is likely to serve an important but yet unknown housekeeping function associated with the human host. PMID:23033471

  20. Purification and characterization of an alkaline protease Prot 1 from Botrytis cinerea : biodetergent catalyst assay.

    PubMed

    Abidi, Ferid; Limam, Ferid; Marzouki, M Nejib

    2007-01-01

    Alkaline thiol protease named Prot 1 was isolated from a culture filtrate of Botrytis cinerea. The enzyme was purified by ammonium sulfate fractionation, gel filtration, and ion-exchange chromatography. Thus, the enzyme was purified to homogeneity with specific activity of 30-fold higher than that of the crude broth. The purified alkaline protease has an apparent molecular mass of 43 kDa under denaturing conditions as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native molecular mass (45 kDa), determined by gel filtration, indicated that the alkaline protease has a monomeric form. The purified protease was biochemically characterized. The enzyme is active at alkaline pH and has a suitable and high thermostability. The optimal pH and temperature for activity were 9.0-10.0 and 60 degrees C, respectively. This protease was stable between pH 5.0 and 12.0. The enzyme retained 85% of its activity by treatment at 50 degrees C over 120 min; it maintained 50% of activity after 60 min of heating at 60 degrees C. Furthermore, the protease retained almost complete activity after 4 wk storage at 25 degrees C. The activity was significantly affected by thiol protease inhibitors, suggesting that the enzyme belongs to the alkaline thiol protease family. With the aim on industrial applications, we focused on studying the stability of the protease in several conditions. Prot 1 activity was not affected by ionic strength and different detergent additives, and, thus, the protease shows remarkable properties as a biodetergent catalyst.

  1. Identification of a mutant locus that bypasses the BsgA protease requirement for social development in Myxococcus xanthus.

    PubMed

    Cusick, John K; Hager, Elizabeth; Gill, Ronald E

    2015-01-01

    The BsgA protease is required for the earliest morphological changes observed in Myxococcus xanthus development. We hypothesize that the BsgA protease is required to cleave an inhibitor of the developmental program, and isolation of genetic bypass suppressors of a bsgA mutant was used to identify signaling components controlling development downstream of the BsgA protease. Strain M955 was created by transposon mutagenesis of a bsgA mutant followed by screening for strains that could develop despite the absence of the BsgA protease. Strain M955 was able to aggregate, form fruiting bodies, and partially restored the production of viable spores in comparison to the parental bsgA mutant. The bsgA Tn5Ω955 strain partially restored developmental expression to a subset of genes normally induced during development, and expressed one developmentally induced fusion at higher amounts during vegetative growth in comparison to wild-type cells. The transposon in strain M955 was localized to a Ribonuclease D homolog that appears to exist in an operon with a downstream aminopeptidase-encoding gene. The identification of a third distinct bypass suppressor of the BsgA protease suggests that the BsgA protease may regulate a potentially complex pathway during the initiation of the M. xanthus developmental program. © FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  2. Understanding serine proteases implications on Leishmania spp lifecycle.

    PubMed

    Alves, Carlos Roberto; Souza, Raquel Santos de; Charret, Karen Dos Santos; Côrtes, Luzia Monteiro de Castro; Sá-Silva, Matheus Pereira de; Barral-Veloso, Laura; Oliveira, Luiz Filipe Gonçalves; da Silva, Franklin Souza

    2018-01-01

    Serine proteases have significant functions over a broad range of relevant biological processes to the Leishmania spp lifecycle. Data gathered here present an update on the Leishmania spp serine proteases and the status of these enzymes as part of the parasite degradome. The serine protease genes (n = 26 to 28) in Leishmania spp, which encode proteins with a wide range of molecular masses (35 kDa-115 kDa), are described along with their degrees of chromosomal and allelic synteny. Amid 17 putative Leishmania spp serine proteases, only ∼18% were experimentally demonstrated, as: signal peptidases that remove the signal peptide from secretory pre-proteins, maturases of other proteins and with metacaspase-like activity. These enzymes include those of clans SB, SC and SF. Classical inhibitors of serine proteases are used as tools for the characterization and investigation of Leishmania spp. Endogenous serine protease inhibitors, which are ecotin-like, can act modulating host actions. However, crude or synthetic based-natural serine protease inhibitors, such as potato tuber extract, Stichodactyla helianthus protease inhibitor I, fukugetin and epoxy-α-lapachone act on parasitic serine proteases and are promising leishmanicidal agents. The functional interrelationship between serine proteases and other Leishmania spp proteins demonstrate essential functions of these enzymes in parasite physiology and therefore their value as targets for leishmaniasis treatment. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Cloning, expression and characterisation of an HtrA-like serine protease produced in vivo by Mycobacterium leprae.

    PubMed

    Ribeiro-Guimarães, Michelle Lopes; Marengo, Eliana Blini; Tempone, Antonio Jorge; Amaral, Julio Jablonski; Klitzke, Clécio F; Silveira, Erika K Xavier da; Portaro, Fernanda Calheta Vieira; Pessolani, Maria Cristina Vidal

    2009-12-01

    Members of the high temperature requirement A (HtrA) family of chaperone proteases have been shown to play a role in bacterial pathogenesis. In a recent report, we demonstrated that the gene ML0176, which codes for a predicted HtrA-like protease, a gene conserved in other species of mycobacteria, is transcribed by Mycobacterium leprae in human leprosy lesions. In the present study, the recombinant ML0176 protein was produced and its enzymatic properties investigated. M. lepraerecombinant ML0176 was able to hydrolyse a variety of synthetic and natural peptides. Similar to other HtrA proteins, this enzyme displayed maximum proteolytic activity at temperatures above 40 degrees C and was completely inactivated by aprotinin, a protease inhibitor with high selectivity for serine proteases. Finally, analysis of M. leprae ML0176 specificity suggested a broader cleavage preference than that of previously described HtrAs homologues. In summary, we have identified an HtrA-like protease in M. lepraethat may constitute a potential new target for the development of novel prophylactic and/or therapeutic strategies against mycobacterial infections.

  4. Network Analyses Reveal Pervasive Functional Regulation Between Proteases in the Human Protease Web

    PubMed Central

    Fortelny, Nikolaus; Cox, Jennifer H.; Kappelhoff, Reinhild; Starr, Amanda E.; Lange, Philipp F.; Pavlidis, Paul; Overall, Christopher M.

    2014-01-01

    Proteolytic processing is an irreversible posttranslational modification affecting a large portion of the proteome. Protease-cleaved mediators frequently exhibit altered activity, and biological pathways are often regulated by proteolytic processing. Many of these mechanisms have not been appreciated as being protease-dependent, and the potential in unraveling a complex new dimension of biological control is increasingly recognized. Proteases are currently believed to act individually or in isolated cascades. However, conclusive but scattered biochemical evidence indicates broader regulation of proteases by protease and inhibitor interactions. Therefore, to systematically study such interactions, we assembled curated protease cleavage and inhibition data into a global, computational representation, termed the protease web. This revealed that proteases pervasively influence the activity of other proteases directly or by cleaving intermediate proteases or protease inhibitors. The protease web spans four classes of proteases and inhibitors and so links both recently and classically described protease groups and cascades, which can no longer be viewed as operating in isolation in vivo. We demonstrated that this observation, termed reachability, is robust to alterations in the data and will only increase in the future as additional data are added. We further show how subnetworks of the web are operational in 23 different tissues reflecting different phenotypes. We applied our network to develop novel insights into biologically relevant protease interactions using cell-specific proteases of the polymorphonuclear leukocyte as a system. Predictions from the protease web on the activity of matrix metalloproteinase 8 (MMP8) and neutrophil elastase being linked by an inactivating cleavage of serpinA1 by MMP8 were validated and explain perplexing Mmp8 −/− versus wild-type polymorphonuclear chemokine cleavages in vivo. Our findings supply systematically derived and

  5. Toxoplasma gondii serine-protease inhibitor-1: A new adjuvant candidate for asthma therapy.

    PubMed

    Soto, Ariadna S; Fenoy, Ignacio M; Sanchez, Vanesa R; March, Florencia; Perrone Sibilia, Matías D; Aldirico, María de Los Angeles; Picchio, Mariano S; Arcon, Nadia; Acosta, Patricio L; Polack, Fernando P; Martin, Valentina; Goldman, Alejandra

    2017-01-01

    Serine-proteases are important players in the pathogenesis of asthma, promoting inflammation and tissue remodeling. It's also known that many serine protease inhibitors display immunomodulatory properties. TgPI-1 is a Toxoplasma gondii protein that exhibits broad spectrum inhibitory activity against serine proteases. In view of the increased prevalence of atopic disorders and the need to develop new treatment strategies we sought to investigate the potential of TgPI-1 for treating respiratory allergies. For this purpose, we developed a therapeutic experimental model. BALB/c mice were rendered allergic by intraperitoneal ovalbumin-alum sensitization and airway-challenged. Once the asthmatic phenotype was achieved, mice were intranasally treated with rTgPI-1 alone or with a mixture of rTgPI-1 and ovalbumin (OVA). A week later mice were given a secondary aerosol challenge. Treatment with rTgPI-1 alone or co-administered with OVA diminished bronchoalveolar eosinophilia, mucus production and peribronchial lung infiltration. This effect was accompanied by a lung resistance reduction of 26.3% and 50.3% respectively. Both treatments resulted in the production of lower levels of IL-4, IL-5, IFN-γ and regulatory IL-10 by thoracic lymph node cells stimulated with OVA. Interestingly, significant decreases in OVA specific IgE and T cell proliferation, and increases in FoxP3+ T cells at local and systemic levels were only detected when the inhibitor was administered along with OVA. These results show that both rTgPI-1 treatments reduced asthma hallmarks. However, co-administration of the inhibitor with the allergen was more effective. Hence, rTgPI-1 emerges as a novel adjuvant candidate for asthma treatment.

  6. Toxoplasma gondii serine-protease inhibitor-1: A new adjuvant candidate for asthma therapy

    PubMed Central

    Soto, Ariadna S.; Fenoy, Ignacio M.; Sanchez, Vanesa R.; March, Florencia; Perrone Sibilia, Matías D.; Aldirico, María de los Angeles; Picchio, Mariano S.; Arcon, Nadia; Acosta, Patricio L.; Polack, Fernando P.; Martin, Valentina

    2017-01-01

    Serine-proteases are important players in the pathogenesis of asthma, promoting inflammation and tissue remodeling. It’s also known that many serine protease inhibitors display immunomodulatory properties. TgPI-1 is a Toxoplasma gondii protein that exhibits broad spectrum inhibitory activity against serine proteases. In view of the increased prevalence of atopic disorders and the need to develop new treatment strategies we sought to investigate the potential of TgPI-1 for treating respiratory allergies. For this purpose, we developed a therapeutic experimental model. BALB/c mice were rendered allergic by intraperitoneal ovalbumin-alum sensitization and airway-challenged. Once the asthmatic phenotype was achieved, mice were intranasally treated with rTgPI-1 alone or with a mixture of rTgPI-1 and ovalbumin (OVA). A week later mice were given a secondary aerosol challenge. Treatment with rTgPI-1 alone or co-administered with OVA diminished bronchoalveolar eosinophilia, mucus production and peribronchial lung infiltration. This effect was accompanied by a lung resistance reduction of 26.3% and 50.3% respectively. Both treatments resulted in the production of lower levels of IL-4, IL-5, IFN-γ and regulatory IL-10 by thoracic lymph node cells stimulated with OVA. Interestingly, significant decreases in OVA specific IgE and T cell proliferation, and increases in FoxP3+ T cells at local and systemic levels were only detected when the inhibitor was administered along with OVA. These results show that both rTgPI-1 treatments reduced asthma hallmarks. However, co-administration of the inhibitor with the allergen was more effective. Hence, rTgPI-1 emerges as a novel adjuvant candidate for asthma treatment. PMID:29073215

  7. An efficient procedure for the expression and purification of HIV-1 protease from inclusion bodies.

    PubMed

    Nguyen, Hong-Loan Thi; Nguyen, Thuy Thi; Vu, Quy Thi; Le, Hang Thi; Pham, Yen; Trinh, Phuong Le; Bui, Thuan Phuong; Phan, Tuan-Nghia

    2015-12-01

    Several studies have focused on HIV-1 protease for developing drugs for treating AIDS. Recombinant HIV-1 protease is used to screen new drugs from synthetic compounds or natural substances. However, large-scale expression and purification of this enzyme is difficult mainly because of its low expression and solubility. In this study, we constructed 9 recombinant plasmids containing a sequence encoding HIV-1 protease along with different fusion tags and examined the expression of the enzyme from these plasmids. Of the 9 plasmids, pET32a(+) plasmid containing the HIV-1 protease-encoding sequence along with sequences encoding an autocleavage site GTVSFNF at the N-terminus and TEV plus 6× His tag at the C-terminus showed the highest expression of the enzyme and was selected for further analysis. The recombinant protein was isolated from inclusion bodies by using 2 tandem Q- and Ni-Sepharose columns. SDS-PAGE of the obtained HIV-1 protease produced a single band of approximately 13 kDa. The enzyme was recovered efficiently (4 mg protein/L of cell culture) and had high specific activity of 1190 nmol min(-1) mg(-1) at an optimal pH of 4.7 and optimal temperature of 37 °C. This procedure for expressing and purifying HIV-1 protease is now being scaled up to produce the enzyme on a large scale for its application. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. The QSAR and docking calculations of fullerene derivatives as HIV-1 protease inhibitors

    NASA Astrophysics Data System (ADS)

    Saleh, Noha A.

    2015-02-01

    The inhibition of HIV-1 protease is considered as one of the most important targets for drug design and the deactivation of HIV-1. In the present work, the fullerene surface (C60) is modified by adding oxygen atoms as well as hydroxymethylcarbonyl (HMC) groups to form 6 investigated fullerene derivative compounds. These compounds have one, two, three, four or five O atoms + HMC groups at different positions on phenyl ring. The effect of the repeating of these groups on the ability of suggested compounds to inhibit the HIV protease is studied by calculating both Quantitative Structure Activity Relationship (QSAR) properties and docking simulation. Based on the QSAR descriptors, the solubility and the hydrophilicity of studied fullerene derivatives increased with increasing the number of oxygen atoms + HMC groups in the compound. While docking calculations indicate that, the compound with two oxygen atoms + HMC groups could interact and binds with HIV-1 protease active site. This is could be attributed to the active site residues of HIV-1 protease are hydrophobic except the two aspartic acids. So that, the increase in the hydrophilicity and polarity of the compound is preventing and/or decreasing the hydrophobic interaction between the compound and HIV-1 protease active site.

  9. Protease nexin 1 is a potent urinary plasminogen activator inhibitor in the presence of collagen type IV.

    PubMed

    Crisp, Robert J; Knauer, Mary F; Knauer, Daniel J

    2002-12-06

    Protease nexin 1 (PN1) in solution forms inhibitory complexes with thrombin or urokinase, which have opposing effects on the blood coagulation cascade. An initial report provided data supporting the idea that PN1 target protease specificity is under the influence of collagen type IV (1). Although collagen type IV demonstrated no effect on the association rate between PN1 and thrombin, the study reported that the association rate between PN1 and urokinase was allosterically reduced 10-fold. This has led to the generally accepted idea that the primary role of PN1 in the brain is to act as a rapid thrombin inhibition and clearance mechanism during trauma and loss of vascular integrity. In studies to identify the structural determinants of PN1 that mediate the allosteric interaction with collagen type IV, we found that protease specificity was only affected after transient exposure of PN1 to acidic conditions that mimic the elution protocol from a monoclonal antibody column. Because PN1 used in previous studies was purified over a monoclonal antibody column, we propose that the allosteric regulation of PN1 target protease specificity by collagen type IV is a result of the purification protocol. We provide both biochemical and kinetic data to support this conclusion. This finding is significant because it implies that PN1 may play a much larger role in the modeling and remodeling of brain tissues during development and is not simply an extravasated thrombin clearance mechanism as previously suggested.

  10. SERPINA2 Is a Novel Gene with a Divergent Function from SERPINA1

    PubMed Central

    Martins, Manuella; Figueiredo, Joana; Silva, Diana Isabel; Castro, Patrícia; Morales-Hojas, Ramiro; Simões-Correia, Joana; Seixas, Susana

    2013-01-01

    Serine protease inhibitors (SERPINs) are a superfamily of highly conserved proteins that play a key role in controlling the activity of proteases in diverse biological processes. The SERPIN cluster located at the 14q32.1 region includes the gene coding for SERPINA1, and a highly homologous sequence, SERPINA2, which was originally thought to be a pseudogene. We have previously shown that SERPINA2 is expressed in different tissues, namely leukocytes and testes, suggesting that it is a functional SERPIN. To investigate the function of SERPINA2, we used HeLa cells stably transduced with the different variants of SERPINA2 and SERPINA1 (M1, S and Z) and leukocytes as the in vivo model. We identified SERPINA2 as a 52 kDa intracellular glycoprotein, which is localized at the endoplasmic reticulum (ER), independently of the variant analyzed. SERPINA2 is not significantly regulated by proteasome, proposing that ER localization is not due to misfolding. Specific features of SERPINA2 include the absence of insoluble aggregates and the insignificant response to cell stress, suggesting that it is a non-polymerogenic protein with divergent activity of SERPINA1. Using phylogenetic analysis, we propose an origin of SERPINA2 in the crown of primates, and we unveiled the overall conservation of SERPINA2 and A1. Nonetheless, few SERPINA2 residues seem to have evolved faster, contributing to the emergence of a new advantageous function, possibly as a chymotrypsin-like SERPIN. Herein, we present evidences that SERPINA2 is an active gene, coding for an ER-resident protein, which may act as substrate or adjuvant of ER-chaperones. PMID:23826168

  11. Singlet Oxygen-Induced Membrane Disruption and Serpin-Protease Balance in Vacuolar-Driven Cell Death1[OPEN

    PubMed Central

    Carmieli, Raanan; Mor, Avishai; Fluhr, Robert

    2016-01-01

    Singlet oxygen plays a role in cellular stress either by providing direct toxicity or through signaling to initiate death programs. It was therefore of interest to examine cell death, as occurs in Arabidopsis, due to differentially localized singlet oxygen photosensitizers. The photosensitizers rose bengal (RB) and acridine orange (AO) were localized to the plasmalemma and vacuole, respectively. Their photoactivation led to cell death as measured by ion leakage. Cell death could be inhibited by the singlet oxygen scavenger histidine in treatments with AO but not with RB. In the case of AO treatment, the vacuolar membrane was observed to disintegrate. Concomitantly, a complex was formed between a vacuolar cell-death protease, RESPONSIVE TO DESSICATION-21 and its cognate cytoplasmic protease inhibitor ATSERPIN1. In the case of RB treatment, the tonoplast remained intact and no complex was formed. Over-expression of AtSerpin1 repressed cell death, only under AO photodynamic treatment. Interestingly, acute water stress showed accumulation of singlet oxygen as determined by fluorescence of Singlet Oxygen Sensor Green, by electron paramagnetic resonance spectroscopy and the induction of singlet oxygen marker genes. Cell death by acute water stress was inhibited by the singlet oxygen scavenger histidine and was accompanied by vacuolar collapse and the appearance of serpin-protease complex. Over-expression of AtSerpin1 also attenuated cell death under this mode of cell stress. Thus, acute water stress damage shows parallels to vacuole-mediated cell death where the generation of singlet oxygen may play a role. PMID:26884487

  12. Structure Determination of Mycobacterium tuberculosis Serine Protease Hip1 (Rv2224c).

    PubMed

    Naffin-Olivos, Jacqueline L; Daab, Andrew; White, Andre; Goldfarb, Nathan E; Milne, Amy C; Liu, Dali; Baikovitz, Jacqueline; Dunn, Ben M; Rengarajan, Jyothi; Petsko, Gregory A; Ringe, Dagmar

    2017-05-02

    The Mycobacterium tuberculosis (Mtb) serine protease Hip1 (hydrolase important for pathogenesis; Rv2224c) promotes tuberculosis (TB) pathogenesis by impairing host immune responses through proteolysis of a protein substrate, Mtb GroEL2. The cell surface localization of Hip1 and its immunomodulatory functions make Hip1 a good drug target for new adjunctive immune therapies for TB. Here, we report the crystal structure of Hip1 to a resolution of 2.6 Å and the kinetic studies of the enzyme against model substrates and the protein GroEL2. The structure shows a two-domain protein, one of which contains the catalytic residues that are the signature of a serine protease. Surprisingly, a threonine is located within the active site close enough to hydrogen bond with the catalytic residues Asp463 and His490. Mutation of this residue, Thr466, to alanine established its importance for function. Our studies provide insights into the structure of a member of a novel family of proteases. Knowledge of the Hip1 structure will aid in designing inhibitors that could block Hip1 activity.

  13. Colocalization and Membrane Association of Murine Hepatitis Virus Gene 1 Products and De Novo-Synthesized Viral RNA in Infected Cells

    PubMed Central

    Shi, Stephanie T.; Schiller, Jennifer J.; Kanjanahaluethai, Amornrat; Baker, Susan C.; Oh, Jong-Won; Lai, Michael M. C.

    1999-01-01

    Murine hepatitis virus (MHV) gene 1, the 22-kb polymerase (pol) gene, is first translated into a polyprotein and subsequently processed into multiple proteins by viral autoproteases. Genetic complementation analyses suggest that the majority of the gene 1 products are required for viral RNA synthesis. However, there is no physical evidence supporting the association of any of these products with viral RNA synthesis. We have now performed immunofluorescent-staining studies with four polyclonal antisera to localize various MHV-A59 gene 1 products in virus-infected cells. Immunoprecipitation experiments showed that these antisera detected proteins representing the two papain-like proteases and the 3C-like protease encoded by open reading frame (ORF) 1a, the putative polymerase (p100) and a p35 encoded by ORF 1b, and their precursors. De novo-synthesized viral RNA was labeled with bromouridine triphosphate in lysolecithin-permeabilized MHV-infected cells. Confocal microscopy revealed that all of the viral proteins detected by these antisera colocalized with newly synthesized viral RNA in the cytoplasm, particularly in the perinuclear region of infected cells. Several cysteine and serine protease inhibitors, i.e., E64d, leupeptin, and zinc chloride, inhibited viral RNA synthesis without affecting the localization of viral proteins, suggesting that the processing of the MHV gene 1 polyprotein is tightly associated with viral RNA synthesis. Dual labeling with antibodies specific for cytoplasmic membrane structures showed that MHV gene 1 products and RNA colocalized with the Golgi apparatus in HeLa cells. However, in murine 17CL-1 cells, the viral proteins and viral RNA did not colocalize with the Golgi apparatus but, instead, partially colocalized with the endoplasmic reticulum. Our results provide clear physical evidence that several MHV gene 1 products, including the proteases and the polymerase, are associated with the viral RNA replication-transcription machinery

  14. Streptomyces flavogriseus HS1: isolation and characterization of extracellular proteases and their compatibility with laundry detergents.

    PubMed

    Ghorbel, Sofiane; Kammoun, Maher; Soltana, Hala; Nasri, Moncef; Hmidet, Noomen

    2014-01-01

    The present study describes the isolation of a new protease producing Streptomyces strain HS1 and the biochemical characterization of the secreted proteases. By sequencing of its noted 16S rDNA, HS1 strain was found to have a 100% identity with Streptomyces flavogriseus. The highest protease production was found using FermII media. In these conditions maximum protease production (99 U/mL) was obtained after 96 h incubation at 30°C and 150 rpm. HS1 strain produced at least five proteases as revealed by zymogram technique. The enzyme preparation exhibited activity over a broad range of pH (5-11) and temperature (25-70°C). Optimum activity was observed at a pH of 7.0 and a temperature of 50°C. Proteolytic activity was significantly unaffected by Ca(2+) and Mg(2+). EDTA and PMSF highly decreased the original activity. The crude extracellular proteases showed high stability when used as a detergent additive. These properties offer an interesting potential for enzymatic hydrolysis at the industrial level.

  15. Streptomyces flavogriseus HS1: Isolation and Characterization of Extracellular Proteases and Their Compatibility with Laundry Detergents

    PubMed Central

    Kammoun, Maher; Soltana, Hala; Nasri, Moncef; Hmidet, Noomen

    2014-01-01

    The present study describes the isolation of a new protease producing Streptomyces strain HS1 and the biochemical characterization of the secreted proteases. By sequencing of its noted 16S rDNA, HS1 strain was found to have a 100% identity with Streptomyces flavogriseus. The highest protease production was found using FermII media. In these conditions maximum protease production (99 U/mL) was obtained after 96 h incubation at 30°C and 150 rpm. HS1 strain produced at least five proteases as revealed by zymogram technique. The enzyme preparation exhibited activity over a broad range of pH (5–11) and temperature (25–70°C). Optimum activity was observed at a pH of 7.0 and a temperature of 50°C. Proteolytic activity was significantly unaffected by Ca2+ and Mg2+. EDTA and PMSF highly decreased the original activity. The crude extracellular proteases showed high stability when used as a detergent additive. These properties offer an interesting potential for enzymatic hydrolysis at the industrial level. PMID:24804214

  16. Molecular characterization of 45 kDa aspartic protease of Trichinella spiralis.

    PubMed

    Park, Jong Nam; Park, Sang Kyun; Cho, Min Kyoung; Park, Mi-Kyung; Kang, Shin Ae; Kim, Dong-Hee; Yu, Hak Sun

    2012-12-21

    In a previous study, we identified an aspartic protease gene (Ts-Asp) from the Trichinella spiralis muscle stage larva cDNA library. The gene sequence of Ts-Asp was 1281 bp long and was found to encode a protein consisting of 405 amino acids, with a molecular mass of 45.248 kD and a pI of 5.95. The deduced Ts-Asp has a conserved catalytic motif with catalytic aspartic acid residues in the active site, a common characteristic of aspartic proteases. In addition, the deduced amino acid sequence of Ts-Asp was found to possess significant homology (above 50%) with aspartic proteases from nematode parasites. Results of phylogenetic analysis indicated a close relationship of Ts-Asp with cathepsin D aspartic proteases. For production of recombinant Ts-Asp (rTs-Asp), the pGEX4T expression system was used. Like other proteases, the purified rTs-Asp was able to digest collagen matrix in vitro. Abundant expression of Ts-Asp was observed in muscle stage larva. Ts-Asp was detected in ES proteins, and was able to elicit the production of specific antibodies. It is the first report of molecular characterization of aspartic protease isolated from T. spiralis. Copyright © 2012 Elsevier B.V. All rights reserved.

  17. Screening and characterization of the alkaline protease isolated from PLI-1, a strain of Brevibacillus sp. collected from Indonesia's hot springs

    NASA Astrophysics Data System (ADS)

    Wang, Shuai; Lin, Xuezheng; Huang, Xiaohang; Zheng, Li; Zilda, Dewi Seswita

    2012-06-01

    A total of 69 strains of thermophilic bacteria were isolated from water, soil and sediment samples from three Indonesia's hot spring areas (Pantai cermin, Kalianda and Banyu wedang) by using Minimal Synthetic Medium (MSM). The extreme thermophile Brevibacillus sp. PLI-1 was found to produce extracellular thermophilic alkaline protease with optimal activity at 70° and pH 8.0-9.0. The molecular weight of the protease was estimated to be around 56 kD by SDS-PAGE. The maximum activity of the protease was 26.54 U mL-1. The protease activity did not decrease after 30 min and still retained more than 70% of relative activity after 60 min at 70°C and pH 8.0. The ion Mg2+ was found to promote protease activity at both low and high concentrations, whereas Cu2+ and Zn2+ could almost completely inhibit the activity. Divalent cation chelator EDTA inhibited the enzyme activity by 55.06% ± 0.27%, while the inhibition caused by PMSF, Leupeptin, Pepstain A and Benzamidine were 66.78% ± 3.25%, 52.37% ± 0.25%, 62.47% ± 2.96% and 50.99% ± 0.24%, respectively. Based on these observations, the enzyme activity was conspicuously sensitive to the serine and cysteine protease inhibitors. All these results indicated that the protease isolated from the strain PLI-1 was a thermophilic protease and had a high-temperature stability and a pH stability.

  18. Emerging roles for diverse intramembrane proteases in plant biology.

    PubMed

    Adam, Zach

    2013-12-01

    Progress in the field of regulated intramembrane proteolysis (RIP) in recent years has made its impact on plant biology as well. Although this field within plant research is still in its infancy, some interesting observations have started to emerge. Gene encoding orthologs of rhomboid proteases, site-2 proteases (S2P), presenilin/γ-secretases, and signal peptide peptidases are found in plant genomes and some of these gene products were identified in different plant cell membranes. The lack of chloroplast-located rhomboid proteases was associated with reduced fertility and aberrations in flower morphology. Mutations in homologues of S2P resulted in chlorophyll deficiency and impaired chloroplast development. An S2P was also implicated in the response to ER stress through cleavage of ER-membrane bZIP transcription factors, allowing their migration to the nucleus and activation of the transcription of BiP chaperones. Other membrane-bound transcription factors of the NAC and PHD families were also demonstrated to undergo RIP and relocalization to the nucleus. These and other new data are expected to shed more light on the roles of intramembrane proteases in plant biology in the future. This article is part of a Special Issue entitled: Intramembrane Proteases. Copyright © 2013 Elsevier B.V. All rights reserved.

  19. Functional diversification upon leader protease domain duplication in the Citrus tristeza virus genome: Role of RNA sequences and the encoded proteins.

    PubMed

    Kang, Sung-Hwan; Atallah, Osama O; Sun, Yong-Duo; Folimonova, Svetlana Y

    2018-01-15

    Viruses from the family Closteroviridae show an example of intra-genome duplications of more than one gene. In addition to the hallmark coat protein gene duplication, several members possess a tandem duplication of papain-like leader proteases. In this study, we demonstrate that domains encoding the L1 and L2 proteases in the Citrus tristeza virus genome underwent a significant functional divergence at the RNA and protein levels. We show that the L1 protease is crucial for viral accumulation and establishment of initial infection, whereas its coding region is vital for virus transport. On the other hand, the second protease is indispensable for virus infection of its natural citrus host, suggesting that L2 has evolved an important adaptive function that mediates virus interaction with the woody host. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Indispensable Role of Proteases in Plant Innate Immunity.

    PubMed

    Balakireva, Anastasia V; Zamyatnin, Andrey A

    2018-02-23

    Plant defense is achieved mainly through the induction of microbe-associated molecular patterns (MAMP)-triggered immunity (MTI), effector-triggered immunity (ETI), systemic acquired resistance (SAR), induced systemic resistance (ISR), and RNA silencing. Plant immunity is a highly complex phenomenon with its own unique features that have emerged as a result of the arms race between plants and pathogens. However, the regulation of these processes is the same for all living organisms, including plants, and is controlled by proteases. Different families of plant proteases are involved in every type of immunity: some of the proteases that are covered in this review participate in MTI, affecting stomatal closure and callose deposition. A large number of proteases act in the apoplast, contributing to ETI by managing extracellular defense. A vast majority of the endogenous proteases discussed in this review are associated with the programmed cell death (PCD) of the infected cells and exhibit caspase-like activities. The synthesis of signal molecules, such as salicylic acid, jasmonic acid, and ethylene, and their signaling pathways, are regulated by endogenous proteases that affect the induction of pathogenesis-related genes and SAR or ISR establishment. A number of proteases are associated with herbivore defense. In this review, we summarize the data concerning identified plant endogenous proteases, their effect on plant-pathogen interactions, their subcellular localization, and their functional properties, if available, and we attribute a role in the different types and stages of innate immunity for each of the proteases covered.

  1. Design, synthesis and evaluation of a potent substrate analog inhibitor identified by scanning Ala/Phe mutagenesis, mimicking substrate co-evolution, against multidrug-resistant HIV-1 protease

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yedidi, Ravikiran S.; Muhuhi, Joseck M.; Liu, Zhigang

    Highlights: •Inhibitors against MDR HIV-1 protease were designed, synthesized and evaluated. •Lead peptide (6a) showed potent inhibition (IC{sub 50}: 4.4 nM) of MDR HIV-1 protease. •(6a) Showed favorable binding isotherms against NL4-3 and MDR proteases. •(6a) Induced perturbations in the {sup 15}N-HSQC spectrum of MDR HIV-1 protease. •Molecular modeling suggested that (6a) may induce total flap closure inMDR protease. -- Abstract: Multidrug-resistant (MDR) clinical isolate-769, human immunodeficiency virus type-1 (HIV-1) protease (PDB ID: (1TW7)), was shown to exhibit wide-open flaps and an expanded active site cavity, causing loss of contacts with protease inhibitors. In the current study, the expanded activemore » site cavity of MDR769 HIV-1 protease was screened with a series of peptide-inhibitors that were designed to mimic the natural substrate cleavage site, capsid/p2. Scanning Ala/Phe chemical mutagenesis approach was incorporated into the design of the peptide series to mimic the substrate co-evolution. Among the peptides synthesized and evaluated, a lead peptide (6a) with potent activity (IC{sub 50}: 4.4 nM) was identified against the MDR769 HIV-1 protease. Isothermal titration calorimetry data showed favorable binding profile for 6aagainst both wild type and MDR769 HIV-1 protease variants. Nuclear magnetic resonance spectrum of {sup 15}N-labeled MDR769 HIV-1 protease in complex with 6a showed some major perturbations in chemical shift, supporting the peptide induced conformational changes in protease. Modeling analysis revealed multiple contacts between 6a and MDR769 HIV-1 protease. The lead peptide-inhibitor, 6a, with high potency and good binding profile can be used as the basis for developing potent small molecule inhibitors against MDR variants of HIV.« less

  2. Structure-Based Design of Novel HIV-1 Protease Inhibitors to Combat Drug Resistance

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ghosh,A.; Sridhar, P.; Leshchenko, S.

    2006-01-01

    Structure-based design and synthesis of novel HIV protease inhibitors are described. The inhibitors are designed specifically to interact with the backbone of HIV protease active site to combat drug resistance. Inhibitor 3 has exhibited exceedingly potent enzyme inhibitory and antiviral potency. Furthermore, this inhibitor maintains impressive potency against a wide spectrum of HIV including a variety of multi-PI-resistant clinical strains. The inhibitors incorporated a stereochemically defined 5-hexahydrocyclopenta[b]furanyl urethane as the P2-ligand into the (R)-(hydroxyethylamino)sulfonamide isostere. Optically active (3aS,5R,6aR)-5-hydroxy-hexahydrocyclopenta[b]furan was prepared by an enzymatic asymmetrization of meso-diacetate with acetyl cholinesterase, radical cyclization, and Lewis acid-catalyzed anomeric reduction as the key steps.more » A protein-ligand X-ray crystal structure of inhibitor 3-bound HIV-1 protease (1.35 Angstroms resolution) revealed extensive interactions in the HIV protease active site including strong hydrogen bonding interactions with the backbone. This design strategy may lead to novel inhibitors that can combat drug resistance.« less

  3. Structure Determination of Mycobacterium tuberculosis Serine Protease Hip1 (Rv2224c)

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Naffin-Olivos, Jacqueline L.; Daab, Andrew; White, Andre

    The Mycobacterium tuberculosis (Mtb) serine protease Hip1 (hydrolase important for pathogenesis; Rv2224c) promotes tuberculosis (TB) pathogenesis by impairing host immune responses through proteolysis of a protein substrate, Mtb GroEL2. The cell surface localization of Hip1 and its immunomodulatory functions make Hip1 a good drug target for new adjunctive immune therapies for TB. Here, we report the crystal structure of Hip1 to a resolution of 2.6 Å and the kinetic studies of the enzyme against model substrates and the protein GroEL2. The structure shows a two-domain protein, one of which contains the catalytic residues that are the signature of a serinemore » protease. Surprisingly, a threonine is located within the active site close enough to hydrogen bond with the catalytic residues Asp463 and His490. Mutation of this residue, Thr466, to alanine established its importance for function. Our studies provide insights into the structure of a member of a novel family of proteases. Knowledge of the Hip1 structure will aid in designing inhibitors that could block Hip1 activity« less

  4. The Mitochondrial m-AAA Protease Prevents Demyelination and Hair Greying.

    PubMed

    Wang, Shuaiyu; Jacquemyn, Julie; Murru, Sara; Martinelli, Paola; Barth, Esther; Langer, Thomas; Niessen, Carien M; Rugarli, Elena I

    2016-12-01

    The m-AAA protease preserves proteostasis of the inner mitochondrial membrane. It ensures a functional respiratory chain, by controlling the turnover of respiratory complex subunits and allowing mitochondrial translation, but other functions in mitochondria are conceivable. Mutations in genes encoding subunits of the m-AAA protease have been linked to various neurodegenerative diseases in humans, such as hereditary spastic paraplegia and spinocerebellar ataxia. While essential functions of the m-AAA protease for neuronal survival have been established, its role in adult glial cells remains enigmatic. Here, we show that deletion of the highly expressed subunit AFG3L2 in mature mouse oligodendrocytes provokes early-on mitochondrial fragmentation and swelling, as previously shown in neurons, but causes only late-onset motor defects and myelin abnormalities. In contrast, total ablation of the m-AAA protease, by deleting both Afg3l2 and its paralogue Afg3l1, triggers progressive motor dysfunction and demyelination, owing to rapid oligodendrocyte cell death. Surprisingly, the mice showed premature hair greying, caused by progressive loss of melanoblasts that share a common developmental origin with Schwann cells and are targeted in our experiments. Thus, while both neurons and glial cells are dependant on the m-AAA protease for survival in vivo, complete ablation of the complex is necessary to trigger death of oligodendrocytes, hinting to cell-autonomous thresholds of vulnerability to m-AAA protease deficiency.

  5. Appearance of drug resistance-associated mutations in human immunodeficiency virus type 1 protease and reverse transcriptase derived from drug-treated Indonesian patients.

    PubMed

    Khairunisa, Siti Qamariyah; Kotaki, Tomohiro; Witaningrum, Adiana Mutamsari; Yunifiar M, Muhammad Qushai; Sukartiningrum, Septhia Dwi; Nasronudin; Kameoka, Masanori

    2015-02-01

    Although HIV-1 drug resistance is a major obstacle in Indonesia, information on drug resistance is limited. In this study, the viral subtype and appearance of drug resistance mutations in the HIV-1 protease (PR) and reverse transcriptase (RT) genes were determined among drug-treated, HIV-1-infected patients in Surabaya. HIV-1 patients who received antiretroviral therapy (ART) more than 2 years were randomly recruited regardless of the viral load or ART failure. Fifty-eight HIV-1 PR genes and 53 RT genes were sequenced. CRF01_AE viruses were identified as the predominant strain. Major drug resistance mutations were not detected in the PR genes. In contrast, 37.7% (20/53) of the participants had one or more major drug resistance mutations in the RT genes, predominantly M184V (28.3%), K103N (11.3%), and thymidine analogue mutations (TAMs) (20.8%). The high prevalence of drug resistance mutations in RT genes indicated the necessity of monitoring the effectiveness of ART in Indonesia.

  6. Insights into the mechanism of drug resistance. X-ray structure analysis of multi-drug resistant HIV-1 protease ritonavir complex

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Liu, Zhigang; Yedidi, Ravikiran S.; Wang, Yong

    2013-01-08

    Ritonavir (RTV) is a first generation HIV-1 protease inhibitor with rapidly emerging drug resistance. Mutations at residues 46, 54, 82 and 84 render the HIV-1 protease drug resistant against RTV. We report the crystal structure of multi-drug resistant (MDR) 769 HIV-1 protease (carrying resistant mutations at residues 10, 36, 46, 54, 62, 63, 71, 82, 84 and 90) complexed with RTV and the in vitro enzymatic IC50 of RTV against MDR HIV-1 protease. The structural and functional studies demonstrate significant drug resistance of MDR HIV-1 protease against RTV, arising from reduced hydrogen bonds and Van der Waals interactions between RTVmore » and MDR HIV-1 protease.« less

  7. HvPap-1 C1A Protease and HvCPI-2 Cystatin Contribute to Barley Grain Filling and Germination.

    PubMed

    Diaz-Mendoza, Mercedes; Dominguez-Figueroa, Jose D; Velasco-Arroyo, Blanca; Cambra, Ines; Gonzalez-Melendi, Pablo; Lopez-Gonzalvez, Angeles; Garcia, Antonia; Hensel, Goetz; Kumlehn, Jochen; Diaz, Isabel; Martinez, Manuel

    2016-04-01

    Proteolysis is an essential process throughout the mobilization of storage proteins in barley (Hordeum vulgare) grains during germination. It involves numerous types of enzymes, with C1A Cys proteases the most abundant key players. Manipulation of the proteolytic machinery is a potential way to enhance grain yield and quality, and it could influence the mobilization of storage compounds along germination. Transgenic barley plants silencing or over-expressing the cathepsin F-like HvPap-1 Cys protease show differential accumulation of storage molecules such as starch, proteins, and free amino acids in the grain. It is particularly striking that the HvPap-1 artificial microRNA lines phenotype show a drastic delay in the grain germination process. Alterations to the proteolytic activities in the over-expressing and knock-down grains associated with changes in the level of expression of several C1A peptidases were also detected. Similarly, down-regulating cystatin Icy-2, one of the proteinaceous inhibitors of the cathepsin F-like protease, also has important effects on grain filling. However, the ultimate physiological influence of manipulating a peptidase or an inhibitor cannot be always predicted, since the plant tries to compensate the modified proteolytic effects by modulating the expression of some other peptidases or their inhibitors. © 2016 American Society of Plant Biologists. All Rights Reserved.

  8. Quantification of Pelvic Organ Prolapse in Mice: Vaginal Protease Activity Precedes Increased MOPQ Scores in Fibulin 5 Knockout Mice1

    PubMed Central

    Wieslander, Cecilia K.; Rahn, David D.; McIntire, Donald D.; Acevedo, Jesús F.; Drewes, Peter G.; Yanagisawa, Hiromi; Word, R. Ann

    2008-01-01

    Two mouse models of pelvic organ prolapse have been generated recently, both of which have null mutations in genes involved in elastic fiber synthesis and assembly (fibulin 5 and lysyl oxidase-like 1). Interestingly, although these mice exhibit elastinopathies early in life, pelvic organ prolapse does not develop until later in life. In this investigation we developed and validated a tool to quantify the severity of pelvic organ prolapse in mice, and we used this tool prospectively to study the role of fibulin 5, aging, and vaginal proteases in the development of pelvic organ prolapse. The results indicate that >90% of Fbln5−/− mice develop prolapse by 6 mo of age, even in the absence of vaginal delivery, and that increased vaginal protease activity precedes the development of prolapse. PMID:18987327

  9. Lung inflammation promotes metastasis through neutrophil protease-mediated degradation of Tsp-1

    PubMed Central

    El Rayes, Tina; Catena, Raúl; Lee, Sharrell; Stawowczyk, Marcin; Joshi, Natasha; Fischbach, Claudia; Powell, Charles A.; Dannenberg, Andrew J.; Altorki, Nasser K.; Gao, Dingcheng; Mittal, Vivek

    2015-01-01

    Inflammation is inextricably associated with primary tumor progression. However, the contribution of inflammation to tumor outgrowth in metastatic organs has remained underexplored. Here, we show that extrinsic inflammation in the lungs leads to the recruitment of bone marrow-derived neutrophils, which degranulate azurophilic granules to release the Ser proteases, elastase and cathepsin G, resulting in the proteolytic destruction of the antitumorigenic factor thrombospondin-1 (Tsp-1). Genetic ablation of these neutrophil proteases protected Tsp-1 from degradation and suppressed lung metastasis. These results provide mechanistic insights into the contribution of inflammatory neutrophils to metastasis and highlight the unique neutrophil protease–Tsp-1 axis as a potential antimetastatic therapeutic target. PMID:26668367

  10. Phage-protease-peptide: a novel trifecta enabling multiplex detection of viable bacterial pathogens.

    PubMed

    Alcaine, S D; Tilton, L; Serrano, M A C; Wang, M; Vachet, R W; Nugen, S R

    2015-10-01

    Bacteriophages represent rapid, readily targeted, and easily produced molecular probes for the detection of bacterial pathogens. Molecular biology techniques have allowed researchers to make significant advances in the bioengineering of bacteriophage to further improve speed and sensitivity of detection. Despite their host specificity, bacteriophages have not been meaningfully leveraged in multiplex detection of bacterial pathogens. We propose a proof-of-principal phage-based scheme to enable multiplex detection. Our scheme involves bioengineering bacteriophage to carry a gene for a specific protease, which is expressed during infection of the target cell. Upon lysis, the protease is released to cleave a reporter peptide, and the signal detected. Here we demonstrate the successful (i) modification of T7 bacteriophage to carry tobacco etch virus (TEV) protease; (ii) expression of TEV protease by Escherichia coli following infection by our modified T7, an average of 2000 units of protease per phage are produced during infection; and (iii) proof-of-principle detection of E. coli in 3 h after a primary enrichment via TEV protease activity using a fluorescent peptide and using a designed target peptide for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis (MALDI-TOF MS) analysis. This proof-of-principle can be translated to other phage-protease-peptide combinations to enable multiplex bacterial detection and readily adopted on multiple platforms, like MALDI-TOF MS or fluorescent readers, commonly found in labs.

  11. Detection of protease and protease activity using a single nanoscrescent SERS probe

    DOEpatents

    Liu, Gang L.; Ellman, Jonathan A.; Lee, Luke P.; Chen, Fanqing Frank

    2013-01-29

    This invention pertains to the in vitro detection of proteases using a single peptide-conjugate nanocrescent surface enhanced Raman scattering (SERS) probes with at least nanomolar sensitivity. The probe enables detection of proteolytic activity in extremely small volume and at low concentration. In certain embodiments the probes comprise an indicator for the detection of an active protease, where the indicator comprises a nanocrescent attached to a peptide, where said peptide comprises a recognition site for the protease and a Raman tag attached to the peptide.

  12. Detection of protease and protease activity using a single nanocrescent SERS probe

    DOEpatents

    Liu, Gang L.; Ellman, Jonathan A.; Lee, Luke P.; Chen, Fanqing Frank

    2015-09-29

    This invention pertains to the in vitro detection of proteases using a single peptide-conjugate nanocrescent surface enhanced Raman scattering (SERS) probes with at least nanomolar sensitivity. The probe enables detection of proteolytic activity in extremely small volume and at low concentration. In certain embodiments the probes comprise an indicator for the detection of an active protease, where the indicator comprises a nanocrescent attached to a peptide, where said peptide comprises a recognition site for the protease and a Raman tag attached to the peptide.

  13. Pretreatment resistance to hepatitis C virus protease inhibitors boceprevir/telaprevir in hepatitis C virus subgenotype 1a-infected patients from Manitoba.

    PubMed

    Andonov, Anton; Kadkhoda, Kamran; Osiowy, Carla; Kaita, Kelly

    2013-07-01

    Traditional therapy with pegylated interferon and ribavirin combined with the new protease inhibitors boceprevir or telaprevir has demonstrated improved outcomes in hepatitis C virus (HCV)-infected patients. Prevalence data regarding pre-existing drug-resistant variants to these two new virus inhibitors in the Canadian population are not available. To detect pre-existing mutations conferring resistance to boceprevir and⁄or telaprevir in Canadian patients infected with HCV genotype 1a. Resistance-associated mutations (RAMs) were evaluated in 85 patients infected with HCV genotype 1a who had not yet received antiviral therapy. The NS3 protease gene was sequenced and common RAMs were identified based on a recently published list. The overall prevalence of pre-existing RAMs to boceprevir and telaprevir was higher compared with other similar studies. All of the observed RAMs were associated with a low level of resistance. A surprisingly high proportion of patients had the V55A RAM (10.6%). None of the mutations associated with a high level of resistance were observed. The simultaneous presence of two low-level resistance mutations (V36L and V55A) was observed in only one patient. Three other patients had both T54S RAM and V55I mutations, which may require a higher concentration of the protease drugs. The prevalence of various mutations in Aboriginal Canadian patients was higher (37.5%) compared with Caucasians (16.39%) (P=0.038). The present study was the first to investigate pre-existing drug resistance to boceprevir⁄telaprevir in Canadian HCV-infected patients. A relatively high proportion of untreated HCV genotype 1a patients in Manitoba harbour low-level RAMs, especially patients of Aboriginal descent, which may contribute to an increased risk of treatment failure.

  14. USP7 Attenuates Hepatic Gluconeogenesis Through Modulation of FoxO1 Gene Promoter Occupancy

    PubMed Central

    Hall, Jessica A.; Tabata, Mitsuhisa; Rodgers, Joseph T.

    2014-01-01

    Hepatic forkhead protein FoxO1 is a key component of systemic glucose homeostasis via its ability to regulate the transcription of rate-limiting enzymes in gluconeogenesis. Important in the regulation of FoxO1 transcriptional activity are the modifying/demodifying enzymes that lead to posttranslational modification. Here, we demonstrate the functional interaction and regulation of FoxO1 by herpesvirus-associated ubiquitin-specific protease 7 (USP7; also known as herpesvirus-associated ubiquitin-specific protease, HAUSP), a deubiquitinating enzyme. We show that USP7-mediated mono-deubiquitination of FoxO1 results in suppression of FoxO1 transcriptional activity through decreased FoxO1 occupancy on the promoters of gluconeogenic genes. Knockdown of USP7 in primary hepatocytes leads to increased expression of FoxO1-target gluconeogenic genes and elevated glucose production. Consistent with this, USP7 gain-of-function suppresses the fasting/cAMP-induced activation of gluconeogenic genes in hepatocyte cells and in mouse liver, resulting in decreased hepatic glucose production. Notably, we show that the effects of USP7 on hepatic glucose metabolism depend on FoxO1. Together, these results place FoxO1 under the intimate regulation of deubiquitination and glucose metabolic control with important implication in diseases such as diabetes. PMID:24694308

  15. Cyclic strain increases protease-activated receptor-1 expression in vascular smooth muscle cells

    NASA Technical Reports Server (NTRS)

    Nguyen, K. T.; Frye, S. R.; Eskin, S. G.; Patterson, C.; Runge, M. S.; McIntire, L. V.

    2001-01-01

    Cyclic strain regulates many vascular smooth muscle cell (VSMC) functions through changing gene expression. This study investigated the effects of cyclic strain on protease-activated receptor-1 (PAR-1) expression in VSMCs and the possible signaling pathways involved, on the basis of the hypothesis that cyclic strain would enhance PAR-1 expression, reflecting increased thrombin activity. Uniaxial cyclic strain (1 Hz, 20%) of cells cultured on elastic membranes induced a 2-fold increase in both PAR-1 mRNA and protein levels. Functional activity of PAR-1, as assessed by cell proliferation in response to thrombin, was also increased by cyclic strain. In addition, treatment of cells with antioxidants or an NADPH oxidase inhibitor blocked strain-induced PAR-1 expression. Preincubation of cells with protein kinase inhibitors (staurosporine or Ro 31-8220) enhanced strain-increased PAR-1 expression, whereas inhibitors of NO synthase, tyrosine kinase, and mitogen-activated protein kinases had no effect. Cyclic strain in the presence of basic fibroblast growth factor induced PAR-1 mRNA levels beyond the effect of cyclic strain alone, whereas no additive effect was observed between cyclic strain and platelet-derived growth factor-AB. Our findings that cyclic strain upregulates PAR-1 mRNA expression but that shear stress downregulates this gene in VSMCs provide an opportunity to elucidate signaling differences by which VSMCs respond to different mechanical forces.

  16. Proteases and protease inhibitors of urinary extracellular vesicles in diabetic nephropathy.

    PubMed

    Musante, Luca; Tataruch, Dorota; Gu, Dongfeng; Liu, Xinyu; Forsblom, Carol; Groop, Per-Henrik; Holthofer, Harry

    2015-01-01

    Diabetic nephropathy (DN) is one of the major complications of diabetes mellitus (DM), leads to chronic kidney disease (CKD), and, ultimately, is the main cause for end-stage kidney disease (ESKD). Beyond urinary albumin, no reliable biomarkers are available for accurate early diagnostics. Urinary extracellular vesicles (UEVs) have recently emerged as an interesting source of diagnostic and prognostic disease biomarkers. Here we used a protease and respective protease inhibitor array to profile urines of type 1 diabetes patients at different stages of kidney involvement. Urine samples were divided into groups based on the level of albuminuria and UEVs isolated by hydrostatic dialysis and screened for relative changes of 34 different proteases and 32 protease inhibitors, respectively. Interestingly, myeloblastin and its natural inhibitor elafin showed an increase in the normo- and microalbuminuric groups. Similarly, a characteristic pattern was observed in the array of protease inhibitors, with a marked increase of cystatin B, natural inhibitor of cathepsins L, H, and B as well as of neutrophil gelatinase-associated Lipocalin (NGAL) in the normoalbuminuric group. This study shows for the first time the distinctive alterations in comprehensive protease profiles of UEVs in diabetic nephropathy and uncovers intriguing mechanistic, prognostic, and diagnostic features of kidney damage in diabetes.

  17. Proteases and Protease Inhibitors of Urinary Extracellular Vesicles in Diabetic Nephropathy

    PubMed Central

    Tataruch, Dorota; Gu, Dongfeng; Liu, Xinyu; Forsblom, Carol; Groop, Per-Henrik; Holthofer, Harry

    2015-01-01

    Diabetic nephropathy (DN) is one of the major complications of diabetes mellitus (DM), leads to chronic kidney disease (CKD), and, ultimately, is the main cause for end-stage kidney disease (ESKD). Beyond urinary albumin, no reliable biomarkers are available for accurate early diagnostics. Urinary extracellular vesicles (UEVs) have recently emerged as an interesting source of diagnostic and prognostic disease biomarkers. Here we used a protease and respective protease inhibitor array to profile urines of type 1 diabetes patients at different stages of kidney involvement. Urine samples were divided into groups based on the level of albuminuria and UEVs isolated by hydrostatic dialysis and screened for relative changes of 34 different proteases and 32 protease inhibitors, respectively. Interestingly, myeloblastin and its natural inhibitor elafin showed an increase in the normo- and microalbuminuric groups. Similarly, a characteristic pattern was observed in the array of protease inhibitors, with a marked increase of cystatin B, natural inhibitor of cathepsins L, H, and B as well as of neutrophil gelatinase-associated Lipocalin (NGAL) in the normoalbuminuric group. This study shows for the first time the distinctive alterations in comprehensive protease profiles of UEVs in diabetic nephropathy and uncovers intriguing mechanistic, prognostic, and diagnostic features of kidney damage in diabetes. PMID:25874235

  18. Analysis of correlated mutations in HIV-1 protease using spectral clustering.

    PubMed

    Liu, Ying; Eyal, Eran; Bahar, Ivet

    2008-05-15

    The ability of human immunodeficiency virus-1 (HIV-1) protease to develop mutations that confer multi-drug resistance (MDR) has been a major obstacle in designing rational therapies against HIV. Resistance is usually imparted by a cooperative mechanism that can be elucidated by a covariance analysis of sequence data. Identification of such correlated substitutions of amino acids may be obscured by evolutionary noise. HIV-1 protease sequences from patients subjected to different specific treatments (set 1), and from untreated patients (set 2) were subjected to sequence covariance analysis by evaluating the mutual information (MI) between all residue pairs. Spectral clustering of the resulting covariance matrices disclosed two distinctive clusters of correlated residues: the first, observed in set 1 but absent in set 2, contained residues involved in MDR acquisition; and the second, included those residues differentiated in the various HIV-1 protease subtypes, shortly referred to as the phylogenetic cluster. The MDR cluster occupies sites close to the central symmetry axis of the enzyme, which overlap with the global hinge region identified from coarse-grained normal-mode analysis of the enzyme structure. The phylogenetic cluster, on the other hand, occupies solvent-exposed and highly mobile regions. This study demonstrates (i) the possibility of distinguishing between the correlated substitutions resulting from neutral mutations and those induced by MDR upon appropriate clustering analysis of sequence covariance data and (ii) a connection between global dynamics and functional substitution of amino acids.

  19. Design and Synthesis of Piperazine Sulfonamide Cores Leading to Highly Potent HIV-1 Protease Inhibitors.

    PubMed

    Bungard, Christopher J; Williams, Peter D; Schulz, Jurgen; Wiscount, Catherine M; Holloway, M Katharine; Loughran, H Marie; Manikowski, Jesse J; Su, Hua-Poo; Bennett, David J; Chang, Lehua; Chu, Xin-Jie; Crespo, Alejandro; Dwyer, Michael P; Keertikar, Kartik; Morriello, Gregori J; Stamford, Andrew W; Waddell, Sherman T; Zhong, Bin; Hu, Bin; Ji, Tao; Diamond, Tracy L; Bahnck-Teets, Carolyn; Carroll, Steven S; Fay, John F; Min, Xu; Morris, William; Ballard, Jeanine E; Miller, Michael D; McCauley, John A

    2017-12-14

    Using the HIV-1 protease binding mode of MK-8718 and PL-100 as inspiration, a novel aspartate binding bicyclic piperazine sulfonamide core was designed and synthesized. The resulting HIV-1 protease inhibitor containing this core showed an 60-fold increase in enzyme binding affinity and a 10-fold increase in antiviral activity relative to MK-8718 .

  20. Subtype-Specific Differences in Gag-Protease-Driven Replication Capacity Are Consistent with Intersubtype Differences in HIV-1 Disease Progression.

    PubMed

    Kiguoya, Marion W; Mann, Jaclyn K; Chopera, Denis; Gounder, Kamini; Lee, Guinevere Q; Hunt, Peter W; Martin, Jeffrey N; Ball, T Blake; Kimani, Joshua; Brumme, Zabrina L; Brockman, Mark A; Ndung'u, Thumbi

    2017-07-01

    There are marked differences in the spread and prevalence of HIV-1 subtypes worldwide, and differences in clinical progression have been reported. However, the biological reasons underlying these differences are unknown. Gag-protease is essential for HIV-1 replication, and Gag-protease-driven replication capacity has previously been correlated with disease progression. We show that Gag-protease replication capacity correlates significantly with that of whole isolates ( r = 0.51; P = 0.04), indicating that Gag-protease is a significant contributor to viral replication capacity. Furthermore, we investigated subtype-specific differences in Gag-protease-driven replication capacity using large well-characterized cohorts in Africa and the Americas. Patient-derived Gag-protease sequences were inserted into an HIV-1 NL4-3 backbone, and the replication capacities of the resulting recombinant viruses were measured in an HIV-1-inducible reporter T cell line by flow cytometry. Recombinant viruses expressing subtype C Gag-proteases exhibited substantially lower replication capacities than those expressing subtype B Gag-proteases ( P < 0.0001); this observation remained consistent when representative Gag-protease sequences were engineered into an HIV-1 subtype C backbone. We identified Gag residues 483 and 484, located within the Alix-binding motif involved in virus budding, as major contributors to subtype-specific replicative differences. In East African cohorts, we observed a hierarchy of Gag-protease-driven replication capacities, i.e., subtypes A/C < D < intersubtype recombinants ( P < 0.0029), which is consistent with reported intersubtype differences in disease progression. We thus hypothesize that the lower Gag-protease-driven replication capacity of subtypes A and C slows disease progression in individuals infected with these subtypes, which in turn leads to greater opportunity for transmission and thus increased prevalence of these subtypes. IMPORTANCE HIV-1 subtypes

  1. Expression and characterization of plant aspartic protease nepenthesin-1 from Nepenthes gracilis.

    PubMed

    Kadek, Alan; Tretyachenko, Vyacheslav; Mrazek, Hynek; Ivanova, Ljubina; Halada, Petr; Rey, Martial; Schriemer, David C; Man, Petr

    2014-03-01

    Carnivorous plants of the genus Nepenthes produce their own aspartic proteases, nepenthesins, to digest prey trapped in their pitchers. Nepenthesins differ significantly in sequence from other aspartic proteases in the animal or even plant kingdoms. This difference, which also brings more cysteine residues into the structure of these proteases, can be a cause of uniquely high temperature and pH stabilities of nepenthesins. Their detailed structure characterization, however, has not previously been possible due to low amounts of protease present in the pitcher fluid and also due to limited accessibility of Nepenthes plants. In the present study we describe a convenient way for obtaining high amounts of nepenthesin-1 from Nepenthes gracilis using heterologous production in Escherichia coli. The protein can be easily refolded in vitro and its characteristics are very close to those described for a natural enzyme isolated from the pitcher fluid. Similarly to the natural enzyme, recombinant nepenthesin-1 is sensitive to denaturing and reducing agents. It also has maximal activity around pH 2.5, shows unusual stability at high pH and its activity is not irreversibly inhibited even after prolonged incubation in the basic pH range. On the other hand, temperature stability of the recombinant enzyme is lower in comparison with the natural enzyme, which can be attributed to missing N-glycosylation in the recombinant protein. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. Plasmodium subtilisin-like protease 1 (SUB1): insights into the active-site structure, specificity and function of a pan-malaria drug target.

    PubMed

    Withers-Martinez, Chrislaine; Suarez, Catherine; Fulle, Simone; Kher, Samir; Penzo, Maria; Ebejer, Jean-Paul; Koussis, Kostas; Hackett, Fiona; Jirgensons, Aigars; Finn, Paul; Blackman, Michael J

    2012-05-15

    Release of the malaria merozoite from its host erythrocyte (egress) and invasion of a fresh cell are crucial steps in the life cycle of the malaria pathogen. Subtilisin-like protease 1 (SUB1) is a parasite serine protease implicated in both processes. In the most dangerous human malarial species, Plasmodium falciparum, SUB1 has previously been shown to have several parasite-derived substrates, proteolytic cleavage of which is important both for egress and maturation of the merozoite surface to enable invasion. Here we have used molecular modelling, existing knowledge of SUB1 substrates, and recombinant expression and characterisation of additional Plasmodium SUB1 orthologues, to examine the active site architecture and substrate specificity of P. falciparum SUB1 and its orthologues from the two other major human malaria pathogens Plasmodium vivax and Plasmodium knowlesi, as well as from the rodent malaria species, Plasmodium berghei. Our results reveal a number of unusual features of the SUB1 substrate binding cleft, including a requirement to interact with both prime and non-prime side residues of the substrate recognition motif. Cleavage of conserved parasite substrates is mediated by SUB1 in all parasite species examined, and the importance of this is supported by evidence for species-specific co-evolution of protease and substrates. Two peptidyl alpha-ketoamides based on an authentic PfSUB1 substrate inhibit all SUB1 orthologues examined, with inhibitory potency enhanced by the presence of a carboxyl moiety designed to introduce prime side interactions with the protease. Our findings demonstrate that it should be possible to develop 'pan-reactive' drug-like compounds that inhibit SUB1 in all three major human malaria pathogens, enabling production of broad-spectrum antimalarial drugs targeting SUB1. Copyright © 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

  3. Comparative one-factor-at-a-time, response surface (statistical) and bench-scale bioreactor level optimization of thermoalkaline protease production from a psychrotrophic Pseudomonas putida SKG-1 isolate.

    PubMed

    Singh, Santosh K; Singh, Sanjay K; Tripathi, Vinayak R; Khare, Sunil K; Garg, Satyendra K

    2011-12-28

    Production of alkaline protease from various bacterial strains using statistical methods is customary now-a-days. The present work is first attempt for the production optimization of a solvent stable thermoalkaline protease by a psychrotrophic Pseudomonas putida isolate using conventional, response surface methods, and fermentor level optimization. The pre-screening medium amended with optimized (w/v) 1.0% glucose, 2.0% gelatin and 0.5% yeast extract, produced 278 U protease ml(-1) at 72 h incubation. Enzyme production increased to 431 Uml(-1) when Mg2+ (0.01%, w/v) was supplemented. Optimization of physical factors further enhanced protease to 514 Uml(-1) at pH 9.0, 25°C and 200 rpm within 60 h. The combined effect of conventionally optimized variables (glucose, yeast extract, MgSO4 and pH), thereafter predicted by response surface methodology yielded 617 U protease ml(-1) at glucose 1.25% (w/v), yeast extract 0.5% (w/v), MgSO4 0.01% (w/v) and pH 8.8. Bench-scale bioreactor level optimization resulted in enhanced production of 882 U protease ml(-1) at 0.8 vvm aeration and 150 rpm agitation during only 48 h incubation. The optimization of fermentation variables using conventional, statistical approaches and aeration/agitation at fermentor level resulted in ~13.5 folds increase (882 Uml(-1)) in protease production compared to un-optimized conditions (65 Uml(-1)). This is the highest level of thermoalkaline protease reported so far by any psychrotrophic bacterium.

  4. Serine proteases SP1 and SP13 mediate the melanization response of Asian corn borer, Ostrinia furnacalis, against entomopathogenic fungus Beauveria bassiana.

    PubMed

    Chu, Yuan; Liu, Yang; Shen, Dongxu; Hong, Fang; Wang, Guirong; An, Chunju

    2015-06-01

    Exposure to entomopathogenic fungi is one approach for insect pest control. Little is known about the immune interactions between fungus and its insect host. Melanization is a prominent immune response in insects in defending against pathogens such as bacteria and fungi. Clip domain serine proteases in insect plasma have been implicated in the activation of prophenoloxidase, a key enzyme in the melanization. The relationship between host melanization and the infection by a fungus needs to be established. We report here that the injection of entomopathogenic fungus Beauveria bassiana induced both melanin synthesis and phenoloxidase activity in its host insect, the Asian corn borer, Ostrinia furnacalis (Guenée). qRT-PCR analysis showed several distinct patterns of expression of 13 clip-domain serine proteases in response to the challenge of fungi, with seven increased, two decreased, and four unchanged. Of special interest among these clip-domain serine protease genes are SP1 and SP13, the orthologs of Manduca sexta HP6 and PAP1 which are involved in the prophenoloxidase activation pathway. Recombinant O. furnacalis SP1 was found to activate proSP13 and induce the phenoloxidase activity in corn borer plasma. Additionally, SP13 was determined to directly cleave prophenoloxidase and therefore act as the prophenoloxidase activating protease. Our work thus reveals a biochemical mechanism in the melanization in corn borer associated with the challenge by B. bassiana injection. These insights could provide valuable information for better understanding the immune responses of Asian corn borer against B. bassiana. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. The Occurrence of Type S1A Serine Proteases in Sponge and Jellyfish

    NASA Technical Reports Server (NTRS)

    Rojas, Ana; Doolittle, Russell F.

    2003-01-01

    Although serine proteases are found in all kinds of cellular organisms and many viruses, the classic "chymotrypsin family" (Group S1A by th e 1998 Barrett nomenclature) has an unusual phylogenetic distribution , being especially common in animals, entirely absent from plants and protists, and rare among fungi. The distribution in Bacteria is larg ely restricted to the genus Streptomyces, although a few isolated occ urrences in other bacteria have been reported. The family may be enti rely absent from Archaea. Although more than a thousand sequences have been reported for enzymes of this type from animals, none of them ha ve been from early diverging phyla like Porifera or Cnidaria, We now report the existence of Group SlA serine proteases in a sponge (phylu m Porifera) and a jellyfish (phylum Cnidaria), making it safe to conc lude that all animal groups possess these enzymes.

  6. Proteases in doping control analysis.

    PubMed

    Thevis, M; Maurer, J; Kohler, M; Geyer, H; Schänzer, W

    2007-07-01

    Urine manipulation in sports drug testing has become a serious problem for doping control laboratories, and recent scandals in elite endurance sports have revealed the problem of urine manipulation presumably using proteases, which will impede the detection of drugs such as erythropoietin (EPO) or other peptide hormones. Using commonly accepted analytical strategies, a protocol was developed enabling the determination of elevated protease activities in doping control specimens followed by the visualization of protein degradation and identification of proteases such as chymotrypsin, trypsin and papain. Therefore, protease detection kits based on fluorescein isothiocyanate-labeled casein were employed, and protease concentrations greater than 15 microg/mL of urine entailed subsequent 1-dimensional gel electrophoretic visualization of urinary proteins. The presence of 20 microg of proteases per mL of urine caused a complete degradation of proteins usually observed in urinary matrices ("trace of burning"), while respective proteases were still detected in spiked urine samples after 10 days of storage at + 4 and - 20 degrees C. Identification of target proteases at respective molecular weights was accomplished using bottom-up sequencing approaches based on in-gel digestion of separated enzymes followed by capillary liquid chromatography--Orbitrap tandem mass spectrometry.

  7. Proteases in agricultural dust induce lung inflammation through PAR-1 and PAR-2 activation.

    PubMed

    Romberger, Debra J; Heires, Art J; Nordgren, Tara M; Souder, Chelsea P; West, William; Liu, Xiang-de; Poole, Jill A; Toews, Myron L; Wyatt, Todd A

    2015-08-15

    Workers exposed to aerosolized dust present in concentrated animal feeding operations (CAFOs) are susceptible to inflammatory lung diseases, such as chronic obstructive pulmonary disease. Extracts of dust collected from hog CAFOs [hog dust extract (HDE)] are potent stimulators of lung inflammatory responses in several model systems. The observation that HDE contains active proteases prompted the present study, which evaluated the role of CAFO dust proteases in lung inflammatory processes and tested whether protease-activated receptors (PARs) are involved in the signaling pathway for these events. We hypothesized that the damaging proinflammatory effect of HDE is due, in part, to the proteolytic activation of PARs, and inhibiting the proteases in HDE or disrupting PAR activation would attenuate HDE-mediated inflammatory indexes in bronchial epithelial cells (BECs), in mouse lung slices in vitro, and in a murine in vivo exposure model. Human BECs and mouse lung slice cultures stimulated with 5% HDE released significantly more of each of the cytokines measured (IL-6, IL-8, TNF-α, keratinocyte-derived chemokine/CXC chemokine ligand 1, and macrophage inflammatory protein-2/CXC chemokine ligand 2) than controls, and these effects were markedly diminished by protease inhibition. Inhibition of PARs also blunted the HDE-induced cytokine release from BECs. In addition, protease depletion inhibited HDE-induced BEC intracellular PKCα and PKCε activation. C57BL/6J mice administered 12.5% HDE intranasally, either once or daily for 3 wk, exhibited increased total cellular and neutrophil influx, bronchial alveolar fluid inflammatory cytokines, lung histopathology, and inflammatory scores compared with mice receiving protease-depleted HDE. These data suggest that proteases in dust from CAFOs are important mediators of lung inflammation, and these proteases and their receptors may provide novel targets for therapeutic intervention in CAFO dust-induced airways disease.

  8. Genome-wide analysis of regulatory proteases sequences identified through bioinformatics data mining in Taenia solium.

    PubMed

    Yan, Hong-Bin; Lou, Zhong-Zi; Li, Li; Brindley, Paul J; Zheng, Yadong; Luo, Xuenong; Hou, Junling; Guo, Aijiang; Jia, Wan-Zhong; Cai, Xuepeng

    2014-06-04

    Cysticercosis remains a major neglected tropical disease of humanity in many regions, especially in sub-Saharan Africa, Central America and elsewhere. Owing to the emerging drug resistance and the inability of current drugs to prevent re-infection, identification of novel vaccines and chemotherapeutic agents against Taenia solium and related helminth pathogens is a public health priority. The T. solium genome and the predicted proteome were reported recently, providing a wealth of information from which new interventional targets might be identified. In order to characterize and classify the entire repertoire of protease-encoding genes of T. solium, which act fundamental biological roles in all life processes, we analyzed the predicted proteins of this cestode through a combination of bioinformatics tools. Functional annotation was performed to yield insights into the signaling processes relevant to the complex developmental cycle of this tapeworm and to highlight a suite of the proteases as potential intervention targets. Within the genome of this helminth parasite, we identified 200 open reading frames encoding proteases from five clans, which correspond to 1.68% of the 11,902 protein-encoding genes predicted to be present in its genome. These proteases include calpains, cytosolic, mitochondrial signal peptidases, ubiquitylation related proteins, and others. Many not only show significant similarity to proteases in the Conserved Domain Database but have conserved active sites and catalytic domains. KEGG Automatic Annotation Server (KAAS) analysis indicated that ~60% of these proteases share strong sequence identities with proteins of the KEGG database, which are involved in human disease, metabolic pathways, genetic information processes, cellular processes, environmental information processes and organismal systems. Also, we identified signal peptides and transmembrane helices through comparative analysis with classes of important regulatory proteases

  9. HIV-1 protease cleavage site prediction based on two-stage feature selection method.

    PubMed

    Niu, Bing; Yuan, Xiao-Cheng; Roeper, Preston; Su, Qiang; Peng, Chun-Rong; Yin, Jing-Yuan; Ding, Juan; Li, HaiPeng; Lu, Wen-Cong

    2013-03-01

    Knowledge of the mechanism of HIV protease cleavage specificity is critical to the design of specific and effective HIV inhibitors. Searching for an accurate, robust, and rapid method to correctly predict the cleavage sites in proteins is crucial when searching for possible HIV inhibitors. In this article, HIV-1 protease specificity was studied using the correlation-based feature subset (CfsSubset) selection method combined with Genetic Algorithms method. Thirty important biochemical features were found based on a jackknife test from the original data set containing 4,248 features. By using the AdaBoost method with the thirty selected features the prediction model yields an accuracy of 96.7% for the jackknife test and 92.1% for an independent set test, with increased accuracy over the original dataset by 6.7% and 77.4%, respectively. Our feature selection scheme could be a useful technique for finding effective competitive inhibitors of HIV protease.

  10. A biotechnology perspective of fungal proteases.

    PubMed

    de Souza, Paula Monteiro; Bittencourt, Mona Lisa de Assis; Caprara, Carolina Canielles; de Freitas, Marcela; de Almeida, Renata Paula Coppini; Silveira, Dâmaris; Fonseca, Yris Maria; Ferreira Filho, Edivaldo Ximenes; Pessoa Junior, Adalberto; Magalhães, Pérola Oliveira

    2015-06-01

    Proteases hydrolyze the peptide bonds of proteins into peptides and amino acids, being found in all living organisms, and are essential for cell growth and differentiation. Proteolytic enzymes have potential application in a wide number of industrial processes such as food, laundry detergent and pharmaceutical. Proteases from microbial sources have dominated applications in industrial sectors. Fungal proteases are used for hydrolyzing protein and other components of soy beans and wheat in soy sauce production. Proteases can be produced in large quantities in a short time by established methods of fermentation. The parameters such as variation in C/N ratio, presence of some sugars, besides several other physical factors are important in the development of fermentation process. Proteases of fungal origin can be produced cost effectively, have an advantage faster production, the ease with which the enzymes can be modified and mycelium can be easily removed by filtration. The production of proteases has been carried out using submerged fermentation, but conditions in solid state fermentation lead to several potential advantages for the production of fungal enzymes. This review focuses on the production of fungal proteases, their distribution, structural-functional aspects, physical and chemical parameters, and the use of these enzymes in industrial applications.

  11. HTRA1, an age-related macular degeneration protease, processes extracellular matrix proteins EFEMP1 and TSP1.

    PubMed

    Lin, Michael K; Yang, Jin; Hsu, Chun Wei; Gore, Anuradha; Bassuk, Alexander G; Brown, Lewis M; Colligan, Ryan; Sengillo, Jesse D; Mahajan, Vinit B; Tsang, Stephen H

    2018-05-05

    High-temperature requirement protein A1 (HTRA1) is a serine protease secreted by a number of tissues including retinal pigment epithelium (RPE). A promoter variant of the gene encoding HTRA1 is part of a mutant allele that causes increased HTRA1 expression and contributed to age-related macular degeneration (AMD) in genomewide association studies. AMD is characterized by pathological development of drusen, extracellular deposits of proteins and lipids on the basal side of RPE. The molecular pathogenesis of AMD is not well understood, and understanding dysregulation of the extracellular matrix may be key. We assess the high-risk genotype at 10q26 by proteomic comparison of protein levels of RPE cells with and without the mutation. We show HTRA1 protein level is increased in high-risk RPE cells along with several extracellular matrix proteins, including known HTRA1 cleavage targets LTBP-1 and clusterin. In addition, two novel targets of HTRA1 have been identified: EFEMP1, an extracellular matrix protein mutated in Doyne honeycomb retinal dystrophy, a genetic eye disease similar to AMD, and thrombospondin 1 (TSP1), an inhibitor of angiogenesis. Our data support the role of RPE extracellular deposition with potential effects in compromised barrier to neovascularization in exudative AMD. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  12. Antimalarial activity of HIV-1 protease inhibitor in chromone series.

    PubMed

    Lerdsirisuk, Pradith; Maicheen, Chirattikan; Ungwitayatorn, Jiraporn

    2014-12-01

    Increasing parasite resistance to nearly all available antimalarial drugs becomes a serious problem to human health and necessitates the need to continue the search for new effective drugs. Recent studies have shown that clinically utilized HIV-1 protease (HIV-1 PR) inhibitors can inhibit the in vitro and in vivo growth of Plasmodium falciparum. In this study, a series of chromone derivatives possessing HIV-1 PR inhibitory activity has been tested for antimalarial activity against P. falciparum (K1 multi-drug resistant strain). Chromone 15, the potent HIV-1 PR inhibitor (IC50=0.65μM), was found to be the most potent antimalarial compound with IC50=0.95μM while primaquine and tafenoquine showed IC50=2.41 and 1.95μM, respectively. Molecular docking study of chromone compounds against plasmepsin II, an aspartic protease enzyme important in hemoglobin degradation, revealed that chromone 15 exhibited the higher binding affinity (binding energy=-13.24kcal/mol) than the known PM II inhibitors. Thus, HIV-1 PR inhibitor in chromone series has the potential to be a new class of antimalarial agent. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Plant cysteine proteases that evoke itch activate protease-activated receptors

    PubMed Central

    Reddy, V.B.; Lerner, E.A.

    2013-01-01

    Background Bromelain, ficin and papain are cysteine proteases from plants that produce itch upon injection into skin. Their mechanism of action has not been considered previously. Objectives To determine the mechanism by which these proteases function. Methods The ability of these proteases to activate protease-activated receptors was determined by ratiometric calcium imaging. Results We show here that bromelain, ficin and papain activate protease-activated receptors 2 and 4. Conclusions Bromelain, ficin and papain function as signalling molecules and activate protease-activated receptors. Activation of these receptors is the likely mechanism by which these proteases evoke itch. PMID:20491769

  14. Molecular Cloning and Optimization for High Level Expression of Cold-Adapted Serine Protease from Antarctic Yeast Glaciozyma antarctica PI12

    PubMed Central

    Ahmad Mazian, Mu'adz; Salleh, Abu Bakar; Basri, Mahiran; Rahman, Raja Noor Zaliha Raja Abd.

    2014-01-01

    Psychrophilic basidiomycete yeast, Glaciozyma antarctica strain PI12, was shown to be a protease-producer. Isolation of the PI12 protease gene from genomic and mRNA sequences allowed determination of 19 exons and 18 introns. Full-length cDNA of PI12 protease gene was amplified by rapid amplification of cDNA ends (RACE) strategy with an open reading frame (ORF) of 2892 bp, coded for 963 amino acids. PI12 protease showed low homology with the subtilisin-like protease from fungus Rhodosporidium toruloides (42% identity) and no homology to other psychrophilic proteases. The gene encoding mature PI12 protease was cloned into Pichia pastoris expression vector, pPIC9, and positioned under the induction of methanol-alcohol oxidase (AOX) promoter. The recombinant PI12 protease was efficiently secreted into the culture medium driven by the Saccharomyces cerevisiae α-factor signal sequence. The highest protease production (28.3 U/ml) was obtained from P. pastoris GS115 host (GpPro2) at 20°C after 72 hours of postinduction time with 0.5% (v/v) of methanol inducer. The expressed protein was detected by SDS-PAGE and activity staining with a molecular weight of 99 kDa. PMID:25093119

  15. H1-A, a compound isolated from Fusarium oxysporum inhibits hepatitis C virus (HCV) NS3 serine protease.

    PubMed

    Yang, Li-Yuan; Lin, Jun; Zhou, Bin; Liu, Yan-Gang; Zhu, Bao-Quan

    2016-04-01

    The present study was aimed to isolate the active compounds from the fermentation products of Fusarium oxysporum, which had hepatitis C virus (HCV) NS3 protease inhibitory activity. A bioactive compound was isolated by reverse-phase silica-gel column chromatography, silica-gel column chromatography, semi-preparative reverse-phase High Performance Liquid Chromatography (HPLC), and then its molecular structure was elucidated based on the spectrosopic analysis. As a result, the compound (H1-A, 1) Ergosta-5, 8 (14), 22-trien-7-one, 3-hydroxy-,(3β, 22E) was isolated and identified. To the best of our knowledge, this was the first report on the isolation of H1-A from microorganisms with the inhibitory activity of NS3 protease. Copyright © 2016 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

  16. Differential susceptibility of Dectin-1 isoforms to functional inactivation by neutrophil and fungal proteases.

    PubMed

    Griffiths, James S; Thompson, Aiysha; Stott, Matthew; Benny, Ankita; Lewis, Natalie A; Taylor, Philip R; Forton, Julian; Herrick, Sarah; Orr, Selinda J; McGreal, Eamon P

    2018-06-01

    Patients with cystic fibrosis (CF) experience chronic or recurrent bacterial and fungal lung infections. Many patients with CF cannot effectively clear Aspergillus from their lungs. This may result in IgE sensitization and the development of allergic bronchopulmonary aspergillosis, or invasive infections, such as Aspergillus bronchitis. Lung disease in patients with CF is associated with neutrophil-dominated inflammation and elevated levels of the serine protease, neutrophil elastase (NE). Various C-type lectin-like receptors (CLRs), including Dectin-1 and Dectin-2, are involved in the immune response to Aspergillus. Here, we show that purified NE cleaves Dectin-1 in an isoform-specific manner. Bronchoalveolar lavage fluid from patients with CF, which contains high NE activity, induces Dectin-1 cleavage. Similarly, filtrate from a protease-producing strain of Aspergillus fumigatus induces isoform-specific cleavage of Dectin-1. Dectin-1 knockout (KO) cells and NE-treated cells demonstrated reduced phagocytosis of zymosan, a fungal cell wall preparation. In addition, NE cleaves 2 other CLRs, Dectin-2 and Mincle, and fungal-induced cytokine production was reduced in Dectin-1 KO cells, Dectin-2 KO cells, and NE-treated cells. Thus, Dectin-1 and Dectin-2 cleavage by NE and/or A. fumigatus-derived proteases results in an aberrant antifungal immune response that likely contributes to disease pathology in patients with CF.-Griffiths, J. S., Thompson, A., Stott, M., Benny, A., Lewis, N. A., Taylor, P. R., Forton, J., Herrick, S., Orr, S. J., McGreal, E. P. Differential susceptibility of Dectin-1 isoforms to functional inactivation by neutrophil and fungal proteases.

  17. Insect response to plant defensive protease inhibitors.

    PubMed

    Zhu-Salzman, Keyan; Zeng, Rensen

    2015-01-07

    Plant protease inhibitors (PIs) are natural plant defense proteins that inhibit proteases of invading insect herbivores. However, their anti-insect efficacy is determined not only by their potency toward a vulnerable insect system but also by the response of the insect to such a challenge. Through the long history of coevolution with their host plants, insects have developed sophisticated mechanisms to circumvent antinutritional effects of dietary challenges. Their response takes the form of changes in gene expression and the protein repertoire in cells lining the alimentary tract, the first line of defense. Research in insect digestive proteases has revealed the crucial roles they play in insect adaptation to plant PIs and has brought about a new appreciation of how phytophagous insects employ this group of molecules in both protein digestion and counterdefense. This review provides researchers in related fields an up-to-date summary of recent advances.

  18. Purification and Characterization of a Protease Produced by a Planomicrobium sp. L-2 from Gut of Octopus vulgaris

    PubMed Central

    Liu, Qing; Sun, Shujing; Piao, Meizi; Yang, Ji Young

    2013-01-01

    Protease widely exists in the digestive tract of animals and humans, playing a very important role in protein digestion and absorption. In this study, a high protease-producing strain Planomicrobium sp. L-2 was isolated and identified from the digestive tract of Octopus variabilis. The strain was identified by physiological and biochemical experiments and 16S rDNA sequences analysis. A protease was obtained from the strain Planomicrobium sp. L-2 through ammonium sulfate precipitation, dialysis and enrichment, DEAE-Sephadex A50 anion-exchange chromatography, and Sephadex G-100 gel chromatography. The molecular weight and properties of the protease were characterized, including optimum temperature and pH, thermal stability, protease inhibitions and metal ions. According to our results, the protease from Planomicrobium sp. L-2 strain designated as F1-1 was obtained by three-step separation and purification from crude enzyme. The molecular weight of the protease was 61.4 kDa and its optimum temperature was 40°C. The protease F1-1 showed a broad pH profile for casein hydrolysis between 5.0~11.0. No residual activity was observed after incubation for 40 min at 60°C and 60 min at 50°C. F1-1 protease was inhibited by Mn2+, Hg2+, Pb2+, Zn2+, and Cu2+ ions, as well as PMSF, indicating that the protease F1-1 was a serine protease. Additionally, research basis provided by this study could be considered for industrial application of octopus intestinal proteases. PMID:24551830

  19. Serine proteases in rodent hippocampus.

    PubMed

    Davies, B J; Pickard, B S; Steel, M; Morris, R G; Lathe, R

    1998-09-04

    Brain serine proteases are implicated in developmental processes, synaptic plasticity, and in disorders including Alzheimer's disease. The spectrum of the major enzymes expressed in brain has not been established previously. We now present a systematic study of the serine proteases expressed in adult rat and mouse hippocampus. Using a combination of techniques including polymerase chain reaction amplification and Northern blotting we show that tissue-type plasminogen activator (t-PA) is the major species represented. Unexpectedly, the next most abundant species were RNK-Met-1, a lymphocyte protease not reported previously in brain, and two new family members, BSP1 (brain serine protease 1) and BSP2. We report full-length sequences of the two new proteases; homologies indicate that these are of tryptic specificity. Although BSP2 is expressed in several brain regions, BSP1 expression is strikingly restricted to hippocampus. Other enzymes represented, but at lower levels, included elastase IV, proteinase 3, complement C2, chymotrypsin B, chymotrypsin-like protein, and Hageman factor. Although thrombin and urokinase-type plasminogen activator were not detected in the primary screen, low level expression was confirmed using specific polymerase chain reaction primers. In contrast, and despite robust expression of t-PA, the usual t-PA substrate plasminogen was not expressed at detectable levels.

  20. Characterization of a novel ADAM protease expressed by Pneumocystis carinii.

    PubMed

    Kennedy, Cassie C; Kottom, Theodore J; Limper, Andrew H

    2009-08-01

    Pneumocystis species are opportunistic fungal pathogens that cause severe pneumonia in immunocompromised hosts. Recent evidence has suggested that unidentified proteases are involved in Pneumocystis life cycle regulation. Proteolytically active ADAM (named for "a disintegrin and metalloprotease") family molecules have been identified in some fungal organisms, such as Aspergillus fumigatus and Schizosaccharomyces pombe, and some have been shown to participate in life cycle regulation. Accordingly, we sought to characterize ADAM-like molecules in the fungal opportunistic pathogen, Pneumocystis carinii (PcADAM). After an in silico search of the P. carinii genomic sequencing project identified a 329-bp partial sequence with homology to known ADAM proteins, the full-length PcADAM sequence was obtained by PCR extension cloning, yielding a final coding sequence of 1,650 bp. Sequence analysis detected the presence of a typical ADAM catalytic active site (HEXXHXXGXXHD). Expression of PcADAM over the Pneumocystis life cycle was analyzed by Northern blot. Southern and contour-clamped homogenous electronic field blot analysis demonstrated its presence in the P. carinii genome. Expression of PcADAM was observed to be increased in Pneumocystis cysts compared to trophic forms. The full-length gene was subsequently cloned and heterologously expressed in Saccharomyces cerevisiae. Purified PcADAMp protein was proteolytically active in casein zymography, requiring divalent zinc. Furthermore, native PcADAMp extracted directly from freshly isolated Pneumocystis organisms also exhibited protease activity. This is the first report of protease activity attributable to a specific, characterized protein in the clinically important opportunistic fungal pathogen Pneumocystis.

  1. Proteases as therapeutics

    PubMed Central

    Craik, Charles S.; Page, Michael J.; Madison, Edwin L.

    2015-01-01

    Proteases are an expanding class of drugs that hold great promise. The U.S. FDA (Food and Drug Administration) has approved 12 protease therapies, and a number of next generation or completely new proteases are in clinical development. Although they are a well-recognized class of targets for inhibitors, proteases themselves have not typically been considered as a drug class despite their application in the clinic over the last several decades; initially as plasma fractions and later as purified products. Although the predominant use of proteases has been in treating cardiovascular disease, they are also emerging as useful agents in the treatment of sepsis, digestive disorders, inflammation, cystic fibrosis, retinal disorders, psoriasis and other diseases. In the present review, we outline the history of proteases as therapeutics, provide an overview of their current clinical application, and describe several approaches to improve and expand their clinical application. Undoubtedly, our ability to harness proteolysis for disease treatment will increase with our understanding of protease biology and the molecular mechanisms responsible. New technologies for rationally engineering proteases, as well as improved delivery options, will expand greatly the potential applications of these enzymes. The recognition that proteases are, in fact, an established class of safe and efficacious drugs will stimulate investigation of additional therapeutic applications for these enzymes. Proteases therefore have a bright future as a distinct therapeutic class with diverse clinical applications. PMID:21406063

  2. Carbonic anhydrases, EPF2 and a novel protease mediate CO2 control of stomatal development

    PubMed Central

    Engineer, Cawas B.; Ghassemian, Majid; Anderson, Jeffrey C.; Peck, Scott C.; Hu, Honghong; Schroeder, Julian I.

    2014-01-01

    Environmental stimuli, including elevated carbon dioxide levels, regulate stomatal development1–3; however, the key mechanisms mediating the perception and relay of the CO2 signal to the stomatal development machinery remain elusive. To adapt CO2 intake to water loss, plants regulate the development of stomatal gas exchange pores in the aerial epidermis. A diverse range of plant species show a decrease in stomatal density in response to the continuing rise in atmospheric CO2 (ref. 4). To date, one mutant that exhibits deregulation of this CO2-controlled stomatal development response, hic (which is defective in cell-wall wax biosynthesis, ref. 5), has been identified. Here we show that recently isolated Arabidopsis thaliana β-carbonic anhydrase double mutants (ca1 ca4)6 exhibit aninversion in their response to elevated CO2, showing increased stomatal development at elevated CO2 levels. We characterized the mechanisms mediating this response and identified an extracellular signalling pathway involved in the regulation of CO2-controlled stomatal development by carbonic anhydrases. RNA-seq analyses of transcripts show that the extracellular pro-peptide-encoding gene EPIDERMAL PATTERNING FACTOR 2 (EPF2)7,8, but not EPF1 (ref. 9), is induced in wild-type leaves but not inca1 ca4 mutant leaves at elevated CO2 levels. Moreover, EPF2 is essential for CO2 control of stomatal development. Using cell-wall proteomic analyses and CO2-dependent transcriptomic analyses, we identified a novel CO2-induced extracellular protease, CRSP (CO2 RESPONSE SECRETED PROTEASE), as a mediator of CO2-controlled stomatal development. Our results identify mechanisms and genes that function in the repression of stomatal development in leaves during atmospheric CO2 elevation, including the carbonic-anhydrase-encoding genes CA1 and CA4 and the secreted protease CRSP, which cleaves the pro-peptide EPF2, in turn repressing stomatal development. Elucidation of these mechanisms advances the

  3. The role played by serine proteases in the development and worsening of vascular complications in type 1 diabetes mellitus.

    PubMed

    Finotti, Paola

    2006-08-01

    Much attention has been given to the role played by serine proteases in the development and worsening of vascular complications in Type 1 diabetes mellitus. A generalized increase in proteolytic activity, either due to a true increase in concentration of specific proteases or defects of their protease inhibitors, represents an early marker of diabetes. However, the precise molecular mechanism whereby an unopposed proteolytic activity leads to overt vascular alterations has not fully been elucidated as yet. The picture is further complicated by the fact that, although sharing the same function, serine proteases constitute a structurally heterogeneous class of molecules. Besides classical proteases, for most part belonging to coagulative and fibrinolytic systems, other unrelated molecules exhibit serine-like protease activity and are capable of triggering both inflammatory and immune reactions. The specific role of these non classical serine proteases in the complex pathogenesis of diabetes and its vascular complications is attracting a new investigative interest, as these molecules may represent additional therapeutic targets. This review will focus on most recent acquisitions on this issue relevant to Type 1 diabetes.

  4. Molecular cloning, sequence and structural analysis of dehairing Mn(2+) dependent alkaline serine protease (MASPT) of Bacillus pumilus TMS55.

    PubMed

    Ibrahim, Kalibulla Syed; Muniyandi, Jeyaraj; Pandian, Shunmugiah Karutha

    2011-10-01

    Leather industries release a large amount of pollution-causing chemicals which creates one of the major industrial pollutions. The development of enzyme based processes as a potent alternative to pollution-causing chemicals is useful to overcome this issue. Proteases are enzymes which have extensive applications in leather processing and in several bioremediation processes due to their high alkaline protease activity and dehairing efficacy. In the present study, we report cloning, characterization of a Mn2+ dependent alkaline serine protease gene (MASPT) of Bacillus pumilus TMS55. The gene encoding the protease from B. pumilus TMS55 was cloned and its nucleotide sequence was determined. This gene has an open reading frame (ORF) of 1,149 bp that encodes a polypeptide of 383 amino acid residues. Our analysis showed that this polypeptide is composed of 29 residues N-terminal signal peptide, a propeptide of 79 residues and a mature protein of 275 amino acids. We performed bioinformatics analysis to compare MASPT enzyme with other proteases. Homology modeling was employed to model three dimensional structure for MASPT. Structural analysis showed that MASPT structure is composed of nine α-helices and nine β-strands. It has 3 catalytic residues and 14 metal binding residues. Docking analysis showed that residues S223, A260, N263, T328 and S329 interact with Mn2+. This study allows initial inferences about the structure of the protease and will allow the rational design of its derivatives for structure-function studies and also for further improvement of the enzyme.

  5. The Chloroplast Protease AMOS1/EGY1 Affects Phosphate Homeostasis under Phosphate Stress1

    PubMed Central

    Yu, Fang Wei; Zhu, Xiao Fang; Li, Guang Jie; Kronzucker, Herbert J.; Shi, Wei Ming

    2016-01-01

    Plastid intramembrane proteases in Arabidopsis (Arabidopsis thaliana) are involved in jasmonic acid biosynthesis, chloroplast development, and flower morphology. Here, we show that Ammonium-Overly-Sensitive1 (AMOS1), a member of the family of plastid intramembrane proteases, plays an important role in the maintenance of phosphate (P) homeostasis under P stress. Loss of function of AMOS1 revealed a striking resistance to P starvation. amos1 plants displayed retarded root growth and reduced P accumulation in the root compared to wild type (Col-0) under P-replete control conditions, but remained largely unaffected by P starvation, displaying comparable P accumulation and root and shoot growth under P-deficient conditions. Further analysis revealed that, under P-deficient conditions, the cell wall, especially the pectin fraction of amos1, released more P than that of wild type, accompanied by a reduction of the abscisic acid (ABA) level and an increase in ethylene production. By using an ABA-insensitive mutant, abi4, and applying ABA and ACC exogenously, we found that ABA inhibits cell wall P remobilization while ethylene facilitates P remobilization from the cell wall by increasing the pectin concentration, suggesting ABA can counteract the effect of ethylene. Furthermore, the elevated ABA level and the lower ethylene production also correlated well with the mimicked P deficiency in amos1. Thus, our study uncovers the role of AMOS1 in the maintenance of P homeostasis through ABA-antagonized ethylene signaling. PMID:27516532

  6. Bioprocess optimization for production of thermoalkali-stable protease from Bacillus subtilis K-1 under solid-state fermentation.

    PubMed

    Singh, Satbir; Bajaj, Bijender Kumar

    2016-10-02

    Cost-effective production of proteases, which are robust enough to function under harsh process conditions, is always sought after due to their wide industrial application spectra. Solid-state production of enzymes using agro-industrial wastes as substrates is an environment-friendly approach, and it has several advantages such as high productivity, cost-effectiveness, being less labor-intensive, and less effluent production, among others. In the current study, different agro-wastes were employed for thermoalkali-stable protease production from Bacillus subtilis K-1 under solid-state fermentation. Agricultural residues such as cotton seed cake supported maximum protease production (728 U ml(-1)), which was followed by gram husk (714 U ml(-1)), mustard cake (680 U ml(-1)), and soybean meal (653 U ml(-1)). Plackett-Burman design of experiment showed that peptone, moisture content, temperature, phosphates, and inoculum size were the significant variables that influenced the protease production. Furthermore, statistical optimization of three variables, namely peptone, moisture content, and incubation temperature, by response surface methodology resulted in 40% enhanced protease production as compared to that under unoptimized conditions (from initial 728 to 1020 U ml(-1)). Thus, solid-state fermentation coupled with design of experiment tools represents a cost-effective strategy for production of industrial enzymes.

  7. The crystal structure of the secreted aspartic protease 1 from Candida parapsilosis in complex with pepstatin A

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Dostál, Jiří; Brynda, Jiří; Hrušková-Heidingsfeldová, Olga

    2010-09-01

    Opportunistic pathogens of the genus Candida cause infections representing a major threat to long-term survival of immunocompromised patients. Virulence of the Candida pathogens is enhanced by production of extracellular proteolytic enzymes and secreted aspartic proteases (Saps) are therefore studied as potential virulence factors and possible targets for therapeutic drug design. Candida parapsilosis is less invasive than C. albicans, however, it is one of the leading causative agents of yeast infections. We report three-dimensional crystal structure of Sapp1p from C. parapsilosis in complex with pepstatin A, the classical inhibitor of aspartic proteases. The structure of Sapp1p was determined from protein isolatedmore » from its natural source and represents the first structure of Sap from C. parapsilosis. Overall fold and topology of Sapp1p is very similar to the archetypic fold of monomeric aspartic protease family and known structures of Sap isoenzymes from C. albicans and Sapt1p from C. tropicalis. Structural comparison revealed noticeable differences in the structure of loops surrounding the active site. This resulted in differential character, shape, and size of the substrate binding site explaining divergent substrate specificities and inhibitor affinities. Determination of structures of Sap isoenzymes from various species might contribute to the development of new Sap-specific inhibitors.« less

  8. Non-ribosomal halogenated protease inhibitors from cyanobacterial isolates as attractive drug targets.

    PubMed

    Silva-Stenico, M E; Rigonato, J; Leal, M G; Vaz, M G M V; Andreote, A P D; Fiore, M F

    2012-01-01

    Cyanobacteria possess the ability to produce compounds with remarkable biological activity, and have thus attracted the attention of the pharmaceutical industry. Cyanopeptides acting as protease inhibitors have shown potential in the field of pharmacotherapy through regulation of abnormal physiological processes in the human body. Despite the already described cyanopeptide protease inhibitors, the search for new congeners is of considerable interest which may pave the way for more efficient molecules. In this study, the presence of the protease inhibitors aeruginosin and cyanopeptolin with non-, mono- and dichlorination and also genes coding for their synthetases was investigated in 90 cyanobacterial strains. Mass spectrometry analyses highlighted production of 91, 19 and 3 non-, mono- and dichlorinated congeners, respectively. The purified extract of Microcystis botrys SPC759 inhibited 61% of pepsin protease. PCR amplifications of aeruginosin and cyanopeptolin synthetase gene regions were observed in 41 and 28% of evaluated strains, respectively. The sequences obtained for the aerA-aerB (aeruginosin) and mcnC-mcnE (cyanopeptolin) gene regions grouped together with their homologues found in other cyanobacterial strains in the phylogenetic analyses with high bootstrap support. Antimicrobial activity assays performed using all intracellular extracts inhibited 31 and 26% of Gram-negative and Gram-positive pathogenic bacterial growth, respectively. The results of this study showed the production of aeruginosin and cyanopeptolin and the presence of their genes in several cyanobacterial genera for the first time besides the discovery of novel congeners.

  9. Carbonic anhydrases, EPF2 and a novel protease mediate CO2 control of stomatal development.

    PubMed

    Engineer, Cawas B; Ghassemian, Majid; Anderson, Jeffrey C; Peck, Scott C; Hu, Honghong; Schroeder, Julian I

    2014-09-11

    Environmental stimuli, including elevated carbon dioxide levels, regulate stomatal development; however, the key mechanisms mediating the perception and relay of the CO2 signal to the stomatal development machinery remain elusive. To adapt CO2 intake to water loss, plants regulate the development of stomatal gas exchange pores in the aerial epidermis. A diverse range of plant species show a decrease in stomatal density in response to the continuing rise in atmospheric CO2 (ref. 4). To date, one mutant that exhibits deregulation of this CO2-controlled stomatal development response, hic (which is defective in cell-wall wax biosynthesis, ref. 5), has been identified. Here we show that recently isolated Arabidopsis thaliana β-carbonic anhydrase double mutants (ca1 ca4) exhibit an inversion in their response to elevated CO2, showing increased stomatal development at elevated CO2 levels. We characterized the mechanisms mediating this response and identified an extracellular signalling pathway involved in the regulation of CO2-controlled stomatal development by carbonic anhydrases. RNA-seq analyses of transcripts show that the extracellular pro-peptide-encoding gene EPIDERMAL PATTERNING FACTOR 2 (EPF2), but not EPF1 (ref. 9), is induced in wild-type leaves but not in ca1 ca4 mutant leaves at elevated CO2 levels. Moreover, EPF2 is essential for CO2 control of stomatal development. Using cell-wall proteomic analyses and CO2-dependent transcriptomic analyses, we identified a novel CO2-induced extracellular protease, CRSP (CO2 RESPONSE SECRETED PROTEASE), as a mediator of CO2-controlled stomatal development. Our results identify mechanisms and genes that function in the repression of stomatal development in leaves during atmospheric CO2 elevation, including the carbonic-anhydrase-encoding genes CA1 and CA4 and the secreted protease CRSP, which cleaves the pro-peptide EPF2, in turn repressing stomatal development. Elucidation of these mechanisms advances the understanding

  10. Cloning, expression and decoding of the cold adaptation of a new widely represented thermolabile subtilisin-like protease.

    PubMed

    Acevedo, J P; Rodriguez, V; Saavedra, M; Muñoz, M; Salazar, O; Asenjo, J A; Andrews, B A

    2013-02-01

    Cloning, expression and characterization of a new cold-adapted protease with potential biotechnological application, isolated from Antarctic bacteria. A subtilisin-like gene was isolated from several Antarctic bacterial genus using CODPEHOP-designed primers and a genome walking method. This gene encodes a precursor protein, which undergoes an autocatalytic cleavage resulting in a 34.6 kDa active cold-adapted protease with a maximum activity at 25-35°C and optimum pH of 8.0-9.0. It showed a higher catalytic efficiency at lower temperatures compared to its mesophilic counterpart. Heat-induced inactivation resulted in a very low melting point. Local packing analysis using the homology model indicated Ala284 as an important cold-adaptation determinant, which was corroborated by the site-directed mutagenesis. A new thermolabile subtilisin-like protease has been successfully cloned and analysed, and an important hot spot in the evolution of the cold adaptation and substrate specificity of this enzyme was identified and tested. This work reports a new cold-adapted protease with a vast representation amongst Antarctic genus, suggesting therefore its evolutionary success in this cold environment. Likewise, important sites for genetic potentiation have been identified, which are extrapolated to other enzymes of the same kind. © 2012 The Society for Applied Microbiology.

  11. Studies on a Novel Serine Protease of a ΔhapAΔprtV Vibrio cholerae O1 Strain and Its Role in Hemorrhagic Response in the Rabbit Ileal Loop Model

    PubMed Central

    Syngkon, Aurelia; Elluri, Sridhar; Koley, Hemanta; Rompikuntal, Pramod K.; Saha, Dhira Rani; Chakrabarti, Manoj K.; Bhadra, Rupak K.; Wai, Sun Nyunt; Pal, Amit

    2010-01-01

    Background Two well-characterized proteases secreted by Vibrio cholerae O1 strains are hemagglutinin protease (HAP) and V. cholerae protease (PrtV). The hapA and prtV knock out mutant, V. cholerae O1 strain CHA6.8ΔprtV, still retains residual protease activity. We initiated this study to characterize the protease present in CHA6.8ΔprtV strain and study its role in pathogenesis in rabbit ileal loop model (RIL). Methodology/Principal Findings We partially purified the residual protease secreted by strain CHA6.8ΔprtV from culture supernatant by anion-exchange chromatography. The major protein band in native PAGE was identified by MS peptide mapping and sequence analysis showed homology with a 59-kDa trypsin-like serine protease encoded by VC1649. The protease activity was partially inhibited by 25 mM PMSF and 10 mM EDTA and completely inhibited by EDTA and PMSF together. RIL assay with culture supernatants of strains C6709 (FA ratio 1.1+/−0.3 n = 3), CHA6.8 (FA ratio 1.08+/−0.2 n = 3), CHA6.8ΔprtV (FA ratio 1.02+/−0.2 n = 3) and partially purified serine protease from CHA6.8ΔprtV (FA ratio 1.2+/−0.3 n = 3) induced fluid accumulation and histopathological studies on rabbit ileum showed destruction of the villus structure with hemorrhage in all layers of the mucosa. RIL assay with culture supernatant of CHA6.8ΔprtVΔVC1649 strain (FA ratio 0.11+/−0.005 n = 3) and with protease incubated with PMSF and EDTA (FA ratio 0.3+/−0.05 n = 3) induced a significantly reduced FA ratio with almost complete normal villus structure. Conclusion Our results show the presence of a novel 59-kDa serine protease in a ΔhapAΔprtV V. cholerae O1 strain and its role in hemorrhagic response in RIL model. PMID:20927349

  12. The role of protease activation of inflammation in allergic respiratory diseases.

    PubMed

    Reed, Charles E; Kita, Hirohito

    2004-11-01

    Extracellular endogenous proteases, as well as exogenous proteases from mites and molds, react with cell-surface receptors in the airways to generate leukocyte infiltration and to amplify the response to allergens. Stimulation leads to increased intracellular Ca ++ and gene transcription. The most thoroughly investigated receptors, protease-activated receptors (PARs), are 7-transmembrane proteins coupled to G proteins. PARs are widely distributed on the cells of the airways, where they contribute to the inflammation characteristic of allergic diseases. PAR stimulation of epithelial cells opens tight junctions, causes desquamation, and produces cytokines, chemokines, and growth factors. They degranulate eosinophils and mast cells. Proteases contract bronchial smooth muscle and cause it to proliferate. PARs also promote maturation, proliferation, and collagen production of fibroblast precursors and mature fibroblasts. PAR-2, apparently the most important of the 4 PARs that have been characterized, is increased on the epithelium of patients with asthma. Trypsin, a product of injured epithelial cells, and mast cell tryptase are potent activators of PAR-2. Mast cell chymase activates PAR-1. Proteases from mites and molds appear to act through similar receptors. They amplify IgE production to allergens, degranulate eosinophils, and can generate inflammation, even in the absence of IgE. Proteases produced by Aspergillus species to support its growth are presumably responsible for the exuberant IgE, IgG, and granulomatous response of allergic bronchopulmonary aspergillosis. Similar proteases from molds germinating on the respiratory mucosa have been recently been implicated in the pathogenesis of chronic hyperplastic rhinitis and polyps and, by extension, of intrinsic asthma. Finally, proteases from mites and fungi growing in damp, water-damaged buildings might be the basis for the increased prevalence in these buildings of rhinitis, asthma, and other respiratory diseases

  13. Activities of Vacuolar Cysteine Proteases in Plant Senescence.

    PubMed

    Martínez, Dana E; Costa, Lorenza; Guiamét, Juan José

    2018-01-01

    Plant senescence is accompanied by a marked increase in proteolytic activities, and cysteine proteases (Cys-protease) represent the prevailing class among the responsible proteases. Cys-proteases predominantly locate to lytic compartments, i.e., to the central vacuole (CV) and to senescence-associated vacuoles (SAVs), the latter being specific to the photosynthetic cells of senescing leaves. Cellular fractionation of vacuolar compartments may facilitate Cys-proteases purification and their concentration for further analysis. Active Cys-proteases may be analyzed by different, albeit complementary approaches: (1) in vivo examination of proteolytic activity by fluorescence microscopy using specific substrates which become fluorescent upon cleavage by Cys-proteases, (2) protease labeling with specific probes that react irreversibly with the active enzymes, and (3) zymography, whereby protease activities are detected in polyacrylamide gels copolymerized with a substrate for proteases. Here we describe the three methods mentioned above for detection of active Cys-proteases and a cellular fractionation technique to isolate SAVs.

  14. Differential Disruption of Nucleocytoplasmic Trafficking Pathways by Rhinovirus 2A Proteases

    PubMed Central

    Watters, Kelly; Inankur, Bahar; Gardiner, Jaye C.; Warrick, Jay; Sherer, Nathan M.; Yin, John

    2017-01-01

    ABSTRACT The RNA rhinoviruses (RV) encode 2A proteases (2Apro) that contribute essential polyprotein processing and host cell shutoff functions during infection, including the cleavage of Phe/Gly-containing nucleoporin proteins (Nups) within nuclear pore complexes (NPC). Within the 3 RV species, multiple divergent genotypes encode diverse 2Apro sequences that act differentially on specific Nups. Since only subsets of Phe/Gly motifs, particularly those within Nup62, Nup98, and Nup153, are recognized by transport receptors (karyopherins) when trafficking large molecular cargos through the NPC, the processing preferences of individual 2Apro predict RV genotype-specific targeting of NPC pathways and cargos. To test this idea, transformed HeLa cell lines were created with fluorescent cargos (mCherry) for the importin α/β, transportin 1, and transportin 3 import pathways and the Crm1-mediated export pathway. Live-cell imaging of single cells expressing recombinant RV 2Apro (A16, A45, B04, B14, B52, C02, and C15) showed disruption of each pathway with measurably different efficiencies and reaction rates. The B04 and B52 proteases preferentially targeted Nups in the import pathways, while B04 and C15 proteases were more effective against the export pathway. Virus-type-specific trends were also observed during infection of cells with A16, B04, B14, and B52 viruses or their chimeras, as measured by NF-κB (p65/Rel) translocation into the nucleus and the rates of virus-associated cytopathic effects. This study provides new tools for evaluating the host cell response to RV infections in real time and suggests that differential 2Apro activities explain, in part, strain-dependent host responses and diverse RV disease phenotypes. IMPORTANCE Genetic variation among human rhinovirus types includes unexpected diversity in the genes encoding viral proteases (2Apro) that help these viruses achieve antihost responses. When the enzyme activities of 7 different 2Apro were measured

  15. Purification and characterization of naturally occurring HIV-1 (South African subtype C) protease mutants from inclusion bodies.

    PubMed

    Maseko, Sibusiso B; Natarajan, Satheesh; Sharma, Vikas; Bhattacharyya, Neelakshi; Govender, Thavendran; Sayed, Yasien; Maguire, Glenn E M; Lin, Johnson; Kruger, Hendrik G

    2016-06-01

    Human immunodeficiency virus (HIV) infections in sub-Saharan Africa represent about 56% of global infections. Many studies have targeted HIV-1 protease for the development of drugs against AIDS. Recombinant HIV-1 protease is used to screen new drugs from synthetic compounds or natural substances. Along with the wild type (C-SA) we also over-expressed and characterized two mutant forms from patients that had shown resistance to protease inhibitors. Using recombinant DNA technology, we constructed three recombinant plasmids in pGEX-6P-1 and expressed them containing a sequence encoding wild type HIV protease and two mutants (I36T↑T contains 100 amino acids and L38L↑N↑L contains 101 amino acids). These recombinant proteins were isolated from inclusion bodies by using QFF anion exchange and GST trap columns. In SDS-PAGE, we obtained these HIV proteases as single bands of approximately 11.5, 11.6 and 11.7 kDa for the wild type, I36T↑Tand L38L↑N↑L mutants, respectively. The enzyme was recovered efficiently (0.25 mg protein/L of Escherichia coli culture) and had high specific activity of 2.02, 2.20 and 1.33 μmol min(-1) mg(-1) at an optimal pH of 5 and temperature of 37 °C for the wild type, I36T↑T and L38L↑N↑L, respectively. The method employed here provides an easy and rapid purification of the HIV-1(C-SA) protease from the inclusion bodies, with high yield and high specific activities. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. A Pseudomonas aeruginosa strain isolated from a contact lens-induced acute red eye (CLARE) is protease-deficient.

    PubMed

    Estrellas, P S; Alionte, L G; Hobden, J A

    2000-03-01

    Pseudomonas aeruginosa proteases are thought to be important virulence factors in the pathogenesis of corneal disease. This study examined protease production from two strains of P. aeruginosa responsible for two very distinct clinical diseases: strain Paer1, isolated from a Contact Lens-induced Acute Red Eye (CLARE), and strain KEI 1025, isolated from a corneal ulcer. Strains were compared to a laboratory strain (ATCC 19660) known to produce severe keratitis in experimentally infected mice for protease production and for ocular virulence. Protease production was examined with colorimetric assays, gelatin zymography and western blots. Elastase A activity was quantitated with a staphylolytic assay. Ocular virulence was examined using a mouse scratch model of keratitis. In contrast to strains KEI 1025 or ATCC 19660, Paer1 was unable to produce enzymatically active elastase A, elastase, and protease IV. All three strains produced active alkaline protease. Strains KEI 1025 and ATCC 19660 produced a fulminant keratitis in mice whereas Paer1 produced a mild transient infection. Restoration of elastase activity in Paer1 via genetic complementation did not result in a virulent phenotype. Co-infection of mouse eyes with strains Paer1 and ATCC 19660 resulted in the eventual loss of Paer1 from corneal tissue. These studies suggest that P. aeruginosa elastase A and/or protease IV, but not alkaline protease or elastase, contribute to the ocular virulence of this organism.

  17. Structural studies of FIV and HIV-1 proteases complexed with an efficient inhibitor of FIV protease.

    PubMed

    Li, M; Morris, G M; Lee, T; Laco, G S; Wong, C H; Olson, A J; Elder, J H; Wlodawer, A; Gustchina, A

    2000-01-01

    Three forms of feline immunodeficiency virus protease (FIV PR), the wild type (wt) and two single point mutants, V59I and Q99V, as well as human immunodeficiency virus type 1 protease (HIV-1 PR), were cocrystallized with the C2-symmetric inhibitor, TL-3. The mutants of FIV PR were designed to replace residues involved in enzyme-ligand interactions by the corresponding HIV-1 PR residues at the structurally equivalent position. TL-3 shows decreased (improved) inhibition constants with these FIV PR mutants relative to wt FIV PR. Despite similar modes of binding of the inhibitor to all PRs (from P3 to P3'), small differences are evident in the conformation of the Phe side chains of TL-3 at the P1 and P1' positions in the complexes with the mutated FIV PRs. The differences mimick the observed binding of TL-3 in HIV-1 PR and correlate with a significant improvement in the inhibition constants of TL-3 with the two mutant FIV PRs. Large differences between the HIV-1 and FIV PR complexes are evident in the binding modes of the carboxybenzyl groups of TL-3 at P4 and P4'. In HIV-1 PR:TL-3, these groups bind over the flap region, whereas in the FIV PR complexes, the rings are located along the major axis of the active site. A significant difference in the location of the flaps in this region of the HIV-1 and FIV PRs correlates with the observed conformational changes in the binding mode of the peptidomimetic inhibitor at the P4 and P4' positions. These findings provide a structural explanation of the observed Ki values for TL-3 with the different PRs and will further assist in the development of improved inhibitors.

  18. Genome-Wide Identification and Immune Response Analysis of Serine Protease Inhibitor Genes in the Silkworm, Bombyx mori

    PubMed Central

    Duan, Jun; Wang, Genhong; Wang, Lingyan; Li, Youshan; Xiang, Zhonghuai; Xia, Qingyou

    2012-01-01

    In most insect species, a variety of serine protease inhibitors (SPIs) have been found in multiple tissues, including integument, gonad, salivary gland, and hemolymph, and are required for preventing unwanted proteolysis. These SPIs belong to different families and have distinct inhibitory mechanisms. Herein, we predicted and characterized potential SPI genes based on the genome sequences of silkworm, Bombyx mori. As a result, a total of eighty SPI genes were identified in B. mori. These SPI genes contain 10 kinds of SPI domains, including serpin, Kunitz_BPTI, Kazal, TIL, amfpi, Bowman-Birk, Antistasin, WAP, Pacifastin, and alpha-macroglobulin. Sixty-three SPIs contain single SPI domain while the others have at least two inhibitor units. Some SPIs also contain non-inhibitor domains for protein-protein interactions, including EGF, ADAM_spacer, spondin_N, reeler, TSP_1 and other modules. Microarray analysis showed that fourteen SPI genes from lineage-specific TIL family and Group F of serpin family had enriched expression in the silk gland. The roles of SPIs in resisting pathogens were investigated in silkworms when they were infected by four pathogens. Microarray and qRT-PCR experiments revealed obvious up-regulation of 8, 4, 3 and 3 SPI genes after infection with Escherichia coli, Bacillus bombysepticus, Beauveria bassiana or B. mori nuclear polyhedrosis virus (BmNPV), respectively. On the contrary, 4, 11, 7 and 9 SPI genes were down-regulated after infection with E. coli, B. bombysepticus, B. bassiana or BmNPV, respectively. These results suggested that these SPI genes may be involved in resistance to pathogenic microorganisms. These findings may provide valuable information for further clarifying the roles of SPIs in the development, immune defence, and efficient synthesis of silk gland protein. PMID:22348050

  19. Purification of a factor from the granules of a rat natural killer cell line (RNK) that reduces tumor cell growth and changes tumor morphology. Molecular identity with a granule serine protease (RNKP-1).

    PubMed

    Sayers, T J; Wiltrout, T A; Sowder, R; Munger, W L; Smyth, M J; Henderson, L E

    1992-01-01

    We have purified a protein from the granules of the rat NK leukemia cell line (RNK) that is cytostatic to a variety of tumor cells. This protein shows no species specificity because certain tumor cell lines of mouse, rat, and human origin were equally sensitive to its growth inhibitory effects. Treatment of sensitive cells resulted in a rounding of the cells followed by homotypic aggregation into large aggregates. The granule protein was distinct from cytolysin, Na-Cbz-Lys-thiobenzylester-esterase, or leukolexin. It had a molecular mass of 29 to 31 kDa, bound strongly to heparin, was inactivated by heating at 70 degrees C for 5 min or reduction, but was stable to trypsin treatment. By using molecular sieve chromatography, heparin agarose chromatography, and reverse phase HPLC, this protein was purified to homogeneity. The first 33 amino acids of the N-terminal amino acid sequence showed complete identity to the sequence predicted from a rat serine protease gene recently cloned and designated RNKP-1. Therefore we have purified a novel serine protease and demonstrated that it has effects on the growth and morphology of certain tumor cells. Other serine proteases that were structurally related and have substantial homology with RNKP-1 at the amino acid level showed neither growth inhibitory properties nor affected the morphology of the tumor target cells we used.

  20. Target-Based Screen Against a Periplasmic Serine Protease That Regulates Intrabacterial pH Homeostasis in Mycobacterium tuberculosis

    PubMed Central

    2015-01-01

    Mycobacterium tuberculosis (Mtb) maintains its intrabacterial pH (pHIB) near neutrality in the acidic environment of phagosomes within activated macrophages. A previously reported genetic screen revealed that Mtb loses this ability when the mycobacterial acid resistance protease (marP) gene is disrupted. In the present study, a high throughput screen (HTS) of compounds against the protease domain of MarP identified benzoxazinones as inhibitors of MarP. A potent benzoxazinone, BO43 (6-chloro-2-(2′-methylphenyl)-4H-1,3-benzoxazin-4-one), acylated MarP and lowered Mtb’s pHIB and survival during incubation at pH 4.5. BO43 had similar effects on MarP-deficient Mtb, suggesting the existence of additional target(s). Reaction of an alkynyl-benzoxazinone, BO43T, with Mycobacterium bovis variant bacille Calmette-Guérin (BCG) followed by click chemistry with azido-biotin identified both the MarP homologue and the high temperature requirement A1 (HtrA1) homologue, an essential protein. Thus, the chemical probe identified through a target-based screen not only reacted with its intended target in the intact cells but also implicated an additional enzyme that had eluded a genetic screen biased against essential genes. PMID:25457457

  1. Secreted aspartic proteases are not required for invasion of reconstituted human epithelia by Candida albicans.

    PubMed

    Lermann, Ulrich; Morschhäuser, Joachim

    2008-11-01

    A well-known virulence attribute of the human-pathogenic yeast Candida albicans is the secretion of aspartic proteases (Saps), which may contribute to colonization and infection of different host niches by degrading tissue barriers, destroying host defence molecules, or digesting proteins for nutrient supply. The role of individual Sap isoenzymes, which are encoded by a large gene family, for the pathogenicity of C. albicans has been investigated by assessing the virulence of mutants lacking specific SAP genes and by studying the expression pattern of the SAP genes in various models of superficial and systemic infections. We used a recombination-based genetic reporter system to detect the induction of the SAP1-SAP6 genes during infection of reconstituted human vaginal epithelium. Only SAP5, but none of the other tested SAP genes, was detectably activated in this in vitro infection model. To directly address the importance of the SAP1-SAP6 genes for invasion of reconstituted human epithelia (RHE), we constructed a set of mutants of the wild-type C. albicans model strain SC5314 in which either single or multiple SAP genes were specifically deleted. Even mutants lacking all of the SAP1-SAP3 or the SAP4-SAP6 genes displayed the same capacity to invade and damage both oral and vaginal RHE as their wild-type parental strain, in contrast to a nonfilamentous efg1Delta mutant that was avirulent under these conditions. We therefore conclude from these results that the secreted aspartic proteases Sap1p-Sap6p are not required for invasion of RHE by C. albicans.

  2. Structure of protease-cleaved Escherichia coli α-2-macroglobulin reveals a putative mechanism of conformational activation for protease entrapment

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Fyfe, Cameron D.; Grinter, Rhys; Josts, Inokentijs

    The X-ray structure of protease-cleaved E. coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. Bacterial α-2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, the X-ray structure of protease-cleaved Escherichia coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native interdomain interactions that maintain E. colimore » α-2-macroglobulin in the inactivated form. The resulting global conformational change results in entrapment of the protease and activation of the thioester bond that covalently links to the attacking protease. Owing to the similarity in structure and domain architecture of Escherichia coli α-2-macroglobulin and human α-2-macroglobulin, this protease-activation mechanism is likely to operate across the diverse members of this group.« less

  3. A lead discovery strategy driven by a comprehensive analysis of proteases in the peptide substrate space

    PubMed Central

    Sukuru, Sai Chetan K; Nigsch, Florian; Quancard, Jean; Renatus, Martin; Chopra, Rajiv; Brooijmans, Natasja; Mikhailov, Dmitri; Deng, Zhan; Cornett, Allen; Jenkins, Jeremy L; Hommel, Ulrich; Davies, John W; Glick, Meir

    2010-01-01

    We present here a comprehensive analysis of proteases in the peptide substrate space and demonstrate its applicability for lead discovery. Aligned octapeptide substrates of 498 proteases taken from the MEROPS peptidase database were used for the in silico analysis. A multiple-category naïve Bayes model, trained on the two-dimensional chemical features of the substrates, was able to classify the substrates of 365 (73%) proteases and elucidate statistically significant chemical features for each of their specific substrate positions. The positional awareness of the method allows us to identify the most similar substrate positions between proteases. Our analysis reveals that proteases from different families, based on the traditional classification (aspartic, cysteine, serine, and metallo), could have substrates that differ at the cleavage site (P1–P1′) but are similar away from it. Caspase-3 (cysteine protease) and granzyme B (serine protease) are previously known examples of cross-family neighbors identified by this method. To assess whether peptide substrate similarity between unrelated proteases could reliably translate into the discovery of low molecular weight synthetic inhibitors, a lead discovery strategy was tested on two other cross-family neighbors—namely cathepsin L2 and matrix metallo proteinase 9, and calpain 1 and pepsin A. For both these pairs, a naïve Bayes classifier model trained on inhibitors of one protease could successfully enrich those of its neighbor from a different family and vice versa, indicating that this approach could be prospectively applied to lead discovery for a novel protease target with no known synthetic inhibitors. PMID:20799349

  4. The roles of cysteine proteases and phytocystatins in development and germination of cereal seeds.

    PubMed

    Szewińska, Joanna; Simińska, Joanna; Bielawski, Wiesław

    2016-12-01

    Proteolysis is an important process for development and germination of cereal seeds. Among the many types of proteases identified in plants are the cysteine proteases (CPs) of the papain and legumain families, which play a crucial role in hydrolysing storage proteins during seed germination as well as in processing the precursors of these proteins and the inactive forms of other proteases. Moreover, all of the tissues of cereal seeds undergo progressive degradation via programed cell death, which is integral to their growth. In view of the important roles played by proteases, their uncontrolled activity could be harmful to the development of seeds and young seedlings. Thus, the activities of these enzymes are regulated by intracellular inhibitors called phytocystatins (PhyCys). The phytocystatins inhibit the activity of proteases of the papain family, and the presence of an additional motif in their C-termini allows them to also regulate the activity of members of the legumain family. A balance between the levels of cysteine proteases and phytocystatins is necessary for proper cereal seed development, and this is maintained through the antagonistic activities of gibberellins (GAs) and abscisic acid (ABA), which regulate the expression of the corresponding genes. Transcriptional regulation of cysteine proteases and phytocystatins is determined by cis-acting elements located in the promoters of these genes and by the expression of their corresponding transcription factors (TFs) and the interactions between different TFs. Copyright © 2016 Elsevier GmbH. All rights reserved.

  5. A serine protease inhibitor attenuates aldosterone-induced kidney injuries via the suppression of plasmin activity.

    PubMed

    Kakizoe, Yutaka; Miyasato, Yoshikazu; Onoue, Tomoaki; Nakagawa, Terumasa; Hayata, Manabu; Uchimura, Kohei; Morinaga, Jun; Mizumoto, Teruhiko; Adachi, Masataka; Miyoshi, Taku; Sakai, Yoshiki; Tomita, Kimio; Mukoyama, Masashi; Kitamura, Kenichiro

    2016-10-01

    Emerging evidence has suggested that aldosterone has direct deleterious effects on the kidney independently of its hemodynamic effects. However, the detailed mechanisms of these direct effects remain to be elucidated. We have previously reported that camostat mesilate (CM), a synthetic serine protease inhibitor, attenuated kidney injuries in Dahl salt-sensitive rats, remnant kidney rats, and unilateral ureteral obstruction rats, suggesting that some serine proteases would be involved in the pathogenesis of kidney injuries. The current study was conducted to investigate the roles of serine proteases and the beneficial effects of CM in aldosterone-related kidney injuries. We observed a serine protease that was activated by aldosterone/salt in rat kidney lysate, and identified it as plasmin with liquid chromatography-tandem mass spectrometry. Plasmin increased pro-fibrotic and inflammatory gene expressions in rat renal fibroblast cells. CM inhibited the protease activity of plasmin and suppressed cell injury markers induced by plasmin in the fibroblast cells. Furthermore, CM ameliorated glomerulosclerosis and interstitial fibrosis in the kidney of aldosterone/salt-treated rats. Our findings indicate that plasmin has important roles in kidney injuries that are induced by aldosterone/salt, and that serine protease inhibitor could provide a new strategy for the treatment of aldosterone-associated kidney diseases in humans. Copyright © 2016 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

  6. A novel organic solvent- and detergent-stable serine alkaline protease from Trametes cingulata strain CTM10101.

    PubMed

    Omrane Benmrad, Maroua; Moujehed, Emna; Ben Elhoul, Mouna; Zaraî Jaouadi, Nadia; Mechri, Sondes; Rekik, Hatem; Kourdali, Sidali; El Hattab, Mohamed; Badis, Abdelmalek; Sayadi, Sami; Bejar, Samir; Jaouadi, Bassem

    2016-10-01

    A protease-producing fungus was isolated from an alkaline wastewater of chemical industries and identified as Trametes cingulata strain CTM10101 on the basis of the ITS rDNA gene-sequencing. It was observed that the fungus strongly produce extracellular protease grown at 30°C in potato-dextrose-broth (PDB) optimized media (13500U/ml). The pure serine protease isolated by Trametes cingulata (designated SPTC) was purified by ammonium sulfate precipitation-dialysis followed by heat-treatment and UNO S-1 FPLC cation-exchange chromatography. The chemical characterization carried on include phisico-chemical determination and spectroscopie analysis. The MALDI-TOF/MS analysis revealed that the purified enzyme was a monomer with a molecular mass of 31405.16-Da. The enzyme had an NH2-terminal sequence of ALTTQTEAPWALGTVSHKGQAST, thus sharing high homology with those of fungal-proteases. The optimum pH and temperature values of its proteolytic activity were pH 9 and 60°C, respectively, and its half-life times at 60 and 70°C were 9 and 5-h, respectively. It was completely inhibited by PMSF and DFP, which strongly suggested its belonging to the serine protease family. Compared to Flavourzyme(®)500L from Aspergillus oryzae and Thermolysin typeX from Geobacillus stearothermophilus, SPTC displayed higher levels of hydrolysis, substrate specificity, and catalytic efficiency as well as elevated organic solvent tolerance and considerable detergent stability. Finally, SPTC could potentially be used in peptide synthesis and detergent formulations. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Effects of drug-resistant mutations on the dynamic properties of HIV-1 protease and inhibition by Amprenavir and Darunavir

    PubMed Central

    Yu, Yuqi; Wang, Jinan; Shao, Qiang; Shi, Jiye; Zhu, Weiliang

    2015-01-01

    Molecular dynamics simulations are performed to investigate the dynamic properties of wild-type HIV-1 protease and its two multi-drug-resistant variants (Flap + (L10I/G48V/I54V/V82A) and Act (V82T/I84V)) as well as their binding with APV and DRV inhibitors. The hydrophobic interactions between flap and 80 s (80’s) loop residues (mainly I50-I84’ and I50’-I84) play an important role in maintaining the closed conformation of HIV-1 protease. The double mutation in Act variant weakens the hydrophobic interactions, leading to the transition from closed to semi-open conformation of apo Act. APV or DRV binds with HIV-1 protease via both hydrophobic and hydrogen bonding interactions. The hydrophobic interactions from the inhibitor is aimed to the residues of I50 (I50’), I84 (I84’), and V82 (V82’) which create hydrophobic core clusters to further stabilize the closed conformation of flaps, and the hydrogen bonding interactions are mainly focused with the active site of HIV-1 protease. The combined change in the two kinds of protease-inhibitor interactions is correlated with the observed resistance mutations. The present study sheds light on the microscopic mechanism underlying the mutation effects on the dynamics of HIV-1 protease and the inhibition by APV and DRV, providing useful information to the design of more potent and effective HIV-1 protease inhibitors. PMID:26012849

  8. Crystal structure of the cysteine protease inhibitor 2 from Entamoeba histolytica: Functional convergence of a common protein fold

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Casados-Vázquez, Luz E.; Lara-González, Samuel; Brieb, Luis G.

    Cysteine proteases (CP) are key pathogenesis and virulence determinants of protozoan parasites. Entamoeba histolytica contains at least 50 cysteine proteases; however, only three (EhCP1, EhCP2 and EhCP5) are responsible for approximately 90% of the cysteine protease activity in this parasite. CPs are expressed as inactive zymogens. Because the processed proteases are potentially cytotoxic, protozoan parasites have developed mechanisms to regulate their activity. Inhibitors of cysteine proteases (ICP) of the chagasin-like inhibitor family (MEROPS family I42) were recently identified in bacteria and protozoan parasites. E. histolytica contains two ICP-encoding genes of the chagasin-like inhibitor family. EhICP1 localizes to the cytosol, whereasmore » EhICP2 is targeted to phagosomes. Herein, we report two crystal structures of EhICP2. The overall structure of EhICP2 consists of eight {beta}-strands and closely resembles the immunoglobulin fold. A comparison between the two crystal forms of EhICP2 indicates that the conserved BC, DE and FG loops form a flexible wedge that may block the active site of CPs. The positively charged surface of the wedge-forming loops in EhICP2 contrasts with the neutral surface of the wedge-forming loops in chagasin. We postulate that the flexibility and positive charge observed in the DE and FG loops of EhICP2 may be important to facilitate the initial binding of this inhibitor to the battery of CPs present in E. histolytica.« less

  9. A comparative analysis of serpin genes in the silkworm genome

    PubMed Central

    Zou, Zhen; Picheng, Zhao; Weng, Hua; Mita, Kazuei; Jiang, Haobo

    2009-01-01

    Serine protease inhibitors (serpins) are a superfamily of proteins, most of which control protease-mediated processes by inhibiting their cognate enzymes. Sequencing of the silkworm genome provides an opportunity to investigate serpin structure, function, and evolution at the genome level. There are thirty-four serpin genes in Bombyx mori. Six are highly similar to their Manduca sexta orthologs that regulate innate immunity. Three alternative exons in serpin1 gene and four in serpin28 encode a variable region including the reactive site loop. Splicing of serpin2 pre-mRNA yields variations in serpin2A, 2A′ and 2B. Sequence similarity and intron positions reveal the evolutionary pathway of seven serpin genes in group C. RT-PCR indicates an increase in the mRNA levels of serpin1, 3, 5, 6, 9, 12, 13, 25, 27, 32 and 34 in fat body and hemocytes of larvae injected with bacteria. These results suggest that the silkworm serpins play regulatory roles in defense responses. PMID:19150649

  10. Secreted fungal aspartic proteases: A review.

    PubMed

    Mandujano-González, Virginia; Villa-Tanaca, Lourdes; Anducho-Reyes, Miguel Angel; Mercado-Flores, Yuridia

    2016-01-01

    The aspartic proteases, also called aspartyl and aspartate proteases or acid proteases (E.C.3.4.23), belong to the endopeptidase family and are characterized by the conserved sequence Asp-Gly-Thr at the active site. These enzymes are found in a wide variety of microorganisms in which they perform important functions related to nutrition and pathogenesis. In addition, their high activity and stability at acid pH make them attractive for industrial application in the food industry; specifically, they are used as milk-coagulating agents in cheese production or serve to improve the taste of some foods. This review presents an analysis of the characteristics and properties of secreted microbial aspartic proteases and their potential for commercial application. Copyright © 2016 Asociación Española de Micología. Published by Elsevier Espana. All rights reserved.

  11. Free energy component analysis for drug design: a case study of HIV-1 protease-inhibitor binding.

    PubMed

    Kalra, P; Reddy, T V; Jayaram, B

    2001-12-06

    A theoretically rigorous and computationally tractable methodology for the prediction of the free energies of binding of protein-ligand complexes is presented. The method formulated involves developing molecular dynamics trajectories of the enzyme, the inhibitor, and the complex, followed by a free energy component analysis that conveys information on the physicochemical forces driving the protein-ligand complex formation and enables an elucidation of drug design principles for a given receptor from a thermodynamic perspective. The complexes of HIV-1 protease with two peptidomimetic inhibitors were taken as illustrative cases. Four-nanosecond-level all-atom molecular dynamics simulations using explicit solvent without any restraints were carried out on the protease-inhibitor complexes and the free proteases, and the trajectories were analyzed via a thermodynamic cycle to calculate the binding free energies. The computed free energies were seen to be in good accord with the reported data. It was noted that the net van der Waals and hydrophobic contributions were favorable to binding while the net electrostatics, entropies, and adaptation expense were unfavorable in these protease-inhibitor complexes. The hydrogen bond between the CH2OH group of the inhibitor at the scissile position and the catalytic aspartate was found to be favorable to binding. Various implicit solvent models were also considered and their shortcomings discussed. In addition, some plausible modifications to the inhibitor residues were attempted, which led to better binding affinities. The generality of the method and the transferability of the protocol with essentially no changes to any other protein-ligand system are emphasized.

  12. Genome-wide identification, evolutionary and expression analysis of the aspartic protease gene superfamily in grape

    PubMed Central

    2013-01-01

    Background Aspartic proteases (APs) are a large family of proteolytic enzymes found in almost all organisms. In plants, they are involved in many biological processes, such as senescence, stress responses, programmed cell death, and reproduction. Prior to the present study, no grape AP gene(s) had been reported, and their research on woody species was very limited. Results In this study, a total of 50 AP genes (VvAP) were identified in the grape genome, among which 30 contained the complete ASP domain. Synteny analysis within grape indicated that segmental and tandem duplication events contributed to the expansion of the grape AP family. Additional analysis between grape and Arabidopsis demonstrated that several grape AP genes were found in the corresponding syntenic blocks of Arabidopsis, suggesting that these genes arose before the divergence of grape and Arabidopsis. Phylogenetic relationships of the 30 VvAPs with the complete ASP domain and their Arabidopsis orthologs, as well as their gene and protein features were analyzed and their cellular localization was predicted. Moreover, expression profiles of VvAP genes in six different tissues were determined, and their transcript abundance under various stresses and hormone treatments were measured. Twenty-seven VvAP genes were expressed in at least one of the six tissues examined; nineteen VvAPs responded to at least one abiotic stress, 12 VvAPs responded to powdery mildew infection, and most of the VvAPs responded to SA and ABA treatments. Furthermore, integrated synteny and phylogenetic analysis identified orthologous AP genes between grape and Arabidopsis, providing a unique starting point for investigating the function of grape AP genes. Conclusions The genome-wide identification, evolutionary and expression analyses of grape AP genes provide a framework for future analysis of AP genes in defining their roles during stress response. Integrated synteny and phylogenetic analyses provide novel insight into the

  13. Elevated Peritoneal Fluid TNF-α Incites Ovarian Early Growth Response Factor 1 Expression and Downstream Protease Mediators

    PubMed Central

    Birt, Julie A.; Nabli, Henda; Stilley, Julie A.; Windham, Emma A.; Frazier, Shellaine R.

    2013-01-01

    Endometriosis-associated infertility manifests itself via multiple, poorly understood mechanisms. Our goal was to characterize signaling pathways, between peritoneal endometriotic lesions and the ovary, leading to failed ovulation. Genome-wide microarray analysis comparing ovarian tissue from an in vivo endometriosis model in the rat (Endo) with controls (Sham) identified 22 differentially expressed genes, including transiently expressed early growth response factor 1 (Egr1). The Egr1 regulates gene requisites for ovulation. The Egr1 promoter is responsive to tumor necrosis factor-alpha (TNF-α) signaling. We hypothesized that altered expression of ovarian EGR1 is induced by elevated peritoneal fluid TNF-α which is upregulated by the presence of peritoneal endometriosis. Endo rats, compared to controls, had more peritoneal fluid TNF-α and quantitative, spatial differences in Egr1 mRNA and EGR1 protein localization in follicular compartments. Interactions between elevated peritoneal fluid TNF-α and overexpression of follicular Egr1/EGR1 expression may affect downstream protease pathways impeding ovulation in endometriosis. Preliminary studies identified similar patterns of EGR1 protein localization in human ovaries from women with endometriosis and compared to those without endometriosis. PMID:23427178

  14. Purification and characterisation of a salt-stable protease from the halophilic archaeon Halogranum rubrum.

    PubMed

    Gao, Ruichang; Shi, Tong; Liu, Xiangdong; Zhao, Mengqin; Cui, Henglin; Yuan, Li

    2017-03-01

    Because proteases play an important role in the fermentation of fish sauce, the purification and characterisation of an extracellular protease from the halophilic archaeon Halogranum rubrum was investigated. The molecular mass of the protease was estimated to be approximately 47 kDa based on sodium dodecyl sulfate-polyacrylamide gel electropheresis (SDS-PAGE) and native-PAGE analysis. The optimum conditions for catalytic activity were pH 8.0 and 50°C. The protease showed alkaline stability (pH 7.0-10.0). The protease also exhibited novel catalytic ability over a broad range of salinity (NaCl 0-3 mol L -1 ). Calcium ion enhanced the proteolytic activity of the enzyme. The K m and V max values of the purified protease for casein were calculated to be 4.89 mg mL -1 and 1111.11 U mL -1 , respectively. The protease was strongly inhibited by ethylenediamine tetraacetic acid (EDTA) and phenylmethanesulfonyl fluoride (PMSF). Meanwhile, the protease was stable in the presence of Triton X-100, isopropanol, ethanol or dithio-bis-nitrobenzoic (DTNB), but was inhibited by sodium dodecyl sulfate (SDS), dimethyl sulfoxide (DMSO) or methanol. MALDI -TOF/TOF MS analysis revealed that the protease shared some functional traits with protease produced by Halogranum salarium. Furthermore, it exhibited high hydrolytic activity on silver carp myosin protein. The protease is an alkaline and salt-tolerant enzyme that hydrolyses silver carp myosin with high efficiency. These excellent characteristics make this protease an attractive candidate for industrial use in low-salt fish sauce fermentation. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  15. Cloning and enhancing production of a detergent- and organic-solvent-resistant nattokinase from Bacillus subtilis VTCC-DVN-12-01 by using an eight-protease-gene-deficient Bacillus subtilis WB800

    PubMed Central

    2013-01-01

    Background Nattokinases/Subtilisins (EC 3.4.21.62) belong to the second large family of serine proteases, which gain significant attention and play important role in many biotechnology processes. Thus, a number of nattokinases/subtilisins from various Bacillus species, especially from B. subtilis strains, extensively have been investigated to understand their biochemical and physical properties as well as to improve the production for industrial application. The purpose of this study was to clone a nattokinase gene from Bacillus subtilis strain VTCC-DVN-12-01, enhance its production in B. subtilis WB800, which is deficient in eight extracellular proteases and characterize its physicochemical properties for potential application in organic synthesis and detergent production. Results A gene coding for the nattokinase (Nk) from B. subtilis strain VTCC-DVN-12-01 consisted of an ORF of 1146 nucleotides, encoding a pre-pro-protein enzyme (30-aa pre-signal peptide, 76-aa pro-peptide and 275-aa mature protein with a predicted molecular mass of 27.7 kDa and pI 6.6). The nattokinase showed 98-99% identity with other nattokinases/subtilisins from B. subtilis strains in GenBank. Nk was expressed in B. subtilis WB800 under the control of acoA promoter at a high level of 600 mg protein per liter culture medium which is highest yield of proteins expressed in any extracellular-protease-deficient B. subtilis system till date. Nk was purified to homogeneity with 3.25 fold purification, a specific activity of 12.7 U/mg, and a recovery of 54.17%. The purified Nk was identified by MALDI-TOF mass spectrometry through three peptides, which showed 100% identity to corresponding peptides of the B. subtilis nattokinase (CAC41625). An optimal activity for Nk was observed at 65°C and pH 9. The nattokinase was stable at temperature up to 50°C and in pH range of 5–11 and retained more than 85% of its initial activity after incubation for 1 h. Mg2+ activated Nk up to 162% of its activity

  16. Cloning and enhancing production of a detergent- and organic-solvent-resistant nattokinase from Bacillus subtilis VTCC-DVN-12-01 by using an eight-protease-gene-deficient Bacillus subtilis WB800.

    PubMed

    Nguyen, Thao Thi; Quyen, Thi Dinh; Le, Hoang Thanh

    2013-09-10

    Nattokinases/Subtilisins (EC 3.4.21.62) belong to the second large family of serine proteases, which gain significant attention and play important role in many biotechnology processes. Thus, a number of nattokinases/subtilisins from various Bacillus species, especially from B. subtilis strains, extensively have been investigated to understand their biochemical and physical properties as well as to improve the production for industrial application. The purpose of this study was to clone a nattokinase gene from Bacillus subtilis strain VTCC-DVN-12-01, enhance its production in B. subtilis WB800, which is deficient in eight extracellular proteases and characterize its physicochemical properties for potential application in organic synthesis and detergent production. A gene coding for the nattokinase (Nk) from B. subtilis strain VTCC-DVN-12-01 consisted of an ORF of 1146 nucleotides, encoding a pre-pro-protein enzyme (30-aa pre-signal peptide, 76-aa pro-peptide and 275-aa mature protein with a predicted molecular mass of 27.7 kDa and pI 6.6). The nattokinase showed 98-99% identity with other nattokinases/subtilisins from B. subtilis strains in GenBank. Nk was expressed in B. subtilis WB800 under the control of acoA promoter at a high level of 600 mg protein per liter culture medium which is highest yield of proteins expressed in any extracellular-protease-deficient B. subtilis system till date. Nk was purified to homogeneity with 3.25 fold purification, a specific activity of 12.7 U/mg, and a recovery of 54.17%. The purified Nk was identified by MALDI-TOF mass spectrometry through three peptides, which showed 100% identity to corresponding peptides of the B. subtilis nattokinase (CAC41625). An optimal activity for Nk was observed at 65 °C and pH 9. The nattokinase was stable at temperature up to 50 °C and in pH range of 5-11 and retained more than 85% of its initial activity after incubation for 1 h. Mg2+ activated Nk up to 162% of its activity. The addition of

  17. Discovery of HIV Type 1 Aspartic Protease Hit Compounds through Combined Computational Approaches.

    PubMed

    Xanthopoulos, Dimitrios; Kritsi, Eftichia; Supuran, Claudiu T; Papadopoulos, Manthos G; Leonis, Georgios; Zoumpoulakis, Panagiotis

    2016-08-05

    A combination of computational techniques and inhibition assay experiments was employed to identify hit compounds from commercial libraries with enhanced inhibitory potency against HIV type 1 aspartic protease (HIV PR). Extensive virtual screening with the aid of reliable pharmacophore models yielded five candidate protease inhibitors. Subsequent molecular dynamics and molecular mechanics Poisson-Boltzmann surface area free-energy calculations for the five ligand-HIV PR complexes suggested a high stability of the systems through hydrogen-bond interactions between the ligands and the protease's flaps (Ile50/50'), as well as interactions with residues of the active site (Asp25/25'/29/29'/30/30'). Binding-energy calculations for the three most promising compounds yielded values between -5 and -10 kcal mol(-1) and suggested that van der Waals interactions contribute most favorably to the total energy. The predicted binding-energy values were verified by in vitro inhibition assays, which showed promising results in the high nanomolar range. These results provide structural considerations that may guide further hit-to-lead optimization toward improved anti-HIV drugs. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Co-evolution of insect proteases and plant protease inhibitors.

    PubMed

    Jongsma, Maarten A; Beekwilder, Jules

    2011-08-01

    Plants are at the basis of the food chain, but there is no such thing as a "free lunch" for herbivores. To promote reproductive success, plants evolved multi-layered defensive tactics to avoid or discourage herbivory. To the detriment of plants, herbivores, in turn, evolved intricate strategies to find, eat, and successfully digest essential plant parts to raise their own offspring. In this battle the digestive tract is the arena determining final victory or defeat as measured by growth or starvation of the herbivore. Earlier, specific molecular opponents were identified as proteases and inhibitors: digestive proteases of herbivores evolved structural motifs to occlude plant protease inhibitors, or alternatively, the insects evolved proteases capable of specifically degrading the host plant inhibitors. In response plant inhibitors evolved hyper-variable and novel protein folds to remain active against potential herbivores. At the level of protease regulation in herbivorous insects, it was shown that inhibition-insensitive digestive proteases are up-regulated when sensitive proteases are inhibited. The way this regulation operates in mammals is known as negative feedback by gut-luminal factors, so-called 'monitor peptides' that are sensitive to the concentration of active enzymes. We propose that regulation of gut enzymes by endogenous luminal factors has been an open invitation to plants to "hijack" this regulation by evolving receptor antagonists, although yet these plant factors have not been identified. In future research the question of the co-evolution of insect proteases and plant inhibitors should, therefore, be better approached from a systems level keeping in mind that evolution is fundamentally opportunistic and that the plant's fitness is primarily improved by lowering the availability of essential amino acids to an herbivore by any available mechanism.

  19. Impact of recombination on polymorphism of genes encoding Kunitz-type protease inhibitors in the genus Solanum.

    PubMed

    Speranskaya, Anna S; Krinitsina, Anastasia A; Kudryavtseva, Anna V; Poltronieri, Palmiro; Santino, Angelo; Oparina, Nina Y; Dmitriev, Alexey A; Belenikin, Maxim S; Guseva, Marina A; Shevelev, Alexei B

    2012-08-01

    The group of Kunitz-type protease inhibitors (KPI) from potato is encoded by a polymorphic family of multiple allelic and non-allelic genes. The previous explanations of the KPI variability were based on the hypothesis of random mutagenesis as a key factor of KPI polymorphism. KPI-A genes from the genomes of Solanum tuberosum cv. Istrinskii and the wild species Solanum palustre were amplified by PCR with subsequent cloning in plasmids. True KPI sequences were derived from comparison of the cloned copies. "Hot spots" of recombination in KPI genes were independently identified by DnaSP 4.0 and TOPALi v2.5 software. The KPI-A sequence from potato cv. Istrinskii was found to be 100% identical to the gene from Solanum nigrum. This fact illustrates a high degree of similarity of KPI genes in the genus Solanum. Pairwise comparison of KPI A and B genes unambiguously showed a non-uniform extent of polymorphism at different nt positions. Moreover, the occurrence of substitutions was not random along the strand. Taken together, these facts contradict the traditional hypothesis of random mutagenesis as a principal source of KPI gene polymorphism. The experimentally found mosaic structure of KPI genes in both plants studied is consistent with the hypothesis suggesting recombination of ancestral genes. The same mechanism was proposed earlier for other resistance-conferring genes in the nightshade family (Solanaceae). Based on the data obtained, we searched for potential motifs of site-specific binding with plant DNA recombinases. During this work, we analyzed the sequencing data reported by the Potato Genome Sequencing Consortium (PGSC), 2011 and found considerable inconsistence of their data concerning the number, location, and orientation of KPI genes of groups A and B. The key role of recombination rather than random point mutagenesis in KPI polymorphism was demonstrated for the first time. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  20. Genome-wide comparative analysis of papain-like cysteine protease family genes in castor bean and physic nut.

    PubMed

    Zou, Zhi; Huang, Qixing; Xie, Guishui; Yang, Lifu

    2018-01-10

    Papain-like cysteine proteases (PLCPs) are a class of proteolytic enzymes involved in many plant processes. Compared with the extensive research in Arabidopsis thaliana, little is known in castor bean (Ricinus communis) and physic nut (Jatropha curcas), two Euphorbiaceous plants without any recent whole-genome duplication. In this study, a total of 26 or 23 PLCP genes were identified from the genomes of castor bean and physic nut respectively, which can be divided into nine subfamilies based on the phylogenetic analysis: RD21, CEP, XCP, XBCP3, THI, SAG12, RD19, ALP and CTB. Although most of them harbor orthologs in Arabidopsis, several members in subfamilies RD21, CEP, XBCP3 and SAG12 form new groups or subgroups as observed in other species, suggesting specific gene loss occurred in Arabidopsis. Recent gene duplicates were also identified in these two species, but they are limited to the SAG12 subfamily and were all derived from local duplication. Expression profiling revealed diverse patterns of different family members over various tissues. Furthermore, the evolution characteristics of PLCP genes were also compared and discussed. Our findings provide a useful reference to characterize PLCP genes and investigate the family evolution in Euphorbiaceae and species beyond.

  1. Computational Study on Substrate Specificity of a Novel Cysteine Protease 1 Precursor from Zea mays

    PubMed Central

    Liu, Huimin; Chen, Liangcheng; Li, Quan; Zheng, Mingzhu; Liu, Jingsheng

    2014-01-01

    Cysteine protease 1 precursor from Zea mays (zmCP1) is classified as a member of the C1A family of peptidases (papain-like cysteine protease) in MEROPS (the Peptidase Database). The 3D structure and substrate specificity of the zmCP1 is still unknown. This study is the first one to build the 3D structure of zmCP1 by computer-assisted homology modeling. In order to determine the substrate specificity of zmCP1, docking study is used for rapid and convenient analysis of large populations of ligand–enzyme complexes. Docking results show that zmCP1 has preference for P1 position and P2 position for Arg and a large hydrophobic residue (such as Phe). Gly147, Gly191, Cys189, and Asp190 are predicted to function as active residues at the S1 subsite, and the S2 subsite contains Leu283, Leu193, Ala259, Met194, and Ala286. SIFt results indicate that Gly144, Arg268, Trp308, and Ser311 play important roles in substrate binding. Then Molecular Mechanics-Poisson-Boltzmann Surface Area (MM-PBSA) method was used to explain the substrate specificity for P1 position of zmCp1. This study provides insights into the molecular basis of zmCP1 activity and substrate specificity. PMID:24921705

  2. A disruption of ctpA encoding carboxy-terminal protease attenuates Burkholderia mallei and induces partial protection in CD1 mice.

    PubMed

    Bandara, Aloka B; DeShazer, David; Inzana, Thomas J; Sriranganathan, Nammalwar; Schurig, Gerhardt G; Boyle, Stephen M

    2008-09-01

    Burkholderia mallei is the etiologic agent of glanders in solipeds (horses, mules and donkeys), and incidentally in carnivores and humans. Little is known about the molecular mechanisms of B. mallei pathogenesis. The putative carboxy-terminal processing protease (CtpA) of B. mallei is a member of a novel family of endoproteases involved in the maturation of proteins destined for the cell envelope. All species and isolates of Burkholderia carry a highly conserved copy of ctpA. We studied the involvement of CtpA on growth, cell morphology, persistence, and pathogenicity of B. mallei. A sucrose-resistant strain of B. mallei was constructed by deleting a major portion of the sacB gene of the wild type strain ATCC 23344 by gene replacement, and designated as strain 23344DeltasacB. A portion of the ctpA gene (encoding CtpA) of strain 23344DeltasacB was deleted by gene replacement to generate strain 23344DeltasacBDeltactpA. In contrast to the wild type ATCC 23344 or the sacB mutant 23344DeltasacB, the ctpA mutant 23344DeltasacBDeltactpA displayed altered cell morphologies with partially or fully disintegrated cell envelopes. Furthermore, relative to the wild type, the ctpA mutant displayed slower growth in vitro and less ability to survive in J774.2 murine macrophages. The expression of mRNA of adtA, the gene downstream of ctpA was similar among the three strains suggesting that disruption of ctpA did not induce any polar effects. As with the wild type or the sacB mutant, the ctpA mutant exhibited a dose-dependent lethality when inoculated intraperitoneally into CD1 mice. The CD1 mice inoculated with a non-lethal dose of the ctpA mutant produced specific serum immunoglobulins IgG1 and IgG2a and were partially protected against challenge with wild type B. mallei ATCC 23344. These findings suggest that CtpA regulates in vitro growth, cell morphology and intracellular survival of B. mallei, and a ctpA mutant protects CD1 mice against glanders.

  3. Membrane protease degradomics: proteomic identification and quantification of cell surface protease substrates.

    PubMed

    Butler, Georgina S; Dean, Richard A; Smith, Derek; Overall, Christopher M

    2009-01-01

    The modification of cell surface proteins by plasma membrane and soluble proteases is important for physiological and pathological processes. Methods to identify shed and soluble substrates are crucial to further define the substrate repertoire, termed the substrate degradome, of individual proteases. Identifying protease substrates is essential to elucidate protease function and involvement in different homeostatic and disease pathways. This characterisation is also crucial for drug target identification and validation, which would then allow the rational design of specific targeted inhibitors for therapeutic intervention. We describe two methods for identifying and quantifying shed cell surface protease targets in cultured cells utilising Isotope-Coded Affinity Tags (ICAT) and Isobaric Tags for Relative and Absolute Quantification (iTRAQ). As a model system to develop these techniques, we chose a cell-membrane expressed matrix metalloproteinase, MMP-14, but the concepts can be applied to proteases of other classes. By over-expression, or conversely inhibition, of a particular protease with careful selection of control conditions (e.g. vector or inactive protease) and differential labelling, shed proteins can be identified and quantified by mass spectrometry (MS), MS/MS fragmentation and database searching.

  4. Purification and characterization of a cysteine protease from corms of freesia, Freesia reflacta.

    PubMed

    Kaneda, M; Yonezawa, H; Uchikoba, T

    1997-09-01

    A protease (freesia protease B) has been purified to electrophoretic homogeneity from corms of freesia, Freesia reflacta by five steps of chromatography. Its M(r) was estimated to be about 26,000 by SDS-PAGE. The optimum pH of the enzyme was 6.0-7.0 at 30 degrees C using casein as a substrate. The enzyme was strongly inhibited by p-chloromercuribenzoic acid but not by phenylmethanesulphonylfluoride and EDTA. These results indicate that freesia protease B is a cysteine protease. Nine sites of oxidized insulin B-chain were cleaved by freesia protease B in 24 h of hydrolysis. The four cleavage sites among them resembled those of papain. From the digestion of five peptidyl substrates the specificity of freesia protease B was found to be approximately broad, but the preferential cleavage sites were negatively charged residues at P1 positions. Freesia protease B preferred also the large hydrophobic amino acid residues at the P2 position, in a similar manner to papain. The amino terminal sequence of freesia protease B was identical with those of papain in regard to the conservative residues of cysteine protease.

  5. Malt1 protease inactivation efficiently dampens immune responses but causes spontaneous autoimmunity

    PubMed Central

    Jaworski, Maike; Marsland, Ben J; Gehrig, Jasmine; Held, Werner; Favre, Stéphanie; Luther, Sanjiv A; Perroud, Mai; Golshayan, Déla; Gaide, Olivier; Thome, Margot

    2014-01-01

    The protease activity of the paracaspase Malt1 has recently gained interest as a drug target for immunomodulation and the treatment of diffuse large B-cell lymphomas. To address the consequences of Malt1 protease inactivation on the immune response in vivo, we generated knock-in mice expressing a catalytically inactive C472A mutant of Malt1 that conserves its scaffold function. Like Malt1-deficient mice, knock-in mice had strong defects in the activation of lymphocytes, NK and dendritic cells, and the development of B1 and marginal zone B cells and were completely protected against the induction of autoimmune encephalomyelitis. Malt1 inactivation also protected the mice from experimental induction of colitis. However, Malt1 knock-in mice but not Malt1-deficient mice spontaneously developed signs of autoimmune gastritis that correlated with an absence of Treg cells, an accumulation of T cells with an activated phenotype and high serum levels of IgE and IgG1. Thus, removal of the enzymatic activity of Malt1 efficiently dampens the immune response, but favors autoimmunity through impaired Treg development, which could be relevant for therapeutic Malt1-targeting strategies. PMID:25319413

  6. Lumenal exposed regions of the D1 protein of PSII are long enough to be degraded by the chloroplast Deg1 protease.

    PubMed

    Knopf, Ronit Rimon; Adam, Zach

    2018-03-27

    Degradation of the D1 protein of photosystem II (PSII) reaction center is a pre-requisite for the repair cycle from photoinhibition. Two types of thylakoid proteases, FtsH and Deg, have been demonstrated to participate in this process. However, the location of the proteolytic sites of the lumenal Deg1 protease within its internal sphere raised the question whether the lumenal-exposed regions of D1 are indeed long enough to reach these sites. Implanting these regions into the stable GFP rendered it sensitive to the presence of Deg1 in vitro, demonstrating that the flexible regions of D1 that protrude into the lumen can penetrate through the three side-openings of Deg1 and reach its internal proteolytic sites. This mode of action, facilitating cooperation between proteases on both sides of the thylakoid membranes, should be applicable to the degradation of other integral thylakoid membrane proteins as well.

  7. Nucleotide sequences encoding a thermostable alkaline protease

    DOEpatents

    Wilson, David B.; Lao, Guifang

    1998-01-01

    Nucleotide sequences, derived from a thermophilic actinomycete microorganism, which encode a thermostable alkaline protease are disclosed. Also disclosed are variants of the nucleotide sequences which encode a polypeptide having thermostable alkaline proteolytic activity. Recombinant thermostable alkaline protease or recombinant polypeptide may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant thermostable alkaline protease or recombinant polypeptide from the culture medium.

  8. Protease-mediated drug delivery

    NASA Astrophysics Data System (ADS)

    Dickson, Eva F.; Goyan, Rebecca L.; Kennedy, James C.; Mackay, M.; Mendes, M. A. K.; Pottier, Roy H.

    2003-12-01

    Drugs used in disease treatment can cause damage to both malignant and normal tissue. This toxicity limits the maximum therapeutic dose. Drug targeting is of high interest to increase the therapeutic efficacy of the drug without increasing systemic toxicity. Certain tissue abnormalities, disease processes, cancers, and infections are characterized by high levels of activity of specific extracellular and/or intracellular proteases. Abnormally high activity levels of specific proteases are present at sites of physical or chemical trauma, blood clots, malignant tumors, rheumatoid arthritis, inflammatory bowel disease, gingival disease, glomerulonerphritis, and acute pancreatitis. Abnormal protease activity is suspected in development of liver thrombosis, pulmonary emphysema, atherosclerosis, and muscular dystrophy. Inactiviating disease-associated proteases by the administration of appropriate protease inhibitors has had limited success. Instead, one could use such proteases to target drugs to treat the condition. Protease mediated drug delivery offers such a possibility. Solubilizing groups are attached to insoluble drugs via a polypeptide chain which is specifically cleavable by certian proteases. When the solubilized drug enounters the protease, the solubilizing moieties are cleaved, and the drug precipitates at the disease location. Thus, a smaller systemic dosage could result in a therapeutic drug concentration at the treatment site with less systemic toxicity.

  9. pH regulation of recombinant glucoamylase production in Fusarium venenatum JeRS 325, a transformant with a Fusarium oxysporum alkaline (trypsin-like) protease promoter.

    PubMed

    Wiebe, M G; Robson, G D; Shuster, J R; Trinci, A P

    1999-08-05

    Fusarium venenatum (formerly Fusarium graminearum) JeRS 325 produces heterologous glucoamylase (GAM) under the regulation of a Fusarium oxysporum alkaline (trypsin-like) protease promoter. The glucoamylase gene was used as a reporter gene to study the effects of ammonium and pH on GAM production under the control of the alkaline protease promoter. Between pH 4.0 and 5.8, GAM production in glucose-limited chemostat cultures of JeRS 325 grown at a dilution rate of 0.10 h-1 (doubling time, 6.9 h) on (NH4)2SO4 medium increased in a linear manner with increase in pH. However, at pH 4.0 and below GAM production was almost completely repressed in glucose-limited chemostat cultures grown on (NH4)2SO4 or NaNO3 medium. Thus GAM production in JeRS 325 is regulated by culture pH, not by the nature of the nitrogen source in the medium. The difficulty of using unbuffered medium when investigating putative ammonium repression is also shown. The study demonstrates the potential for use of the alkaline protease promoter in F. graminearum for the production of recombinant proteins in a pH dependent man ner. Copyright 1999 John Wiley & Sons, Inc.

  10. Room temperature neutron crystallography of drug resistant HIV-1 protease uncovers limitations of X-ray structural analysis at 100K

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Gerlits, Oksana O.; Keen, David A.; Blakeley, Matthew P.

    HIV-1 protease inhibitors are crucial for treatment of HIV-1/AIDS, but their effectiveness is thwarted by rapid emergence of drug resistance. To better understand binding of clinical inhibitors to resistant HIV-1 protease, we used room-temperature joint X-ray/neutron (XN) crystallography to obtain an atomic-resolution structure of the protease triple mutant (V32I/I47V/V82I) in complex with amprenavir. The XN structure reveals a D+ ion located midway between the inner Oδ1 oxygen atoms of the catalytic aspartic acid residues. Comparison of the current XN structure with our previous XN structure of the wild-type HIV-1 protease-amprenavir complex suggests that the three mutations do not significantly altermore » the drug–enzyme interactions. This is in contrast to the observations in previous 100 K X-ray structures of these complexes that indicated loss of interactions by the drug with the triple mutant protease. These findings, thus, uncover limitations of structural analysis of drug binding using X-ray structures obtained at 100 K.« less

  11. Room temperature neutron crystallography of drug resistant HIV-1 protease uncovers limitations of X-ray structural analysis at 100K

    DOE PAGES

    Gerlits, Oksana O.; Keen, David A.; Blakeley, Matthew P.; ...

    2017-02-14

    HIV-1 protease inhibitors are crucial for treatment of HIV-1/AIDS, but their effectiveness is thwarted by rapid emergence of drug resistance. To better understand binding of clinical inhibitors to resistant HIV-1 protease, we used room-temperature joint X-ray/neutron (XN) crystallography to obtain an atomic-resolution structure of the protease triple mutant (V32I/I47V/V82I) in complex with amprenavir. The XN structure reveals a D+ ion located midway between the inner Oδ1 oxygen atoms of the catalytic aspartic acid residues. Comparison of the current XN structure with our previous XN structure of the wild-type HIV-1 protease-amprenavir complex suggests that the three mutations do not significantly altermore » the drug–enzyme interactions. This is in contrast to the observations in previous 100 K X-ray structures of these complexes that indicated loss of interactions by the drug with the triple mutant protease. These findings, thus, uncover limitations of structural analysis of drug binding using X-ray structures obtained at 100 K.« less

  12. Chronic pancreatitis associated with the p.G208A variant of PRSS1 gene in a European patient.

    PubMed

    Hegyi, Eszter; Cierna, Iveta; Vavrova, Ludmila; Ilencikova, Denisa; Konecny, Michal; Kovacs, Laszlo

    2014-01-10

    The major etiologic factor of chronic pancreatitis in adults is excessive alcohol consumption, whereas among children structural anomalies, systemic and metabolic disorders, and genetic factors are prevalent. Mutations in the cationic trypsinogen gene (PRSS1) cause hereditary pancreatitis, while mutations in serine protease inhibitor Kazal type 1 (SPINK1), cystic fibrosis transmembrane conductance regulator (CFTR) and chymotrypsin C (CTRC) genes have been shown to associate with chronic pancreatitis as independent risk factors. We present a case of 13-year-old boy with idiopathic chronic pancreatitis. Given the unexplained attacks of pancreatitis since early childhood and despite the negative family history, molecular-genetic analysis of four pancreatitis susceptibility genes (PRSS1, SPINK1, CTRC and CFTR) was performed. The boy was found to carry the c.623G>C (p.G208A) mutation of the PRSS1 gene and the c.180C>T (p.G60G) mutation of the CTRC gene, both in heterozygous state. These mutations are considered as contributing risk factors for chronic pancreatitis. In children with idiopathic chronic pancreatitis genetic causes should be considered, even in absence of positive family history. To the best of our knowledge, this is the first description of a European patient with chronic pancreatitis associated with the p.G208A mutation of PRSS1 gene. This mutation was previously reported only in Asian subjects and is thought to be a unique genetic cause of pancreatitis in Asia.

  13. Heterocyclic HIV-protease inhibitors.

    PubMed

    Calugi, C; Guarna, A; Trabocchi, A

    2013-01-01

    In the panorama of HIV protease inhibitors (HIV PIs), many efforts have been devoted to the development of new compounds with reduced peptidic nature in order to improve pharmacokinetics and pharmacodynamics features. The introduction of cyclic scaffolds in the design of new chemical entities reduces flexibility and affords more rigid inhibitors. Specifically, common dipeptide isosteres are replaced by a central cyclic scaffold designed to address the key interactions with catalytic aspartic acids and residues belonging to the flap region of the active site. The current interest in cyclic chemotypes addressing key interactions of HIV protease is motivated by the different nature of interactions formed with the enzyme, although maintaining key structural resemblance to a peptide substrate, hopefully giving rise to novel HIV-1 PIs displaying an improved profile towards multidrug resistant strains. This approach has been demonstrated for Tipranavir, which is a potent FDA approved HIV-1 PI representing the most famous example of heterocyclic aspartic protease inhibitors.

  14. Genetic Polymorphism of SUMO-Specific Cysteine Proteases - SENP1 and SENP2 in Breast Cancer.

    PubMed

    Mirecka, Alicja; Morawiec, Zbigniew; Wozniak, Katarzyna

    2016-10-01

    SENP proteases take part in post-translational modification of proteins known as sumoylation. They catalyze three distinct processes during sumoylation: processing of SUMO protein, deconjugation of SUMO from the target protein, and chain editing which mentions to the dismantling of SUMO chain. Many proteins that are involved in the basic processes of cells, such as regulation of transcription, DNA repair or cell cycle control, are sumoylated. The aim of these studies was to investigate an association between polymorphic variants (SNPs) of the SENP1 gene (c.1691 + 36C > T, rs12297820) and SENP2 gene (c.902C > A, p.Thr301Lys, rs6762208) and a risk of breast cancer occurrence. We performed a case-control study in 324 breast cancer cases and 335 controls using PCR-RLFP. In the case of the SENP1 gene polymorphism we did not find any association between this polymorphism and breast cancer risk. In the case of SENP2 gene polymorphism we observed higher risk of breast cancer for carriers of the A allele (OR =1.33; 95 % CI 1.04-1.69). Our analysis also showed the genotype C/C (OR =0.67, 95 % CI 0.48-0.93) and the allele C (OR =0.75, 95 % CI 0.59-0.69) of this polymorphism decrease a risk of breast cancer. We also checked the distribution of genotypes and frequency of alleles of the SENP1 and SENP2 genes polymorphisms in groups of patients with different hormone receptor status, patients with positive and negative lymph node status and patients with different tumor grade. Odds ratio analysis showed a higher risk of metastases in women with the genotype C/C (OR =2.07, 95 % CI 1.06-4.05) and allele C (OR =2.10 95 % CI 1.10-4.01) of the c.1691 + 36C > T SENP1 gene polymorphism. Moreover, we observed reduced risk in women with the allele T (OR =0.48, 95 % CI 0.25-0.91) in this polymorphic site. In the case of SENP2 gene polymorphism we observed that the A/A genotype correlated with the lack of estrogen receptor (OR =1.94, 95 % CI 1.04-3.62). Our results suggest

  15. Nucleotide sequences encoding a thermostable alkaline protease

    DOEpatents

    Wilson, D.B.; Lao, G.

    1998-01-06

    Nucleotide sequences, derived from a thermophilic actinomycete microorganism, which encode a thermostable alkaline protease are disclosed. Also disclosed are variants of the nucleotide sequences which encode a polypeptide having thermostable alkaline proteolytic activity. Recombinant thermostable alkaline protease or recombinant polypeptide may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant thermostable alkaline protease or recombinant polypeptide from the culture medium. 3 figs.

  16. Structures of a bi-functional Kunitz-type STI family inhibitor of serine and aspartic proteases: Could the aspartic protease inhibition have evolved from a canonical serine protease-binding loop?

    PubMed

    Guerra, Yasel; Valiente, Pedro A; Pons, Tirso; Berry, Colin; Rudiño-Piñera, Enrique

    2016-08-01

    Bi-functional inhibitors from the Kunitz-type soybean trypsin inhibitor (STI) family are glycosylated proteins able to inhibit serine and aspartic proteases. Here we report six crystal structures of the wild-type and a non-glycosylated mutant of the bifunctional inhibitor E3Ad obtained at different pH values and space groups. The crystal structures show that E3Ad adopts the typical β-trefoil fold of the STI family exhibiting some conformational changes due to pH variations and crystal packing. Despite the high sequence identity with a recently reported potato cathepsin D inhibitor (PDI), three-dimensional structures obtained in this work show a significant conformational change in the protease-binding loop proposed for aspartic protease inhibition. The E3Ad binding loop for serine protease inhibition is also proposed, based on structural similarity with a novel non-canonical conformation described for the double-headed inhibitor API-A from the Kunitz-type STI family. In addition, structural and sequence analyses suggest that bifunctional inhibitors of serine and aspartic proteases from the Kunitz-type STI family are more similar to double-headed inhibitor API-A than other inhibitors with a canonical protease-binding loop. Copyright © 2016. Published by Elsevier Inc.

  17. Crystal structures of inhibitor complexes of human T-cell leukemia virus (HTLV-1) protease

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Satoh, Tadashi; Li, Mi; Nguyen, Jeffrey-Tri

    2010-09-28

    Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus associated with several serious diseases, such as adult T-cell leukemia and tropical spastic paraparesis/myelopathy. For a number of years, the protease (PR) encoded by HTLV-1 has been a target for designing antiviral drugs, but that effort was hampered by limited available structural information. We report a high-resolution crystal structure of HTLV-1 PR complexed with a statine-containing inhibitor, a significant improvement over the previously available moderate-resolution structure. We also report crystal structures of the complexes of HTLV-1 PR with five different inhibitors that are more compact and more potent. A detailedmore » study of structure-activity relationships was performed to interpret in detail the influence of the polar and hydrophobic interactions between the inhibitors and the protease.« less

  18. Crystal Structures of Inhibitir Complexes of Human T-Cell Leukemia Virus (HTLV-1) Protease

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Satoh, Tadashi; Li, Mi; Nguyen, Jeffrey-Tri

    2010-09-17

    Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus associated with several serious diseases, such as adult T-cell leukemia and tropical spastic paraparesis/myelopathy. For a number of years, the protease (PR) encoded by HTLV-1 has been a target for designing antiviral drugs, but that effort was hampered by limited available structural information. We report a high-resolution crystal structure of HTLV-1 PR complexed with a statine-containing inhibitor, a significant improvement over the previously available moderate-resolution structure. We also report crystal structures of the complexes of HTLV-1 PR with five different inhibitors that are more compact and more potent. A detailedmore » study of structure-activity relationships was performed to interpret in detail the influence of the polar and hydrophobic interactions between the inhibitors and the protease.« less

  19. Targeting cysteine residues of human immunodeficiency virus type 1 protease by reactive free radical species.

    PubMed

    Basu, A; Sehajpal, P K; Ogiste, J S; Lander, H M

    1999-01-01

    Nitric oxide (NO) is a naturally occurring free radical with many functions. The oxidized form of NO, the nitrosonium ion, reacts with the thiol group of cysteine residues resulting in their modification to S-nitrosothiols. The human immunodeficiency virus type 1 (HIV-1) protease (HIV-PR) has two cysteine residues that are conserved amongst different viral isolates found in patients with acquired immunodeficiency syndrome (AIDS). In an active dimer, these residues are located near the surface of the protease. We have found that treatment of HIV-PR with different NO congeners results in loss of its proteolytic activity and simultaneous formation of S-nitrosothiols. Sodium nitroprusside inhibited HIV-PR up to 70% and S-nitroso-N-acetylpenicillamine completely inhibited the protease within 5 min of treatment. The pattern of inhibition by NO donors is comparable to its inhibition by N-acetyl pepstatin. Using electrospray ionization-mass spectrometry, we identified the modification of HIV-PR by NO as that of S-nitrosation. Our findings point towards a possible role of NO in mediating resistance to HIV-1 infection.

  20. Cell entry by a novel European filovirus requires host endosomal cysteine proteases and Niemann-Pick C1

    PubMed Central

    Ng, Melinda; Ndungo, Esther; Jangra, Rohit K.; Cai, Yingyun; Postnikova, Elena; Radoshitzky, Sheli R.; Dye, John M.; de Arellano, Eva Ramírez; Negredo, Ana; Palacios, Gustavo; Kuhn, Jens H.; Chandran, Kartik

    2014-01-01

    Lloviu virus (LLOV), a phylogenetically divergent filovirus, is the proposed etiologic agent of die-offs of Schreiber’s long-fingered bats (Miniopterus schreibersii) in western Europe. Studies of LLOV remain limited because the infectious agent has not yet been isolated. Here, we generated a recombinant vesicular stomatitis virus expressing the LLOV spike glycoprotein (GP) and used it to show that LLOV GP resembles other filovirus GP proteins in structure and function. LLOV GP must be cleaved by endosomal cysteine proteases during entry, but is much more protease-sensitive than EBOV GP. The EBOV/MARV receptor, Niemann Pick C1 (NPC1), is also required for LLOV entry, and its second luminal domain is recognized with high affinity by a cleaved form of LLOV GP, suggesting that receptor binding would not impose a barrier to LLOV infection of humans and non-human primates. The use of NPC1 as an intracellular entry receptor may be a universal property of filoviruses. PMID:25310500

  1. Subfamily-Specific Fluorescent Probes for Cysteine Proteases Display Dynamic Protease Activities during Seed Germination.

    PubMed

    Lu, Haibin; Chandrasekar, Balakumaran; Oeljeklaus, Julian; Misas-Villamil, Johana C; Wang, Zheming; Shindo, Takayuki; Bogyo, Matthew; Kaiser, Markus; van der Hoorn, Renier A L

    2015-08-01

    Cysteine proteases are an important class of enzymes implicated in both developmental and defense-related programmed cell death and other biological processes in plants. Because there are dozens of cysteine proteases that are posttranslationally regulated by processing, environmental conditions, and inhibitors, new methodologies are required to study these pivotal enzymes individually. Here, we introduce fluorescence activity-based probes that specifically target three distinct cysteine protease subfamilies: aleurain-like proteases, cathepsin B-like proteases, and vacuolar processing enzymes. We applied protease activity profiling with these new probes on Arabidopsis (Arabidopsis thaliana) protease knockout lines and agroinfiltrated leaves to identify the probe targets and on other plant species to demonstrate their broad applicability. These probes revealed that most commercially available protease inhibitors target unexpected proteases in plants. When applied on germinating seeds, these probes reveal dynamic activities of aleurain-like proteases, cathepsin B-like proteases, and vacuolar processing enzymes, coinciding with the remobilization of seed storage proteins. © 2015 American Society of Plant Biologists. All Rights Reserved.

  2. Heterogeneity of heat-resistant proteases from milk Pseudomonas species.

    PubMed

    Marchand, Sophie; Vandriesche, Gonzalez; Coorevits, An; Coudijzer, Katleen; De Jonghe, Valerie; Dewettinck, Koen; De Vos, Paul; Devreese, Bart; Heyndrickx, Marc; De Block, Jan

    2009-07-31

    Pseudomonas fragi, Pseudomonas lundensis and members of the Pseudomonas fluorescens group may spoil Ultra High Temperature (UHT) treated milk and dairy products, due to the production of heat-stable proteases in the cold chain of raw milk. Since the aprX gene codes for a heat-resistant protease in P. fluorescens, the presence of this gene has also been investigated in other members of the genus. For this purpose an aprX-screening PCR test has been developed. Twenty-nine representatives of important milk Pseudomonas species and thirty-five reference strains were screened. In 42 out of 55 investigated Pseudomonas strains, the aprX gene was detected, which proves the potential of the aprX-PCR test as a screening tool for potentially proteolytic Pseudomonas strains in milk samples. An extensive study of the obtained aprX-sequences on the DNA and the amino acid level, however, revealed a large heterogeneity within the investigated milk isolates. Although this heterogeneity sets limitations to a general detection method for all proteolytic Pseudomonas strains in milk, it offers a great potential for the development of a multiplex PCR screening test targeting individual aprX-genes. Furthermore, our data illustrated the potential use of the aprX gene as a taxonomic marker, which may help in resolving the current taxonomic deadlock in the P. fluorescens group.

  3. Isolation, activity and immunological characterisation of a secreted aspartic protease, CtsD, from Aspergillus fumigatus.

    PubMed

    Vickers, Imelda; Reeves, Emer P; Kavanagh, Kevin A; Doyle, Sean

    2007-05-01

    Aspergillus fumigatus is an opportunistic fungal pathogen that infects immunocompromised patients. A putative aspartic protease gene (ctsD; 1425 bp; intron-free) was identified and cloned. CtsD is evolutionarily distinct from all previously identified A. fumigatus aspartic proteases. Recombinant CtsD was expressed in inclusion bodies in Escherichia coli (0.2mg/g cells) and subjected to extensive proteolysis in the baculovirus expression system. Activation studies performed on purified, refolded, recombinant CtsD resulted in protease activation with a pH(opt)4.0 and specific activity=10 U/mg. Pepstatin A also inhibited recombinant CtsD activity by up to 72% thereby confirming classification as an aspartic protease. Native CtsD was also immunologically identified in culture supernatants and purified from fungal cultures using pepstatin-agarose affinity chromatography (7.8 microg CtsD/g mycelia). In A. fumigatus, semi-quantitative RT-PCR analysis revealed expression of ctsD in minimal and proteinaceous media only. Expression of ctsD was absent under nutrient-rich conditions. Expression of ctsD was also detected, in vivo, in the Galleria mellonella virulence model following A. fumigatus infection.

  4. Tripeptidyl Peptidase II. An Oligomeric Protease Complex from Arabidopsis1

    PubMed Central

    Book, Adam J.; Yang, Peizhen; Scalf, Mark; Smith, Lloyd M.; Vierstra, Richard D.

    2005-01-01

    The breakdown of most nuclear and cytoplasmic proteins involves their partial cleavage by the 26S proteasome followed by further disassembly to free amino acids by the combined action of endo- and exopeptidases. In animals, one important intermediate exopeptidase is tripeptidyl peptidase (TPP)II, which digests peptide products of the 26S proteasome and other endopeptidases into tripeptides. Here, we describe the purification and characterization of TPPII from Arabidopsis (Arabidopsis thaliana). Like its animal counterparts, Arabidopsis TPPII exists as a soluble, approximately 5- to 9-MD complex. Two related species of 153 and 142 kD are present in the purified preparations that are derived from a single TPP2 gene. Sequencing by Edman degradation of the intact polypeptides and mass spectrometry of proteolytic fragments demonstrated that the 142-kD form mainly differs from the 153-kD form by a truncation at the C-terminal end. This serine protease is a member of the subtilisin superfamily and is sensitive to the inhibitors alanine-alanine-phenylalanine-chloromethylketone and butabindide, which are diagnostic for the TPPII subfamily. The Arabidopsis TPP2 gene is widely expressed in many tissue types with related genes evident in other plant genomes. Whereas the 26S proteasome is essential, TPPII appears not as important for plant physiology. An Arabidopsis T-DNA mutant defective in TPP2 expression displays no phenotypic abnormalities and is not hypersensitive to either amino acid analogs or the 26S proteasome inhibitor MG132. As a consequence, plants likely contain other intermediate exopeptidases that assist in amino acid recycling. PMID:15908606

  5. NS3 protease resistance-associated substitutions in liver tissue and plasma samples from patients infected by hepatitis C virus genotype 1A or 1B.

    PubMed

    Morsica, Giulia; Andolina, Andrea; Merli, Marco; Messina, Emanuela; Hasson, Hamid; Lazzarin, Adriano; Uberti-Foppa, Caterina; Bagaglio, Sabrina

    2017-08-01

    The presence of naturally occurring resistance-associated substitutions (RASs) in the HCV-protease domain has been poorly investigated in the liver, the main site of HCV replication. We evaluated the natural resistance of the virus to NS3 protease inhibitors in liver tissue and plasma samples taken from HCV-infected patients. RASs were investigated by means of viral population sequencing in liver tissue samples from 18 HCV-infected patients harbouring genotype 1a or genotype 1b; plasma samples from 12 of these patients were also available for virological investigation. A discordant genotype was found in two of the 12 patients (16.6%) who provided samples from both compartments. Sequence analysis of the NS3 protease domain showed the presence of RASs in four of the 18 liver tissue samples (22.2%), two of which showed cross-resistance to protease inhibitors in clinical use or phase 2-3 trials. The analysis of the 12 paired tissues and plasma samples excluded the presence of RASs in the plasma compartment. The dominance of discordant genotypes in the paired liver and plasma samples of some HCV-infected patients suggests mixed infection possibly leading to the selective advantage of different genotype in the two compartments. The presence of RASs at intra-hepatic level is not uncommon and may lead to the early emergence of cross-resistant strains.

  6. Activation of the classical complement pathway by mannose-binding protein in association with a novel C1s-like serine protease

    PubMed Central

    1992-01-01

    Serum mannose-binding protein (MBP) is a C-type lectin that binds to terminal mannose and N-acetylglucosamine moieties present on surfaces of certain pathogens and activates the classical complement pathway. In the present study, we describe the mechanism underlying the activation triggered by MBP. The human serum MBP fraction was obtained by sequential affinity chromatography on mannan-Sepharose, anti-IgM- Sepharose and anti-MBP-Sepharose in the presence of calcium ions. This fraction contained a C1s-like serine protease as assessed by C4 consumption. The C1s-like serine protease, designated MBP-associated serine protease (MASP), was separated from MBP by rechromatography on anti-MBP-Sepharose in the presence of ethylenediaminetetraacetic acid. MASP exhibited both C4- and C2-consuming activities. The molecular mass of MASP was estimated to be 83 kD with two polypeptides of heavy (66 kD) and light (L) (31 kD) chains linked by disulfide bonds. The serine residue responsible for protease activity is located on the L chain. Reconstitution experiments using MASP and MBP revealed that combination of the two components restores C4- and C2-activating capacity on mannan. Based on analyses of molecular size, antigenicity, and 11 NH2- terminal amino acid sequences of the L chain, we conclude that MASP is a novel protein different from C1r or C1s. Our findings are not in accord with a proposed mechanism by which MBP utilizes the C1r2-C1s2 complex to initiate the classical complement pathway. PMID:1460414

  7. Alteration of Substrate and Inhibitor Specificity of Feline Immunodeficiency Virus Protease

    PubMed Central

    Lin, Ying-Chuan; Beck, Zachary; Lee, Taekyu; Le, Van-Duc; Morris, Garrett M.; Olson, Arthur J.; Wong, Chi-Huey; Elder, John H.

    2000-01-01

    Feline immunodeficiency virus (FIV) protease is structurally very similar to human immunodeficiency virus (HIV) protease but exhibits distinct substrate and inhibitor specificities. We performed mutagenesis of subsite residues of FIV protease in order to define interactions that dictate this specificity. The I37V, N55M, M56I, V59I, and Q99V mutants yielded full activity. The I37V, N55M, V59I, and Q99V mutants showed a significant increase in activity against the HIV-1 reverse transcriptase/integrase and P2/nucleocapsid junction peptides compared with wild-type (wt) FIV protease. The I37V, V59I, and Q99V mutants also showed an increase in activity against two rapidly cleaved peptides selected by cleavage of a phage display library with HIV-1 protease. Mutations at Q54K, I98P, and L101I dramatically reduced activity. Mutants containing a I35D or I57G substitution showed no activity against either FIV or HIV substrates. FIV proteases all failed to cut HIV-1 matrix/capsid, P1/P6, P6/protease, and protease/reverse transcriptase junctions, indicating that none of the substitutions were sufficient to change the specificity completely. The I37V, N55M, M56I, V59I, and Q99V mutants, compared with wt FIV protease, all showed inhibitor specificity more similar to that of HIV-1 protease. The data also suggest that FIV protease prefers a hydrophobic P2/P2′ residue like Val over Asn or Glu, which are utilized by HIV-1 protease, and that S2/S2′ might play a critical role in distinguishing FIV and HIV-1 protease by specificity. The findings extend our observations regarding the interactions involved in substrate binding and aid in the development of broad-based inhibitors. PMID:10775609

  8. LuxO controls extracellular protease, haemolytic activities and siderophore production in fish pathogen Vibrio alginolyticus.

    PubMed

    Wang, Q; Liu, Q; Ma, Y; Rui, H; Zhang, Y

    2007-11-01

    To characterize the luxO gene in fish pathogen Vibrio alginolyticus MVP01 and investigate its roles in regulation of extracellular products (ECP) and siderophore production. The luxO gene was cloned from V. alginolyticus MVP01. Genetic analysis revealed that it encoded a protein with high similarity to other LuxO homologues. The luxO in-frame deletion mutant and rpoN null mutant were constructed with suicide plasmids. We demonstrated that sole deletion in LuxO increased the secretion of extracellular protease and haemolytic products, but decreased siderophore production for V. alginolyticus MVP01. Mutants with null rpoN displayed significantly enhanced protease level and siderophore production while notable reduction in haemolytic activities of ECP. Vibrio alginolyticus harbours functional luxO gene that regulates the secretion of extracellular protease and haemolytic materials as well as siderophore production in either sigma(54) dependent or independent manners. The current study demonstrated that V. alginolyticus MVP01 produces extracellular protease and haemolytic activity material as well as siderophore, which may be characteristics of the virulence of the strain. Revelations that secretion of these products is under the regulation of LuxO and sigma(54) as well as the potential quorum sensing systems in V. alginolyticus MVP01 will expedite the understanding of vibriosis pathogenesis.

  9. Synthetic, structural mimetics of the β-hairpin flap of HIV-1 protease inhibit enzyme function.

    PubMed

    Chauhan, Jay; Chen, Shen-En; Fenstermacher, Katherine J; Naser-Tavakolian, Aurash; Reingewertz, Tali; Salmo, Rosene; Lee, Christian; Williams, Emori; Raje, Mithun; Sundberg, Eric; DeStefano, Jeffrey J; Freire, Ernesto; Fletcher, Steven

    2015-11-01

    Small-molecule mimetics of the β-hairpin flap of HIV-1 protease (HIV-1 PR) were designed based on a 1,4-benzodiazepine scaffold as a strategy to interfere with the flap-flap protein-protein interaction, which functions as a gated mechanism to control access to the active site. Michaelis-Menten kinetics suggested our small-molecules are competitive inhibitors, which indicates the mode of inhibition is through binding the active site or sterically blocking access to the active site and preventing flap closure, as designed. More generally, a new bioactive scaffold for HIV-1PR inhibition has been discovered, with the most potent compound inhibiting the protease with a modest K(i) of 11 μM. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Computational design and experimental study of tighter binding peptides to an inactivated mutant of HIV-1 protease

    PubMed Central

    Altman, Michael D.; Nalivaika, Ellen A.; Prabu-Jeyabalan, Moses; Schiffer, Celia A.; Tidor, Bruce

    2009-01-01

    Drug resistance in HIV-1 protease, a barrier to effective treatment, is generally caused by mutations in the enzyme that disrupt inhibitor binding but still allow for substrate processing. Structural studies with mutant, inactive enzyme, have provided detailed information regarding how the substrates bind to the protease yet avoid resistance mutations; insights obtained inform the development of next generation therapeutics. Although structures have been obtained of complexes between substrate peptide and inactivated (D25N) protease, thermodynamic studies of peptide binding have been challenging due to low affinity. Peptides that bind tighter to the inactivated protease than the natural substrates would be valuable for thermodynamic studies as well as to explore whether the structural envelope observed for substrate peptides is a function of weak binding. Here, two computational methods — namely, charge optimization and protein design — were applied to identify peptide sequences predicted to have higher binding affinity to the inactivated protease, starting from an RT–RH derived substrate peptide. Of the candidate designed peptides, three were tested for binding with isothermal titration calorimetry, with one, containing a single threonine to valine substitution, measured to have more than a ten-fold improvement over the tightest binding natural substrate. Crystal structures were also obtained for the same three designed peptide complexes; they show good agreement with computational prediction. Thermodynamic studies show that binding is entropically driven, more so for designed affinity enhanced variants than for the starting substrate. Structural studies show strong similarities between natural and tighter-binding designed peptide complexes, which may have implications in understanding the molecular mechanisms of drug resistance in HIV-1 protease. PMID:17729291

  11. A new fusion protein platform for quantitatively measuring activity of multiple proteases

    PubMed Central

    2014-01-01

    Background Recombinant proteins fused with specific cleavage sequences are widely used as substrate for quantitatively analyzing the activity of proteases. Here we propose a new fusion platform for multiple proteases, by using diaminopropionate ammonia-lyase (DAL) as the fusion protein. It was based on the finding that a fused His6-tag could significantly decreases the activities of DAL from E. coli (eDAL) and Salmonella typhimurium (sDAL). Previously, we have shown that His6GST-tagged eDAL could be used to determine the activity of tobacco etch virus protease (TEVp) under different temperatures or in the denaturant at different concentrations. In this report, we will assay different tags and cleavage sequences on DAL for expressing yield in E. coli, stability of the fused proteins and performance of substrate of other common proteases. Results We tested seven different protease cleavage sequences (rhinovirus 3C, TEV protease, factor Xa, Ssp DnaB intein, Sce VMA1 intein, thrombin and enterokinase), three different tags (His6, GST, CBD and MBP) and two different DALs (eDAL and sDAL), for their performance as substrate to the seven corresponding proteases. Among them, we found four active DAL-fusion substrates suitable for TEVp, factor Xa, thrombin and DnaB intein. Enterokinase cleaved eDAL at undesired positions and did not process sDAL. Substitution of GST with MBP increase the expression level of the fused eDAL and this fusion protein was suitable as a substrate for analyzing activity of rhinovirus 3C. We demonstrated that SUMO protease Ulp1 with a N-terminal His6-tag or MBP tag displayed different activity using the designed His6SUMO-eDAL as substrate. Finally, owing to the high level of the DAL-fusion protein in E. coli, these protein substrates can also be detected directly from the crude extract. Conclusion The results show that our designed DAL-fusion proteins can be used to quantify the activities of both sequence- and conformational-specific proteases, with

  12. Extracellular Protease Inhibition Alters the Phenotype of Chondrogenically Differentiating Human Mesenchymal Stem Cells (MSCs) in 3D Collagen Microspheres.

    PubMed

    Han, Sejin; Li, Yuk Yin; Chan, Barbara Pui

    2016-01-01

    Matrix remodeling of cells is highly regulated by proteases and their inhibitors. Nevertheless, how would the chondrogenesis of mesenchymal stem cells (MSCs) be affected, when the balance of the matrix remodeling is disturbed by inhibiting matrix proteases, is incompletely known. Using a previously developed collagen microencapsulation platform, we investigated whether exposing chondrogenically differentiating MSCs to intracellular and extracellular protease inhibitors will affect the extracellular matrix remodeling and hence the outcomes of chondrogenesis. Results showed that inhibition of matrix proteases particularly the extracellular ones favors the phenotype of fibrocartilage rather than hyaline cartilage in chondrogenically differentiating hMSCs by upregulating type I collagen protein deposition and type II collagen gene expression without significantly altering the hypertrophic markers at gene level. This study suggests the potential of manipulating extracellular proteases to alter the outcomes of hMSC chondrogenesis, contributing to future development of differentiation protocols for fibrocartilage tissues for intervertebral disc and meniscus tissue engineering.

  13. Extracellular Protease Inhibition Alters the Phenotype of Chondrogenically Differentiating Human Mesenchymal Stem Cells (MSCs) in 3D Collagen Microspheres

    PubMed Central

    Han, Sejin; Li, Yuk Yin; Chan, Barbara Pui

    2016-01-01

    Matrix remodeling of cells is highly regulated by proteases and their inhibitors. Nevertheless, how would the chondrogenesis of mesenchymal stem cells (MSCs) be affected, when the balance of the matrix remodeling is disturbed by inhibiting matrix proteases, is incompletely known. Using a previously developed collagen microencapsulation platform, we investigated whether exposing chondrogenically differentiating MSCs to intracellular and extracellular protease inhibitors will affect the extracellular matrix remodeling and hence the outcomes of chondrogenesis. Results showed that inhibition of matrix proteases particularly the extracellular ones favors the phenotype of fibrocartilage rather than hyaline cartilage in chondrogenically differentiating hMSCs by upregulating type I collagen protein deposition and type II collagen gene expression without significantly altering the hypertrophic markers at gene level. This study suggests the potential of manipulating extracellular proteases to alter the outcomes of hMSC chondrogenesis, contributing to future development of differentiation protocols for fibrocartilage tissues for intervertebral disc and meniscus tissue engineering. PMID:26760956

  14. A unique serpin P1' glutamate and a conserved β-sheet C arginine are key residues for activity, protease recognition and stability of serpinA12 (vaspin).

    PubMed

    Ulbricht, David; Pippel, Jan; Schultz, Stephan; Meier, René; Sträter, Norbert; Heiker, John T

    2015-09-15

    SerpinA12 (vaspin) is thought to be mainly expressed in adipose tissue and has multiple beneficial effects on metabolic, inflammatory and atherogenic processes related to obesity. KLK7 (kallikrein 7) is the only known protease target of vaspin to date and is inhibited with a moderate inhibition rate. In the crystal structure, the cleavage site (P1-P1') of the vaspin reactive centre loop is fairly rigid compared with the flexible residues before P2, possibly supported by an ionic interaction of P1' glutamate (Glu(379)) with an arginine residue (Arg(302)) of the β-sheet C. A P1' glutamate seems highly unusual and unfavourable for the protease KLK7. We characterized vaspin mutants to investigate the roles of these two residues in protease inhibition and recognition by vaspin. Reactive centre loop mutations changing the P1' residue or altering the reactive centre loop conformation significantly increased inhibition parameters, whereas removal of the positive charge within β-sheet C impeded the serpin-protease interaction. Arg(302) is a crucial contact to enable vaspin recognition by KLK7 and it supports moderate inhibition of the serpin despite the presence of the detrimental P1' Glu(379), which clearly represents a major limiting factor for vaspin-inhibitory activity. We also show that the vaspin-inhibition rate for KLK7 can be modestly increased by heparin and demonstrate that vaspin is a heparin-binding serpin. Noteworthily, we observed vaspin as a remarkably thermostable serpin and found that Glu(379) and Arg(302) influence heat-induced polymerization. These structural and functional results reveal the mechanistic basis of how reactive centre loop sequence and exosite interaction in vaspin enable KLK7 recognition and regulate protease inhibition as well as stability of this adipose tissue-derived serpin. © 2015 Authors; published by Portland Press Limited.

  15. Do alterations in follicular fluid proteases contribute to human infertility?

    PubMed

    Cookingham, Lisa Marii; Van Voorhis, Bradley J; Ascoli, Mario

    2015-05-01

    Cathepsin L and ADAMTS-1 are known to play critical roles in follicular rupture, ovulation, and fertility in mice. Similar studies in humans are limited; however, both are known to increase during the periovulatory period. No studies have examined either protease in the follicular fluid of women with unexplained infertility or infertility related to advanced maternal age (AMA). We sought to determine if alterations in cathepsin L and/or ADAMTS-1 existed in these infertile populations. Patients undergoing in vitro fertilization (IVF) for unexplained infertility or AMA-related infertility were prospectively recruited for the study; patients with tubal or male factor infertility were recruited as controls. Follicular fluid was collected to determine gene expression (via quantitative polymerase chain reaction), enzyme concentrations (via enzyme-linked immunosorbent assays), and enzymatic activities (via fluorogenic enzyme cleavage assay or Western blot analysis) of cathepsin L and ADAMTS-1. The analysis included a total of 42 patients (14 per group). We found no statistically significant difference in gene expression, enzyme concentration, or enzymatic activity of cathepsin L or ADAMTS-1 in unexplained infertility or AMA-related infertility as compared to controls. We also found no statistically significant difference in expression or concentration with advancing age. Cathepsin L and ADAMTS-1 are not altered in women with unexplained infertility or AMA-related infertility undergoing IVF, and they do not decline with advancing age. It is possible that differences exist in natural cycles, contributing to infertility; however, our findings do not support a role for protease alterations as a common cause of infertility.

  16. Two Paralogous Families of a Two-Gene Subtilisin Operon Are Widely Distributed in Oral Treponemes

    PubMed Central

    Correia, Frederick F.; Plummer, Alvin R.; Ellen, Richard P.; Wyss, Chris; Boches, Susan K.; Galvin, Jamie L.; Paster, Bruce J.; Dewhirst, Floyd E.

    2003-01-01

    Certain oral treponemes express a highly proteolytic phenotype and have been associated with periodontal diseases. The periodontal pathogen Treponema denticola produces dentilisin, a serine protease of the subtilisin family. The two-gene operon prcA-prtP is required for expression of active dentilisin (PrtP), a putative lipoprotein attached to the treponeme's outer membrane or sheath. The purpose of this study was to examine the diversity and structure of treponemal subtilisin-like proteases in order to better understand their distribution and function. The complete sequences of five prcA-prtP operons were determined for Treponema lecithinolyticum, “Treponema vincentii,” and two canine species. Partial operon sequences were obtained for T. socranskii subsp. 04 as well as 450- to 1,000-base fragments of prtP genes from four additional treponeme strains. Phylogenetic analysis demonstrated that the sequences fall into two paralogous families. The first family includes the sequence from T. denticola. Treponemes possessing this operon family express chymotrypsin-like protease activity and can cleave the substrate N-succinyl-alanyl-alanyl-prolyl-phenylalanine-p-nitroanilide (SAAPFNA). Treponemes possessing the second paralog family do not possess chymotrypsin-like activity or cleave SAAPFNA. Despite examination of a range of protein and peptide substrates, the specificity of the second protease family remains unknown. Each of the fully sequenced prcA and prtP genes contains a 5′ hydrophobic leader sequence with a treponeme lipobox. The two paralogous families of treponeme subtilisins represent a new subgroup within the subtilisin family of proteases and are the only subtilisin lipoprotein family. The present study demonstrated that the subtilisin paralogs comprising a two-gene operon are widely distributed among treponemes. PMID:14617650

  17. RNAi-mediated knockdown of serine protease inhibitor genes increases the mortality of Plutella xylostella challenged by destruxin A.

    PubMed

    Han, Pengfei; Fan, Jiqiao; Liu, Yu; Cuthbertson, Andrew G S; Yan, Shaoqiao; Qiu, Bao-Li; Ren, Shunxiang

    2014-01-01

    Destruxin A is a mycotoxin that is secreted by entomopathogenic fungi which has a broad-spectrum insecticidal effect. Previous transcript and protein profiling analysis showed that destruxin A has significant effects on the expression of serine protease inhibitor genes (serpin-2, 4, 5) in the larvae of Plutella xylostella. In the current study, we aimed to understand the role of serpins under application of destruxin A. We obtained two full-length cDNA sequences of P. xylostella serpins, named serpin-4 and serpin-5, and cloned the serpin-2 gene whose full-length has already been published. Phylogenetic analysis indicated that these two serpin genes were highly clustered with other serpins associated with the immune response in other insects. The temporal and spatial expression of serpin-2, serpin-4 and serpin-5 were determined to be the highest in the fat body and hemolymph of 4th larval stage using qRT-PCR and western blot detection techniques. RNA interference (RNAi) mediated knockdown of P. xylostella serpin genes was carried out by microinjection of double-stranded RNA (dsRNA). The expression levels of serpins decreased significantly after RNAi. Results showed that the depletion of serpins induced cecropins expression, increased phenoloxidase (PO) activity, body melanization and mortality in the larvae of P. xylostella under the same lethal concentration of destruxin A. The superimposed effects of serpins RNAi were similar with the destruxin A treatment upon mortality of P. xylostella larvae. We discovered for the first time that serpins play indispensable role in P. xylostella when challenged by destruxin A and deduced the possible function mechanism of destruxin A. Our findings are conducive to fully understanding the potential insecticidal mechanism of destruxin A and constitute a well-defined potential molecular target for novel insecticides.

  18. RNAi-Mediated Knockdown of Serine Protease Inhibitor Genes Increases the Mortality of Plutella xylostella Challenged by Destruxin A

    PubMed Central

    Han, Pengfei; Fan, Jiqiao; Liu, Yu; Cuthbertson, Andrew G. S.; Yan, Shaoqiao; Qiu, Bao-Li; Ren, Shunxiang

    2014-01-01

    Destruxin A is a mycotoxin that is secreted by entomopathogenic fungi which has a broad-spectrum insecticidal effect. Previous transcript and protein profiling analysis showed that destruxin A has significant effects on the expression of serine protease inhibitor genes (serpin-2, 4, 5) in the larvae of Plutella xylostella. In the current study, we aimed to understand the role of serpins under application of destruxin A. We obtained two full-length cDNA sequences of P. xylostella serpins, named serpin-4 and serpin-5, and cloned the serpin-2 gene whose full-length has already been published. Phylogenetic analysis indicated that these two serpin genes were highly clustered with other serpins associated with the immune response in other insects. The temporal and spatial expression of serpin-2, serpin-4 and serpin-5 were determined to be the highest in the fat body and hemolymph of 4th larval stage using qRT-PCR and western blot detection techniques. RNA interference (RNAi) mediated knockdown of P. xylostella serpin genes was carried out by microinjection of double-stranded RNA (dsRNA). The expression levels of serpins decreased significantly after RNAi. Results showed that the depletion of serpins induced cecropins expression, increased phenoloxidase (PO) activity, body melanization and mortality in the larvae of P. xylostella under the same lethal concentration of destruxin A. The superimposed effects of serpins RNAi were similar with the destruxin A treatment upon mortality of P. xylostella larvae. We discovered for the first time that serpins play indispensable role in P. xylostella when challenged by destruxin A and deduced the possible function mechanism of destruxin A. Our findings are conducive to fully understanding the potential insecticidal mechanism of destruxin A and constitute a well-defined potential molecular target for novel insecticides. PMID:24837592

  19. Association of frailty with the serine protease HtrA1 in older adults.

    PubMed

    Lorenzi, Maria; Lorenzi, Teresa; Marzetti, Emanuele; Landi, Francesco; Vetrano, Davide L; Settanni, Silvana; Antocicco, Manuela; Bonassi, Stefano; Valdiglesias, Vanessa; Bernabei, Roberto; Onder, Graziano

    2016-08-01

    Frailty is a geriatric syndrome characterized by multi system dysregulation. It has been suggested that chronic inflammation may be involved in the pathogenesis of frailty. No study so far has identified accurate, specific and sensitive molecular biomarkers for frailty. High-temperature requirement serine protease A1 (HtrA1) is a secreted multidomain serine protease implicated in the inhibition of signaling of active transforming growth factor-β (TGF-β)1, a cytokine which has an important anti-inflammation role. The aim of the present study was to investigate the association of circulating levels of HtrA1 with frailty in a sample of older adults. The study was performed in 120 older adults aged >65years and admitted to a geriatric outpatient clinic. The frailty status of participants was assessed by both the Fried's criteria (physical frailty, PF) and a modified Rockwood's frailty index (FI). Plasma HtrA1 concentration was measured using commercial ELISA kit. Frailty was identified in 61/120 participants (50.8%) using PF, and in 60/118 subjects (50.8%) using FI. Plasma levels of HtrA1 were significantly higher in individuals classified as frail according to PF (75.9ng/mL, 95% CI 67.4-85.6) as compared with non-frail participants (48.4ng/mL, 95% CI 42.5-54.6, p<0.001). A significant association was also observed between frailty, assessed by FI, and HtrA1 levels (72.2ng/mL, 95% CI 63.4-82.3, vs. 50.4ng/mL, 95% CI 44.3-58.0, p<0.001). These associations were confirmed after adjusting for potential confounders. This study demonstrates for the first time the association of plasma levels of HtrA1 with frailty status. Future investigations are needed to validate the potential value of HtrA1 as possible biomarker for frailty. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Identification of a Degradation Signal Sequence within Substrates of the Mitochondrial i-AAA Protease.

    PubMed

    Rampello, Anthony J; Glynn, Steven E

    2017-03-24

    The i-AAA protease is a component of the mitochondrial quality control machinery that regulates respiration, mitochondrial dynamics, and protein import. The protease is required to select specific substrates for degradation from among the diverse complement of proteins present in mitochondria, yet the rules that govern this selection are unclear. Here, we reconstruct the yeast i-AAA protease, Yme1p, to examine the in vitro degradation of two intermembrane space chaperone subunits, Tim9 and Tim10. Yme1p degrades Tim10 more rapidly than Tim9 despite high sequence and structural similarity, and loss of Tim10 is accelerated by the disruption of conserved disulfide bonds within the substrate. An unstructured N-terminal region of Tim10 is necessary and sufficient to target the substrate to the protease through recognition of a short phenylalanine-rich motif, and the presence of similar motifs in other small Tim proteins predicts robust degradation by the protease. Together, these results identify the first specific degron sequence within a native i-AAA protease substrate. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. A novel serine protease, Sep1, from Bacillus firmus DS-1 has nematicidal activity and degrades multiple intestinal-associated nematode proteins.

    PubMed

    Geng, Ce; Nie, Xiangtao; Tang, Zhichao; Zhang, Yuyang; Lin, Jian; Sun, Ming; Peng, Donghai

    2016-04-27

    Plant-parasitic nematodes (PPNs) cause serious harm to agricultural production. Bacillus firmus shows excellent control of PPNs and has been produced as a commercial nematicide. However, its nematicidal factors and mechanisms are still unknown. In this study, we showed that B. firmus strain DS-1 has high toxicity against Meloidogyne incognita and soybean cyst nematode. We sequenced the whole genome of DS-1 and identified multiple potential virulence factors. We then focused on a peptidase S8 superfamily protein called Sep1 and demonstrated that it had toxicity against the nematodes Caenorhabditis elegans and M. incognita. The Sep1 protein exhibited serine protease activity and degraded the intestinal tissues of nematodes. Thus, the Sep1 protease of B. firmus is a novel biocontrol factor with activity against a root-knot nematode. We then used C. elegans as a model to elucidate the nematicidal mechanism of Sep1, and the results showed that Sep1 could degrade multiple intestinal and cuticle-associated proteins and destroyed host physical barriers. The knowledge gained in our study will lead to a better understanding of the mechanisms of B. firmus against PPNs and will aid in the development of novel bio-agents with increased efficacy for controlling PPNs.

  2. Serpin1 and WSCP differentially regulate the activity of the cysteine protease RD21 during plant development in Arabidopsis thaliana

    PubMed Central

    Rustgi, Sachin; Boex-Fontvieille, Edouard; Reinbothe, Christiane; von Wettstein, Diter; Reinbothe, Steffen

    2017-01-01

    Proteolytic enzymes (proteases) participate in a vast range of physiological processes, ranging from nutrient digestion to blood coagulation, thrombosis, and beyond. In plants, proteases are implicated in host recognition and pathogen infection, induced defense (immunity), and the deterrence of insect pests. Because proteases irreversibly cleave peptide bonds of protein substrates, their activity must be tightly controlled in time and space. Here, we report an example of how nature evolved alternative mechanisms to fine-tune the activity of a cysteine protease dubbed RD21 (RESPONSIVE TO DESICCATION-21). One mechanism in the model plant Arabidopsis thaliana studied here comprises irreversible inhibition of RD21’s activity by Serpin1, whereas the other mechanism is a result of the reversible inhibition of RD21 activity by a Kunitz protease inhibitor named water-soluble chlorophyll-binding protein (WSCP). Activity profiling, complex isolation, and homology modeling data revealed unique interactions of RD21 with Serpin1 and WSCP, respectively. Expression studies identified only partial overlaps in Serpin1 and WSCP accumulation that explain how RD21 contributes to the innate immunity of mature plants and arthropod deterrence of seedlings undergoing skotomorphogenesis and greening. PMID:28179567

  3. Serpin1 and WSCP differentially regulate the activity of the cysteine protease RD21 during plant development in Arabidopsis thaliana.

    PubMed

    Rustgi, Sachin; Boex-Fontvieille, Edouard; Reinbothe, Christiane; von Wettstein, Diter; Reinbothe, Steffen

    2017-02-28

    Proteolytic enzymes (proteases) participate in a vast range of physiological processes, ranging from nutrient digestion to blood coagulation, thrombosis, and beyond. In plants, proteases are implicated in host recognition and pathogen infection, induced defense (immunity), and the deterrence of insect pests. Because proteases irreversibly cleave peptide bonds of protein substrates, their activity must be tightly controlled in time and space. Here, we report an example of how nature evolved alternative mechanisms to fine-tune the activity of a cysteine protease dubbed RD21 (RESPONSIVE TO DESICCATION-21). One mechanism in the model plant Arabidopsis thaliana studied here comprises irreversible inhibition of RD21's activity by Serpin1, whereas the other mechanism is a result of the reversible inhibition of RD21 activity by a Kunitz protease inhibitor named water-soluble chlorophyll-binding protein (WSCP). Activity profiling, complex isolation, and homology modeling data revealed unique interactions of RD21 with Serpin1 and WSCP, respectively. Expression studies identified only partial overlaps in Serpin1 and WSCP accumulation that explain how RD21 contributes to the innate immunity of mature plants and arthropod deterrence of seedlings undergoing skotomorphogenesis and greening.

  4. Structural determinants of tobacco vein mottling virus protease substrate specificity

    PubMed Central

    Sun, Ping; Austin, Brian P; Tözsér, József; Waugh, David S

    2010-01-01

    Tobacco vein mottling virus (TVMV) is a member of the Potyviridae, one of the largest families of plant viruses. The TVMV genome is translated into a single large polyprotein that is subsequently processed by three virally encoded proteases. Seven of the nine cleavage events are carried out by the NIa protease. Its homolog from the tobacco etch virus (TEV) is a widely used reagent for the removal of affinity tags from recombinant proteins. Although TVMV protease is a close relative of TEV protease, they exhibit distinct sequence specificities. We report here the crystal structure of a catalytically inactive mutant TVMV protease (K65A/K67A/C151A) in complex with a canonical peptide substrate (Ac-RETVRFQSD) at 1.7-Å resolution. As observed in several crystal structures of TEV protease, the C-terminus (∼20 residues) of TVMV protease is disordered. Unexpectedly, although deleting the disordered residues from TEV protease reduces its catalytic activity by ∼10-fold, an analogous truncation mutant of TVMV protease is significantly more active. Comparison of the structures of TEV and TVMV protease in complex with their respective canonical substrate peptides reveals that the S3 and S4 pockets are mainly responsible for the differing substrate specificities. The structure of TVMV protease suggests that it is less tolerant of variation at the P1′ position than TEV protease. This conjecture was confirmed experimentally by determining kinetic parameters kcat and Km for a series of oligopeptide substrates. Also, as predicted by the cocrystal structure, we confirm that substitutions in the P6 position are more readily tolerated by TVMV than TEV protease. PMID:20862670

  5. Variably Protease-Sensitive Prionopathy: A New Sporadic Disease of the Prion Protein

    PubMed Central

    Zou, Wen-Quan; Puoti, Gianfranco; Xiao, Xiangzhu; Yuan, Jue; Qing, Liuting; Cali, Ignazio; Shimoji, Miyuki; Langeveld, Jan P. M.; Castellani, Rudy; Notari, Silvio; Crain, Barbara; Schmidt, Robert E.; Geschwind, Michael; DeArmond, Stephen J.; Cairns, Nigel J.; Dickson, Dennis; Honig, Lawrence; Torres, Juan Maria; Mastrianni, James; Capellari, Sabina; Giaccone, Giorgio; Belay, Ermias D.; Schonberger, Lawrence B.; Cohen, Mark; Perry, George; Kong, Qingzhong; Parchi, Piero; Tagliavini, Fabrizio; Gambetti, Pierluigi

    2011-01-01

    Objective The objective of the study is to report 2 new genotypic forms of protease-sensitive prionopathy (PSPr), a novel prion disease described in 2008, in 11 subjects all homozygous for valine at codon 129 of the prion protein (PrP) gene (129VV). The 2 new PSPr forms affect individuals who are either homozygous for methionine (129MM) or heterozygous for methionine/valine (129MV). Methods Fifteen affected subjects with 129MM, 129MV, and 129VV underwent comparative evaluation at the National Prion Disease Pathology Surveillance Center for clinical, histopathologic, immunohistochemical, genotypical, and PrP characteristics. Results Disease duration (between 22 and 45 months) was significantly different in the 129VV and 129MV subjects. Most other phenotypic features along with the PrP electrophoretic profile were similar but distinguishable in the 3 129 genotypes. A major difference laid in the sensitivity to protease digestion of the disease-associated PrP, which was high in 129VV but much lower, or altogether lacking, in 129MV and 129MM. This difference prompted the substitution of the original designation with “variably protease-sensitive prionopathy” (VPSPr). None of the subjects had mutations in the PrP gene coding region. Interpretation Because all 3 129 genotypes are involved, and are associated with distinguishable phenotypes, VPSPr becomes the second sporadic prion protein disease with this feature after Creutzfeldt-Jakob disease, originally reported in 1920. However, the characteristics of the abnormal prion protein suggest that VPSPr is different from typical prion diseases, and perhaps more akin to subtypes of Gerstmann-Sträussler-Scheinker disease. PMID:20695009

  6. Effects of protease and non-starch polysaccharide enzyme on performance, digestive function, activity and gene expression of endogenous enzyme of broilers.

    PubMed

    Yuan, Lin; Wang, Mingfa; Zhang, Xiaotu; Wang, Zhixiang

    2017-01-01

    Three hundred one-day-old male broiler chickens (Ross-308) were fed corn-soybean basal diets containing non-starch polysaccharide (NSP) enzyme and different levels of acid protease from 1 to 42 days of age to investigate the effects of exogenous enzymes on growth performance, digestive function, activity of endogenous digestive enzymes in the pancreas and mRNA expression of pancreatic digestive enzymes. For days 1-42, compared to the control chickens, average daily feed intake (ADFI) and average daily gain (ADG) were significantly enhanced by the addition of NSP enzyme in combination with protease supplementation at 40 or 80 mg/kg (p<0.05). Feed-to-gain ratio (FGR) was significantly improved by supplementation with NSP enzymes or NSP enzyme combined with 40 or 80 mg/kg protease compared to the control diet (p<0.05). Apparent digestibility of crude protein (ADCP) was significantly enhanced by the addition of NSP enzyme or NSP enzyme combined with 40 or 80 mg/kg protease (p<0.05). Cholecystokinin (CCK) level in serum was reduced by 31.39% with NSP enzyme combined with protease supplementation at 160 mg/kg (p<0.05), but the CCK level in serum was increased by 26.51% with NSP enzyme supplementation alone. After 21 days, supplementation with NSP enzyme and NSP enzyme combined with 40 or 80 mg/kg protease increased the activity of pancreatic trypsin by 74.13%, 70.66% and 42.59% (p<0.05), respectively. After 42 days, supplementation with NSP enzyme and NSP enzyme combined with 40 mg/kg protease increased the activity of pancreatic trypsin by 32.45% and 27.41%, respectively (p<0.05). However, supplementation with NSP enzyme and 80 or 160 mg/kg protease decreased the activity of pancreatic trypsin by 10.75% and 25.88%, respectively (p<0.05). The activities of pancreatic lipase and amylase were significantly higher in treated animals than they were in the control group (p<0.05). Supplementation with NSP enzyme, NSP enzyme combined with 40 or 80 mg/kg protease increased

  7. Effects of protease and non-starch polysaccharide enzyme on performance, digestive function, activity and gene expression of endogenous enzyme of broilers

    PubMed Central

    Wang, Mingfa; Zhang, Xiaotu; Wang, Zhixiang

    2017-01-01

    Three hundred one-day-old male broiler chickens (Ross-308) were fed corn-soybean basal diets containing non-starch polysaccharide (NSP) enzyme and different levels of acid protease from 1 to 42 days of age to investigate the effects of exogenous enzymes on growth performance, digestive function, activity of endogenous digestive enzymes in the pancreas and mRNA expression of pancreatic digestive enzymes. For days 1-42, compared to the control chickens, average daily feed intake (ADFI) and average daily gain (ADG) were significantly enhanced by the addition of NSP enzyme in combination with protease supplementation at 40 or 80 mg/kg (p<0.05). Feed-to-gain ratio (FGR) was significantly improved by supplementation with NSP enzymes or NSP enzyme combined with 40 or 80 mg/kg protease compared to the control diet (p<0.05). Apparent digestibility of crude protein (ADCP) was significantly enhanced by the addition of NSP enzyme or NSP enzyme combined with 40 or 80 mg/kg protease (p<0.05). Cholecystokinin (CCK) level in serum was reduced by 31.39% with NSP enzyme combined with protease supplementation at 160 mg/kg (p<0.05), but the CCK level in serum was increased by 26.51% with NSP enzyme supplementation alone. After 21 days, supplementation with NSP enzyme and NSP enzyme combined with 40 or 80 mg/kg protease increased the activity of pancreatic trypsin by 74.13%, 70.66% and 42.59% (p<0.05), respectively. After 42 days, supplementation with NSP enzyme and NSP enzyme combined with 40 mg/kg protease increased the activity of pancreatic trypsin by 32.45% and 27.41%, respectively (p<0.05). However, supplementation with NSP enzyme and 80 or 160 mg/kg protease decreased the activity of pancreatic trypsin by 10.75% and 25.88%, respectively (p<0.05). The activities of pancreatic lipase and amylase were significantly higher in treated animals than they were in the control group (p<0.05). Supplementation with NSP enzyme, NSP enzyme combined with 40 or 80 mg/kg protease increased

  8. The Lon protease homologue LonA, not LonC, contributes to the stress tolerance and biofilm formation of Actinobacillus pleuropneumoniae.

    PubMed

    Xie, Fang; Li, Gang; Zhang, Yanhe; Zhou, Long; Liu, Shuanghong; Liu, Siguo; Wang, Chunlai

    2016-04-01

    Lon proteases are a family of ATP-dependent proteases that are involved in the degradation of abnormal proteins in bacteria exposed to adverse environmental stress. An analysis of the genome sequence of Actinobacillus pleuropneumoniae revealed the unusual presence of two putative ATP-dependent Lon homologues, LonA and LonC. Sequence comparisons indicated that LonA has the classical domain organization of the LonA subfamily, which includes the N-terminal domain, central ATPase (AAA) domain, and C-terminal proteolytic (P) domain. LonC belongs to the recently classified LonC subfamily, which includes Lon proteases that contain neither the N-terminal domain of LonA nor the transmembrane region that is present only in LonB subfamily members. To investigate the roles of LonA and LonC in A. pleuropneumoniae, mutants with deletions in the lonA and lonC genes were constructed. The impaired growth of the △lonA mutant exposed to low and high temperatures and osmotic and oxidative stress conditions indicates that the LonA protease is required for the stress tolerance of A. pleuropneumoniae. Furthermore, the △lonA mutant exhibited significantly reduced biofilm formation compared to the wild-type strain. However, no significant differences in stress responses or biofilm formation were observed between the △lonC mutant and the wild-type strain. The △lonA mutant exhibited reduced colonization ability and attenuated virulence of A. pleuropneumoniae in the BALB/c mouse model compared to the wild-type strain. Disruption of lonC gene did not significantly influence the colonization and virulence of A. pleuropneumoniae. The data presented in this study illustrate that the LonA protease, but not the LonC protease, is required for the stress tolerance, biofilm formation and pathogenicity of A. pleuropneumoniae. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Pressure-induced structural transition of mature HIV-1 protease from a combined NMR/MD simulation approach.

    PubMed

    Roche, Julien; Louis, John M; Bax, Ad; Best, Robert B

    2015-12-01

    We investigate the pressure-induced structural changes in the mature human immunodeficiency virus type 1 protease dimer, using residual dipolar coupling (RDC) measurements in a weakly oriented solution. (1)DNH RDCs were measured under high-pressure conditions for an inhibitor-free PR and an inhibitor-bound complex, as well as for an inhibitor-free multidrug resistant protease bearing 20 mutations (PR20). While PR20 and the inhibitor-bound PR were little affected by pressure, inhibitor-free PR showed significant differences in the RDCs measured at 600 bar compared with 1 bar. The structural basis of such changes was investigated by MD simulations using the experimental RDC restraints, revealing substantial conformational perturbations, specifically a partial opening of the flaps and the penetration of water molecules into the hydrophobic core of the subunits at high pressure. This study highlights the exquisite sensitivity of RDCs to pressure-induced conformational changes and illustrates how RDCs combined with MD simulations can be used to determine the structural properties of metastable intermediate states on the folding energy landscape. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.

  10. Discovery and characterization of a novel plant pathogen protease

    USDA-ARS?s Scientific Manuscript database

    Chitinase modifying proteins are fungal proteases that attack specific plant defense chitinases. At least three unrelated types of proteases have evolved to have this function. They all truncate the targeted chitinases by cleaving near their amino termini, but each protease type targets a different ...

  11. Identification of a New cry1I-Type Gene as a Candidate for Gene Pyramiding in Corn To Control Ostrinia Species Larvae

    PubMed Central

    Zhao, Can; Abdelgaffar, Heba M.; Pan, Hongyu; Song, Fuping

    2015-01-01

    Pyramiding of diverse cry toxin genes from Bacillus thuringiensis with different modes of action is a desirable strategy to delay the evolution of resistance in the European corn borer (Ostrinia nubilalis). Considering the dependency of susceptibility to Cry toxins on toxin binding to receptors in the midgut of target pests, a diverse mode of action is commonly defined as recognition of unique binding sites in the target insect. In this study, we present a novel cry1Ie toxin gene (cry1Ie2) as a candidate for pyramiding with Cry1Ab or Cry1Fa in corn to control Ostrinia species larvae. The new toxin gene encodes an 81-kDa protein that is processed to a protease-resistant core form of approximately 55 kDa by trypsin digestion. The purified protoxin displayed high toxicity to Ostrinia furnacalis and O. nubilalis larvae but low to no activity against Spodoptera or heliothine species or the coleopteran Tenebrio molitor. Results of binding assays with 125I-labeled Cry1Ab toxin and brush border membrane vesicles from O. nubilalis larvae demonstrated that Cry1Ie2 does not recognize the Cry1Ab binding sites in that insect. Reciprocal competition binding assays with biotin-labeled Cry1Ie2 confirmed the lack of shared sites with Cry1Ab or Cry1Fa in O. nubilalis brush border membrane vesicles. These data support Cry1Ie2 as a good candidate for pyramiding with Cry1Ab or Cry1Fa in corn to increase the control of O. nubilalis and reduce the risk of resistance evolution. PMID:25795679

  12. Identification of a New cry1I-Type Gene as a Candidate for Gene Pyramiding in Corn To Control Ostrinia Species Larvae.

    PubMed

    Zhao, Can; Jurat-Fuentes, Juan Luis; Abdelgaffar, Heba M; Pan, Hongyu; Song, Fuping; Zhang, Jie

    2015-06-01

    Pyramiding of diverse cry toxin genes from Bacillus thuringiensis with different modes of action is a desirable strategy to delay the evolution of resistance in the European corn borer (Ostrinia nubilalis). Considering the dependency of susceptibility to Cry toxins on toxin binding to receptors in the midgut of target pests, a diverse mode of action is commonly defined as recognition of unique binding sites in the target insect. In this study, we present a novel cry1Ie toxin gene (cry1Ie2) as a candidate for pyramiding with Cry1Ab or Cry1Fa in corn to control Ostrinia species larvae. The new toxin gene encodes an 81-kDa protein that is processed to a protease-resistant core form of approximately 55 kDa by trypsin digestion. The purified protoxin displayed high toxicity to Ostrinia furnacalis and O. nubilalis larvae but low to no activity against Spodoptera or heliothine species or the coleopteran Tenebrio molitor. Results of binding assays with (125)I-labeled Cry1Ab toxin and brush border membrane vesicles from O. nubilalis larvae demonstrated that Cry1Ie2 does not recognize the Cry1Ab binding sites in that insect. Reciprocal competition binding assays with biotin-labeled Cry1Ie2 confirmed the lack of shared sites with Cry1Ab or Cry1Fa in O. nubilalis brush border membrane vesicles. These data support Cry1Ie2 as a good candidate for pyramiding with Cry1Ab or Cry1Fa in corn to increase the control of O. nubilalis and reduce the risk of resistance evolution. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  13. Activation of mas-related G-protein-coupled receptors by the house dust mite cysteine protease Der p1 provides a new mechanism linking allergy and inflammation.

    PubMed

    Reddy, Vemuri B; Lerner, Ethan A

    2017-10-20

    Cysteine and serine proteases function via protease-activated and mas-related G-protein-coupled receptors (Mrgprs) to contribute to allergy and inflammation. Der p1 is a cysteine protease and major allergen from the house dust mite and is associated with allergic rhinitis and allergic asthma. Der p1 activates protease-activated receptor 2 and induces the release of the pro-inflammatory cytokine IL-6 from cells. However, the possibility that Der p1 acts on Mrgprs has not been considered. We report here that ratiometric calcium imaging reveals that Der p1 activates the human receptor MRGPRX1 and the mouse homolog MrgprC11, implicated previously in itch. Der p1 cleavage of N-terminal receptor peptides followed by site-directed mutagenesis of the cleavage sites links receptor activation to specific amino acid residues. Der p1 also induced the release of IL-6 from heterologous cells expressing MRGPRX1. In summary, activation of Mrgprs by the allergen Der p1 may contribute to inflammation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Design, synthesis, X-ray studies, and biological evaluation of novel macrocyclic HIV-1 protease inhibitors involving the P1'-P2' ligands

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ghosh, Arun K.; Sean Fyvie, W.; Brindisi, Margherita

    Design, synthesis, and evaluation of a new class of HIV-1 protease inhibitors containing diverse flexible macrocyclic P1'-P2' tethers are reported. Inhibitor 5a with a pyrrolidinone-derived macrocycle exhibited favorable enzyme inhibitory and antiviral activity (Ki = 13.2 nM, IC50 = 22 nM). Further incorporation of heteroatoms in the macrocyclic skeleton provided macrocyclic inhibitors 5m and 5o. These compounds showed excellent HIV-1 protease inhibitory (Ki = 62 pM and 14 pM, respectively) and antiviral activity (IC50 = 5.3 nM and 2.0 nM, respectively). Inhibitor 5o also remained highly potent against a DRV-resistant HIV-1 variant.

  15. Protease digestion from wheat stillage within a dry grind ethanol facility.

    PubMed

    Bals, Bryan; Brehmer, Ben; Dale, Bruce; Sanders, Johan

    2011-01-01

    As the current starch based ethanol market increases at its rapid pace, finding new markets for the primary coproduct, distiller's grains, has gained considerable interest. One possibility is to isolate the protein-rich fraction for use as precursors to biochemicals and bioplastics, further decreasing fossil fuel consumption. This research focuses on enzymatic extraction of protein peptides from wheat heavy stillage using commercially available proteases. The energy saved due to this process ranged from ∼ 1.5 to 3.0 GJ/ton wheat stillage compared to fossil fuel-based chemicals. Using Protex 6L (Genencor), ∼ 57% of the protein in the stillage was soluble 24 h after protease addition at 0.1% w/w loading. Of these proteins, ∼ 32% were already soluble, indicating the importance of using wet heavy stillage as the feedstock rather than dried distiller's grains. Peptide size was less than 6 kDa. Further improvements in protein removal may be obtained through a fed batch addition of protease and improved protease cocktails. Copyright © 2011 American Institute of Chemical Engineers (AIChE).

  16. PAR-1 mediated apoptosis of breast cancer cells by V. cholerae hemagglutinin protease.

    PubMed

    Ray, Tanusree; Pal, Amit

    2016-05-01

    Bacterial toxins have emerged as promising agents in cancer treatment strategy. Hemagglutinin (HAP) protease secreted by Vibrio cholerae induced apoptosis in breast cancer cells and regresses tumor growth in mice model. The success of novel cancer therapies depends on their selectivity for cancer cells with limited toxicity for normal tissues. Increased expression of Protease Activated Receptor-1 (PAR-1) has been reported in different malignant cells. In this study we report that HAP induced activation and over expression of PAR-1 in breast cancer cells (EAC). Immunoprecipitation studies have shown that HAP specifically binds with PAR-1. HAP mediated activation of PAR-1 caused nuclear translocation of p50-p65 and the phosphorylation of p38 which triggered the activation of NFκB and MAP kinase signaling pathways. These signaling pathways enhanced the cellular ROS level in malignant cells that induced the intrinsic pathway of cell apoptosis. PAR-1 mediated apoptosis by HAP of malignant breast cells without effecting normal healthy cells in the same environment makes it a good therapeutic agent for treatment of cancer.

  17. Theoretical study on the mechanism of a ring-opening reaction of oxirane by the active-site aspartic dyad of HIV-1 protease.

    PubMed

    Kóna, Juraj

    2008-01-21

    Two possible mechanisms of the irreversible inhibition of HIV-1 protease by epoxide inhibitors are investigated on an enzymatic model using ab initio (MP2) and density functional theory (DFT) methods (B3LYP, MPW1K and M05-2X). The calculations predict the inhibition as a general acid-catalyzed nucleophilic substitution reaction proceeding by a concerted SN2 mechanism with a reaction barrier of ca. 15-21 kcal mol(-1). The irreversible nature of the inhibition is characterized by a large negative reaction energy of ca. -17-(-24) kcal mol(-1). A mechanism with a direct proton transfer from an aspartic acid residue of the active site onto the epoxide ring has been shown to be preferred compared to one with the proton transfer from the acid catalyst facilitated by a bridging catalytic water molecule. Based on the geometry of the transition state, structural data important for the design of irreversible epoxide inhibitors of HIV-1 protease were defined. Here we also briefly discuss differences between the epoxide ring-opening reaction in HIV-1 protease and epoxide hydrolase, and the accuracy of the DFT method used.

  18. OMP Peptides Activate the DegS Stress-Sensor Protease by a Relief of Inhibition Mechanism

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sohn, Jungsan; Grant, Robert A.; Sauer, Robert T.

    2010-03-19

    In the E. coli periplasm, C-terminal peptides of misfolded outer-membrane porins (OMPs) bind to the PDZ domains of the trimeric DegS protease, triggering cleavage of a transmembrane regulator and transcriptional activation of stress genes. We show that an active-site DegS mutation partially bypasses the requirement for peptide activation and acts synergistically with mutations that disrupt contacts between the protease and PDZ domains. Biochemical results support an allosteric model, in which these mutations, active-site modification, and peptide/substrate binding act in concert to stabilize proteolytically active DegS. Cocrystal structures of DegS in complex with different OMP peptides reveal activation of the proteasemore » domain with varied conformations of the PDZ domain and without specific contacts from the bound OMP peptide. Taken together, these results indicate that the binding of OMP peptides activates proteolysis principally by relieving inhibitory contacts between the PDZ domain and the protease domain of DegS.« less

  19. Structural determinants of tobacco vein mottling virus protease substrate specificity

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sun, Ping; Austin, Brian P.; Tozer, Jozsef

    2010-10-28

    Tobacco vein mottling virus (TVMV) is a member of the Potyviridae, one of the largest families of plant viruses. The TVMV genome is translated into a single large polyprotein that is subsequently processed by three virally encoded proteases. Seven of the nine cleavage events are carried out by the NIa protease. Its homolog from the tobacco etch virus (TEV) is a widely used reagent for the removal of affinity tags from recombinant proteins. Although TVMV protease is a close relative of TEV protease, they exhibit distinct sequence specificities. We report here the crystal structure of a catalytically inactive mutant TVMVmore » protease (K65A/K67A/C151A) in complex with a canonical peptide substrate (Ac-RETVRFQSD) at 1.7-{angstrom} resolution. As observed in several crystal structures of TEV protease, the C-terminus ({approx}20 residues) of TVMV protease is disordered. Unexpectedly, although deleting the disordered residues from TEV protease reduces its catalytic activity by {approx}10-fold, an analogous truncation mutant of TVMV protease is significantly more active. Comparison of the structures of TEV and TVMV protease in complex with their respective canonical substrate peptides reveals that the S3 and S4 pockets are mainly responsible for the differing substrate specificities. The structure of TVMV protease suggests that it is less tolerant of variation at the P1{prime} position than TEV protease. This conjecture was confirmed experimentally by determining kinetic parameters k{sub cat} and K{sub m} for a series of oligopeptide substrates. Also, as predicted by the cocrystal structure, we confirm that substitutions in the P6 position are more readily tolerated by TVMV than TEV protease.« less

  20. Effects of eye rubbing on the levels of protease, protease activity and cytokines in tears: relevance in keratoconus.

    PubMed

    Balasubramanian, Sivaraman A; Pye, David C; Willcox, Mark D P

    2013-03-01

    Proteases, protease activity and inflammatory molecules in tears have been found to be relevant in the pathogenesis of keratoconus. We sought to determine the influence of eye rubbing on protease expression, protease activity and concentration of inflammatory molecules in tears. Basal tears were collected from normal volunteers before and after 60 seconds of experimental eye rubbing. The total amount of matrix metalloproteinase (MMP)-13 and inflammatory molecules interleukin (IL)-6 and tumour necrosis factor (TNF)-α in the tear samples were measured using specific enzyme-linked immunosorbent assays (ELISA). Tear collagenase activity was investigated using a specific activity assay. The concentrations of MMP-13 (51.9 ± 34.3 versus 63 ± 36.8 pg/ml, p = 0.006), IL-6 (1.24 ± 0.98 versus 2.02 ± 1.52 pg/ml, p = 0.004) and TNF-α (1.16 ± 0.74 versus 1.44 ± 0.66 pg/ml, p = 0.003) were significantly increased in normal subjects after eye rubbing. The experimental eye rub did not alter significantly the collagenase activity (5.02 ± 3 versus 7.50 ± 3.90 fluorescent intensity units, p = 0.14) of tears. Eye rubbing for 60 seconds increased the level of tear MMP-13, IL-6 and TNF-α in normal study subjects. This increase in protease, protease activity and inflammatory mediators in tears after eye rubbing may be exacerbated even further during persistent and forceful eye rubbing seen in people with keratoconus and this in turn may contribute to the progression of the disease. © 2013 The Authors. Clinical and Experimental Optometry © 2013 Optometrists Association Australia.

  1. Gene encoding a novel extracellular metalloprotease in Bacillus subtilis.

    PubMed Central

    Sloma, A; Rudolph, C F; Rufo, G A; Sullivan, B J; Theriault, K A; Ally, D; Pero, J

    1990-01-01

    The gene for a novel extracellular metalloprotease was cloned, and its nucleotide sequence was determined. The gene (mpr) encodes a primary product of 313 amino acids that has little similarity to other known Bacillus proteases. The amino acid sequence of the mature protease was preceded by a signal sequence of approximately 34 amino acids and a pro sequence of 58 amino acids. Four cysteine residues were found in the deduced amino acid sequence of the mature protein, indicating the possible presence of disulfide bonds. The mpr gene mapped in the cysA-aroI region of the chromosome and was not required for growth or sporulation. Images FIG. 2 FIG. 7 PMID:2105291

  2. Characterization of an alkaline protease associated with a granulosis virus of Plodia interpunctella.

    PubMed

    Tweeten, K A; Bulla, L A; Consigli, R A

    1978-06-01

    An alkaline protease was found to be associated with the granulosis virus of the Indian meal moth. Plodia interpunctella. The protease was located within the protein matrix of the occluded virus and hydrolyzed the major constituent of this matrix, a 28,000-dalton protein (granulin), to a mixture of polypeptides ranging in molecular weight from 10,000 to 27,000. A rapid, sensitive assay for the protease was developed using radioactively labeled granulosis virus as substrate. With this assay, the proteolytic activity could be detected by measuring the release of acid-soluble peptides from the labeled virus. The protease had a pH optimum of 10.5 and a temperature optimum of 40 degrees C and was inhibited by diisopropyl phosphorofluoridate, phenylmethylsulfonyl fluoride, and L-(1-tosylamido-2-phenyl) ethyl chloromethyl ketone. Purification of the protease from matrix protein was achieved by anion-exchange and gel permeation chromatography. The molecular weight of the isolated protease, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, was approximately 14,000.

  3. Nanoplatforms for highly sensitive fluorescence detection of cancer-related proteases.

    PubMed

    Wang, Hongwang; Udukala, Dinusha N; Samarakoon, Thilani N; Basel, Matthew T; Kalita, Mausam; Abayaweera, Gayani; Manawadu, Harshi; Malalasekera, Aruni; Robinson, Colette; Villanueva, David; Maynez, Pamela; Bossmann, Leonie; Riedy, Elizabeth; Barriga, Jenny; Wang, Ni; Li, Ping; Higgins, Daniel A; Zhu, Gaohong; Troyer, Deryl L; Bossmann, Stefan H

    2014-02-01

    Numerous proteases are known to be necessary for cancer development and progression including matrix metalloproteinases (MMPs), tissue serine proteases, and cathepsins. The goal of this research is to develop an Fe/Fe3O4 nanoparticle-based system for clinical diagnostics, which has the potential to measure the activity of cancer-associated proteases in biospecimens. Nanoparticle-based "light switches" for measuring protease activity consist of fluorescent cyanine dyes and porphyrins that are attached to Fe/Fe3O4 nanoparticles via consensus sequences. These consensus sequences can be cleaved in the presence of the correct protease, thus releasing a fluorescent dye from the Fe/Fe3O4 nanoparticle, resulting in highly sensitive (down to 1 × 10(-16) mol l(-1) for 12 proteases), selective, and fast nanoplatforms (required time: 60 min).

  4. Biochemical characterization of a halophilic, alkalithermophilic protease from Alkalibacillus sp. NM-Da2.

    PubMed

    Abdel-Hamed, Asmaa R; Abo-Elmatty, Dina M; Wiegel, Juergen; Mesbah, Noha M

    2016-11-01

    An extracellular, halophilic, alkalithermophilic serine protease from the halo-alkaliphilic Alkalibacillus sp. NM-Da2 was purified to homogeneity by ethanol precipitation and anion-exchange chromatography. The purified protease was a monomeric enzyme with an approximate molecular mass of 35 kDa and exhibited maximal activity at 2.7 M NaCl, pH 55 °C 9 and 56 °C. The protease showed great temperature stability, retaining greater than 80 % of initial activity after 2 h incubation at 55 °C. The protease was also extremely pH tolerant, retaining 80 % of initial activity at pH 55 °C 10.5 after 30 min incubation. Protease hydrolyzed complex substrates, displaying activity on yeast extract, tryptone, casein, gelatin and peptone. Protease activity was inhibited at casein concentrations greater than 1.2 mg/mL. The enzyme was stable and active in 40 % (v/v) solutions of isopropanol, ethanol and benzene and was stable in the presence of the polysorbate surfactant Tween 80. Activity was stimulated with the oxidizing agent hydrogen peroxide. Inhibition with phenyl methylsulfonylfluoride indicates it is a serine protease. Synthetic saline wastewater treated with the protease showed 50 % protein removal after 5 h. Being halophilic, alkaliphilic and thermophilic, in addition to being resistant to organic solvents, this protease has potential for various applications in biotechnological and pharmaceutical industries.

  5. Cysteine Protease Inhibitors as Chemotherapy: Lessons from a Parasite Target

    NASA Astrophysics Data System (ADS)

    Selzer, Paul M.; Pingel, Sabine; Hsieh, Ivy; Ugele, Bernhard; Chan, Victor J.; Engel, Juan C.; Bogyo, Matthew; Russell, David G.; Sakanari, Judy A.; McKerrow, James H.

    1999-09-01

    Papain family cysteine proteases are key factors in the pathogenesis of cancer invasion, arthritis, osteoporosis, and microbial infections. Targeting this enzyme family is therefore one strategy in the development of new chemotherapy for a number of diseases. Little is known, however, about the efficacy, selectivity, and safety of cysteine protease inhibitors in cell culture or in vivo. We now report that specific cysteine protease inhibitors kill Leishmania parasites in vitro, at concentrations that do not overtly affect mammalian host cells. Inhibition of Leishmania cysteine protease activity was accompanied by defects in the parasite's lysosome/endosome compartment resembling those seen in lysosomal storage diseases. Colocalization of anti-protease antibodies with biotinylated surface proteins and accumulation of undigested debris and protease in the flagellar pocket of treated parasites were consistent with a pathway of protease trafficking from flagellar pocket to the lysosome/endosome compartment. The inhibitors were sufficiently absorbed and stable in vivo to ameliorate the pathology associated with a mouse model of Leishmania infection.

  6. Integrase inhibitor versus protease inhibitor based regimen for HIV-1 infected women (WAVES): a randomised, controlled, double-blind, phase 3 study

    PubMed Central

    Squires, Kathleen; Kityo, Cissy; Hodder, Sally; Johnson, Margaret; Voronin, Evgeny; Hagins, Debbie; Avihingsanon, Anchalee; Koenig, Ellen; Jiang, Shuping; White, Kirsten; Cheng, Andrew; Szwarcberg, Javier; Cao, Huyen

    2018-01-01

    Summary Background Women are under-represented in HIV antiretroviral therapy (ART) studies. Guidelines for selection of ART as initial therapy in patients with HIV-1 infection do not contain sex-specific treatment. We aimed to assess the safety and efficacy of the single tablet integrase inhibitor regimen containing elvitegravir, cobicistat, emtricitabine, and tenofovir disoproxil fumarate compared with a boosted protease inhibitor regimen of ritonavir-boosted atazanavir with emtricitabine and tenofovir disoproxil fumarate. Methods In this international, randomised, controlled, double-blind, phase 3 study (Women AntiretroViral Efficacy and Safety study [WAVES]), we recruited treatment-naive HIV-infected women with an estimated creatinine clearance of 70 mL/min or higher from 80 centres in 11 countries. Women were randomly assigned (1:1) to receive elvitegravir, cobicistat, emtricitabine, and tenofovir disoproxil fumarate (integrase inhibitor regimen) or ritonavir-boosted atazanavir with emtricitabine and tenofovir disoproxil fumarate (protease inhibitor based regimen); regimens were masked with matching placebos. Randomisation was done by a computer-generated allocation sequence (block size four) and was stratified by HIV-1 RNA viral load and race. Investigators, patients, study staff, and those assessing outcomes were masked to treatment group. All participants who received one dose of study drug were included in the primary efficacy and safety analyses. The main outcome was the proportion of patients with plasma HIV-1 RNA less than 50 copies per mL at week 48 as defined by US Food and Drug Administration snapshot algorithm (prespecified non-inferiority margin of 12%). This study is registered with ClinicalTrials.gov, number NCT01705574. Findings Between Nov 28, 2012, and March 12, 2014, 575 women were enrolled. 289 were randomly assigned to receive the integrase inhibitor regimen and 286 to receive the protease inhibitor based regimen. 252 (87%) women in the

  7. In vitro protease cleavage and computer simulations reveal the HIV-1 capsid maturation pathway

    NASA Astrophysics Data System (ADS)

    Ning, Jiying; Erdemci-Tandogan, Gonca; Yufenyuy, Ernest L.; Wagner, Jef; Himes, Benjamin A.; Zhao, Gongpu; Aiken, Christopher; Zandi, Roya; Zhang, Peijun

    2016-12-01

    HIV-1 virions assemble as immature particles containing Gag polyproteins that are processed by the viral protease into individual components, resulting in the formation of mature infectious particles. There are two competing models for the process of forming the mature HIV-1 core: the disassembly and de novo reassembly model and the non-diffusional displacive model. To study the maturation pathway, we simulate HIV-1 maturation in vitro by digesting immature particles and assembled virus-like particles with recombinant HIV-1 protease and monitor the process with biochemical assays and cryoEM structural analysis in parallel. Processing of Gag in vitro is accurate and efficient and results in both soluble capsid protein and conical or tubular capsid assemblies, seemingly converted from immature Gag particles. Computer simulations further reveal probable assembly pathways of HIV-1 capsid formation. Combining the experimental data and computer simulations, our results suggest a sequential combination of both displacive and disassembly/reassembly processes for HIV-1 maturation.

  8. Amprenavir, new protease inhibitor, approved.

    PubMed

    James, J S

    1999-05-07

    A new protease inhibitor, amprenavir (Agenerase), has received FDA marketing approval. The approval was based on two 24-week controlled trials and safety data in more than 1,400 patients under FDA accelerated-approval rules. Amprenavir is approved for patients 4 years of age and older. The drug is taken twice daily, with or without food. Side effects include gastrointestinal disturbances, rashes, and oral paresthesia. Severe or life-threatening rashes have occurred in 1 percent of all patients. Pregnant women should not use the drug unless necessary. The drug was developed by Vertex Pharmaceuticals Inc. and is being marketed by Glaxo Wellcome. Some studies suggest that amprenavir is less likely than other protease inhibitors to be associated with lipid metabolism problems. It may have a resistance profile different from that of other protease inhibitors, and therefore may cause different cross resistance problems. Amprenavir appears to be synergistic with abacavir (Ziagen) in laboratory tests.

  9. Stability of thermostable alkaline protease from Bacillus licheniformis RP1 in commercial solid laundry detergent formulations.

    PubMed

    Sellami-Kamoun, Alya; Haddar, Anissa; Ali, Nedra El-Hadj; Ghorbel-Frikha, Basma; Kanoun, Safia; Nasri, Moncef

    2008-01-01

    The stability of crude extracellular protease produced by Bacillus licheniformis RP1, isolated from polluted water, in various solid laundry detergents was investigated. The enzyme had an optimum pH and temperature at pH 10.0-11.0 and 65-70 degrees C. Enzyme activity was inhibited by PMSF, suggesting that the preparation contains a serine-protease. The alkaline protease showed extreme stability towards non-ionic (5% Tween 20% and 5% Triton X-100) and anionic (0.5% SDS) surfactants, which retained 100% and above 73%, respectively, of its initial activity after preincubation 60 min at 40 degrees C. The RP1 protease showed excellent stability and compatibility with a wide range of commercial solid detergents at temperatures from 40 to 50 degrees C, suggesting its further application in detergent industry. The enzyme retained 95% of its initial activity with Ariel followed by Axion (94%) then Dixan (93.5%) after preincubation 60 min at 40 degrees C in the presence of 7 mg/ml of detergents. In the presence of Nadhif and New Det, the enzyme retained about 83.5% of the original activity. The effects of additives such as maltodextrin, sucrose and PEG 4000 on the stability of the enzyme during spray-drying and during subsequent storage in New Det detergent were also examined. All additives tested enhanced stability of the enzyme.

  10. A computational module assembled from different protease family motifs identifies PI PLC from Bacillus cereus as a putative prolyl peptidase with a serine protease scaffold.

    PubMed

    Rendón-Ramírez, Adela; Shukla, Manish; Oda, Masataka; Chakraborty, Sandeep; Minda, Renu; Dandekar, Abhaya M; Ásgeirsson, Bjarni; Goñi, Félix M; Rao, Basuthkar J

    2013-01-01

    Proteolytic enzymes have evolved several mechanisms to cleave peptide bonds. These distinct types have been systematically categorized in the MEROPS database. While a BLAST search on these proteases identifies homologous proteins, sequence alignment methods often fail to identify relationships arising from convergent evolution, exon shuffling, and modular reuse of catalytic units. We have previously established a computational method to detect functions in proteins based on the spatial and electrostatic properties of the catalytic residues (CLASP). CLASP identified a promiscuous serine protease scaffold in alkaline phosphatases (AP) and a scaffold recognizing a β-lactam (imipenem) in a cold-active Vibrio AP. Subsequently, we defined a methodology to quantify promiscuous activities in a wide range of proteins. Here, we assemble a module which encapsulates the multifarious motifs used by protease families listed in the MEROPS database. Since APs and proteases are an integral component of outer membrane vesicles (OMV), we sought to query other OMV proteins, like phospholipase C (PLC), using this search module. Our analysis indicated that phosphoinositide-specific PLC from Bacillus cereus is a serine protease. This was validated by protease assays, mass spectrometry and by inhibition of the native phospholipase activity of PI-PLC by the well-known serine protease inhibitor AEBSF (IC50 = 0.018 mM). Edman degradation analysis linked the specificity of the protease activity to a proline in the amino terminal, suggesting that the PI-PLC is a prolyl peptidase. Thus, we propose a computational method of extending protein families based on the spatial and electrostatic congruence of active site residues.

  11. Delimitation of the Earliness per se D1 (Eps-D1) flowering gene to a subtelomeric chromosomal deletion in bread wheat (Triticum aestivum).

    PubMed

    Zikhali, Meluleki; Wingen, Luzie U; Griffiths, Simon

    2016-01-01

    Earliness per se (Eps) genes account for the variation in flowering time when vernalization and photoperiod requirements are satisfied. Genomics and bioinformatics approaches were used to describe allelic variation for 40 Triticum aestivum genes predicted, by synteny with Brachypodium distachyon, to be in the 1DL Eps region. Re-sequencing 1DL genes revealed that varieties carrying early heading alleles at this locus, Spark and Cadenza, carry a subtelomeric deletion including several genes. The equivalent region in Rialto and Avalon is intact. A bimodal distribution in the segregating Spark X Rialto single seed descent (SSD) populations enabled the 1DL QTL to be defined as a discrete Mendelian factor, which we named Eps-D1. Near isogenic lines (NILs) and NIL derived key recombinants between markers flanking Eps-D1 suggest that the 1DL deletion contains the gene(s) underlying Eps-D1. The deletion spans the equivalent of the Triticum monoccocum Eps-A (m) 1 locus, and hence includes MODIFIER OF TRANSCRIPTION 1 (MOT1) and FTSH PROTEASE 4 (FTSH4), the candidates for Eps-A (m) 1. The deletion also contains T. aestivum EARLY FLOWERING 3-D1 (TaELF3-D1) a homologue of the Arabidopsis thaliana circadian clock gene EARLY FLOWERING 3. Eps-D1 is possibly a homologue of Eps-B1 on chromosome 1BL. NILs carrying the Eps-D1 deletion have significantly reduced total TaELF3 expression and altered TaGIGANTEA (TaGI) expression compared with wild type. Altered TaGI expression is consistent with an ELF3 mutant, hence we propose TaELF3-D1 as the more likely candidate for Eps-D1. This is the first direct fine mapping of Eps effect in bread wheat. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  12. TIL-type protease inhibitors may be used as targeted resistance factors to enhance silkworm defenses against invasive fungi.

    PubMed

    Li, Youshan; Zhao, Ping; Liu, Huawei; Guo, Xiaomeng; He, Huawei; Zhu, Rui; Xiang, Zhonghuai; Xia, Qingyou

    2015-02-01

    Entomopathogenic fungi penetrate the insect cuticle using their abundant hydrolases. These hydrolases, which include cuticle-degrading proteases and chitinases, are important virulence factors. Our recent findings suggest that many serine protease inhibitors, especially TIL-type protease inhibitors, are involved in insect resistance to pathogenic microorganisms. To clarify the molecular mechanism underlying this resistance to entomopathogenic fungi and identify novel genes to improve the silkworm antifungal capacity, we conducted an in-depth study of serine protease inhibitors. Here, we cloned and expressed a novel silkworm TIL-type protease inhibitor, BmSPI39. In activity assays, BmSPI39 potently inhibited the virulence protease CDEP-1 of Beauveria bassiana, suggesting that it might suppress the fungal penetration of the silkworm integument by inhibiting the cuticle-degrading proteases secreted by the fungus. Phenol oxidase activation studies showed that melanization is involved in the insect immune response to fungal invasion, and that fungus-induced excessive melanization is suppressed by BmSPI39 by inhibiting the fungal cuticle-degrading proteases. To better understand the mechanism involved in the inhibition of fungal virulence by protease inhibitors, their effects on the germination of B. bassiana conidia was examined. BmSPI38 and BmSPI39 significantly inhibited the germination of B. bassiana conidia. Survival assays showed that BmSPI38 and BmSPI39 markedly improved the survival rates of silkworms, and can therefore be used as targeted resistance proteins in the silkworm. These results provided new insight into the molecular mechanisms whereby insect protease inhibitors confer resistance against entomopathogenic fungi, suggesting their potential application in medicinal or agricultural fields. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. A Modified P1 Moiety Enhances in vitro Antiviral Activity against Various Multi-Drug-Resistant HIV-1 Variants and in vitro CNS Penetration Properties of a Novel Nonpeptidic Protease Inhibitor, GRL-10413

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Amano, Masayuki; Salcedo-Gómez, Pedro Miguel; Zhao, Rui

    We here report that GRL-10413, a novel non-peptidic HIV-1 protease inhibitor (PI) containing a modified P1 moiety and a sulfonamide isostere, is highly active against laboratory HIV-1 strains and primary clinical isolates (EC 50: 0.00035 - 0.0018 μM) with minimal cytotoxicity (CC 50: 35.7 μM). GRL-10413 blocked the infectivity and replication of HIV-1 NL4-3variants selected by up to 5 μM concentrations of atazanavir, lopinavir, or amprenavir (EC 50: 0.0021 - 0.0023 μM). GRL-10413 also maintained its strong antiviral activity against multi-drug-resistant clinical HIV-1 variants isolated from patients, who no longer responded to various antiviral regimens after long-term antiretroviral therapy. Themore » development of resistance against GRL-10413 was significantly delayed compared to that of APV. In addition, GRL-10413 showed a favorable central nervous system (CNS) penetration property as assessed with anin vitroblood brain barrier (BBB) reconstruction system. Analysis of the crystal structure of HIV-1 protease in complex with GRL-10413 demonstrated that the modified P1 moiety of GRL-10413 has a greater hydrophobic surface area and makes greater van der Waals contacts with active-site amino acids of protease than in the case of darunavir. Moreover, the chlorine substituent in the P1 moiety interacts with protease in two distinct configurations. The present data demonstrate that GRL-10413 has desirable features for treating patients infected with wild-type and/or multi-drug-resistant HIV-1 variants with favorable CNS-penetration capability and that the newly modified P1-moiety may confer desirable features in designing novel anti-HIV-1 PIs.« less

  14. Optimization studies on production of a salt-tolerant protease from Pseudomonas aeruginosa strain BC1 and its application on tannery saline wastewater treatment

    PubMed Central

    Sivaprakasam, Senthilkumar; Dhandapani, Balaji; Mahadevan, Surianarayanan

    2011-01-01

    Treatment and safe disposal of tannery saline wastewater, a primary effluent stream that is generated by soaking salt-laden hides and skin is one of the major problems faced by the leather manufacturing industries. Conventional treatment methods like solar evaporation ponds and land composting are not eco-friendly as they deteriorate the ground water quality. Though, this waste stream is comprised of high concentration of dissolved proteins the presence of high salinity (1–6 % NaCl by wt) makes it non-biodegradable. Enzymatic treatment is one of the positive alternatives for management of such kind of waste streams. A novel salt-tolerant alkaline protease obtained from P.aeruginosa (isolated from tannery saline wastewater) was used for enzymatic degradation studies. The effect of various physical factors including pH, temperature, incubation time, protein source and salinity on the activity of identified protease were investigated. Kinetic parameters (Km , Vmax) were calculated for the identified alkaline protease at varying substrate concentrations. Tannery saline wastewater treated with identified salt tolerant protease showed 75 % protein removal at 6 h duration and 2 % (v/v) protease addition was found to be the optimum dosage value. PMID:24031785

  15. Highly Potent HIV-1 Protease Inhibitors with Novel Tricyclic P2-ligands: Design, Synthesis, and Protein-ligand X-Ray Studies

    PubMed Central

    Ghosh, Arun K.; Parham, Garth L.; Martyr, Cuthbert D.; Nyalapatla, Prasanth R.; Osswald, Heather L.; Agniswamy, Johnson; Wang, Yuan-Fang; Amano, Masayuki; Weber, Irene T.; Mitsuya, Hiroaki

    2013-01-01

    The design, synthesis, and biological evaluation of a series of HIV-1 protease inhibitors incorporating stereochemically defined fused tricyclic P2-ligands are described. Various substituent effects were investigated in order to maximize the ligand-binding site interactions in the protease active site. Inhibitors 16a and 16f showed excellent enzyme inhibitory and antiviral activity while incorporation of sulfone functionality resulted in a decrease in potency. Both inhibitors 16a and 16f have maintained activity against a panel of multidrug resistant HIV-1 variants. A high-resolution X-ray crystal structure of 16a-bound HIV-1 protease revealed important molecular insights into the ligand-binding site interactions which may account for the inhibitor’s potent antiviral activity and excellent resistance profiles. PMID:23947685

  16. A recombinant fusion protein-based, fluorescent protease assay for high throughput-compatible substrate screening.

    PubMed

    Bozóki, Beáta; Gazda, Lívia; Tóth, Ferenc; Miczi, Márió; Mótyán, János András; Tőzsér, József

    2018-01-01

    In connection with the intensive investigation of proteases, several methods have been developed for analysis of the substrate specificity. Due to the great number of proteases and the expected target molecules to be analyzed, time- and cost-efficient high-throughput screening (HTS) methods are preferred. Here we describe the development and application of a separation-based HTS-compatible fluorescent protease assay, which is based on the use of recombinant fusion proteins as substrates of proteases. The protein substrates used in this assay consists of N-terminal (hexahistidine and maltose binding protein) fusion tags, cleavage sequences of the tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein (mApple or mTurquoise2). The assay is based on the fluorimetric detection of the fluorescent proteins, which are released from the magnetic bead-attached substrates by the proteolytic cleavage. The protease assay has been applied for activity measurements of TEV and HIV-1 proteases to test the suitability of the system for enzyme kinetic measurements, inhibition studies, and determination of pH optimum. We also found that denatured fluorescent proteins can be renatured after SDS-PAGE of denaturing conditions, but showed differences in their renaturation abilities. After in-gel renaturation both substrates and cleavage products can be identified by in-gel UV detection. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. A Look Inside HIV Resistance through Retroviral Protease Interaction Maps

    PubMed Central

    Kontijevskis, Aleksejs; Prusis, Peteris; Petrovska, Ramona; Yahorava, Sviatlana; Mutulis, Felikss; Mutule, Ilze; Komorowski, Jan; Wikberg, Jarl E. S

    2007-01-01

    Retroviruses affect a large number of species, from fish and birds to mammals and humans, with global socioeconomic negative impacts. Here the authors report and experimentally validate a novel approach for the analysis of the molecular networks that are involved in the recognition of substrates by retroviral proteases. Using multivariate analysis of the sequence-based physiochemical descriptions of 61 retroviral proteases comprising wild-type proteases, natural mutants, and drug-resistant forms of proteases from nine different viral species in relation to their ability to cleave 299 substrates, the authors mapped the physicochemical properties and cross-dependencies of the amino acids of the proteases and their substrates, which revealed a complex molecular interaction network of substrate recognition and cleavage. The approach allowed a detailed analysis of the molecular–chemical mechanisms involved in substrate cleavage by retroviral proteases. PMID:17352531

  18. Understanding the basis of I50V-induced affinity decrease in HIV-1 protease via molecular dynamics simulations using polarized force field.

    PubMed

    Duan, Rui; Lazim, Raudah; Zhang, Dawei

    2015-09-30

    Human immunodeficiency virus (HIV)-1 protease is one of the most promising drug target commonly utilized to combat Acquired Immune Deficiency Syndrome (AIDS). However, with the emergence of drug resistance arising from mutations, the efficiency of protease inhibitors (PIs) as a viable treatment for AIDS has been greatly reduced. I50V mutation as one of the most significant mutations occurring in HIV-1 protease will be investigated in this study. Molecular dynamics (MD) simulation was utilized to examine the effect of I50V mutation on the binding of two PIs namely indinavir and amprenavir to HIV-1 protease. Prior to the simulations conducted, the electron density distributions of the PI and each residue in HIV-1 protease are derived by combining quantum fragmentation approach molecular fractionation with conjugate caps and Poisson-Boltzmann solvation model based on polarized protein-specific charge scheme. The atomic charges of the binding complex are subsequently fitted using delta restrained electrostatic potential (delta-RESP) method to overcome the poor charge determination of buried atom. This way, both intraprotease polarization and the polarization between protease and the PI are incorporated into partial atomic charges. Through this study, the mutation-induced affinity variations were calculated and significant agreement between experiments and MD simulations conducted was observed for both HIV-1 protease-drug complexes. In addition, the mechanism governing the decrease in the binding affinity of PI in the presence of I50V mutation was also explored to provide insights pertaining to the design of the next generation of anti-HIV drugs. © 2015 Wiley Periodicals, Inc.

  19. [Analysis of salivary protease spectrum in chronic periodontitis].

    PubMed

    Qian, Li; Xuedong, Zhou; Yaping, Fan; Tengyu, Yang; Songtao, Wu; Yu, Yu; Jiao, Chen; Ping, Zhang; Yun, Feng

    2017-02-01

    This study aimed to investigate the difference in salivary protease expression in patients with chronic periodontitis and normal individuals. The stimulating saliva in patients with chronic periodontitis and normal individuals were collected. Protein chip technology was adapted to analyze salivary protease spectrum. Among the 34 proteases in the chip, disintegrin and metalloproteinase (ADAM)8, matrix metalloproteinase (MMP)-8, MMP-12, neprilysin/CD10, and uridylyl phosphate adenosine/urokinase showed a significantly increased concentration in the saliva of chronic periodontitis patients compared with those in the saliva of normal individuals (P<0.01). By contrast, the concentrations of ADAM9, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)1, ADAMTS13, cathepsin B, E, L, V, X/Z/P, kallikrein 6, 7, 11, 13, MMP-9, proteinase 3, presenilin-1, and proprotein convertase 9 sharply decreased (P<0.05). The results demonstrated that protease spectrum in the saliva of chronic periodontitis patients and normal individuals significantly differed. Analysis of salivary protease spectrum is a potential clinical method to examine, diagnose, and monitor chronic periodontitis.

  20. The IL-8 Protease SpyCEP/ScpC of Group A Streptococcus Promotes Resistance to Neutrophil Killing

    PubMed Central

    Zinkernagel, Annelies S.; Timmer, Anjuli M.; Pence, Morgan A.; Locke, Jeffrey B.; Buchanan, John T.; Turner, Claire E.; Mishalian, Inbal; Sriskandan, Shiranee; Hanski, Emanuel; Nizet, Victor

    2009-01-01

    SUMMARY Interleukin-8 (IL-8) promotes neutrophil-mediated host defense through its chemoattractant and immunostimulatory activities. The Group A Streptococcus (GAS) protease SpyCEP (also called ScpC) cleaves IL-8, and SpyCEP expression is strongly upregulated in vivo in the M1T1 GAS strains associated with life-threatening systemic disease including necrotizing fasciitis. Coupling allelic replacement with heterologous gene expression, we show that SpyCEP is necessary and sufficient for IL-8 degradation. SpyCEP decreased IL-8-dependent neutrophil endothelial transmigration and bacterial killing, the latter by reducing neutrophil extracellular trap formation. The knockout mutant lacking SpyCEP was attenuated for virulence in murine infection models, and SpyCEP expression conferred protection to coinfecting bacteria. We also show that the zoonotic pathogen Streptococcus iniae possesses a functional homolog of SpyCEP (Cepl) that cleaves IL-8, promotes neutrophil resistance, and contributes to virulence. By inactivating the multifunctional host defense peptide IL-8, the SpyCEP protease impairs neutrophil clearance mechanisms, contributing to the pathogenesis of invasive streptococcal infection. PMID:18692776

  1. The Inflammasome Drives GSDMD-Independent Secondary Pyroptosis and IL-1 Release in the Absence of Caspase-1 Protease Activity.

    PubMed

    Schneider, Katharina S; Groß, Christina J; Dreier, Roland F; Saller, Benedikt S; Mishra, Ritu; Gorka, Oliver; Heilig, Rosalie; Meunier, Etienne; Dick, Mathias S; Ćiković, Tamara; Sodenkamp, Jan; Médard, Guillaume; Naumann, Ronald; Ruland, Jürgen; Kuster, Bernhard; Broz, Petr; Groß, Olaf

    2017-12-26

    Inflammasomes activate the protease caspase-1, which cleaves interleukin-1β and interleukin-18 to generate the mature cytokines and controls their secretion and a form of inflammatory cell death called pyroptosis. By generating mice expressing enzymatically inactive caspase-1 C284A , we provide genetic evidence that caspase-1 protease activity is required for canonical IL-1 secretion, pyroptosis, and inflammasome-mediated immunity. In caspase-1-deficient cells, caspase-8 can be activated at the inflammasome. Using mice either lacking the pyroptosis effector gasdermin D (GSDMD) or expressing caspase-1 C284A , we found that GSDMD-dependent pyroptosis prevented caspase-8 activation at the inflammasome. In the absence of GSDMD-dependent pyroptosis, the inflammasome engaged a delayed, alternative form of lytic cell death that was accompanied by the release of large amounts of mature IL-1 and contributed to host protection. Features of this cell death modality distinguished it from apoptosis, suggesting it may represent a distinct form of pro-inflammatory regulated necrosis. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  2. The effect of desolvation on the binding of inhibitors to HIV-1 protease and cyclin-dependent kinases: Causes of resistance.

    PubMed

    Fong, Clifford W

    2016-08-01

    Studies of the cyclin-dependent kinase inhibitors and HIV-1 protease inhibitors have confirmed that ligand-protein binding is dependent on desolvation effects. It has been found that a four parameter linear model incorporating desolvation energy, lipophilicity, dipole moment and molecular volume of the ligands is a good model to describe the binding between ligands and kinases or proteases. The resistance shown by MDR proteases to the anti-viral drugs is multi-faceted involving varying changes in desolvation, lipophilicity and dipole moment interaction compared to the non-resistant protease. Desolvation has been shown to be the dominant factor influencing the effect of inhibitors against the cyclin-dependent kinases, but lipophilicity and dipole moment are also significant factors. The model can differentiate between the inhibitory activity of CDK2/cycE, CDK1/cycB and CDK4/cycD enzymes. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. In Vitro Evolution of the Human Immunodeficiency Virus Type 1 Gag-Protease Region and Maintenance of Reverse Transcriptase Resistance following Prolonged Drug Exposure†

    PubMed Central

    La Seta Catamancio, Simona; De Pasquale, Maria Pia; Citterio, Paola; Kurtagic, Semir; Galli, Massimo; Rusconi, Stefano

    2001-01-01

    We studied the human immunodeficiency virus type 1 phenotypic and genotypic profiles of a dual drug-resistant isolate (isolate 14aPost-DR) selected for zidovudine (ZDV) and lamivudine (3TC) resistance and then cultured in the presence of 3TC and a protease inhibitor: indinavir (IDV), ritonavir, or KNI-272. The IDV-treated virus was highly resistant to 3TC, ZDV, and IDV and accumulated protease mutations at positions M46I and V82F. A change from alanine to valine was observed in 4 of 10 clones in the P2 position of the p7-p1 Gag-protease cleavage site, linked to position M46I in the dominant viral quasispecies. Previous 3TC resistance did not impair the development of additional mutations in the protease and Gag-protease cleavage regions. PMID:11230439

  4. Analysis of Milk from Mothers Who Delivered Prematurely Reveals Few Changes in Proteases and Protease Inhibitors across Gestational Age at Birth and Infant Postnatal Age.

    PubMed

    Demers-Mathieu, Veronique; Nielsen, Søren Drud; Underwood, Mark A; Borghese, Robyn; Dallas, David C

    2017-06-01

    Background: Peptidomics research has demonstrated that protease activity is higher in breast milk from preterm-delivering mothers than from term-delivering mothers. However, to our knowledge, the effect of the degree of prematurity and postnatal age on proteases and protease inhibitors in human milk remains unknown. Objective: We aimed to determine the change of proteases and protease inhibitors in milk from mothers who delivered prematurely across gestational age (GA) and postnatal age. Methods: Milk samples were collected from 18 mothers aged 26-40 y who delivered preterm infants and who lacked mastitis. For analysis, samples were separated into 2 groups: 9 from early GA (EGA) (24-26 wk GA)-delivering mothers and 9 from late GA (LGA) (27-32 wk GA)-delivering mothers. Within the 9 samples in each group, the collection time ranged from postnatal days 2 to 47. The activity and predicted activity of proteases in preterm milk were determined with the use of fluorometric and spectrophotometric assays and peptidomics, respectively. Protease and protease inhibitor concentrations were determined with the use of ELISA. Linear mixed models were applied to compare enzymes across GA and postnatal age. Results: Carboxypeptidase B2, kallikrein, plasmin, elastase, thrombin, and cytosol aminopeptidase were present and active in the milk of preterm-delivering mothers. Most milk protease and antiprotease concentrations did not change with GA or postnatal age. However, the concentration and activity of kallikrein, the most abundant and active protease in preterm milk, increased by 25.4 ng · mL -1 · d -1 and 0.454 μg · mL -1 · d -1 postnatally, respectively, in EGA milk samples while remaining stable in LGA milk samples. Conclusions: This research demonstrates that proteases are active in human milk and begin to degrade milk protein within the mammary gland before consumption by infants. Proteases and protease inhibitors in milk from mothers of premature infants mostly did not

  5. The Membrane-anchored Serine Protease Prostasin (CAP1/PRSS8) Supports Epidermal Development and Postnatal Homeostasis Independent of Its Enzymatic Activity*

    PubMed Central

    Peters, Diane E.; Szabo, Roman; Friis, Stine; Shylo, Natalia A.; Uzzun Sales, Katiuchia; Holmbeck, Kenn; Bugge, Thomas H.

    2014-01-01

    The membrane-anchored serine protease prostasin (CAP1/PRSS8) is part of a cell surface proteolytic cascade that is essential for epithelial barrier formation and homeostasis. Here, we report the surprising finding that prostasin executes these functions independent of its own enzymatic activity. Prostasin null (Prss8−/−) mice lack barrier formation and display fatal postnatal dehydration. In sharp contrast, mice homozygous for a point mutation in the Prss8 gene, which causes the substitution of the active site serine within the catalytic histidine-aspartate-serine triad with alanine and renders prostasin catalytically inactive (Prss8Cat−/Cat− mice), develop barrier function and are healthy when followed for up to 20 weeks. This striking difference could not be explained by genetic modifiers or by maternal effects, as these divergent phenotypes were displayed by Prss8−/− and Prss8Cat−/Cat− mice born within the same litter. Furthermore, Prss8Cat−/Cat− mice were able to regenerate epidermal covering following cutaneous wounding. This study provides the first demonstration that essential in vivo functions of prostasin are executed by a non-enzymatic activity of this unique membrane-anchored serine protease. PMID:24706745

  6. Design, Synthesis, and Biological and Structural Evaluations of Novel HIV-1 Protease Inhibitors To Combat Drug Resistance

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Parai, Maloy Kumar; Huggins, David J.; Cao, Hong

    2012-09-11

    A series of new HIV-1 protease inhibitors (PIs) were designed using a general strategy that combines computational structure-based design with substrate-envelope constraints. The PIs incorporate various alcohol-derived P2 carbamates with acyclic and cyclic heteroatomic functionalities into the (R)-hydroxyethylamine isostere. Most of the new PIs show potent binding affinities against wild-type HIV-1 protease and three multidrug resistant (MDR) variants. In particular, inhibitors containing the 2,2-dichloroacetamide, pyrrolidinone, imidazolidinone, and oxazolidinone moieties at P2 are the most potent with Ki values in the picomolar range. Several new PIs exhibit nanomolar antiviral potencies against patient-derived wild-type viruses from HIV-1 clades A, B, and Cmore » and two MDR variants. Crystal structure analyses of four potent inhibitors revealed that carbonyl groups of the new P2 moieties promote extensive hydrogen bond interactions with the invariant Asp29 residue of the protease. These structure-activity relationship findings can be utilized to design new PIs with enhanced enzyme inhibitory and antiviral potencies.« less

  7. Preliminary crystallographic analysis of avian infectious bronchitis virus main protease

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Li, Jun; Shen, Wei; Liao, Ming, E-mail: mliao@scau.edu.cn

    The avian infectious bronchitis virus main protease has been crystallized; crystals diffract to 2.7 Å resolution. Infectious bronchitis virus (IBV) is the prototype of the genus Coronavirus. It causes a highly contagious disease which affects the respiratory, reproductive, neurological and renal systems of chickens, resulting great economic losses in the poultry industry worldwide. The coronavirus (CoV) main protease (M{sup pro}), which plays a pivotal role in viral gene expression and replication through a highly complex cascade involving the proteolytic processing of replicase polyproteins, is an attractive target for antiviral drug design. In this study, IBV M{sup pro} was overexpressed inmore » Escherichia coli. Crystals suitable for X-ray crystallography have been obtained using microseeding techniques and belong to space group P6{sub 1}22. X-ray diffraction data were collected in-house to 2.7 Å resolution from a single crystal. The unit-cell parameters were a = b = 119.1, c = 270.7 Å, α = β = 90, γ = 120°. Three molecules were predicted to be present in the asymmetric unit from a calculated self-rotation function.« less

  8. Prevalence, mutation patterns, and effects on protease inhibitor susceptibility of the L76V mutation in HIV-1 protease.

    PubMed

    Young, Thomas P; Parkin, Neil T; Stawiski, Eric; Pilot-Matias, Tami; Trinh, Roger; Kempf, Dale J; Norton, Michael

    2010-11-01

    Patterns of HIV-1 protease inhibitor (PI) resistance-associated mutations (RAMs) and effects on PI susceptibility associated with the L76V mutation were studied in a large database. Of 20,501 sequences with ≥1 PI RAM, 3.2% contained L76V; L76V was alone in 0.04%. Common partner mutations included M46I, I54V, V82A, I84V, and L90M. L76V was associated with a 2- to 6-fold decrease in susceptibility to lopinavir, darunavir, amprenavir, and indinavir and a 7- to 8-fold increase in susceptibility to atazanavir and saquinavir.

  9. Identification and characterization of MOR-CP, a cysteine protease induced by ozone and developmental senescence in maize (Zea mays L.) leaves.

    PubMed

    Ahmad, Rafiq; Zuily-Fodil, Yasmine; Passaquet, Chantal; Bethenod, Olivier; Roche, Romain; Repellin, Anne

    2014-08-01

    Among the different classes of endoproteases, cysteine proteases are consistently associated with senescence, defense signaling pathways and cellular responses to abiotic stresses. The objectives of this work were to study the effects of various concentrations of ozone on gene expression and enzymatic activity for papain-like cysteine proteases (PLCPs), in the leaves of maize plants grown under field conditions. Leaves from ranks 12 and 10 (cob leaf) were harvested regularly over a long-term artificial ozone fumigation experiment (50 d). Tissues were tested for transcriptional and activity changes concerning cysteine proteases, using qRT-PCR for the newly identified ozone-responsive PLCP gene (Mor-CP) and synthetic oligopeptide Boc-Val-Leu-Lys-AMC as a PLCP-specific substrate, respectively. Results showed that developmental senescence induced a significant and progressive rise in CP activity, only in the older leaves 10 and had no effect on Mor-CP gene expression levels. On the other hand, ozone dramatically enhanced Mor-CP mRNA levels and global PLCP enzymatic activity in leaves 12 and 10, particularly toward the end of the treatment. Ozone impact was more pronounced in the older leaves 10. Together, these observations concurred to conclude that ozone stress enhances natural senescence processes, such as those related to proteolysis. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. Comparison of the genomes and transcriptomes associated with the different protease secretions of Aspergillus oryzae 100-8 and 3.042.

    PubMed

    Zhao, Guozhong; Yao, Yunping; Hou, Lihua; Wang, Chunling; Cao, Xiaohong

    2014-10-01

    Aspergillus oryzae is used to produce traditional fermented foods and beverages. A. oryzae 3.042 produces a neutral protease and an alkaline protease but rarely an acid protease, which is unfavourable to soy-sauce fermentation. A. oryzae 100-8 was obtained by N(+) ion implantation mutagenesis of A. oryzae 3.042, and the protease secretions of these two strains are different. Sequencing the genome of A. oryzae 100-8 and comparing it to the genomes of A. oryzae 100-8 and 3.042 revealed some differences, such as single nucleotide polymorphisms, nucleotide deletion or insertion. Some of these differences may reflect the ability of A. oryzae to secrete proteases. Transcriptional sequencing and analysis of the two strains during the same growth processes provided further insights into the genes and pathways involved in protease secretion.

  11. Recombinant protease inhibitors for herbivore pest control: a multitrophic perspective.

    PubMed

    Schlüter, Urte; Benchabane, Meriem; Munger, Aurélie; Kiggundu, Andrew; Vorster, Juan; Goulet, Marie-Claire; Cloutier, Conrad; Michaud, Dominique

    2010-10-01

    Protease inhibitors are a promising complement to Bt toxins for the development of insect-resistant transgenic crops, but their limited specificity against proteolytic enzymes and the ubiquity of protease-dependent processes in living organisms raise questions about their eventual non-target effects in agroecosystems. After a brief overview of the main factors driving the impacts of insect-resistant transgenic crops on non-target organisms, the possible effects of protease inhibitors are discussed from a multitrophic perspective, taking into account not only the target herbivore proteases but also the proteases of other organisms found along the trophic chain, including the plant itself. Major progress has been achieved in recent years towards the design of highly potent broad-spectrum inhibitors and the field deployment of protease inhibitor-expressing transgenic plants resistant to major herbivore pests. A thorough assessment of the current literature suggests that, whereas the non-specific inhibitory effects of recombinant protease inhibitors in plant food webs could often be negligible and their 'unintended' pleiotropic effects in planta of potential agronomic value, the innocuity of these proteins might always remain an issue to be assessed empirically, on a case-by-case basis.

  12. Purification and characterization of a milk-clotting aspartic protease from Withania coagulans fruit.

    PubMed

    Salehi, Mahmoud; Aghamaali, Mahmoud Reza; Sajedi, Reza H; Asghari, S Mohsen; Jorjani, Eisa

    2017-05-01

    Withania coagulans fruit has traditionally been used as milk coagulant. The present study reports the purification and characterization of an aspartic protease from W. coagulans fruit. The enzyme was purified via fractional ammonium sulfate precipitation and cation exchange chromatography. SDS-PAGE analysis revealed the presence of a monomeric protein with molecular weight of 31kDa. Proteolytic activity (PA) of the protease was evaluated using casein, and the milk-clotting activity (MCA) was analyzed by skim milk. The K m and V max values of the enzyme for casein were obtained to be 1.29mg/ml and 0.035μmol Tyr/min, respectively. Optimal temperature and pH were 65°C and 5.5, respectively. After incubation of enzyme at 65°C for 1h, 73% of PA was remained which demonstrated high thermal stability of the enzyme. Mass spectrometry analysis of the purified protease and enzyme assays in the presence of protease inhibitors indicated that aspartic protease was the only responsible enzyme in milk coagulation. Furthermore, by investigating the effect of salts on enzyme activity, it was observed that both NaCl and CaCl 2 reduced enzyme activity. These characteristics of the protease suggest that the enzyme may be suitable for producing low salt content cheeses. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Functional analysis of a missense mutation in the serine protease inhibitor SPINT2 associated with congenital sodium diarrhea.

    PubMed

    Faller, Nicolas; Gautschi, Ivan; Schild, Laurent

    2014-01-01

    Membrane-bound serine proteases play important roles in different biological processes. Their regulation by endogenous inhibitors is poorly understood. A Y163C mutation in the SPINT2 gene encoding the serine protease inhibitor Hepatocyte Growth Factor Inhibitor HAI-2 is associated with a congenital sodium diarrhea. The functional consequences of this mutation on HAI-2 activity and its physiological targets are unknown. We established a cellular assay in Xenopus laevis oocytes to study functional interactions between HAI-2 and candidate membrane-bound serine proteases expressed in the gastro-intestinal tract. We found that the wild-type form of HAI-2 is a potent inhibitor of nine gastro-intestinal serine proteases. The Y163C mutation in the second Kunitz domain of HAI-2 resulted in a complete loss of inhibitory activity on two intestinal proteases, prostasin and tmprss13. The effect of the mutation of the homologous Y68C in the first Kunitz domain of HAI-2 is consistent with a differential contribution of the two Kunitz domains of HAI-2 in the inhibition of serine proteases. By contrast to the Tyr to Cys, the Tyr to Ser substitution did not change the inhibitory potency of HAI-2, indicating that the thiol-group of the cysteine rather than the Tyr deletion is responsible for the HAI-2 loss of function. Our functional assay allowed us to identify membrane-bound serine proteases as cellular target for inhibition by HAI-2 wild type and mutants, and to better define the role of the Tyr in the second Kunitz domain in the inhibitory activity of HAI-2.

  14. Evolution of inhibitor-resistant natural mutant forms of HIV-1 protease probed by pre-steady state kinetic analysis.

    PubMed

    Zakharova, Maria Yu; Kuznetsova, Alexandra A; Kaliberda, Elena N; Dronina, Maria A; Kolesnikov, Alexander V; Kozyr, Arina V; Smirnov, Ivan V; Rumsh, Lev D; Fedorova, Olga S; Knorre, Dmitry G; Gabibov, Alexander G; Kuznetsov, Nikita A

    2017-11-01

    Pre-steady state kinetic analysis of mechanistic features of substrate binding and processing is crucial for insight into the evolution of inhibitor-resistant forms of HIV-1 protease. These data may provide a correct vector for rational drug design assuming possible intrinsic dynamic effects. These data should also give some clues to the molecular mechanism of protease action and resistance to inhibitors. Here we report pre-steady state kinetics of the interaction of wild type or mutant forms of HIV-1 protease with a FRET-labeled peptide. The three-stage "minimal" kinetic scheme with first and second reversible steps of substrate binding and with following irreversible peptide cleavage step adequately described experimental data. For the first time, a set of "elementary" kinetic parameters of wild type HIV-1 protease and its natural mutant inhibitor-resistant forms MDR-HM, ANAM-11 and prDRV4 were compared. Inhibitors of the first and second generation were used to estimate the inhibitory effects on HIV-1 protease activity. The resulting set of kinetic data supported that the mutant forms are kinetically unaffected by inhibitors of the first generation, proving their functional resistance to these compounds. The second generation inhibitor darunavir inhibited mutant forms MDR-HM and ANAM-11, but was ineffective against prDRV4. Our kinetic data revealed that these inhibitors induced different conformational changes in the enzyme and, thereby they have different mode of binding in the enzyme active site. These data confirmed hypothesis that the driving force of the inhibitor-resistance evolution is disruption of enzyme-inhibitor complex by changing of the contact network in the inhibitor binding site. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  15. Production of alkaline protease from Cellulosimicrobium cellulans

    PubMed Central

    Ferracini-Santos, Luciana; Sato, Hélia H

    2009-01-01

    Cellulosimicrobium cellulans is one of the microorganisms that produces a wide variety of yeast cell wall-degrading enzymes, β-1,3-glucanase, protease and chitinase. Dried cells of Saccharomyces cerevisiae were used as carbon and nitrogen source for cell growth and protease production. The medium components KH2PO4, KOH and dried yeast cells showed a significant effect (p<0.05) on the factorial fractional design. A second design was prepared using two factors: pH and percentage of dried yeast cells. The results showed that the culture medium for the maximum production of protease was 0.2 g/l of MgSO4.7H2O, 2.0 g/l of (NH4)2SO4 and 8% of dried yeast cells in 0.15M phosphate buffer at pH 8.0. The maximum alkaline protease production was 7.0 ± 0.27 U/ml over the center point. Crude protease showed best activity at 50ºC and pH 7.0-8.0, and was stable at 50ºC. PMID:24031317

  16. Activities of the human immunodeficiency virus type 1 (HIV-1) protease inhibitor nelfinavir mesylate in combination with reverse transcriptase and protease inhibitors against acute HIV-1 infection in vitro.

    PubMed

    Patick, A K; Boritzki, T J; Bloom, L A

    1997-10-01

    Nelfinavir mesylate (formerly AG1343) is a potent and selective, nonpeptidic inhibitor of human immunodeficiency virus type 1 (HIV-1) protease that was discovered by protein structure-based design methodologies. We evaluated the antiviral and cytotoxic effects of two-drug combinations of nelfinavir with the clinically approved antiretroviral therapeutics zidovudine (ZDV), lamivudine (3TC), dideoxycytidine (ddC; zalcitabine), stavudine (d4T), didanosine (ddI), indinavir, saquinavir, and ritonavir and a three-drug combination of nelfinavir with ZDV and 3TC against an acute HIV-1 strain RF infection of CEM-SS cells in vitro. Quantitative assessment of drug interaction was evaluated by a universal response surface approach (W. R. Greco, G. Bravo, and J. C. Parsons, Pharm. Rev. 47:331-385, 1995) and by the method of M. N. Prichard and C. Shipman (Antiviral Res. 14:181-206, 1990). Both analytical methods yielded similar results and showed that the two-drug combinations of nelfinavir with the reverse transcriptase inhibitors ZDV, 3TC, ddI, d4T, and ddC and the three-drug combination with ZDV and 3TC resulted in additive to statistically significant synergistic interactions. In a similar manner, the combination of nelfinavir with the three protease inhibitors resulted in additive (ritonavir and saquinavir) to slightly antagonistic (indinavir) interactions. In all combinations, minimal cellular cytotoxicity was observed with any drug alone and in combination. These results suggest that administration of combinations of the appropriate doses of nelfinavir with other currently approved antiretroviral therapeutic agents in vivo may result in enhanced antiviral activity with no associated increase in cellular cytotoxicity.

  17. A novel serine protease highly expressed in the pancreas is expressed in various kinds of cancer cells.

    PubMed

    Mitsui, Shinichi; Okui, Akira; Kominami, Katsuya; Konishi, Eiichi; Uemura, Hidetoshi; Yamaguchi, Nozomi

    2005-10-01

    We have isolated a cDNA that encodes a novel serine protease, prosemin, from human brain. The cDNA of human prosemin is 1306 bp, encoding 317 amino acids. It showed significant homology with the sequence of a chromosome 16 cosmid clone (accession no. NT_037887.4). The prosemin gene contains six exons and five introns. The amino acid sequence of prosemin shows significant homology to prostasin, gamma-tryptase, and testisin (43%, 41%, and 38% identity, respectively), the genes of which are also located on chromosome 16. Northern hybridization showed that prosemin is expressed predominantly in the pancreas and weakly in the prostate and cerebellum. However, western blot and RT-PCR analyses showed that prosemin is expressed and secreted from various kinds of cancer cells, such as glioma, pancreas, prostate, and ovarian cell lines. Prosemin is secreted in the cystic fluid of clinical ovarian cancers. Furthermore, immunohistochemistry showed prosemin protein localized in the apical parts of ovarian carcinomas. Recombinant prosemin was expressed in COS cells and was purified by immunoaffinity chromatography. Recombinant prosemin preferentially cleaved benzyloxycarbonyl (Z)-His-Glu-Lys-methylcoumaryl amidide (MCA) and t-butyloxycarbonyl (Boc)-Gln-Ala-Arg-MCA. Our results suggest that prosemin is a novel serine protease of the chromosome 16 cluster that is highly expressed in the pancreas. The usefulness of this serine protease as a candidate tumor marker should be further examined.

  18. Identification of Proteases and Protease Inhibitors in Allergenic and Non-Allergenic Pollen.

    PubMed

    Höllbacher, Barbara; Schmitt, Armin O; Hofer, Heidi; Ferreira, Fatima; Lackner, Peter

    2017-06-05

    Pollen is one of the most common causes of allergy worldwide, making the study of their molecular composition crucial for the advancement of allergy research. Despite substantial efforts in this field, it is not yet clear why some plant pollens strongly provoke allergies while others do not. However, proteases and protease inhibitors from allergen sources are known to play an important role in the development of pollen allergies. In this study, we aim to uncover differences in the transcriptional pattern of proteases and protease inhibitors in Betula verrucosa and Pinus sylvestris pollen as models for high and low allergenic potential, respectively. We applied RNA sequencing to Betula verrucosa and Pinus sylvestris pollen. After de-novo assembly we derived general functional profiles of the protein coding transcripts. By utilization of domain based functional annotation we identified potential proteases and protease inhibitors and compared their expression in the two types of pollen. Functional profiles are highly similar between Betula verrucosa and Pinus sylvestris pollen. Both pollen contain proteases and inhibitors from 53 and 7 Pfam families, respectively. Some of the members comprised within those families are implicated in facilitating allergen entry, while others are known allergens themselves. Our work revealed several candidate proteins which, with further investigation, represent exciting new leads in elucidating the process behind allergic sensitization.

  19. Purification and characterization of a serine protease (CESP) from mature coconut endosperm

    PubMed Central

    Panicker, Leelamma M; Usha, Rajamma; Roy, Samir; Mandal, Chhabinath

    2009-01-01

    Background In plants, proteases execute an important role in the overall process of protein turnover during seed development, germination and senescence. The limited knowledge on the proteolytic machinery that operates during seed development in coconut (Cocos nucifera L.) prompted us to search for proteases in the coconut endosperm. Findings We have identified and purified a coconut endosperm protease (CESP) to apparent homogeneity. CESP is a single polypeptide enzyme of approximate molecular mass of 68 kDa and possesses pH optimum of 8.5 for the hydrolysis of BAPNA. Studies relating to substrate specificity and pattern of inhibition by various protease inhibitors indicated that CESP is a serine protease with cleavage specificity to peptide bonds after arginine. Purified CESP was often autolysed to two polypeptides of 41.6 kDa (CESP1) and 26.7 kDa (CESP2) and is confirmed by immunochemistry. We have shown the expression of CESP in all varieties of coconut and in all stages of coconut endosperm development with maximum amount in fully matured coconut. Conclusion Since the involvement of proteases in the processing of pre-proteins and maintenance of intracellular protein levels in seeds are well known, we suspect this CESP might play an important role in the coconut endosperm development. However this need to be confirmed using further studies. PMID:19426537

  20. Activation of Influenza A Viruses by Host Proteases from Swine Airway Epithelium

    PubMed Central

    Peitsch, Catharina; Klenk, Hans-Dieter; Garten, Wolfgang

    2014-01-01

    Pigs are important natural hosts of influenza A viruses, and due to their susceptibility to swine, avian, and human viruses, they may serve as intermediate hosts supporting adaptation and genetic reassortment. Cleavage of the influenza virus surface glycoprotein hemagglutinin (HA) by host cell proteases is essential for viral infectivity. Most influenza viruses, including human and swine viruses, are activated at a monobasic HA cleavage site, and we previously identified TMPRSS2 and HAT to be relevant proteases present in human airways. We investigated the proteolytic activation of influenza viruses in primary porcine tracheal and bronchial epithelial cells (PTEC and PBEC, respectively). Human H1N1 and H3N2 viruses replicated efficiently in PTECs and PBECs, and viruses containing cleaved HA were released from infected cells. Moreover, the cells supported the proteolytic activation of HA at the stage of entry. We found that swine proteases homologous to TMPRSS2 and HAT, designated swTMPRSS2 and swAT, respectively, were expressed in several parts of the porcine respiratory tract. Both proteases cloned from primary PBECs were shown to activate HA with a monobasic cleavage site upon coexpression and support multicycle replication of influenza viruses. swAT was predominantly localized at the plasma membrane, where it was present as an active protease that mediated activation of incoming virus. In contrast, swTMPRSS2 accumulated in the trans-Golgi network, suggesting that it cleaves HA in this compartment. In conclusion, our data show that HA activation in porcine airways may occur by similar proteases and at similar stages of the viral life cycle as in human airways. PMID:24155384

  1. Full-length and defective enterovirus G genomes with distinct torovirus protease insertions are highly prevalent on a Chinese pig farm.

    PubMed

    Wang, Yan; Zhang, Wen; Liu, Zhijian; Fu, Xingli; Yuan, Jiaqi; Zhao, Jieji; Lin, Yuan; Shen, Quan; Wang, Xiaochun; Deng, Xutao; Delwart, Eric; Shan, Tongling; Yang, Shixing

    2018-05-21

    Recombination occurs frequently between enteroviruses (EVs) which are classified within the same species of the Picornaviridae family. Here, using viral metagenomics, the genomes of two recombinant EV-Gs (strains EVG 01/NC_CHI/2014 and EVG 02/NC_CHI/2014) found in the feces of pigs from a swine farm in China are described. The two strains are characterized by distinct insertion of a papain-like protease gene from toroviruses classified within the Coronaviridae family. According to recent reports the site of the torovirus protease insertion was located at the 2C/3A junction region in EVG 02/NC_CHI/2014. For the other variant EVG 01/NC_CHI/2014, the inserted protease sequence replaced the entire viral capsid protein region up to the VP1/2A junction. These two EV-G strains were highly prevalent in the same pig farm with all animals shedding the full-length genome (EVG 02/NC_CHI/2014) while 65% also shed the capsid deletion mutant (EVG 01/NC_CHI/2014). A helper-defective virus relationship between the two co-circulating EV-G recombinants is hypothesized.

  2. Analysis of Milk from Mothers Who Delivered Prematurely Reveals Few Changes in Proteases and Protease Inhibitors across Gestational Age at Birth and Infant Postnatal Age123

    PubMed Central

    Demers-Mathieu, Veronique; Nielsen, Søren Drud; Underwood, Mark A; Borghese, Robyn

    2017-01-01

    Background: Peptidomics research has demonstrated that protease activity is higher in breast milk from preterm-delivering mothers than from term-delivering mothers. However, to our knowledge, the effect of the degree of prematurity and postnatal age on proteases and protease inhibitors in human milk remains unknown. Objective: We aimed to determine the change of proteases and protease inhibitors in milk from mothers who delivered prematurely across gestational age (GA) and postnatal age. Methods: Milk samples were collected from 18 mothers aged 26–40 y who delivered preterm infants and who lacked mastitis. For analysis, samples were separated into 2 groups: 9 from early GA (EGA) (24–26 wk GA)-delivering mothers and 9 from late GA (LGA) (27–32 wk GA)-delivering mothers. Within the 9 samples in each group, the collection time ranged from postnatal days 2 to 47. The activity and predicted activity of proteases in preterm milk were determined with the use of fluorometric and spectrophotometric assays and peptidomics, respectively. Protease and protease inhibitor concentrations were determined with the use of ELISA. Linear mixed models were applied to compare enzymes across GA and postnatal age. Results: Carboxypeptidase B2, kallikrein, plasmin, elastase, thrombin, and cytosol aminopeptidase were present and active in the milk of preterm-delivering mothers. Most milk protease and antiprotease concentrations did not change with GA or postnatal age. However, the concentration and activity of kallikrein, the most abundant and active protease in preterm milk, increased by 25.4 ng · mL−1 · d−1 and 0.454 μg · mL−1 · d−1 postnatally, respectively, in EGA milk samples while remaining stable in LGA milk samples. Conclusions: This research demonstrates that proteases are active in human milk and begin to degrade milk protein within the mammary gland before consumption by infants. Proteases and protease inhibitors in milk from mothers of premature infants mostly

  3. Isolation and characterization of a cysteine protease of freesia corms.

    PubMed

    Uchikoba, Tetsuya; Okubo, Michiko; Arima, Kazunari; Yonezawa, Hiroo

    2002-02-01

    A protease, freesia protease (FP)-A, was purified to electrophoretic homogeneity from regular freesia (Freesia reflacta) corms in harvest time. The Mr of FP-A was estimated to be 24 k by SDS-PAGE. The optimum pH of the enzyme was 8.0 using a casein substrate. These enzymes were strongly inhibited by p-chloromercuribenzoic acid but not by phenylmethane-sulfonylfluoride and EDTA. These results indicate that FP-A belongs to the cysteine proteases. The amino terminal sequence of FP-A was similar to that of papain, and the sequences was regarded to the conservative residues of cysteine protease. From the hydrolysis of peptidyl-p-NAs, the specificity of FP-A was found to be broad. It was thought that FP-A was a new protease from freesia corms.

  4. Expression of exogenous human hepatic nuclear factor-1α by a lentiviral vector and its interactions with Plasmodium falciparum subtilisin-like protease 2.

    PubMed

    Liao, Shunyao; Liu, Yunqiang; Zheng, Bing; Cho, Pyo Yun; Song, Hyun Ok; Lee, Yun-Seok; Jung, Suk-Yul; Park, Hyun

    2011-12-01

    The onset, severity, and ultimate outcome of malaria infection are influenced by parasite-expressed virulence factors as well as by individual host responses to these determinants. In both humans and mice, liver injury follows parasite entry, persisting to the erythrocytic stage in the case of infection with the fatal strain of Plasmodium falciparum. Hepatic nuclear factor (HNF)-1α is a master regulator of not only the liver damage and adaptive responses but also diverse metabolic functions. In this study, we analyzed the expression of host HNF-1α in relation to malaria infection and evaluated its interaction with the 5'-untranslated region of subtilisin-like protease 2 (subtilase, Sub2). Recombinant human HNF-1α expressed by a lentiviral vector (LV HNF-1α) was introduced into mice. Interestingly, differences in the activity of the 5'-untranslated region of the Pf-Sub2 promoter were detected in 293T cells, and LV HNF-1α was observed to influence promoter activity, suggesting that host HNF-1α interacts with the Sub2 gene.

  5. Drug resistance conferred by mutations outside the active site through alterations in the dynamic and structural ensemble of HIV-1 protease.

    PubMed

    Ragland, Debra A; Nalivaika, Ellen A; Nalam, Madhavi N L; Prachanronarong, Kristina L; Cao, Hong; Bandaranayake, Rajintha M; Cai, Yufeng; Kurt-Yilmaz, Nese; Schiffer, Celia A

    2014-08-27

    HIV-1 protease inhibitors are part of the highly active antiretroviral therapy effectively used in the treatment of HIV infection and AIDS. Darunavir (DRV) is the most potent of these inhibitors, soliciting drug resistance only when a complex combination of mutations occur both inside and outside the protease active site. With few exceptions, the role of mutations outside the active site in conferring resistance remains largely elusive. Through a series of DRV-protease complex crystal structures, inhibition assays, and molecular dynamics simulations, we find that single and double site mutations outside the active site often associated with DRV resistance alter the structure and dynamic ensemble of HIV-1 protease active site. These alterations correlate with the observed inhibitor binding affinities for the mutants, and suggest a network hypothesis on how the effect of distal mutations are propagated to pivotal residues at the active site and may contribute to conferring drug resistance.

  6. HIV-1 Protease Function and Structure Studies with the Simplicial Neighborhood Analysis of Protein Packing (SNAPP) Method

    PubMed Central

    Zhang, Shuxing; Kaplan, Andrew H.; Tropsha, Alexander

    2009-01-01

    The Simplicial Neighborhood Analysis of Protein Packing (SNAPP) method was used to predict the effect of mutagenesis on the enzymatic activity of the HIV-1 protease (HIVP). SNAPP relies on a four-body statistical scoring function derived from the analysis of spatially nearest neighbor residue compositional preferences in a diverse and representative subset of protein structures from the Protein Data Bank. The method was applied to the analysis of HIVP mutants with residue substitutions in the hydrophobic core as well as at the interface between the two protease monomers. Both wild type and tethered structures were employed in the calculations. We obtained a strong correlation, with R2 as high as 0.96, between ΔSNAPP score (i.e., the difference in SNAPP scores between wild type and mutant proteins) and the protease catalytic activity for tethered structures. A weaker but significant correlation was also obtained for non-tethered structures as well. Our analysis identified residues both in the hydrophobic core and at the dimeric interface (DI) that are very important for the protease function. This study demonstrates a potential utility of the SNAPP method for rational design of mutagenesis studies and protein engineering. PMID:18498108

  7. MBL-associated serine proteases (MASPs) and infectious diseases.

    PubMed

    Beltrame, Marcia H; Boldt, Angelica B W; Catarino, Sandra J; Mendes, Hellen C; Boschmann, Stefanie E; Goeldner, Isabela; Messias-Reason, Iara

    2015-09-01

    The lectin pathway of the complement system has a pivotal role in the defense against infectious organisms. After binding of mannan-binding lectin (MBL), ficolins or collectin 11 to carbohydrates or acetylated residues on pathogen surfaces, dimers of MBL-associated serine proteases 1 and 2 (MASP-1 and MASP-2) activate a proteolytic cascade, which culminates in the formation of the membrane attack complex and pathogen lysis. Alternative splicing of the pre-mRNA encoding MASP-1 results in two other products, MASP-3 and MAp44, which regulate activation of the cascade. A similar mechanism allows the gene encoding MASP-2 to produce the truncated MAp19 protein. Polymorphisms in MASP1 and MASP2 genes are associated with protein serum levels and functional activity. Since the first report of a MASP deficiency in 2003, deficiencies in lectin pathway proteins have been associated with recurrent infections and several polymorphisms were associated with the susceptibility or protection to infectious diseases. In this review, we summarize the findings on the role of MASP polymorphisms and serum levels in bacterial, viral and protozoan infectious diseases. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Loss of Drosophila i-AAA protease, dYME1L, causes abnormal mitochondria and apoptotic degeneration.

    PubMed

    Qi, Y; Liu, H; Daniels, M P; Zhang, G; Xu, H

    2016-02-01

    Mitochondrial AAA (ATPases Associated with diverse cellular Activities) proteases i-AAA (intermembrane space-AAA) and m-AAA (matrix-AAA) are closely related and have major roles in inner membrane protein homeostasis. Mutations of m-AAA proteases are associated with neuromuscular disorders in humans. However, the role of i-AAA in metazoans is poorly understood. We generated a deletion affecting Drosophila i-AAA, dYME1L (dYME1L(del)). Mutant flies exhibited premature aging, progressive locomotor deficiency and neurodegeneration that resemble some key features of m-AAA diseases. dYME1L(del) flies displayed elevated mitochondrial unfolded protein stress and irregular cristae. Aged dYME1L(del) flies had reduced complex I (NADH/ubiquinone oxidoreductase) activity, increased level of reactive oxygen species (ROS), severely disorganized mitochondrial membranes and increased apoptosis. Furthermore, inhibiting apoptosis by targeting dOmi (Drosophila Htra2/Omi) or DIAP1, or reducing ROS accumulation suppressed retinal degeneration. Our results suggest that i-AAA is essential for removing unfolded proteins and maintaining mitochondrial membrane architecture. Loss of i-AAA leads to the accumulation of oxidative damage and progressive deterioration of membrane integrity, which might contribute to apoptosis upon the release of proapoptotic molecules such as dOmi. Containing ROS level could be a potential strategy to manage mitochondrial AAA protease deficiency.

  9. Staphylococcus aureus secretes a unique class of neutrophil serine protease inhibitors

    PubMed Central

    Stapels, Daphne A. C.; Ramyar, Kasra X.; Bischoff, Markus; von Köckritz-Blickwede, Maren; Milder, Fin J.; Ruyken, Maartje; Eisenbeis, Janina; McWhorter, William J.; Herrmann, Mathias; van Kessel, Kok P. M.; Geisbrecht, Brian V.; Rooijakkers, Suzan H. M.

    2014-01-01

    Neutrophils are indispensable for clearing infections with the prominent human pathogen Staphylococcus aureus. Here, we report that S. aureus secretes a family of proteins that potently inhibits the activity of neutrophil serine proteases (NSPs): neutrophil elastase (NE), proteinase 3, and cathepsin G. The NSPs, but not related serine proteases, are specifically blocked by the extracellular adherence protein (Eap) and the functionally orphan Eap homologs EapH1 and EapH2, with inhibitory-constant values in the low-nanomolar range. Eap proteins are together essential for NSP inhibition by S. aureus in vitro and promote staphylococcal infection in vivo. The crystal structure of the EapH1/NE complex showed that Eap molecules constitute a unique class of noncovalent protease inhibitors that occlude the catalytic cleft of NSPs. These findings increase our insights into the complex pathogenesis of S. aureus infections and create opportunities to design novel treatment strategies for inflammatory conditions related to excessive NSP activity. PMID:25161283

  10. Protease production by fermentation of fish solubles from salmon canning processes.

    PubMed

    Wah-On, H C; Branion, R M; Strasdine, G A

    1980-09-01

    Production of protease by fermentation, using Sorangium 495, of a substrate based on condensed fish solubles is demonstrated. The effects of carbohydrate addition, pH, fish solubles concentration, scale-up, agitation, and air flow rate on protease yields are described. While the fish solubles medium alone could give rise to measurable yields of protease, these were, at worst, doubled when 1% glucose was added to the medium. pH 7 was optimal for protease yield. Although the concentration of fish solubles in the basic medium showed no significant effect on cell yield, maximum protease yield was observed at a protein concentration equivalent to 3.85 mg/mL of bovine serum albumin. Protease production rates decreased as medium protein fermentor showed no significant effect on maximum protease yields. The effects of agitator speed and air flow rate on protease yield suggested that the rate of O2 transfer from air to medium could limit the rate of protease production. It was also noted that protease production is not growth associated.

  11. 2-D zymographic analysis of Broccoli (Brassica oleracea L. var. Italica) florets proteases: follow up of cysteine protease isotypes in the course of post-harvest senescence.

    PubMed

    Rossano, Rocco; Larocca, Marilena; Riccio, Paolo

    2011-09-01

    Zymographic analysis of Broccoli florets (Brassica oleracea L. var. Italica) revealed the presence of acidic metallo-proteases, serine proteases and cysteine proteases. Under conditions which were denaturing for the other proteases, the study was restricted to cysteine proteases. 2-D zymography, a technique that combines IEF and zymography was used to show the presence of 11 different cysteine protease spots with molecular mass of 44 and 47-48kDa and pIs ranging between 4.1 and 4.7. pI differences could be ascribed to different degrees of phosphorylation that partly disappeared in the presence of alkaline phosphatase. Post-harvest senescence of Broccoli florets was characterized by decrease in protein and chlorophyll contents and increase of protease activity. In particular, as determined by 2-D zymography, the presence of cysteine protease clearly increased during senescence, a finding that may represent a useful tool for the control of the aging process. Copyright © 2011 Elsevier GmbH. All rights reserved.

  12. Role of tissue factor and protease-activated receptors in a mouse model of endotoxemia.

    PubMed

    Pawlinski, Rafal; Pedersen, Brian; Schabbauer, Gernot; Tencati, Michael; Holscher, Todd; Boisvert, William; Andrade-Gordon, Patricia; Frank, Rolf Dario; Mackman, Nigel

    2004-02-15

    Sepsis is associated with a systemic activation of coagulation and an excessive inflammatory response. Anticoagulants have been shown to inhibit both coagulation and inflammation in sepsis. In this study, we used both genetic and pharmacologic approaches to analyze the role of tissue factor and protease-activated receptors in coagulation and inflammation in a mouse endotoxemia model. We used mice expressing low levels of the procoagulant molecule, tissue factor (TF), to analyze the effects of TF deficiency either in all tissues or selectively in hematopoietic cells. Low TF mice had reduced coagulation, inflammation, and mortality compared with control mice. Similarly, a deficiency of TF expression by hematopoietic cells reduced lipopolysaccharide (LPS)-induced coagulation, inflammation, and mortality. Inhibition of the down-stream coagulation protease, thrombin, reduced fibrin deposition and prolonged survival without affecting inflammation. Deficiency of either protease activated receptor-1 (PAR-1) or protease activated receptor-2 (PAR-2) alone did not affect inflammation or survival. However, a combination of thrombin inhibition and PAR-2 deficiency reduced inflammation and mortality. These data demonstrate that hematopoietic cells are the major pathologic site of TF expression during endotoxemia and suggest that multiple protease-activated receptors mediate crosstalk between coagulation and inflammation.

  13. Aspartic proteases involved in Alzheimer's disease.

    PubMed

    Schmidt, Boris

    2003-05-09

    Alzheimer's disease afflicts every tenth human aged over 65. Despite the dramatic progress that has been made in understanding the disease, the exact cause of Alzheimer's disease is still unknown. Most gene mutations associated with Alzheimer's disease point at the same culprits: amyloid precursor protein and ultimately amyloid beta. The enigmatic proteases alpha-,beta-, and gamma-secretase are the three executioners of amyloid precursor protein processing, and disruption of their delicate balance is suspected to result in Alzheimer's disease. Significant progress has been made in the selective control of these proteases, regardless of the availability of structural information. Not even the absence of a robust cell-free assay for gamma-secretase could hamper the identification of nonpeptidic inhibitors of this enzyme for long. Within five years, four distinctly different structural moieties were developed and the first drug candidates are in clinical trials. Unfortunately, selective inhibition of amyloid beta formation remains a crucial issue because fundamental fragments of the gamma-secretase complex are important for other signaling events. This problem makes beta-secretase inhibition and alpha-secretase induction even more appealing.

  14. Protease nexin-1 promotes secretory granule biogenesis by preventing granule protein degradation.

    PubMed

    Kim, Taeyoon; Loh, Y Peng

    2006-02-01

    Dense-core secretory granule (DCG) biogenesis is a prerequisite step for the sorting, processing, and secretion of neuropeptides and hormones in (neuro)endocrine cells. Previously, chromogranin A (CgA) has been shown to play a key role in the regulation of DCG biogenesis in vitro and in vivo. However, the underlying mechanism of CgA-mediated DCG biogenesis has not been explored. In this study, we have uncovered a novel mechanism for the regulation of CgA-mediated DCG biogenesis. Transfection of CgA into endocrine 6T3 cells lacking CgA and DCGs not only recovered DCG formation and regulated secretion but also prevented granule protein degradation. Genetic profiling of CgA-expressing 6T3 versus CgA- and DCG-deficient 6T3 cells, followed by real-time reverse transcription-polymerase chain reaction and Western blotting analyses, revealed that a serine protease inhibitor, protease nexin-1 (PN-1), was significantly up-regulated in CgA-expressing 6T3 cells. Overexpression of PN-1 in CgA-deficient 6T3 cells prevented degradation of DCG proteins at the Golgi apparatus, enhanced DCG biogenesis, and recovered regulated secretion. Moreover, depletion of PN-1 by antisense RNAs in CgA-expressing 6T3 cells resulted in the specific degradation of DCG proteins. We conclude that CgA increases DCG biogenesis in endocrine cells by up-regulating PN-1 expression to stabilize granule proteins against degradation.

  15. Protease Nexin-1 Promotes Secretory Granule Biogenesis by Preventing Granule Protein Degradation

    PubMed Central

    Kim, Taeyoon; Loh, Y. Peng

    2006-01-01

    Dense-core secretory granule (DCG) biogenesis is a prerequisite step for the sorting, processing, and secretion of neuropeptides and hormones in (neuro)endocrine cells. Previously, chromogranin A (CgA) has been shown to play a key role in the regulation of DCG biogenesis in vitro and in vivo. However, the underlying mechanism of CgA-mediated DCG biogenesis has not been explored. In this study, we have uncovered a novel mechanism for the regulation of CgA-mediated DCG biogenesis. Transfection of CgA into endocrine 6T3 cells lacking CgA and DCGs not only recovered DCG formation and regulated secretion but also prevented granule protein degradation. Genetic profiling of CgA-expressing 6T3 versus CgA- and DCG-deficient 6T3 cells, followed by real-time reverse transcription-polymerase chain reaction and Western blotting analyses, revealed that a serine protease inhibitor, protease nexin-1 (PN-1), was significantly up-regulated in CgA-expressing 6T3 cells. Overexpression of PN-1 in CgA-deficient 6T3 cells prevented degradation of DCG proteins at the Golgi apparatus, enhanced DCG biogenesis, and recovered regulated secretion. Moreover, depletion of PN-1 by antisense RNAs in CgA-expressing 6T3 cells resulted in the specific degradation of DCG proteins. We conclude that CgA increases DCG biogenesis in endocrine cells by up-regulating PN-1 expression to stabilize granule proteins against degradation. PMID:16319172

  16. Plant proteases for bioactive peptides release: A review.

    PubMed

    Mazorra-Manzano, M A; Ramírez-Suarez, J C; Yada, R Y

    2017-04-10

    Proteins are a potential source of health-promoting biomolecules with medical, nutraceutical, and food applications. Nowadays, bioactive peptides production, its isolation, characterization, and strategies for its delivery to target sites are a matter of intensive research. In vitro and in vivo studies regarding the bioactivity of peptides has generated strong evidence of their health benefits. Dairy proteins are considered the richest source of bioactive peptides, however proteins from animal and vegetable origin also have been shown to be important sources. Enzymatic hydrolysis has been the process most commonly used for bioactive peptide production. Most commercial enzymatic preparations frequently used are from animal (e.g., trypsin and pepsin) and microbial (e.g., Alcalase® and Neutrase®) sources. Although the use of plant proteases is still relatively limited to papain and bromelain from papaya and pineapple, respectively, the application of new plant proteases is increasing. This review presents the latest knowledge in the use and diversity of plant proteases for bioactive peptides release from food proteins including both available commercial plant proteases as well as new potential plant sources. Furthermore, the properties of peptides released by plant proteases and health benefits associated in the control of disorders such as hypertension, diabetes, obesity, and cancer are reviewed.

  17. Generation of an antibody that recognizes Plasmodium chabaudi cysteine protease (chabaupain-1) in both sexual and asexual parasite life cycle and evaluation of chabaupain-1 vaccine potential.

    PubMed

    Armada, Ana; Gazarini, Marcos L; Gonçalves, Lídia M; Antunes, Sandra; Custódio, Ana; Rodrigues, Armanda; Almeida, António J; Silveira, Henrique; Rosário, Virgílio do; Santos-Gomes, Gabriela; Domingos, Ana

    2013-09-01

    Malaria cysteine proteases have been shown to be immunogenic and are being exploited as serodiagnostic markers, drug and vaccine targets. Several Plasmodium spp. cysteine proteases have been described and the best characterized of these are the falcipains, a family of papain-family enzymes. Falcipain-2 and falcipain-3 act in concert with other proteases to hydrolyze host erythrocyte hemoglobin in the parasite food vacuole. Falcipain-1 has less similarity to the other falcipains and its physiological role in parasite asexual blood stage still remains uncertain. Immunolocalization studies using an antibody developed against the Plasmodium chabaudi recombinant chabaupain-1, the falcipain-1 ortholog, were performed confirming its cellular localization in both erythrocyte and mosquito ookinete stage. Immunostaining of chabaupain-1 preferentially in apical portion of parasite ookinete suggests that this protease may be related with parasite egression from mosquito midgut. Immune responses to chabaupain-1 were evaluated using two different adjuvants, chitosan nanoparticles and hydroxide aluminum. Mice immunized with the recombinant protein alone or in association with nanoparticles were challenged with P. chabaudi showing that immunization with the recombinant protein confers partial protection to blood stage infection in BALB/c animal model. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. Cleavage Entropy as Quantitative Measure of Protease Specificity

    PubMed Central

    Fuchs, Julian E.; von Grafenstein, Susanne; Huber, Roland G.; Margreiter, Michael A.; Spitzer, Gudrun M.; Wallnoefer, Hannes G.; Liedl, Klaus R.

    2013-01-01

    A purely information theory-guided approach to quantitatively characterize protease specificity is established. We calculate an entropy value for each protease subpocket based on sequences of cleaved substrates extracted from the MEROPS database. We compare our results with known subpocket specificity profiles for individual proteases and protease groups (e.g. serine proteases, metallo proteases) and reflect them quantitatively. Summation of subpocket-wise cleavage entropy contributions yields a measure for overall protease substrate specificity. This total cleavage entropy allows ranking of different proteases with respect to their specificity, separating unspecific digestive enzymes showing high total cleavage entropy from specific proteases involved in signaling cascades. The development of a quantitative cleavage entropy score allows an unbiased comparison of subpocket-wise and overall protease specificity. Thus, it enables assessment of relative importance of physicochemical and structural descriptors in protease recognition. We present an exemplary application of cleavage entropy in tracing substrate specificity in protease evolution. This highlights the wide range of substrate promiscuity within homologue proteases and hence the heavy impact of a limited number of mutations on individual substrate specificity. PMID:23637583

  19. Molecular Basis for the Relative Substrate Specificity of Human Immunodeficiency Virus Type 1 and Feline Immunodeficiency Virus Proteases

    PubMed Central

    Beck, Zachary Q.; Lin, Ying-Chuan; Elder, John H.

    2001-01-01

    We have used a random hexamer phage library to delineate similarities and differences between the substrate specificities of human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus (FIV) proteases (PRs). Peptide sequences were identified that were specifically cleaved by each protease, as well as sequences cleaved equally well by both enzymes. Based on amino acid distinctions within the P3-P3′ region of substrates that appeared to correlate with these cleavage specificities, we prepared a series of synthetic peptides within the framework of a peptide sequence cleaved with essentially the same efficiency by both HIV-1 and FIV PRs, Ac-KSGVF↓VVNGLVK-NH2 (arrow denotes cleavage site). We used the resultant peptide set to assess the influence of specific amino acid substitutions on the cleavage characteristics of the two proteases. The findings show that when Asn is substituted for Val at the P2 position, HIV-1 PR cleaves the substrate at a much greater rate than does FIV PR. Likewise, Glu or Gln substituted for Val at the P2′ position also yields peptides specifically susceptible to HIV-1 PR. In contrast, when Ser is substituted for Val at P1′, FIV PR cleaves the substrate at a much higher rate than does HIV-1 PR. In addition, Asn or Gln at the P1 position, in combination with an appropriate P3 amino acid, Arg, also strongly favors cleavage by FIV PR over HIV PR. Structural analysis identified several protease residues likely to dictate the observed specificity differences. Interestingly, HIV PR Asp30 (Ile-35 in FIV PR), which influences specificity at the S2 and S2′ subsites, and HIV-1 PR Pro-81 and Val-82 (Ile-98 and Gln-99 in FIV PR), which influence specificity at the S1 and S1′ subsites, are residues which are often involved in development of drug resistance in HIV-1 protease. The peptide substrate KSGVF↓VVNGK, cleaved by both PRs, was used as a template for the design of a reduced amide inhibitor, Ac-GSGVFΨ(CH2NH)VVNGL-NH2

  20. Gene trapping in differentiating cell lines: regulation of the lysosomal protease cathepsin B in skeletal myoblast growth and fusion.

    PubMed

    Gogos, J A; Thompson, R; Lowry, W; Sloane, B F; Weintraub, H; Horwitz, M

    1996-08-01

    To identify genes regulated during skeletal muscle differentiation, we have infected mouse C2C12 myoblasts with retroviral gene trap vectors, containing a promoterless marker gene with a 5' splice acceptor signal. Integration of the vector adjacent to an actively transcribed gene places the marker under the transcriptional control of the endogenous gene, while the adjacent vector sequences facilitate cloning. The vector insertionally mutates the trapped locus and may also form fusion proteins with the endogenous gene product. We have screened several hundred clones, each containing a trapping vector integrated into a different endogenous gene. In agreement with previous estimates based on hybridization kinetics, we find that a large proportion of all genes expressed in myoblasts are regulated during differentiation. Many of these genes undergo unique temporal patterns of activation or repression during cell growth and myotube formation, and some show specific patterns of subcellular localization. The first gene we have identified with this strategy is the lysosomal cysteine protease cathepsin B. Expression from the trapped allele is upregulated during early myoblast fusion and downregulated in myotubes. A direct role for cathepsin B in myoblast growth and fusion is suggested by the observation that the trapped cells deficient in cathepsin B activity have an unusual morphology and reduced survival in low-serum media and undergo differentiation with impaired cellular fusion. The phenotype is reproduced by antisense cathepsin B expression in parental C2C12 myoblasts. The cellular phenotype is similar to that observed in cultured myoblasts from patients with I cell disease, in which there is diminished accumulation of lysosomal enzymes. This suggests that a specific deficiency of cathepsin B could contribute to the myopathic component of this illness.

  1. Nematode sperm maturation triggered by protease involves sperm-secreted serine protease inhibitor (Serpin)

    PubMed Central

    Zhao, Yanmei; Sun, Wei; Zhang, Pan; Chi, Hao; Zhang, Mei-Jun; Song, Chun-Qing; Ma, Xuan; Shang, Yunlong; Wang, Bin; Hu, Youqiao; Hao, Zhiqi; Hühmer, Andreas F.; Meng, Fanxia; L'Hernault, Steven W.; He, Si-Min; Dong, Meng-Qiu; Miao, Long

    2012-01-01

    Spermiogenesis is a series of poorly understood morphological, physiological and biochemical processes that occur during the transition of immotile spermatids into motile, fertilization-competent spermatozoa. Here, we identified a Serpin (serine protease inhibitor) family protein (As_SRP-1) that is secreted from spermatids during nematode Ascaris suum spermiogenesis (also called sperm activation) and we showed that As_SRP-1 has two major functions. First, As_SRP-1 functions in cis to support major sperm protein (MSP)-based cytoskeletal assembly in the spermatid that releases it, thereby facilitating sperm motility acquisition. Second, As_SRP-1 released from an activated sperm inhibits, in trans, the activation of surrounding spermatids by inhibiting vas deferens-derived As_TRY-5, a trypsin-like serine protease necessary for sperm activation. Because vesicular exocytosis is necessary to create fertilization-competent sperm in many animal species, components released during this process might be more important modulators of the physiology and behavior of surrounding sperm than was previously appreciated. PMID:22307610

  2. Crimean-Congo Hemorrhagic Fever Virus Suppresses Innate Immune Responses via a Ubiquitin and ISG15 Specific Protease.

    PubMed

    Scholte, Florine E M; Zivcec, Marko; Dzimianski, John V; Deaton, Michelle K; Spengler, Jessica R; Welch, Stephen R; Nichol, Stuart T; Pegan, Scott D; Spiropoulou, Christina F; Bergeron, Éric

    2017-09-05

    Antiviral responses are regulated by conjugation of ubiquitin (Ub) and interferon-stimulated gene 15 (ISG15) to proteins. Certain classes of viruses encode Ub- or ISG15-specific proteases belonging to the ovarian tumor (OTU) superfamily. Their activity is thought to suppress cellular immune responses, but studies demonstrating the function of viral OTU proteases during infection are lacking. Crimean-Congo hemorrhagic fever virus (CCHFV, family Nairoviridae) is a highly pathogenic human virus that encodes an OTU with both deubiquitinase and deISGylase activity as part of the viral RNA polymerase. We investigated CCHFV OTU function by inactivating protease catalytic activity or by selectively disrupting its deubiquitinase and deISGylase activity using reverse genetics. CCHFV OTU inactivation blocked viral replication independently of its RNA polymerase activity, while deubiquitinase activity proved critical for suppressing the interferon responses. Our findings provide insights into viral OTU functions and support the development of therapeutics and vaccines. Published by Elsevier Inc.

  3. [In vitro function of outer membrane protease T of Escherichia coli K1 pathogenic strain].

    PubMed

    Hui, Changye; Guo, Yan; Wu, Shuchi; Peng, Liang; Cao, Hong; Huang, Shenghe

    2010-01-01

    Plasminogen activation and antimicrobial peptide hydrolysis contribute to pathogens invasion and survival in vivo. To demonstrate the expression of outer membrane protease T in E. coli K1 pathogenic strain E44, its activity of plasminogen activator and protamine hydrolysis. After Benzamidine Sepharose Fast Flow and SOURCE 30Q chromatography, we got E44 outer membrane mixed fraction, and examined its activity of plasminogen activation with chromogenic substrate S-2251 method. An ompT deletion mutant of E44 was constructed by using the suicide vector pCVD442, termed as E44ompT. We examined 0.1 mg/mL cationic antimicrobial peptide protamine susceptibility of E44, ompT mutant strain E44ompT and E44ompT harboring pUCT, which was constructed by inserting complete ompT open reading frame into pUC13. We got about 37 kDa E44 membrane extract, which could activate plasminogen, and activation was membrane extract dose dependent. This confirmed the expression of outer membrane protease T in the outer membrane of E44. E44ompT was, more susceptible to 0.1 mg/mL protamine than E44, and E440mpT was partially complemented by pUCT. Outer membrane protease T is expressed in E. coli K1 pathogenic strain E44, and can activate plasminogen and hydrolyze protamine.

  4. Proteases and caspase-like activity in the yeast Saccharomyces cerevisiae.

    PubMed

    Wilkinson, Derek; Ramsdale, Mark

    2011-10-01

    A variety of proteases have been implicated in yeast PCD (programmed cell death) including the metacaspase Mca1 and the separase Esp1, the HtrA-like serine protease Nma111, the cathepsin-like serine carboxypeptideases and a range of vacuolar proteases. Proteasomal activity is also shown to have an important role in determining cell fate, with both pro- and anti-apoptotic roles. Caspase 3-, 6- and 8-like activities are detected upon stimulation of yeast PCD, but not all of this activity is associated with Mca1, implicating other proteases with caspase-like activity in the yeast cell death response. Global proteolytic events that accompany PCD are discussed alongside a consideration of the conservation of the death-related degradome (both at the level of substrate choice and cleavage site). The importance of both gain-of-function changes in the degradome as well as loss-of-function changes are highlighted. Better understanding of both death-related proteases and their substrates may facilitate the design of future antifungal drugs or the manipulation of industrial yeasts for commercial exploitation.

  5. Proteolytic crosstalk in multi-protease networks

    NASA Astrophysics Data System (ADS)

    Ogle, Curtis T.; Mather, William H.

    2016-04-01

    Processive proteases, such as ClpXP in E. coli, are conserved enzyme assemblies that can recognize and rapidly degrade proteins. These proteases are used for a number of purposes, including degrading mistranslated proteins and controlling cellular stress response. However, proteolytic machinery within the cell is limited in capacity and can lead to a bottleneck in protein degradation, whereby many proteins compete (‘queue’) for proteolytic resources. Previous work has demonstrated that such queueing can lead to pronounced statistical relationships between different protein counts when proteins compete for a single common protease. However, real cells contain many different proteases, e.g. ClpXP, ClpAP, and Lon in E. coli, and it is not clear how competition between proteins for multiple classes of protease would influence the dynamics of cellular networks. In the present work, we theoretically demonstrate that a multi-protease proteolytic bottleneck can substantially couple the dynamics for both simple and complex (oscillatory) networks, even between substrates with substantially different affinities for protease. For these networks, queueing often leads to strong positive correlations between protein counts, and these correlations are strongest near the queueing theoretic point of balance. Furthermore, we find that the qualitative behavior of these networks depends on the relative size of the absolute affinity of substrate to protease compared to the cross affinity of substrate to protease, leading in certain regimes to priority queue statistics.

  6. Synthesis and disulfide bond connectivity-activity studies of a kalata B1-inspired cyclopeptide against dengue NS2B-NS3 protease.

    PubMed

    Gao, Yaojun; Cui, Taian; Lam, Yulin

    2010-02-01

    Kalata B1 is a plant protein with remarkable thermal, chemical and enzymatic stability. Its potential applications could be centered on the possibility of using its cyclic structure and cystine knot motif as a scaffold for the design of stable pharmaceuticals. To discover potent dengue NS2B-NS3 protease inhibitors, we have prepared various kalata B1 analogues by varying the amino acid sequence. Mass spectrometric and biochemical investigations of these analogues revealed a cyclopeptide whose two fully oxidized forms are substrate-competitive inhibitors of the dengue viral NS2B-NS3 protease. Both oxidized forms showed potent inhibition with K(i) of 1.39+/-0.35 and 3.03+/-0.75 microM, respectively. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

  7. Mast cell proteases as pharmacological targets

    PubMed Central

    Caughey, George H.

    2015-01-01

    Mast cells are rich in proteases, which are the major proteins of intracellular granules and are released with histamine and heparin by activated cells. Most of these proteases are active in the granule as well outside of the mast cell when secreted, and can cleave targets near degranulating mast cells and in adjoining tissue compartments. Some proteases released from mast cells reach the bloodstream and may have far-reaching actions. In terms of relative amounts, the major mast cell proteases include the tryptases, chymases, cathepsin G, carboxypeptidase A3, dipeptidylpeptidase I/cathepsin C, and cathepsins L and S. Some mast cells also produce granzyme B, plasminogen activators, and matrix metalloproteinases. Tryptases and chymases are almost entirely mast cell-specific, whereas other proteases, such as cathepsins G, C, and L are expressed by a variety of inflammatory cells. Carboxypeptidase A3 expression is a property shared by basophils and mast cells. Other proteases, such as mastins, are largely basophil-specific, although human basophils are protease-deficient compared with their murine counterparts. The major classes of mast cell proteases have been targeted for development of therapeutic inhibitors. Also, a human β-tryptase has been proposed as a potential drug itself, to inactivate of snake venins. Diseases linked to mast cell proteases include allergic diseases, such as asthma, eczema, and anaphylaxis, but also include non-allergic diseases such inflammatory bowel disease, autoimmune arthritis, atherosclerosis, aortic aneurysms, hypertension, myocardial infarction, heart failure, pulmonary hypertension and scarring diseases of lungs and other organs. In some cases, studies performed in mouse models suggest protective or homeostatic roles for specific proteases (or groups of proteases) in infections by bacteria, worms and other parasites, and even in allergic inflammation. At the same time, a clearer picture has emerged of differences in the properties

  8. Antiacanthain A: New proteases isolated from Bromelia antiacantha Bertol. (Bromeliaceae).

    PubMed

    Vallés, Diego; Cantera, Ana M B

    2018-07-01

    Crude extract (CE) from pulp of Bromelia antiacantha Bertol. mature fruit, contains at least 3 cysteine proteases with proteolytic activity. By single step cation exchange chromatography (Hi-trap SP-HP) of partially purified CE, the protease with the lowest pI, Antiacanthain A (AntA), was isolated. It showed maximum activity at pH9, and 75% of remaining activity was maintained over a wide pH range (pH6-10). The AntA activity exhibits a constant increase up to 70°C. Maintains almost 100% of its activity at 45 at pH6 and 9. A 60% of AntA was active by titration with specific inhibitor, E64. Amidasic activity was studied with pyroglutamyl-phenyl-leucyl-paranitroaniline (PFLNA) substrate having higher AntA catalytic efficiency of (k cat /K m =470s -1 M -1 ) relative to stem bromelain (k cat /K m =305s -1 M -1 ). Esterase activity using p-nitrophenyl esters of N-α-CBZ-l-Lysine (z-L-LysONp) showed a 10-fold higher catalytic efficiency for AntA (k cat /K m =6376s -1 M -1 ) relative to stem bromelain (k cat /K m =688s -1 M -1 ). Incubation with 8M Urea did not affect AntA activity and remained unchanged for 18h, with 6M GndHCl resulted in a 41% decrease in activity after 30min incubation, maintained this activity 18h. AntA exhibits high sequence identity with proteases of the Bromeliaceae family. Copyright © 2018 Elsevier B.V. All rights reserved.

  9. Heterologous expression of the plant cysteine protease bromelain and its inhibitor in Pichia pastoris.

    PubMed

    Luniak, Nora; Meiser, Peter; Burkart, Sonja; Müller, Rolf

    2017-01-01

    Expression of proteases in heterologous hosts remains an ambitious challenge due to severe problems associated with digestion of host proteins. On the other hand, proteases are broadly used in industrial applications and resemble promising drug candidates. Bromelain is an herbal drug that is medicinally used for treatment of oedematous swellings and inflammatory conditions and consists in large part of proteolytic enzymes. Even though various experiments underline the requirement of active cysteine proteases for biological activity, so far no investigation succeeded to clearly clarify the pharmacological mode of action of bromelain. The potential role of proteases themselves and other molecules of this multi-component extract currently remain largely unknown or ill defined. Here, we set out to express several bromelain cysteine proteases as well as a bromelain inhibitor molecule in order to gain defined molecular entities for subsequent studies. After cloning the genes from its natural source Ananas comosus (pineapple plant) into Pichia pastoris and subsequent fermentation and purification, we obtained active protease and inhibitor molecules which were subsequently biochemically characterized. Employing purified bromelain fractions paves the way for further elucidation of pharmacological activities of this natural product. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:54-65, 2017. © 2016 American Institute of Chemical Engineers.

  10. Activities of the human immunodeficiency virus type 1 (HIV-1) protease inhibitor nelfinavir mesylate in combination with reverse transcriptase and protease inhibitors against acute HIV-1 infection in vitro.

    PubMed Central

    Patick, A K; Boritzki, T J; Bloom, L A

    1997-01-01

    Nelfinavir mesylate (formerly AG1343) is a potent and selective, nonpeptidic inhibitor of human immunodeficiency virus type 1 (HIV-1) protease that was discovered by protein structure-based design methodologies. We evaluated the antiviral and cytotoxic effects of two-drug combinations of nelfinavir with the clinically approved antiretroviral therapeutics zidovudine (ZDV), lamivudine (3TC), dideoxycytidine (ddC; zalcitabine), stavudine (d4T), didanosine (ddI), indinavir, saquinavir, and ritonavir and a three-drug combination of nelfinavir with ZDV and 3TC against an acute HIV-1 strain RF infection of CEM-SS cells in vitro. Quantitative assessment of drug interaction was evaluated by a universal response surface approach (W. R. Greco, G. Bravo, and J. C. Parsons, Pharm. Rev. 47:331-385, 1995) and by the method of M. N. Prichard and C. Shipman (Antiviral Res. 14:181-206, 1990). Both analytical methods yielded similar results and showed that the two-drug combinations of nelfinavir with the reverse transcriptase inhibitors ZDV, 3TC, ddI, d4T, and ddC and the three-drug combination with ZDV and 3TC resulted in additive to statistically significant synergistic interactions. In a similar manner, the combination of nelfinavir with the three protease inhibitors resulted in additive (ritonavir and saquinavir) to slightly antagonistic (indinavir) interactions. In all combinations, minimal cellular cytotoxicity was observed with any drug alone and in combination. These results suggest that administration of combinations of the appropriate doses of nelfinavir with other currently approved antiretroviral therapeutic agents in vivo may result in enhanced antiviral activity with no associated increase in cellular cytotoxicity. PMID:9333041

  11. Evidence that the recA441 (tif-1) mutant of Escherichia coli K-12 contains a thermosensitive intragenic suppressor of RecA constitutive protease activity.

    PubMed

    Wang, W B; Tessman, E S

    1985-07-01

    The recA441 mutant of Escherichia coli, which has been thought to have thermoinducible constitutive RecA protease activity, is known to have two mutations within recA. We show here that the mutation that alters codon 38 actually confers temperature-independent constitutive protease activity; the second mutation in recA441, which is at codon 298, appears to be acting as a temperature-sensitive suppressor of the protease activity.

  12. An Escherichia coli Expression Assay and Screen for Human Immunodeficiency Virus Protease Variants with Decreased Susceptibility to Indinavir

    PubMed Central

    Melnick, Laurence; Yang, Shiow-Shong; Rossi, Rick; Zepp, Charlie; Heefner, Donald

    1998-01-01

    We have developed a recombinant Escherichia coli screening system for the rapid detection and identification of amino acid substitutions in the human immunodeficiency virus (HIV) protease associated with decreased susceptibility to the protease inhibitor indinavir (MK-639; Merck & Co.). The assay depends upon the correct processing of a segment of the HIV-1 HXB2 gag-pol polyprotein followed by detection of HIV reverse transcriptase activity by a highly sensitive, colorimetric enzyme-linked immunosorbent assay. The highly sensitive system detects the contributions of single substitutions such as I84V, L90M, and L63P. The combination of single substitutions further decreases the sensitivity to indinavir. We constructed a library of HIV protease variant genes containing dispersed mutations and, using the E. coli recombinant system, screened for mutants with decreased indinavir sensitivity. The discovered HIV protease variants contain amino acid substitutions commonly associated with indinavir resistance in clinical isolates, including the substitutions L90M, L63P, I64V, V82A, L24I, and I54T. One substitution, W6R, is also frequently found by the screen and has not been reported elsewhere. Of a total of 12,000 isolates that were screened, 12 protease variants with decreased sensitivity to indinavir were found. The L63P substitution, which is also associated with indinavir resistance, increases the stability of the isolated protease relative to that of the native HXB2 protease. The rapidity, sensitivity, and accuracy of this screen also make it useful for screening for novel inhibitors. We have found the approach described here to be useful for the detection of amino acid substitutions in HIV protease that have been associated with drug resistance as well as for the screening of novel compounds for inhibitory activity. PMID:9835523

  13. A real-time fluorescence assay for protease activity and inhibitor screening based on the aggregation-caused quenching of a perylene probe.

    PubMed

    Wang, Yan; Zhang, Zhifang; Zhang, Ya; Yu, Cong

    2018-06-01

    We have established a real-time and label-free fluorescence turn-on strategy for protease activity detection and inhibitor screening via peptide-induced aggregation-caused quenching of a perylene probe. Because of electrostatic interactions and high hydrophilicity, poly-l-glutamic acid sodium salt (PGA; a negatively charged peptide) could induce aggregation of a positively charged perylene probe (probe 1) and the monomer fluorescence of probe 1 was effectively quenched. After a protease was added, PGA was enzymatically hydrolyzed into small fragments and probe 1 disaggregated. The fluorescence recovery of probe 1 was found to be proportional to the concentration of protease in the range from 0 to 1 mU/ml. The detection limit was down to 0.1 mU/ml. In the presence of a protease inhibitor, protease activity was inhibited and fluorescence recovery reduced. Moreover, we demonstrated the potential application of our method in a complex mixture sample including 1% human serum. Our method is simple, fast and cost effective. Copyright © 2018 John Wiley & Sons, Ltd.

  14. The Tla protein of Porphyromonas gingivalis W50: a homolog of the RI protease precursor (PrpRI) is an outer membrane receptor required for growth on low levels of hemin.

    PubMed

    Aduse-Opoku, J; Slaney, J M; Rangarajan, M; Muir, J; Young, K A; Curtis, M A

    1997-08-01

    The prpR1 gene of Porphyromonas gingivalis W50 encodes the polyprotein precursor (PrpRI) of an extracellular arginine-specific protease. PrpRI is organized into four distinct domains (pro, alpha, beta, and gamma) and is processed to a heterodimeric protease (RI) which comprises the alpha and beta components in a noncovalent association. The alpha component contains the protease active site, whereas the beta component appears to have a role in adherence and hemagglutination processes. DNA sequences homologous to the coding region for the RI beta component are present at multiple loci on the P. gingivalis chromosome and may represent a family of related genes. In this report, we describe the cloning, sequence analysis, and characterization of one of these homologous loci isolated in plasmid pJM7. The 6,041-bp P. gingivalis DNA fragment in pJM7 contains a major open reading frame of 3,291 bp with coding potential for a protein with an Mr 118,700. An internal region of the deduced sequence (V304 to N768) shows 98% identity to the beta domain of PrpRI, and the recombinant product of pJM7 is immunoreactive with an antibody specific to the RI beta component. The N terminus of the deduced sequence has regional similarity to TonB-linked receptors which are frequently involved in periplasmic translocation of hemin, iron, colicins, or vitamin B12 in other bacteria. We have therefore designated this gene tla (TonB-linked adhesin). In contrast to the parent strain, an isogenic mutant of P. gingivalis W50 in which the tla was insertionally inactivated was unable to grow in medium containing low concentrations of hemin (<2.5 mg liter(-1)), and hemin-depleted cells of this mutant failed to respond to hemin in an agar diffusion plate assay. These data suggest a role for this gene product in hemin acquisition and utilization. Furthermore, the mutant produced significantly less arginine- and lysine-specific protease activities than the parent strain, indicating that there may be a

  15. The Tla protein of Porphyromonas gingivalis W50: a homolog of the RI protease precursor (PrpRI) is an outer membrane receptor required for growth on low levels of hemin.

    PubMed Central

    Aduse-Opoku, J; Slaney, J M; Rangarajan, M; Muir, J; Young, K A; Curtis, M A

    1997-01-01

    The prpR1 gene of Porphyromonas gingivalis W50 encodes the polyprotein precursor (PrpRI) of an extracellular arginine-specific protease. PrpRI is organized into four distinct domains (pro, alpha, beta, and gamma) and is processed to a heterodimeric protease (RI) which comprises the alpha and beta components in a noncovalent association. The alpha component contains the protease active site, whereas the beta component appears to have a role in adherence and hemagglutination processes. DNA sequences homologous to the coding region for the RI beta component are present at multiple loci on the P. gingivalis chromosome and may represent a family of related genes. In this report, we describe the cloning, sequence analysis, and characterization of one of these homologous loci isolated in plasmid pJM7. The 6,041-bp P. gingivalis DNA fragment in pJM7 contains a major open reading frame of 3,291 bp with coding potential for a protein with an Mr 118,700. An internal region of the deduced sequence (V304 to N768) shows 98% identity to the beta domain of PrpRI, and the recombinant product of pJM7 is immunoreactive with an antibody specific to the RI beta component. The N terminus of the deduced sequence has regional similarity to TonB-linked receptors which are frequently involved in periplasmic translocation of hemin, iron, colicins, or vitamin B12 in other bacteria. We have therefore designated this gene tla (TonB-linked adhesin). In contrast to the parent strain, an isogenic mutant of P. gingivalis W50 in which the tla was insertionally inactivated was unable to grow in medium containing low concentrations of hemin (<2.5 mg liter(-1)), and hemin-depleted cells of this mutant failed to respond to hemin in an agar diffusion plate assay. These data suggest a role for this gene product in hemin acquisition and utilization. Furthermore, the mutant produced significantly less arginine- and lysine-specific protease activities than the parent strain, indicating that there may be a

  16. Cysteine proteases and cell differentiation: excystment of the ciliated protist Sterkiella histriomuscorum.

    PubMed

    Villalobo, Eduardo; Moch, Clara; Fryd-Versavel, Ghislaine; Fleury-Aubusson, Anne; Morin, Loïc

    2003-12-01

    The process of excystment of Sterkiella histriomuscorum (Ciliophora, Oxytrichidae) leads in a few hours, through a massive influx of water and the resorption of the cyst wall, from an undifferentiated resting cyst to a highly differentiated and dividing vegetative cell. While studying the nature of the genes involved in this process, we isolated three different cysteine proteases genes, namely, a cathepsin B gene, a cathepsin L-like gene, and a calpain-like gene. Excystation was selectively inhibited at a precise differentiating stage by cysteine proteases inhibitors, suggesting that these proteins are specifically required during the excystment process. Reverse transcription-PCR experiments showed that both genes display differential expression between the cyst and the vegetative cells. A phylogenetic analysis showed for the first time that the cathepsin B tree is paraphyletic and that the diverging S. histriomuscorum cathepsin B is closely related to its Giardia homologues, which take part in the cyst wall breakdown process. The deduced cathepsin L-like protein sequence displays the structural signatures and phylogenetic relationships of cathepsin H, a protein that is known only in plants and animals and that is involved in the degradation of extracellular matrix components in cancer diseases. The deduced calpain-like protein sequence does not display the calcium-binding domain of conventional calpains; it belongs to a diverging phylogenetic cluster that includes Aspergillus palB, a protein which is involved in a signal transduction pathway that is sensitive to ambient pH.

  17. Protease-Mediated Suppression of DRG Neuron Excitability by Commensal Bacteria.

    PubMed

    Sessenwein, Jessica L; Baker, Corey C; Pradhananga, Sabindra; Maitland, Megan E; Petrof, Elaine O; Allen-Vercoe, Emma; Noordhof, Curtis; Reed, David E; Vanner, Stephen J; Lomax, Alan E

    2017-11-29

    Peripheral pain signaling reflects a balance of pronociceptive and antinociceptive influences; the contribution by the gastrointestinal microbiota to this balance has received little attention. Disorders, such as inflammatory bowel disease and irritable bowel syndrome, are associated with exaggerated visceral nociceptive actions that may involve altered microbial signaling, particularly given the evidence for bacterial dysbiosis. Thus, we tested whether a community of commensal gastrointestinal bacteria derived from a healthy human donor (microbial ecosystem therapeutics; MET-1) can affect the excitability of male mouse DRG neurons. MET-1 reduced the excitability of DRG neurons by significantly increasing rheobase, decreasing responses to capsaicin (2 μm) and reducing action potential discharge from colonic afferent nerves. The increase in rheobase was accompanied by an increase in the amplitude of voltage-gated K + currents. A mixture of bacterial protease inhibitors abrogated the effect of MET-1 effects on DRG neuron rheobase. A serine protease inhibitor but not inhibitors of cysteine proteases, acid proteases, metalloproteases, or aminopeptidases abolished the effects of MET-1. The serine protease cathepsin G recapitulated the effects of MET-1 on DRG neurons. Inhibition of protease-activated receptor-4 (PAR-4), but not PAR-2, blocked the effects of MET-1. Furthermore, Faecalibacterium prausnitzii recapitulated the effects of MET-1 on excitability of DRG neurons. We conclude that serine proteases derived from commensal bacteria can directly impact the excitability of DRG neurons, through PAR-4 activation. The ability of microbiota-neuronal interactions to modulate afferent signaling suggests that therapies that induce or correct microbial dysbiosis may impact visceral pain. SIGNIFICANCE STATEMENT Commercially available probiotics have the potential to modify visceral pain. Here we show that secretory products from gastrointestinal microbiota derived from a human

  18. Activity-based mass spectrometric characterization of proteases and inhibitors in human saliva

    PubMed Central

    Sun, Xiuli; Salih, Erdjan; Oppenheim, Frank G.; Helmerhorst, Eva J.

    2009-01-01

    Proteases present in oral fluid effectively modulate the structure and function of some salivary proteins and have been implicated in tissue destruction in oral disease. To identify the proteases operating in the oral environment, proteins in pooled whole saliva supernatant were separated by anion-exchange chromatography and individual fractions were analyzed for proteolytic activity by zymography using salivary histatins as the enzyme substrates. Protein bands displaying proteolytic activity were particularly prominent in the 50–75 kDa region. Individual bands were excised, in-gel trypsinized and subjected to LC/ESI-MS/MS. The data obtained were searched against human, oral microbial and protease databases. A total of 13 proteases were identified all of which were of mammalian origin. Proteases detected in multiple fractions with cleavage specificities toward arginine and lysine residues, were lactotransferrin, kallikrein-1, and human airway trypsin-like protease. Unexpectedly, ten protease inhibitors were co-identified suggesting they were associated with the proteases in the same fractions. The inhibitors found most frequently were alpha-2-macroglobulin-like protein 1, alpha-1-antitrypsin, and leukocyte elastase inhibitor. Regulation of oral fluid proteolysis is highly important given that an inbalance in such activities has been correlated to a variety of pathological conditions including oral cancer. PMID:20011683

  19. Recombinant expression and antigenic properties of a 32-kilodalton extracellular alkaline protease, representing a possible virulence factor from Aspergillus fumigatus.

    PubMed Central

    Moser, M; Menz, G; Blaser, K; Crameri, R

    1994-01-01

    A 32-kDa nonglycosylated alkaline protease (EC 3.4.1.14) with elastolytic activity, secreted by the opportunistic pathogen Aspergillus fumigatus ATCC 42202, is suggested to be a virulence factor of this fungus. The enzyme is a serine protease of the subtilisin family, and its cDNA nucleotide sequence has recently been reported. We have cloned the cDNA encoding the mature protease into a high-level Escherichia coli expression plasmid and produced the recombinant protease as a fusion protein with a six-adjacent-histidine affinity tag at the carboxy terminus. Subsequently, the recombinant protease was purified to homogeneity, with affinity chromatography yielding 30 to 40 mg of recombinant protease per liter of E. coli culture. Refolded recombinant protease, in comparison with native protease, demonstrated weak enzymatic activity but similar immunochemical characteristics as analyzed by antigen-specific enzyme-linked immunosorbent assay (ELISA), competition ELISA, and immunoblotting assays. To assess the allergenic potential of the protease, sera from patients with allergic bronchopulmonary aspergillosis and sera from healthy control individuals were analyzed by ELISA and immunoblotting techniques. Sera from patients with allergic bronchopulmonary aspergillosis did not have protease-specific immunoglobulin E (IgE) antibodies and, remarkably, did not show significantly elevated protease-specific IgG antibody levels compared with those in sera from healthy control individuals. This suggests that the alkaline protease from A. fumigatus does not elicit IgE antibodies and has weak immunogenicity, a property which may explain fungus persistence in allergic individuals. Images PMID:8112866

  20. [Isolation and characteristics of IgA1 and its use for detecting bacterial IgA1 proteases].

    PubMed

    Amelina, I P; Zakharova, N A

    1984-12-01

    Sufficiently purified IgA, subclass I, has been isolated from the defibrinated plasma of a myeloma patient by chromatography on columns packed with DEAE-Sephadex A-50 or Sephadex G-200, and rabbit antiserum to this immunoglobulin has been obtained. These preparations have been used for detecting specific protease in Bordetella pertussis. The tested B. pertussis strains have been shown to induce, as revealed by immunoelectrophoretic methods, the proteolysis of human IgA, subclass I.

  1. Structure of the aspartic protease from Rous sarcoma retrovirus refined at 2-A resolution.

    PubMed

    Jaskólski, M; Miller, M; Rao, J K; Leis, J; Wlodawer, A

    1990-06-26

    The structure of Rous sarcoma virus protease has been solved by multiple isomorphous replacement in the crystal form belonging to space group P3(1)21, with unit-cell parameters a = 88.95 A and c = 78.90 A. The enzyme belongs to the family of aspartic proteases with two identical subunits composing the active homodimer. The noncrystallographic dyad relating these two subunits was identified after preliminary tracing in the MIR map and was used for phase improvement by electron-density averaging. Structure refinement resulted in a model that included 1772 protein atoms and 252 water molecules, with an R factor of 0.144 for data extending to 2 A. The secondary structure of a retroviral protease molecule closely resembles that of a single domain in pepsin-like aspartic proteases and consists of several beta-strands and of one well-defined and one distorted alpha-helix. The dimer interface is composed of the N- and C-terminal chains from both subunits which are intertwined to form a well-ordered four-stranded antiparallel beta-sheet. In each monomer, the catalytic triad (Asp-Ser-Gly) is located in a loop that forms a part of the psi-structure characteristic to all aspartic proteases. The position of a water molecule between the active-site aspartate residues and the general scheme of H bonding within the active site bear close resemblance to those in pepsin-like aspartic proteases and therefore suggest a similar enzymatic mechanism. The binding cleft over the active site is covered by two flap arms, one from each monomer, which are partially disordered. The retroviral protease dimer has been compared with several enzymes of cellular origin, with chains aligning to an rms deviation of 1.90 A or better.

  2. A Novel Fission Yeast Gene, tht1 +, Is Required for the Fusion of Nuclear Envelopes during Karyogamy

    PubMed Central

    Tange, Yoshie; Horio, Tetsuya; Shimanuki, Mizuki; Ding, Da-Qiao; Hiraoka, Yasushi; Niwa, Osami

    1998-01-01

    We have isolated a fission yeast karyogamy mutant, tht1, in which nuclear congression and the association of two spindle pole bodies occurs but the subsequent fusion of nuclear envelopes is blocked. The tht1 mutation does not prevent meiosis, so cells execute meiosis with two unfused nuclei, leading to the production of aberrant asci. The tht1 + gene was cloned and sequenced. Predicted amino acid sequence has no significant homology to previously known proteins but strongly suggests that it is a type I membrane protein. The tht1 + gene is dispensable for vegetative growth and expressed only in conjugating cells. Tht1p is a glycoprotein susceptible to endoglycosilase H digestion. Site- directed mutagenesis showed that the N-glycosylation site, as well as the COOH-terminal region of Tht1p, is essential for its function. A protease protection assay indicated that the COOH terminus is cytoplasmic. Immunocytological analysis using a HA-tagged Tht1p suggested that the protein is localized in nuclear envelopes and in the ER during karyogamy and that its levels are reduced in cells containing fused nuclei. PMID:9442101

  3. Evidence that the recA441 (tif-1) mutant of Escherichia coli K-12 contains a thermosensitive intragenic suppressor of RecA constitutive protease activity.

    PubMed Central

    Wang, W B; Tessman, E S

    1985-01-01

    The recA441 mutant of Escherichia coli, which has been thought to have thermoinducible constitutive RecA protease activity, is known to have two mutations within recA. We show here that the mutation that alters codon 38 actually confers temperature-independent constitutive protease activity; the second mutation in recA441, which is at codon 298, appears to be acting as a temperature-sensitive suppressor of the protease activity. Images PMID:3891740

  4. The Flavivirus Protease As a Target for Drug Discovery

    PubMed Central

    Brecher, Matthew; Zhang, Jing; Li, Hongmin

    2014-01-01

    Many flaviviruses are significant human pathogens causing considerable disease burdens, including encephalitis and hemorrhagic fever, in the regions in which they are endemic. A paucity of treatments for flaviviral infections has driven interest in drug development targeting proteins essential to flavivirus replication, such as the viral protease. During viral replication, the flavivirus genome is translated as a single polyprotein precursor, which must be cleaved into individual proteins by a complex of the viral protease, NS3, and its cofactor, NS2B. Because this cleavage is an obligate step of the viral life-cycle, the flavivirus protease is an attractive target for antiviral drug development. In this review, we will survey recent drug development studies targeting the NS3 active site, as well as studies targeting an NS2B/NS3 interaction site determined from flavivirus protease crystal structures. PMID:24242363

  5. The flavivirus protease as a target for drug discovery.

    PubMed

    Brecher, Matthew; Zhang, Jing; Li, Hongmin

    2013-12-01

    Many flaviviruses are significant human pathogens causing considerable disease burdens, including encephalitis and hemorrhagic fever, in the regions in which they are endemic. A paucity of treatments for flaviviral infections has driven interest in drug development targeting proteins essential to flavivirus replication, such as the viral protease. During viral replication, the flavivirus genome is translated as a single polyprotein precursor, which must be cleaved into individual proteins by a complex of the viral protease, NS3, and its cofactor, NS2B. Because this cleavage is an obligate step of the viral life-cycle, the flavivirus protease is an attractive target for antiviral drug development. In this review, we will survey recent drug development studies targeting the NS3 active site, as well as studies targeting an NS2B/NS3 interaction site determined from flavivirus protease crystal structures.

  6. Targeted gene disruption in Koji mold Aspergillus oryzae.

    PubMed

    Maruyama, Jun-Ichi; Kitamoto, Katsuhiko

    2011-01-01

    Filamentous fungi have received attentions as hosts for heterologous protein production because of their high secretion capability and eukaryotic post-translational modifications. One of the safest hosts for heterologous protein production is Koji mold Aspergillus oryzae since it has been used in the production of Japanese fermented foods for over 1,000 years. The production levels of proteins from higher eukaryotes are much lower than those of homologous (fungal) proteins. Bottlenecks in the heterologous protein production are suggested to be proteolytic degradation of the produced protein in the medium and the secretory pathway. For construction of excellent host strains, many genes causing the bottlenecks should be disrupted rapidly and efficiently. We developed a marker recycling system with the highly efficient gene-targeting background in A. oryzae. By employing this technique, we performed multiple gene disruption of the ten protease genes. The decuple protease gene disruptant showed fourfold production level of a heterologous protein compared with the wild-type strain.

  7. Comparisons of Transcriptional Profiles of Gut Genes between Cry1Ab-Resistant and Susceptible Strains of Ostrinia nubilalis Revealed Genes Possibly Related to the Adaptation of Resistant Larvae to Transgenic Cry1Ab Corn.

    PubMed

    Yao, Jianxiu; Zhu, Yu-Cheng; Lu, Nanyan; Buschman, Lawrent L; Zhu, Kun Yan

    2017-01-30

    A microarray developed on the basis of 2895 unique transcripts from larval gut was used to compare gut gene expression profiles between a laboratory-selected Cry1Ab-resistant (R) strain and its isoline susceptible (S) strain of the European corn borer (Ostrinia nubilalis) after the larvae were fed the leaves of transgenic corn (MON810) expressing Cry1Ab or its non-transgenic isoline for 6 h. We revealed 398 gut genes differentially expressed (i.e., either up- or down-regulated genes with expression ratio ≥2.0) in S-strain, but only 264 gut genes differentially expressed in R-strain after being fed transgenic corn leaves. Although the percentages of down-regulated genes among the total number of differentially expressed genes (50% in S-strain and 45% in R-strain) were similar between the R- and S-strains, the expression ratios of down-regulated genes were much higher in S-strain than in R-strain. We revealed that 17 and 9 significantly up- or down-regulated gut genes from S and R-strain, respectively, including serine proteases and aminopeptidases. These genes may be associated with Cry1Ab toxicity by degradation, binding, and cellular defense. Overall, our study suggests enhanced adaptation of Cry1Ab-resistant larvae on transgenic Cry1Ab corn as revealed by lower number and lower ratios of differentially expressed genes in R-strain than in S-strain of O. nubilalis.

  8. Hepatitis C virus NS3 protease genotyping and drug concentration determination during triple therapy with telaprevir or boceprevir for chronic infection with genotype 1 viruses, southeastern France.

    PubMed

    Aherfi, Sarah; Solas, Caroline; Motte, Anne; Moreau, Jacques; Borentain, Patrick; Mokhtari, Saadia; Botta-Fridlund, Danielle; Dhiver, Catherine; Portal, Isabelle; Ruiz, Jean-Marie; Ravaux, Isabelle; Bregigeon, Sylvie; Poizot-Martin, Isabelle; Stein, Andreas; Gérolami, René; Brouqui, Philippe; Tamalet, Catherine; Colson, Philippe

    2014-11-01

    Telaprevir and boceprevir, the two first hepatitis C virus (HCV) NS3 protease inhibitors (PIs), considerably increase rates of sustained virologic response in association with pegylated interferon and ribavirin in chronic HCV genotype 1 infections. The 30 first patients treated by telaprevir or boceprevir including anti-HCV therapies since 2011 in Marseille University hospitals, France, were monitored. HCV loads and plasmatic concentrations of telaprevir and boceprevir were determined on sequential blood samples. HCV NS3 protease gene population sequencing was performed at baseline of treatment and in case of treatment failure. Fifteen patients (including 7 co-infected with HIV) received telaprevir and the other 15 patients (including 4 co-infected with HIV) received boceprevir. At baseline, HCV NS3 protease from six patients harbored amino acid substitutions associated with PI-resistance. Treatment failure occurred at week 12 for 7 patients. Amino acid substitutions associated with PI-resistance were observed in six of these cases. HCV NS3 R155K and T54A/S mutants, all of genotype 1a, were found from four patients. Median (interquartile range) plasma concentrations were 3,092 ng/ml (2,320-3,525) for telaprevir and 486 ng/ml (265-619) for boceprevir. For HIV-HCV co-infected patients, median concentrations were 3,162 ng/ml (2,270-4,232) for telaprevir and 374 ng/ml (229-519) for boceprevir. Plasma drug concentration monitoring revealed undetectable concentrations for two patients at week 4, and probable non-adherence to therapy for another patient. These findings indicate that routine HCV NS3 protease sequencing and plasma PI concentration monitoring might be helpful to characterize cases of therapy failure, at a cost dramatically low compared to that of anti-HCV therapy. © 2014 Wiley Periodicals, Inc.

  9. Targeting host calpain proteases decreases influenza A virus infection.

    PubMed

    Blanc, Fany; Furio, Laetitia; Moisy, Dorothée; Yen, Hui-Ling; Chignard, Michel; Letavernier, Emmanuel; Naffakh, Nadia; Mok, Chris Ka Pun; Si-Tahar, Mustapha

    2016-04-01

    Influenza A viruses (IAV) trigger contagious acute respiratory diseases. A better understanding of the molecular mechanisms of IAV pathogenesis and host immune responses is required for the development of more efficient treatments of severe influenza. Calpains are intracellular proteases that participate in diverse cellular responses, including inflammation. Here, we used in vitro and in vivo approaches to investigate the role of calpain signaling in IAV pathogenesis. Calpain expression and activity were found altered in IAV-infected bronchial epithelial cells. With the use of small-interfering RNA (siRNA) gene silencing, specific synthetic inhibitors of calpains, and mice overexpressing calpastatin, we found that calpain inhibition dampens IAV replication and IAV-triggered secretion of proinflammatory mediators and leukocyte infiltration. Remarkably, calpain inhibition has a protective impact in IAV infection, since it significantly reduced mortality of mice challenged not only by seasonal H3N2- but also by hypervirulent H5N1 IAV strains. Hence, our study suggests that calpains are promising therapeutic targets for treating IAV acute pneumonia. Copyright © 2016 the American Physiological Society.

  10. Aspartyl proteases in Candida glabrata are required for suppression of the host innate immune response.

    PubMed

    Rasheed, Mubashshir; Battu, Anamika; Kaur, Rupinder

    2018-04-27

    A family of 11 cell surface-associated aspartyl proteases (CgYps1-11), also referred as yapsins, is a key virulence factor in the pathogenic yeast Candida glabrata However, the mechanism by which CgYapsins modulate immune response and facilitate survival in the mammalian host remains to be identified. Here, using RNA-Seq analysis, we report that genes involved in cell wall metabolism are differentially regulated in the Cgyps1-11 Δ mutant. Consistently, the mutant contained lower β-glucan and mannan levels and exhibited increased chitin content in the cell wall. As cell wall components are known to regulate the innate immune response, we next determined the macrophage transcriptional response to C. glabrata infection and observed differential expression of genes implicated in inflammation, chemotaxis, ion transport, and the tumor necrosis factor signaling cascade. Importantly, the Cgyps1-11 Δ mutant evoked a different immune response, resulting in an enhanced release of the pro-inflammatory cytokine IL-1β in THP-1 macrophages. Further, Cgyps1-11 Δ-induced IL-1β production adversely affected intracellular proliferation of co-infected WT cells and depended on activation of spleen tyrosine kinase (Syk) signaling in the host cells. Accordingly, the Syk inhibitor R406 augmented intracellular survival of the Cgyps1-11 Δ mutant. Finally, we demonstrate that C. glabrata infection triggers elevated IL-1β production in mouse organs and that the CgYPS genes are required for organ colonization and dissemination in the murine model of systemic infection. Altogether, our results uncover the basis for macrophage-mediated killing of Cgyps1-11 Δ cells and provide the first evidence that aspartyl proteases in C. glabrata are required for suppression of IL-1β production in macrophages. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Aspartyl proteases in Candida glabrata are required for suppression of the host innate immune response

    PubMed Central

    Rasheed, Mubashshir; Battu, Anamika; Kaur, Rupinder

    2018-01-01

    A family of 11 cell surface-associated aspartyl proteases (CgYps1–11), also referred as yapsins, is a key virulence factor in the pathogenic yeast Candida glabrata. However, the mechanism by which CgYapsins modulate immune response and facilitate survival in the mammalian host remains to be identified. Here, using RNA-Seq analysis, we report that genes involved in cell wall metabolism are differentially regulated in the Cgyps1–11Δ mutant. Consistently, the mutant contained lower β-glucan and mannan levels and exhibited increased chitin content in the cell wall. As cell wall components are known to regulate the innate immune response, we next determined the macrophage transcriptional response to C. glabrata infection and observed differential expression of genes implicated in inflammation, chemotaxis, ion transport, and the tumor necrosis factor signaling cascade. Importantly, the Cgyps1–11Δ mutant evoked a different immune response, resulting in an enhanced release of the pro-inflammatory cytokine IL-1β in THP-1 macrophages. Further, Cgyps1–11Δ–induced IL-1β production adversely affected intracellular proliferation of co-infected WT cells and depended on activation of spleen tyrosine kinase (Syk) signaling in the host cells. Accordingly, the Syk inhibitor R406 augmented intracellular survival of the Cgyps1–11Δ mutant. Finally, we demonstrate that C. glabrata infection triggers elevated IL-1β production in mouse organs and that the CgYPS genes are required for organ colonization and dissemination in the murine model of systemic infection. Altogether, our results uncover the basis for macrophage-mediated killing of Cgyps1–11Δ cells and provide the first evidence that aspartyl proteases in C. glabrata are required for suppression of IL-1β production in macrophages. PMID:29491142

  12. Protease-Mediated Maturation of HIV: Inhibitors of Protease and the Maturation Process.

    PubMed

    Adamson, Catherine S

    2012-01-01

    Protease-mediated maturation of HIV-1 virus particles is essential for virus infectivity. Maturation occurs concomitant with immature virus particle release and is mediated by the viral protease (PR), which sequentially cleaves the Gag and Gag-Pol polyproteins into mature protein domains. Maturation triggers a second assembly event that generates a condensed conical capsid core. The capsid core organizes the viral RNA genome and viral proteins to facilitate viral replication in the next round of infection. The fundamental role of proteolytic maturation in the generation of mature infectious particles has made it an attractive target for therapeutic intervention. Development of small molecules that target the PR active site has been highly successful and nine protease inhibitors (PIs) have been approved for clinical use. This paper provides an overview of their development and clinical use together with a discussion of problems associated with drug resistance. The second-half of the paper discusses a novel class of antiretroviral drug termed maturation inhibitors, which target cleavage sites in Gag not PR itself. The paper focuses on bevirimat (BVM) the first-in-class maturation inhibitor: its mechanism of action and the implications of naturally occurring polymorphisms that confer reduced susceptibility to BVM in phase II clinical trials.

  13. Understanding the HIV-1 protease nelfinavir resistance mutation D30N in subtypes B and C through molecular dynamics simulations.

    PubMed

    Soares, Rosemberg O; Batista, Paulo R; Costa, Mauricio G S; Dardenne, Laurent E; Pascutti, Pedro G; Soares, Marcelo A

    2010-09-01

    A major concern in the antiretroviral (ARV) treatment of HIV infections with protease inhibitors (PI) is the emergence of resistance, which results from the selection of distinct mutations within the viral protease (PR) gene. Among patients who do not respond to treatment with the PI nelfinavir (NFV), the D30N mutation is often observed. However, several reports have shown that D30N emerges with different frequencies in distinct HIV-1 genetic forms or subtypes. In the present work, we analyzed the binding of NFV and the Gag substrate CA/p2 to PR from HIV-1 subtypes B and C through molecular dynamics (MD) simulations. The wild-type and drug-resistant D30N mutants were investigated in both subtypes. The compensatory mutations N83T and N88D, observed in vitro and in vivo when subtype C acquires D30N, were also studied. D30N appears to facilitate conformational changes in subtype B PR, but not in that from subtype C, and this could be associated with disestablishment of an alpha-helical region of the PR. Furthermore, the total contact areas of NFV or the CA/p2 substrate with the mutant PR correlated with changes in the resistance patterns and replicative capacity. Finally, we observed in our MD simulations that mutant PR proteins show different patterns for hydrophobic/van der Waals contact. These findings suggest that different molecular mechanisms contribute to resistance, and we propose that a single mutation has distinct impacts on different HIV-1 subtypes. (c) 2010 Elsevier Inc. All rights reserved.

  14. Exposure to Ozone Modulates Human Airway Protease/Antiprotease Balance Contributing to Increased Influenza A Infection

    PubMed Central

    Kesic, Matthew J.; Meyer, Megan; Bauer, Rebecca; Jaspers, Ilona

    2012-01-01

    Exposure to oxidant air pollution is associated with increased respiratory morbidities and susceptibility to infections. Ozone is a commonly encountered oxidant air pollutant, yet its effects on influenza infections in humans are not known. The greater Mexico City area was the primary site for the spring 2009 influenza A H1N1 pandemic, which also coincided with high levels of environmental ozone. Proteolytic cleavage of the viral membrane protein hemagglutinin (HA) is essential for influenza virus infectivity. Recent studies suggest that HA cleavage might be cell-associated and facilitated by the type II transmembrane serine proteases (TTSPs) human airway trypsin-like protease (HAT) and transmembrane protease, serine 2 (TMPRSS2), whose activities are regulated by antiproteases, such as secretory leukocyte protease inhibitor (SLPI). Based on these observations, we sought to determine how acute exposure to ozone may modulate cellular protease/antiprotease expression and function, and to define their roles in a viral infection. We utilized our in vitro model of differentiated human nasal epithelial cells (NECs) to determine the effects of ozone on influenza cleavage, entry, and replication. We show that ozone exposure disrupts the protease/antiprotease balance within the airway liquid. We also determined that functional forms of HAT, TMPRSS2, and SLPI are secreted from human airway epithelium, and acute exposure to ozone inversely alters their expression levels. We also show that addition of antioxidants significantly reduces virus replication through the induction of SLPI. In addition, we determined that ozone-induced cleavage of the viral HA protein is not cell-associated and that secreted endogenous proteases are sufficient to activate HA leading to a significant increase in viral replication. Our data indicate that pre-exposure to ozone disrupts the protease/antiprotease balance found in the human airway, leading to increased influenza susceptibility. PMID

  15. Novel Mycosin Protease MycP1 Inhibitors Identified by Virtual Screening and 4D Fingerprints

    PubMed Central

    2015-01-01

    The rise of drug-resistant Mycobacterium tuberculosis lends urgency to the need for new drugs for the treatment of tuberculosis (TB). The identification of a serine protease, mycosin protease-1 (MycP1), as the crucial agent in hydrolyzing the virulence factor, ESX-secretion-associated protein B (EspB), potentially opens the door to new tuberculosis treatment options. Using the crystal structure of mycobacterial MycP1 in the apo form, we performed an iterative ligand- and structure-based virtual screening (VS) strategy to identify novel, nonpeptide, small-molecule inhibitors against MycP1 protease. Screening of ∼485 000 ligands from databases at the Genomics Research Institute (GRI) at the University of Cincinnati and the National Cancer Institute (NCI) using our VS approach, which integrated a pharmacophore model and consensus molecular shape patterns of active ligands (4D fingerprints), identified 81 putative inhibitors, and in vitro testing subsequently confirmed two of them as active inhibitors. Thereafter, the lead structures of each VS round were used to generate a new 4D fingerprint that enabled virtual rescreening of the chemical libraries. Finally, the iterative process identified a number of diverse scaffolds as lead compounds that were tested and found to have micromolar IC50 values against the MycP1 target. This study validated the efficiency of the SABRE 4D fingerprints as a means of identifying novel lead compounds in each screening round of the databases. Together, these results underscored the value of using a combination of in silico iterative ligand- and structure-based virtual screening of chemical libraries with experimental validation for the identification of promising structural scaffolds, such as the MycP1 inhibitors. PMID:24628123

  16. Purification and characterization of a newly serine protease inhibitor from Rhamnus frangula with potential for use as therapeutic drug.

    PubMed

    Bacha, Abir Ben; Jemel, Ikram; Moubayed, Nadine M S; Abdelmalek, Imen Ben

    2017-06-01

    Protease inhibitors from plants are well known to be potent inhibitors of the growth of bacteria, fungi, and even certain viruses which make them excellent candidates for use as the lead compounds for the development of novel antimicrobial agents for applications in medicine. In this study, Rhamnus frangula was selected as a protease inhibitor source. The maximum recovery of the protease inhibitor against trypsin was recorded in the crude extract made in 0.1 M phosphate buffer (pH 7.0) and isolated from the mature leaves. Then, the protease inhibitor designated as RfIP1 was purified to homogeneity by Sephadex G50 with an apparent molecular mass of 22.5 kDa and its N-terminal sequence exhibited a high degree of homology with known serine protease inhibitor sequences. The RfIP1 displayed maximal activity at pH 7 and 37 °C. It maintained almost 80% of its maximal activity through a large pH range. The thermo-stability of RfIP1 was markedly enhanced by BSA, CaCl 2, and sorbitol, whereas the addition of Mg 2+ , Zn 2+ , NaTDC, SDS, DTT, and β-ME significantly promoted inhibitory activity. The protease inhibitor displayed high inhibitory activity toward some known proteases (cathepsin B, chymotrypsin, collagenase, thrombin, and trypsin) that have more importance in pharmaceutical industry and it acted as potent inhibitor of some commercially proteases from Aspergillus oryzae, Bacillus sp, and Bacillus licheniformis. The protease inhibitor also possessed an appreciable antibacterial effect against both Gram-positive and Gram-negative bacteria.

  17. A screen for immunity genes evolving under positive selection in Drosophila.

    PubMed

    Jiggins, F M; Kim, K W

    2007-05-01

    Genes involved in the immune system tend to have higher rates of adaptive evolution than other genes in the genome, probably because they are coevolving with pathogens. We have screened a sample of Drosophila genes to identify those evolving under positive selection. First, we identified rapidly evolving immunity genes by comparing 140 loci in Drosophila erecta and D. yakuba. Secondly, we resequenced 23 of the fastest evolving genes from the independent species pair D. melanogaster and D. simulans, and identified those under positive selection using a McDonald-Kreitman test. There was strong evidence of adaptive evolution in two serine proteases (persephone and spirit) and a homolog of the Anopheles serpin SRPN6, and weaker evidence in another serine protease and the death domain protein dFADD. These results add to mounting evidence that immune signalling pathway molecules often evolve rapidly, possibly because they are sites of host-parasite coevolution.

  18. Serine Protease PrtA from Streptococcus pneumoniae Plays a Role in the Killing of S. pneumoniae by Apolactoferrin ▿

    PubMed Central

    Mirza, Shaper; Wilson, Landon; Benjamin, William H.; Novak, Jan; Barnes, Stephen; Hollingshead, Susan K.; Briles, David E.

    2011-01-01

    It is known that apolactoferrin, the iron-free form of human lactoferrin, can kill many species of bacteria, including Streptococcus pneumoniae. Lactoferricin, an N-terminal peptide of apolactoferrin, and fragments of it are even more bactericidal than apolactoferrin. In this study we found that apolactoferrin must be cleaved by a serine protease in order for it to kill pneumococci. The serine protease inhibitors were able to block killing by apolactoferrin but did not block killing by a lactoferrin-derived peptide. Thus, the killing of pneumococci by apolactoferrin appears to require a protease to release a lactoferricin-like peptide(s). Incubation of apolactoferrin with growing pneumococci resulted in a 12-kDa reduction in its molecular mass, of which about 7 to 8 kDa of the reduction was protease dependent. Capsular type 2 and 19F strains with mutations in the gene encoding the major cell wall-associated serine protease, prtA, lost much of their ability to degrade apolactoferrin and were relatively resistant to killing by apolactoferrin (P < 0.001). Recombinant PrtA was also able to cleave apolactoferrin, reducing its mass by about 8 kDa, and greatly enhance the killing activity of the solution containing the apolactoferrin and its cleavage products. Mass spectroscopy revealed that PrtA makes a major cut between amino acids 78 and 79 of human lactoferrin, removing the N-terminal end of the molecule (about 8.6 kDa). The simplest interpretation of these data is that the mechanism by which apolactoferrin kills Streptococcus pneumoniae requires the release of a lactoferricin-like peptide(s) and that it is this peptide(s), and not the intact apolactoferrin, which kills pneumococci. PMID:21422179

  19. Detergent protease exposure and respiratory disease: case-referent analysis of a retrospective cohort.

    PubMed

    Brant, A; Upchurch, S; van Tongeren, M; Zekveld, C; Helm, J; Barnes, F; Newman Taylor, A J; Cullinan, P

    2009-11-01

    To examine the relationship between protease exposure and respiratory disease in a cohort of detergent enzyme manufacturers. Case-referent analysis of a cohort of employees working in a European detergent factory between 1989 and 2002. Cases with new lower or upper respiratory disease were ascertained by examination of occupational health records and matched to referents on date of first employment. Personal exposures to airborne detergent protease were estimated, using a job exposure matrix, from >12,000 measurements taken in the factory during the period of study. We found clear, monotonic relationships between estimated protease exposure and both lower and upper respiratory disease. After control for age, sex and smoking, the odds ratio of lower respiratory disease was significantly elevated (1.98, 95% CI 1.04 to 3.79) in those employees working in jobs in the highest quartile of protease exposure (geometric mean 7.9 ng x m(-3)). For employees with upper respiratory disease, the risk was significantly elevated at a lower level of estimated protease exposure (geometric mean 2.3 ng x m(-3)). These findings provide strong evidence of an association between detergent enzyme exposure and the development of respiratory disease in an occupational setting. Using the routinely collected information on specific sensitisation and the close attention to workplace exposures that are characteristic of this industry, it should be possible to derive meaningful occupational exposure standards for most detergent enzymes.

  20. Chimeric exchange of coronavirus nsp5 proteases (3CLpro) identifies common and divergent regulatory determinants of protease activity.

    PubMed

    Stobart, Christopher C; Sexton, Nicole R; Munjal, Havisha; Lu, Xiaotao; Molland, Katrina L; Tomar, Sakshi; Mesecar, Andrew D; Denison, Mark R

    2013-12-01

    Human coronaviruses (CoVs) such as severe acute respiratory syndrome CoV (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV) cause epidemics of severe human respiratory disease. A conserved step of CoV replication is the translation and processing of replicase polyproteins containing 16 nonstructural protein domains (nsp's 1 to 16). The CoV nsp5 protease (3CLpro; Mpro) processes nsp's at 11 cleavage sites and is essential for virus replication. CoV nsp5 has a conserved 3-domain structure and catalytic residues. However, the intra- and intermolecular determinants of nsp5 activity and their conservation across divergent CoVs are unknown, in part due to challenges in cultivating many human and zoonotic CoVs. To test for conservation of nsp5 structure-function determinants, we engineered chimeric betacoronavirus murine hepatitis virus (MHV) genomes encoding nsp5 proteases of human and bat alphacoronaviruses and betacoronaviruses. Exchange of nsp5 proteases from HCoV-HKU1 and HCoV-OC43, which share the same genogroup, genogroup 2a, with MHV, allowed for immediate viral recovery with efficient replication albeit with impaired fitness in direct competition with wild-type MHV. Introduction of MHV nsp5 temperature-sensitive mutations into chimeric HKU1 and OC43 nsp5 proteases resulted in clear differences in viability and temperature-sensitive phenotypes compared with MHV nsp5. These data indicate tight genetic linkage and coevolution between nsp5 protease and the genomic background and identify differences in intramolecular networks regulating nsp5 function. Our results also provide evidence that chimeric viruses within coronavirus genogroups can be used to test nsp5 determinants of function and inhibition in common isogenic backgrounds and cell types.

  1. Evaluation of pre-rigor injection of beef with proteases on cooked meat volatile profile after 1 day and 21 days post-mortem storage.

    PubMed

    Ma, Q L; Hamid, N; Bekhit, A E D; Robertson, J; Law, T F

    2012-12-01

    This research was carried out to determine the effects of pre-rigor injection of beef semimembranosus muscle with nine proteases from plant and microbial sources, on the volatile profile of cooked beef after 1 day and 21 days post-mortem (PM) storage using Solid-phase microextraction gas chromatography mass spectrometry analysis. A total of 23 aldehydes, 5 ketones, 3 furans, 8 nitrogen and sulphur compounds, 4 alkanes, 7 alcohols and 6 terpenes were detected. Eleven volatile compounds characteristic of ginger flavour were detected in zingibain-treated meat. Benzaldehyde significantly increased (p<0.05) only in kiwifruit juice (KJ), fungal 31 protease and Asparagus protease (ASP) treated samples from 1 day to 21 days PM storage. A significant increase (p<0.05) in 3-methylbutanal was observed in KJ, bacterial and fungal protease treated samples at 21 days PM storage. Treatments with bromelain, papain, ASP, actinidin, and KJ (except KJ 21 days) proteases resulted in flavour profiles closer to that of the control beef sample. Copyright © 2012 Elsevier Ltd. All rights reserved.

  2. Boosted protease inhibitor monotherapy versus boosted protease inhibitor plus lamivudine dual therapy as second-line maintenance treatment for HIV-1-infected patients in sub-Saharan Africa (ANRS12 286/MOBIDIP): a multicentre, randomised, parallel, open-label, superiority trial.

    PubMed

    Ciaffi, Laura; Koulla-Shiro, Sinata; Sawadogo, Adrien Bruno; Ndour, Cheik Tidiane; Eymard-Duvernay, Sabrina; Mbouyap, Pretty Rosereine; Ayangma, Liliane; Zoungrana, Jacques; Gueye, Ndeye Fatou Ngom; Diallo, Mohamadou; Izard, Suzanne; Bado, Guillaume; Kane, Coumba Toure; Aghokeng, Avelin Fobang; Peeters, Martine; Girard, Pierre Marie; Le Moing, Vincent; Reynes, Jacques; Delaporte, Eric

    2017-09-01

    Despite satisfactory efficacy of WHO-recommended second-line antiretroviral treatment for patients with HIV in low-income countries, the need for simplified, low-cost, and less-toxic maintenance strategies remains high. We compared boosted protease inhibitor monotherapy with dual therapy with boosted protease inhibitor plus lamivudine in patients on second-line antiretrovial therapy (ART). We did a multicentre, randomised, parallel, open-label, superiority, trial in the HIV services of five hospitals in sub-Saharan Africa (Yaoundé, Cameroon; Dakar, Senegal; and Bobo Dioulasso, Burkina Faso). We recruited patients from the long-term, post-trial cohort of the ANRS 12169/2LADY study that compared the efficacy of three second-line combinations based on boosted protease inhibitors. Participants for our study were HIV-1 infected with multiple mutations including M184V, at first-line failure, aged 18 years and older, on boosted protease inhibitor plus two nucleoside reverse transcriptase inhibitors (NRTI) for at least 48 weeks with at least 48 weeks follow-up in the 2LADY trial, with two viral load measurements of less than 200 copies per mL in the previous 6 months, CD4 counts of more than 100 cells per μL, adherence of at least 90%, and no change to ART in the past 3 months. We randomly assigned participants (1:1) to receive either monotherapy with their boosted protease inhibitor (once-daily darunavir 800 mg [two 400 mg tablets] boosted with ritonavir 100 mg [one tablet] or coformulation of lopinavir 200 mg with ritonavir 50 mg [two tablets taken twice per day]) or to boosted protease inhibitor plus once-daily lamivudine 300 mg (one 300 mg tablet or two 150 mg tablets). Computer-generated randomisation was stratified by study site and viral load at screening (< 50 copies per mL, and 50-200 copies per mL), and concealed from study personnel throughout the inclusion period. After randomisation, treatment allocation was not masked from clinicians or patients]. Patients

  3. The multiple functions of plant serine protease inhibitors

    PubMed Central

    Giri, Ashok P; Kaur, Harleen; Baldwin, Ian T

    2011-01-01

    Plant protease inhibitors (PIs) are a diverse group of proteins which have been intensely investigated due to their potential function in protecting plants against herbivorous insects by inhibiting digestive proteases. Although this mechanism has been well documented for a number of single PIs and their target enzymes, whether this mechanism protects plants in nature remains unclear. Moreover, many plants express a number of different PIs and it was unknown if these proteins work synergistically as defenses or if they also have other functions. We recently identified four serine PIs (SPI) of Solanum nigrum and demonstrated that they differ substantially in substrate specificity, accumulation patterns, and their effect against different natural herbivorous insects in field- and glasshouse experiments. These differences suggest that SPIs have at least partially diversified to provide protection against different attackers. Although we could not detect effects on plant development or growth when silencing SPIs, gene- and tissue-specific expression patterns suggest multiple functions in generative tissues, including a possible involvement in development. PMID:22004998

  4. Immobilization of Bacillus amyloliquefaciens SP1 and its alkaline protease in various matrices for effective hydrolysis of casein.

    PubMed

    Guleria, Shiwani; Walia, Abhishek; Chauhan, Anjali; Shirkot, C K

    2016-12-01

    An extracellular alkaline protease producing B. amyloliquefaciens SP1 was isolated from apple rhizosphere having multifarious plant growth-promoting activities. B. amyloliquefaciens SP1 protease was immobilized using various concentrations of calcium alginate, agar and polyacrylamide to determine the optimum concentration for formation of the beads. Enzyme activity before immobilization (at 60 °C, pH 8.0 for 5 min) was 3580 µg/ml/min. The results of immobilization with various matrices revealed that 3 % calcium alginate (2829.92 µg/ml/min), 2 % agar (2600 µg/ml/min) and 10 % polyacrylamide (5698.99 µg/ml/min) were optimum concentrations for stable bead formation. Immobilized enzyme reusability results indicated that calcium alginate, agar and polyacrylamide beads retained 25.63, 22.05 and 34.04 % activity in their fifth repeated cycle, respectively. In cell immobilization technique, the free movement of microorganisms is restricted in the process, and a semi-continuous system of fermentation can be used. In the present work, this technique has been used for alkaline protease production using different matrices. Polyacrylamide (10 %) was found with the highest total alkaline protease titer, i.e., 24,847 µg/ml/min semi-continuously for 18 days as compared to agar (total enzyme titer: 5800 in 10 days) and calcium alginate (total enzyme titer: 13,010 in 15 days). This present study reported that polyacrylamide (10 %) among different matrices has maximum potential of immobilization of B. amyloliquefaciens SP1 and its detergent stable alkaline protease with effective application in bloodstain removal.

  5. Molecular adaptation of a plant-bacterium outer membrane protease towards plague virulence factor Pla

    PubMed Central

    2011-01-01

    Background Omptins are a family of outer membrane proteases that have spread by horizontal gene transfer in Gram-negative bacteria that infect vertebrates or plants. Despite structural similarity, the molecular functions of omptins differ in a manner that reflects the life style of their host bacteria. To simulate the molecular adaptation of omptins, we applied site-specific mutagenesis to make Epo of the plant pathogenic Erwinia pyrifoliae exhibit virulence-associated functions of its close homolog, the plasminogen activator Pla of Yersinia pestis. We addressed three virulence-associated functions exhibited by Pla, i.e., proteolytic activation of plasminogen, proteolytic degradation of serine protease inhibitors, and invasion into human cells. Results Pla and Epo expressed in Escherichia coli are both functional endopeptidases and cleave human serine protease inhibitors, but Epo failed to activate plasminogen and to mediate invasion into a human endothelial-like cell line. Swapping of ten amino acid residues at two surface loops of Pla and Epo introduced plasminogen activation capacity in Epo and inactivated the function in Pla. We also compared the structure of Pla and the modeled structure of Epo to analyze the structural variations that could rationalize the different proteolytic activities. Epo-expressing bacteria managed to invade human cells only after all extramembranous residues that differ between Pla and Epo and the first transmembrane β-strand had been changed. Conclusions We describe molecular adaptation of a protease from an environmental setting towards a virulence factor detrimental for humans. Our results stress the evolvability of bacterial β-barrel surface structures and the environment as a source of progenitor virulence molecules of human pathogens. PMID:21310089

  6. A genetic screen for modifiers of Drosophila caspase Dcp-1 reveals caspase involvement in autophagy and novel caspase-related genes.

    PubMed

    Kim, Young-Il; Ryu, Taewoo; Lee, Judong; Heo, Young-Shin; Ahnn, Joohong; Lee, Seung-Jae; Yoo, OokJoon

    2010-01-25

    Caspases are cysteine proteases with essential functions in the apoptotic pathway; their proteolytic activity toward various substrates is associated with the morphological changes of cells. Recent reports have described non-apoptotic functions of caspases, including autophagy. In this report, we searched for novel modifiers of the phenotype of Dcp-1 gain-of-function (GF) animals by screening promoter element- inserted Drosophila melanogaster lines (EP lines). We screened approximately 15,000 EP lines and identified 72 Dcp-1-interacting genes that were classified into 10 groups based on their functions and pathways: 4 apoptosis signaling genes, 10 autophagy genes, 5 insulin/IGF and TOR signaling pathway genes, 6 MAP kinase and JNK signaling pathway genes, 4 ecdysone signaling genes, 6 ubiquitination genes, 11 various developmental signaling genes, 12 transcription factors, 3 translation factors, and 11 other unclassified genes including 5 functionally undefined genes. Among them, insulin/IGF and TOR signaling pathway, MAP kinase and JNK signaling pathway, and ecdysone signaling are known to be involved in autophagy. Together with the identification of autophagy genes, the results of our screen suggest that autophagy counteracts Dcp-1-induced apoptosis. Consistent with this idea, we show that expression of eGFP-Atg5 rescued the eye phenotype caused by Dcp-1 GF. Paradoxically, we found that over-expression of full-length Dcp-1 induced autophagy, as Atg8b-GFP, an indicator of autophagy, was increased in the eye imaginal discs and in the S2 cell line. Taken together, these data suggest that autophagy suppresses Dcp-1-mediated apoptotic cell death, whereas Dcp-1 positively regulates autophagy, possibly through feedback regulation. We identified a number of Dcp-1 modifiers that genetically interact with Dcp-1-induced cell death. Our results showing that Dcp-1 and autophagy-related genes influence each other will aid future investigations of the complicated relationships

  7. New types of metacaspases in phytoplankton reveal diverse origins of cell death proteases

    PubMed Central

    Choi, C J; Berges, J A

    2013-01-01

    Metacaspases are evolutionarily distant homologs of caspases that are found outside the metazoan and are known to have key roles in programmed cell death (PCD). Two types of metacaspases (types I and II) have been defined in plants based on their domain structures; these have similarities to metazoan ‘initiator' and ‘executioner' caspases. However, we know little about metacaspases in unicellular organisms and even less about their roles in cell death. We identified a novel group of metacaspases in sequenced phytoplanktonic protists that show domain architectures distinct from either type I or II enzymes; we designate them as type III. Type III metacaspases exhibit a rearrangement of domain structures between N- and C-terminus. In addition, we found a group of metacaspase-like proteases in phytoplankton that show sequence homology with other metacaspases, but defy classification in conventional schemes. These metacaspase-like proteases exist in bacteria alongside a variant of type I metacaspases and we propose these bacterial metacaspases are the origins of eukaryotic metacaspases. Type II and III metacaspases were not detected in bacteria and they might be variants of bacterial type I metacaspases that evolved in plants and phytoplanktonic protists, respectively, during the establishment of plastids through the primary and secondary endosymbiotic events. A complete absence of metacaspases in protists that lost plastids, such as oömycetes and ciliates indicates the gene loss during the plastid-to-nucleus gene transfer. Taken together, our findings suggest endosymbiotic gene transfer (EGT) is a key mechanism resulting in the evolutionary diversity of cell death proteases. PMID:23412383

  8. Energetic basis for drug resistance of HIV-1 protease mutants against amprenavir

    NASA Astrophysics Data System (ADS)

    Kar, Parimal; Knecht, Volker

    2012-02-01

    Amprenavir (APV) is a high affinity (0.15 nM) HIV-1 protease (PR) inhibitor. However, the affinities of the drug resistant protease variants V32I, I50V, I54V, I54M, I84V and L90M to amprenavir are decreased 3 to 30-fold compared to the wild-type. In this work, the popular molecular mechanics Poisson-Boltzmann surface area method has been used to investigate the effectiveness of amprenavir against the wild-type and these mutated protease variants. Our results reveal that the protonation state of Asp25/Asp25' strongly affects the dynamics, the overall affinity and the interactions of the inhibitor with individual residues. We emphasize that, in contrast to what is often assumed, the protonation state may not be inferred from the affinities but requires pKa calculations. At neutral pH, Asp25 and Asp25' are ionized or protonated, respectively, as suggested from pKa calculations. This protonation state was thus mainly considered in our study. Mutation induced changes in binding affinities are in agreement with the experimental findings. The decomposition of the binding free energy reveals the mechanisms underlying binding and drug resistance. Drug resistance arises from an increase in the energetic contribution from the van der Waals interactions between APV and PR (V32I, I50V, and I84V mutant) or a rise in the energetic contribution from the electrostatic interactions between the inhibitor and its target (I54M and I54V mutant). For the V32I mutant, also an increased free energy for the polar solvation contributes to the drug resistance. For the L90M mutant, a rise in the van der Waals energy for APV-PR interactions is compensated by a decrease in the polar solvation free energy such that the net binding affinity remains unchanged. Detailed understanding of the molecular forces governing binding and drug resistance might assist in the design of new inhibitors against HIV-1 PR variants that are resistant against current drugs.

  9. High-resolution structure of a retroviral protease folded as a monomer

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Gilski, Miroslaw; Polish Academy of Sciences, 61-704 Poznan; Kazmierczyk, Maciej

    2011-11-01

    The crystal structure of Mason–Pfizer monkey virus protease folded as a monomer has been solved by molecular replacement using a model generated by players of the online game Foldit. The structure shows at high resolution the details of a retroviral protease folded as a monomer which can guide rational design of protease dimerization inhibitors as retroviral drugs. Mason–Pfizer monkey virus (M-PMV), a D-type retrovirus assembling in the cytoplasm, causes simian acquired immunodeficiency syndrome (SAIDS) in rhesus monkeys. Its pepsin-like aspartic protease (retropepsin) is an integral part of the expressed retroviral polyproteins. As in all retroviral life cycles, release and dimerizationmore » of the protease (PR) is strictly required for polyprotein processing and virion maturation. Biophysical and NMR studies have indicated that in the absence of substrates or inhibitors M-PMV PR should fold into a stable monomer, but the crystal structure of this protein could not be solved by molecular replacement despite countless attempts. Ultimately, a solution was obtained in mr-rosetta using a model constructed by players of the online protein-folding game Foldit. The structure indeed shows a monomeric protein, with the N- and C-termini completely disordered. On the other hand, the flap loop, which normally gates access to the active site of homodimeric retropepsins, is clearly traceable in the electron density. The flap has an unusual curled shape and a different orientation from both the open and closed states known from dimeric retropepsins. The overall fold of the protein follows the retropepsin canon, but the C{sup α} deviations are large and the active-site ‘DTG’ loop (here NTG) deviates up to 2.7 Å from the standard conformation. This structure of a monomeric retropepsin determined at high resolution (1.6 Å) provides important extra information for the design of dimerization inhibitors that might be developed as drugs for the treatment of retroviral

  10. Cathepsin L plays a major role in cholecystokinin production in mouse brain cortex and in pituitary AtT-20 cells: protease gene knockout and inhibitor studies.

    PubMed

    Beinfeld, Margery C; Funkelstein, Lydiane; Foulon, Thierry; Cadel, Sandrine; Kitagawa, Kouki; Toneff, Thomas; Reinheckel, Thomas; Peters, Christoph; Hook, Vivian

    2009-10-01

    Cholecystokinin (CCK) is a peptide neurotransmitter whose production requires proteolytic processing of the proCCK precursor to generate active CCK8 neuropeptide in brain. This study demonstrates the significant role of the cysteine protease cathepsin L for CCK8 production. In cathepsin L knockout (KO) mice, CCK8 levels were substantially reduced in brain cortex by an average of 75%. To evaluate the role of cathepsin L in producing CCK in the regulated secretory pathway of neuroendocrine cells, pituitary AtT-20 cells that stably produce CCK were treated with the specific cathepsin L inhibitor, CLIK-148. CLIK-148 inhibitor treatment resulted in decreased amounts of CCK secreted from the regulated secretory pathway of AtT-20 cells. CLIK-148 also reduced cellular levels of CCK9 (Arg-CCK8), consistent with CCK9 as an intermediate product of cathepsin L, shown by the decreased ratio of CCK9/CCK8. The decreased CCK9/CCK8 ratio also suggests a shift in the production to CCK8 over CCK9 during inhibition of cathepsin L. During reduction of the PC1/3 processing enzyme by siRNA, the ratio of CCK9/CCK8 was increased, suggesting a shift to the cathepsin L pathway for the production of CCK9. The changes in ratios of CCK9 compared to CCK8 are consistent with dual roles of the cathepsin L protease pathway that includes aminopeptidase B to remove NH2-terminal Arg or Lys, and the PC1/3 protease pathway. These results suggest that cathepsin L functions as a major protease responsible for CCK8 production in mouse brain cortex, and participates with PC1/3 for CCK8 production in pituitary cells.

  11. Diversity of Both the Cultivable Protease-Producing Bacteria and Bacterial Extracellular Proteases in the Coastal Sediments of King George Island, Antarctica

    PubMed Central

    Zhou, Ming-Yang; Wang, Guang-Long; Li, Dan; Zhao, Dian-Li; Qin, Qi-Long; Chen, Xiu-Lan; Chen, Bo; Zhou, Bai-Cheng; Zhang, Xi-Ying; Zhang, Yu-Zhong

    2013-01-01

    Protease-producing bacteria play a vital role in degrading sedimentary organic nitrogen. However, the diversity of these bacteria and their extracellular proteases in most regions remain unknown. In this paper, the diversity of the cultivable protease-producing bacteria and of bacterial extracellular proteases in the sediments of Maxwell Bay, King George Island, Antarctica was investigated. The cultivable protease-producing bacteria reached 105 cells/g in all 8 sediment samples. The cultivated protease-producing bacteria were mainly affiliated with the phyla Actinobacteria, Firmicutes, Bacteroidetes, and Proteobacteria, and the predominant genera were Bacillus (22.9%), Flavobacterium (21.0%) and Lacinutrix (16.2%). Among these strains, Pseudoalteromonas and Flavobacteria showed relatively high protease production. Inhibitor analysis showed that nearly all the extracellular proteases from the bacteria were serine proteases or metalloproteases. These results begin to address the diversity of protease-producing bacteria and bacterial extracellular proteases in the sediments of the Antarctic Sea. PMID:24223990

  12. Identification of Mycoparasitism-Related Genes in Trichoderma atroviride ▿ † ‡

    PubMed Central

    Reithner, Barbara; Ibarra-Laclette, Enrique; Mach, Robert L.; Herrera-Estrella, Alfredo

    2011-01-01

    A high-throughput sequencing approach was utilized to carry out a comparative transcriptome analysis of Trichoderma atroviride IMI206040 during mycoparasitic interactions with the plant-pathogenic fungus Rhizoctonia solani. In this study, transcript fragments of 7,797 Trichoderma genes were sequenced, 175 of which were host responsive. According to the functional annotation of these genes by KOG (eukaryotic orthologous groups), the most abundant group during direct contact was “metabolism.” Quantitative reverse transcription (RT)-PCR confirmed the differential transcription of 13 genes (including swo1, encoding an expansin-like protein; axe1, coding for an acetyl xylan esterase; and homologs of genes encoding the aspartyl protease papA and a trypsin-like protease, pra1) in the presence of R. solani. An additional relative gene expression analysis of these genes, conducted at different stages of mycoparasitism against Botrytis cinerea and Phytophthora capsici, revealed a synergistic transcription of various genes involved in cell wall degradation. The similarities in expression patterns and the occurrence of regulatory binding sites in the corresponding promoter regions suggest a possible analog regulation of these genes during the mycoparasitism of T. atroviride. Furthermore, a chitin- and distance-dependent induction of pra1 was demonstrated. PMID:21531825

  13. A limited role for gene duplications in the evolution of platypus venom.

    PubMed

    Wong, Emily S W; Papenfuss, Anthony T; Whittington, Camilla M; Warren, Wesley C; Belov, Katherine

    2012-01-01

    Gene duplication followed by adaptive selection is believed to be the primary driver of venom evolution. However, to date, no studies have evaluated the importance of gene duplications for venom evolution using a genomic approach. The availability of a sequenced genome and a venom gland transcriptome for the enigmatic platypus provides a unique opportunity to explore the role that gene duplication plays in venom evolution. Here, we identify gene duplication events and correlate them with expressed transcripts in an in-season venom gland. Gene duplicates (1,508) were identified. These duplicated pairs (421), including genes that have undergone multiple rounds of gene duplications, were expressed in the venom gland. The majority of these genes are involved in metabolism and protein synthesis not toxin functions. Twelve secretory genes including serine proteases, metalloproteinases, and protease inhibitors likely to produce symptoms of envenomation such as vasodilation and pain were detected. Only 16 of 107 platypus genes with high similarity to known toxins evolved through gene duplication. Platypus venom C-type natriuretic peptides and nerve growth factor do not possess lineage-specific gene duplicates. Extensive duplications, believed to increase the potency of toxic content and promote toxin diversification, were not found. This is the first study to take a genome-wide approach in order to examine the impact of gene duplication on venom evolution. Our findings support the idea that adaptive selection acts on gene duplicates to drive the independent evolution and functional diversification of similar venom genes in venomous species. However, gene duplications alone do not explain the "venome" of the platypus. Other mechanisms, such as alternative splicing and mutation, may be important in venom innovation.

  14. A Limited Role for Gene Duplications in the Evolution of Platypus Venom

    PubMed Central

    Wong, Emily S. W.; Papenfuss, Anthony T.; Whittington, Camilla M.; Warren, Wesley C.; Belov, Katherine

    2012-01-01

    Gene duplication followed by adaptive selection is believed to be the primary driver of venom evolution. However, to date, no studies have evaluated the importance of gene duplications for venom evolution using a genomic approach. The availability of a sequenced genome and a venom gland transcriptome for the enigmatic platypus provides a unique opportunity to explore the role that gene duplication plays in venom evolution. Here, we identify gene duplication events and correlate them with expressed transcripts in an in-season venom gland. Gene duplicates (1,508) were identified. These duplicated pairs (421), including genes that have undergone multiple rounds of gene duplications, were expressed in the venom gland. The majority of these genes are involved in metabolism and protein synthesis not toxin functions. Twelve secretory genes including serine proteases, metalloproteinases, and protease inhibitors likely to produce symptoms of envenomation such as vasodilation and pain were detected. Only 16 of 107 platypus genes with high similarity to known toxins evolved through gene duplication. Platypus venom C-type natriuretic peptides and nerve growth factor do not possess lineage-specific gene duplicates. Extensive duplications, believed to increase the potency of toxic content and promote toxin diversification, were not found. This is the first study to take a genome-wide approach in order to examine the impact of gene duplication on venom evolution. Our findings support the idea that adaptive selection acts on gene duplicates to drive the independent evolution and functional diversification of similar venom genes in venomous species. However, gene duplications alone do not explain the “venome” of the platypus. Other mechanisms, such as alternative splicing and mutation, may be important in venom innovation. PMID:21816864

  15. Apoplastic effectors secreted by two unrelated eukaryotic plant pathogens target the tomato defense protease Rcr3.

    PubMed

    Song, Jing; Win, Joe; Tian, Miaoying; Schornack, Sebastian; Kaschani, Farnusch; Ilyas, Muhammad; van der Hoorn, Renier A L; Kamoun, Sophien

    2009-02-03

    Current models of plant-pathogen interactions stipulate that pathogens secrete effector proteins that disable plant defense components known as virulence targets. Occasionally, the perturbations caused by these effectors trigger innate immunity via plant disease resistance proteins as described by the "guard hypothesis." This model is nicely illustrated by the interaction between the fungal plant pathogen Cladosporium fulvum and tomato. C. fulvum secretes a protease inhibitor Avr2 that targets the tomato cysteine protease Rcr3(pim). In plants that carry the resistance protein Cf2, Rcr3(pim) is required for resistance to C. fulvum strains expressing Avr2, thus fulfilling one of the predictions of the guard hypothesis. Another prediction of the guard hypothesis has not yet been tested. Considering that virulence targets are important components of defense, different effectors from unrelated pathogens are expected to evolve to disable the same host target. In this study we confirm this prediction using a different pathogen of tomato, the oomycete Phytophthora infestans that is distantly related to fungi such as C. fulvum. This pathogen secretes an array of protease inhibitors including EPIC1 and EPIC2B that inhibit tomato cysteine proteases. Here we show that, similar to Avr2, EPIC1 and EPIC2B bind and inhibit Rcr3(pim). However, unlike Avr2, EPIC1 and EPIC2B do not trigger hypersensitive cell death or defenses on Cf-2/Rcr3(pim) tomato. We also found that the rcr3-3 mutant of tomato that carries a premature stop codon in the Rcr3 gene exhibits enhanced susceptibility to P. infestans, suggesting a role for Rcr3(pim) in defense. In conclusion, our findings fulfill a key prediction of the guard hypothesis and suggest that the effectors Avr2, EPIC1, and EPIC2B secreted by two unrelated pathogens of tomato target the same defense protease Rcr3(pim). In contrast to C. fulvum, P. infestans appears to have evolved stealthy effectors that carry inhibitory activity without

  16. Degradation of recombinant proteins by CHO host cell proteases is prevented by Matriptase-1 knock-out.

    PubMed

    Laux, Holger; Romand, Sandrine; Nuciforo, Sandro; Farady, Christopher J; Tapparel, Joel; Buechmann-Moeller, Stine; Sommer, Benjamin; Oakeley, Edward J; Bodendorf, Ursula

    2018-05-19

    An increasing number of non-antibody format proteins are entering the clinical development. However, one of the major hurdles for the production of non-antibody glycoproteins is host cell-related proteolytic degradation, which can drastically impact developability and timelines of pipeline projects. Chinese hamster ovary (CHO) cells are the preferred production host for recombinant therapeutic proteins. Using protease inhibitors, transcriptomics and genetic knockdowns we have identified, out of the more than 700 known proteases in rodents, Matriptase-1 as the major protease involved in degradation of recombinant proteins expressed in CHO-K1 cells. Subsequently Matriptase-1 was deleted in CHO-K1 cells using "Transcription Activator-Like Effector Nucleases" (TALENs) as well as zinc-finger nucleases (ZFNs). This resulted in a superior CHO-K1 matriptase knockout (KO) cell line with strongly reduced or no proteolytic degradation activity towards a panel of recombinantly-expressed proteins. The matriptase KO cell line was evaluated in spike-in experiments, and showed little or no degradation of proteins incubated in culture supernatant derived from the KO cells. This effect was confirmed when the same proteins were recombinantly expressed in the KO cell line. In summary, the combination of novel cell line engineering tools, next generation sequencing screening methods and the recently published Chinese hamster genome has enabled the development of this novel matriptase KO CHO cell line capable of improving expression yields of intact therapeutic proteins. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  17. Crystal structure of a complex of HIV-1 protease with a dihydroxyethylene-containing inhibitor: comparisons with molecular modeling.

    PubMed Central

    Thanki, N.; Rao, J. K.; Foundling, S. I.; Howe, W. J.; Moon, J. B.; Hui, J. O.; Tomasselli, A. G.; Heinrikson, R. L.; Thaisrivongs, S.; Wlodawer, A.

    1992-01-01

    The structure of a crystal complex of recombinant human immunodeficiency virus type 1 (HIV-1) protease with a peptide-mimetic inhibitor containing a dihydroxyethylene isostere insert replacing the scissile bond has been determined. The inhibitor is Noa-His-Hch psi [CH(OH)CH(OH)]Vam-Ile-Amp (U-75875), and its Ki for inhibition of the HIV-1 protease is < 1.0 nM (Noa = 1-naphthoxyacetyl, Hch = a hydroxy-modified form of cyclohexylalanine, Vam = a hydroxy-modified form of valine, Amp = 2-pyridylmethylamine). The structure of the complex has been refined to a crystallographic R factor of 0.169 at 2.0 A resolution by using restrained least-squares procedures. Root mean square deviations from ideality are 0.02 A and 2.4 degrees, for bond lengths and angles, respectively. The bound inhibitor diastereomer has the R configurations at both of the hydroxyl chiral carbon atoms. One of the diol hydroxyl groups is positioned such that it forms hydrogen bonds with both the active site aspartates, whereas the other interacts with only one of them. Comparison of this X-ray structure with a model-built structure of the inhibitor, published earlier, reveals similar positioning of the backbone atoms and of the side-chain atoms in the P2-P2' region, where the interaction with the protein is strongest. However, the X-ray structure and the model differ considerably in the location of the P3 and P3' end groups, and also in the positioning of the second of the two central hydroxyl groups. Reconstruction of the central portion of the model revealed the source of the hydroxyl discrepancy, which, when corrected, provided a P1-P1' geometry very close to that seen in the X-ray structure. PMID:1304383

  18. Characterization of Bactrocera dorsalis Serine Proteases and Evidence for Their Indirect Role in Insecticide Tolerance

    PubMed Central

    Hou, Ming-Zhe; Shen, Guang-Mao; Wei, Dong; Li, Ya-Li; Dou, Wei; Wang, Jin-Jun

    2014-01-01

    The oriental fruit fly Bactrocera dorsalis (Hendel) causes devastating losses to agricultural crops world-wide and is considered to be an economically important pest. Little is known about the digestive enzymes such as serine proteases (SPs) in B. dorsalis, which are important both for energy supply and mitigation of fitness cost associated with insecticide tolerance. In this study, we identified five SP genes in the midgut of B. dorsalis, and the alignments of their deduced amino acid sequences revealed the presence of motifs conserved in the SP superfamily. Phylogenetic analyses with known SPs from other insect species suggested that three of them were trypsin-like proteases. Analyses of the expression profiles among the different developmental stages showed that all five genes were most abundant in larvae than in other stages. When larvae were continuously fed on diet containing 0.33 μg/g β-Cypermethrin, expression of all five genes were upregulated in the midgut but the larval development was delayed. Biochemical assays were consistent with the increased protease activity exhibited by SPs in the midgut after treatment with β-Cypermethrin. Taken together, these findings provide evidence for the hypothesis that enhanced SP activity may play an indirect role in relieving the toxicity stress of insecticide in B. dorsalis. PMID:24566149

  19. A novel serine protease from strawberry (Fragaria ananassa): Purification and biochemical characterization.

    PubMed

    Alici, Esma Hande; Arabaci, Gulnur

    2018-03-27

    In this study, a protease enzyme was purified from strawberry by using Sepharose-4B-l-tyrosine-p-amino benzoic acid affinity chromatography. The molecular weight of pure protease was determined 65.8 kDa by SDS-PAGE. The single band observed on the gel showed that the enzyme had a single polypeptide chain and was successfully purified. Purification of the protease by the chromatographic method resulted in a 395.6-fold increase in specific activity (3600 U/mg). Optimum pH and temperature for the enzyme were 6 and 40 °C, respectively. The protease was stable at a wide temperature range of 40 to 70 °C and a pH range of 3.0 to 9.0. Co 2+ ions stimulated protease activity very strongly. Cu 2+ , Hg 2+ , Cd 2+ and Mn 2+ ions significantly inhibited protease activity. While 2-propanol completely inhibited the enzyme, the enzyme maintained its activity better in the presence of ethanol and methanol. The strawberry protease showed the highest specificity towards hemoglobin among all the natural substrates tested. The specificity of the enzyme towards synthetic substrates was also investigated and it was concluded that it has broad substrate specificity. The obtained results indicated that this purified protease was highly-likely a serine protease and its activity was significantly affected by the presence of metal ions. Copyright © 2018. Published by Elsevier B.V.

  20. kdgREcc Negatively Regulates Genes for Pectinases, Cellulase, Protease, HarpinEcc, and a Global RNA Regulator in Erwinia carotovora subsp. carotovora†

    PubMed Central

    Liu, Yang; Jiang, Guoqiao; Cui, Yaya; Mukherjee, Asita; Ma, Wei Lei; Chatterjee, Arun K.

    1999-01-01

    Erwinia carotovora subsp. carotovora produces extracellular pectate lyase (Pel), polygalacturonase (Peh), cellulase (Cel), and protease (Prt). The concerted actions of these enzymes largely determine the virulence of this plant-pathogenic bacterium. E. carotovora subsp. carotovora also produces HarpinEcc, the elicitor of the hypersensitive reaction. We document here that KdgREcc (Kdg, 2-keto-3-deoxygluconate; KdgR, general repressor of genes involved in pectin and galacturonate catabolism), a homolog of the E. chrysanthemi repressor, KdgREch and the Escherichia coli repressor, KdgREco, negatively controls not only the pectinases, Pel and Peh, but also Cel, Prt, and HarpinEcc production in E. carotovora subsp. carotovora. The levels of pel-1, peh-1, celV, and hrpNEcc transcripts are markedly affected by KdgREcc. The KdgREcc− mutant is more virulent than the KdgREcc+ parent. Thus, our data for the first time establish a global regulatory role for KdgREcc in E. carotovora subsp. carotovora. Another novel observation is the negative effect of KdgREcc on the transcription of rsmB (previously aepH), which specifies an RNA regulator controlling exoenzyme and HarpinEcc production. The levels of rsmB RNA are higher in the KdgREcc− mutant than in the KdgREcc+ parent. Moreover, by DNase I protection assays we determined that purified KdgREcc protected three 25-bp regions within the transcriptional unit of rsmB. Alignment of the protected sequences revealed the 21-mer consensus sequence of the KdgREcc-binding site as 5′-G/AA/TA/TGAAA[N6]TTTCAG/TG/TA-3′. Two such KdgREcc-binding sites occur in rsmB DNA in a close proximity to each other within nucleotides +79 and +139 and the third KdgREcc-binding site within nucleotides +207 and +231. Analysis of lacZ transcriptional fusions shows that the KdgR-binding sites negatively affect the expression of rsmB. KdgREcc also binds the operator DNAs of pel-1 and peh-1 genes and represses expression of a pel1-lacZ and a peh1-lac

  1. Substantial decrease in cell wall α-1,3-glucan caused by disruption of the kexB gene encoding a subtilisin-like processing protease in Aspergillus oryzae.

    PubMed

    Mizutani, Osamu; Shiina, Matsuko; Yoshimi, Akira; Sano, Motoaki; Watanabe, Takeshi; Yamagata, Youhei; Nakajima, Tasuku; Gomi, Katsuya; Abe, Keietsu

    2016-09-01

    Disruption of the kexB encoding a subtilisin-like processing protease in Aspergillus oryzae (ΔkexB) leads to substantial morphological defects when the cells are grown on Czapek-Dox agar plates. We previously found that the disruption of kexB causes a constitutive activation of the cell wall integrity pathway. To understand how the disruption of the kexB affects cell wall organization and components, we analyzed the cell wall of ΔkexB grown on the plates. The results revealed that both total N-acetylglucosamine content, which constitutes chitin, and chitin synthase activities were increased. Whereas total glucose content, which constitutes β-1,3-glucan and α-1,3-glucan, was decreased; this decrease was attributed to a remarkable decrease in α-1,3-glucan. Additionally, the β-1,3-glucan in the alkali-insoluble fraction of the ΔkexB showed a high degree of polymerization. These results suggested that the loss of α-1,3-glucan in the ΔkexB was compensated by increases in the chitin content and the average degree of β-1,3-glucan polymerization.

  2. Thermostable Acid Protease Produced by Penicillium duponti K1014, a True Thermophilic Fungus Newly Isolated from Compost

    PubMed Central

    Hashimoto, Hikotaka; Iwaasa, Takashi; Yokotsuka, Tamotsu

    1972-01-01

    A thermophilic fungus, K1014, newly derived from a compost was selected on the basis of protease productivity as the only one of 81 isolates to produce high levels of acid protease. The fungus was named Penicillium duponti K1014 based on taxonomical studies. It grew in the temperature range of 28 to 58 C, and the optimum was 45 to 50 C. These temperature characteristics showed that the fungus was the most strongly thermophilic of all the fungi next to Humicola lanuginosa. When P. duponti K1014 was grown on moistened wheat bran, maximal accumulation of acid protease occurred after 2 days at 45 to 50 C. The addition of ammonium salts, but not nitrate, was effective for the production of the acid protease. The acid protease of P. duponti K1014 was stable at 60 C for 1 hr and retained more than 65% of original activity after the treatment for 1 hr at 70 C at pH 4.7. This thermal property was different from those of the ordinary acid proteases, indicating that the enzyme is a thermostable protein. Images PMID:4650601

  3. Biocontrol activity of an alkaline serine protease from Aureobasidium pullulans expressed in Pichia pastoris against four postharvest pathogens on apple.

    PubMed

    Banani, Houda; Spadaro, Davide; Zhang, Dianpeng; Matic, Slavica; Garibaldi, Angelo; Gullino, Maria Lodovica

    2014-07-16

    The yeast-like fungus Aureobasidium pullulans PL5 is a microbial antagonist against postharvest pathogens of fruits. The strain is able to produce hydrolases, including glucanases, chitinases and proteases. The alkaline serine protease gene ALP5 from A. pullulans was cloned, inserted into the vector pPIC9 to construct pPIC9/ALP5, and then expressed in Pichia pastoris strain KM71. ALP5 had a molecular mass of 42.9kDa after 5days growth with 1% methanol induction at 28°C. The recombinant protease expressed in P. pastoris showed its highest activity under alkaline conditions (at pH10) and a temperature of 50°C. The antifungal activity of the recombinant protease was investigated against Penicillium expansum, Botrytis cinerea, Monilinia fructicola and Alternaria alternata in vitro and on apple. The recombinant protease reduced significantly the spore germination and the germ tube length of the tested pathogens in PDB medium. The highest level of protease efficacy was observed against M. fructicola and B. cinerea, whereas a lower efficacy was observed against P. expansum and A. alternata indicating a possible effect of the pathogen cell wall composition on the proteolytic activity of the recombinant protease. The presence of protease was able to cause the swelling of the hyphae of B. cinerea, under an optical microscope. The recombinant protease expressed in P. pastoris was more active against the pathogens in vitro than the same enzyme expressed in E. coli in previous studies. The efficacy of ALP5 was also evaluated against the pathogens in vivo on cv Golden Delicious apples. The protease was more efficient in controlling M. fructicola, B. cinerea and P. expansum than A. alternata. However, the extent of the activity was dependent on the enzyme concentration and the length of fruit storage. This study demonstrated the capacity of the alkaline serine protease to keep its enzymatic activity for some days in the unfavorable environment of the fruit wounds. The alkaline

  4. Significance of Cuscutain, a cysteine protease from Cuscuta reflexa, in host-parasite interactions.

    PubMed

    Bleischwitz, Marc; Albert, Markus; Fuchsbauer, Hans-Lothar; Kaldenhoff, Ralf

    2010-10-22

    Plant infestation with parasitic weeds like Cuscuta reflexa induces morphological as well as biochemical changes in the host and the parasite. These modifications could be caused by a change in protein or gene activity. Using a comparative macroarray approach Cuscuta genes specifically upregulated at the host attachment site were identified. One of the infestation specific Cuscuta genes encodes a cysteine protease. The protein and its intrinsic inhibitory peptide were heterologously expressed, purified and biochemically characterized. The haustoria specific enzyme was named cuscutain in accordance with similar proteins from other plants, e.g. papaya. The role of cuscutain and its inhibitor during the host parasite interaction was studied by external application of an inhibitor suspension, which induced a significant reduction of successful infection events. The study provides new information about molecular events during the parasitic plant--host interaction. Inhibition of cuscutain cysteine proteinase could provide means for antagonizing parasitic plants.

  5. Metabolic effects of the HIV protease inhibitor--saquinavir in differentiating human preadipocytes.

    PubMed

    Bociąga-Jasik, Monika; Polus, Anna; Góralska, Joanna; Czech, Urszula; Gruca, Anna; Śliwa, Agnieszka; Garlicki, Aleksander; Mach, Tomasz; Dembińska-Kieć, Aldona

    2013-01-01

    The iatrogenic, HIV-related lipodystrophy is associated with development of the significant metabolic and cardiovascular complications. The underlying mechanisms of antiretroviral (ARV) drugs are not completely explored. The aim of the study was to characterize effects of the protease inhibitor (PI)--saquinavir (SQV) on metabolic functions, and gene expression during differentiation in cells (Chub-S7) culture. SQV in concentrations observed during antiretroviral therapy (ART) significantly decreased mitochondrial membrane potential (MMP), oxygen consumption and ATP generation. The effects were greater in already differentiated cells. This was accompanied by characteristic changes in the expression of the genes involved in endoplasmic reticulum (ER) stress, and differentiation (lipid droplet formation) process such as: WNT10a, C/EBPa, AFT4, CIDEC, ADIPOQ, LPIN1. The results indicate that SQV affects not only metabolic (mitochondrial) activity of adipocytes, but affects the expression of genes related to differentiation and to a lesser extent to cell apoptosis.

  6. [Fish ovarian fluid contains protease inhibitors].

    PubMed

    Minin, A A; Ozerova, S G

    2015-01-01

    Studies of the conditions under which fish egg is activated spontaneously without the sperm showed that the egg retains the ability for fertilization in the ovarian (coelomic) fluid, which surrounds it in the gonad cavity after ovulation. Earlier, we showed that, in artificial media, the spontaneous activation is suppressed by protease inhibitors. In this study, we investigated the presence of natural protease inhibitors in the ovarian fluid and showed that the ovarian fluid of zebrafish and loach contains protease inhibitors, in particular, type I serpin a, a protein inhibitor of trypsin proteases.

  7. Electron cryomicroscopy structure of a membrane-anchored mitochondrial AAA protease.

    PubMed

    Lee, Sukyeong; Augustin, Steffen; Tatsuta, Takashi; Gerdes, Florian; Langer, Thomas; Tsai, Francis T F

    2011-02-11

    FtsH-related AAA proteases are conserved membrane-anchored, ATP-dependent molecular machines, which mediate the processing and turnover of soluble and membrane-embedded proteins in eubacteria, mitochondria, and chloroplasts. Homo- and hetero-oligomeric proteolytic complexes exist, which are composed of homologous subunits harboring an ATPase domain of the AAA family and an H41 metallopeptidase domain. Mutations in subunits of mitochondrial m-AAA proteases have been associated with different neurodegenerative disorders in human, raising questions on the functional differences between homo- and hetero-oligomeric AAA proteases. Here, we have analyzed the hetero-oligomeric yeast m-AAA protease composed of homologous Yta10 and Yta12 subunits. We combined genetic and structural approaches to define the molecular determinants for oligomer assembly and to assess functional similarities between Yta10 and Yta12. We demonstrate that replacement of only two amino acid residues within the metallopeptidase domain of Yta12 allows its assembly into homo-oligomeric complexes. To provide a molecular explanation, we determined the 12 Å resolution structure of the intact yeast m-AAA protease with its transmembrane domains by electron cryomicroscopy (cryo-EM) and atomic structure fitting. The full-length m-AAA protease has a bipartite structure and is a hexamer in solution. We found that residues in Yta12, which facilitate homo-oligomerization when mutated, are located at the interface between neighboring protomers in the hexamer ring. Notably, the transmembrane and intermembrane space domains are separated from the main body, creating a passage on the matrix side, which is wide enough to accommodate unfolded but not folded polypeptides. These results suggest a mechanism regarding how proteins are recognized and degraded by m-AAA proteases.

  8. Proteases and the gut barrier.

    PubMed

    Biancheri, Paolo; Di Sabatino, Antonio; Corazza, Gino R; MacDonald, Thomas T

    2013-02-01

    Serine proteases, cysteine proteases, aspartic proteases and matrix metalloproteinases play an essential role in extracellular matrix remodeling and turnover through their proteolytic action on collagens, proteoglycans, fibronectin, elastin and laminin. Proteases can also act on chemokines, receptors and anti-microbial peptides, often potentiating their activity. The intestinal mucosa is the largest interface between the external environment and the tissues of the human body and is constantly exposed to proteolytic enzymes from many sources, including bacteria in the intestinal lumen, fibroblasts and immune cells in the lamina propria and enterocytes. Controlled proteolytic activity is crucial for the maintenance of gut immune homeostasis, for normal tissue turnover and for the integrity of the gut barrier. However, in intestinal immune-mediated disorders, pro-inflammatory cytokines induce the up-regulation of proteases, which become the end-stage effectors of mucosal damage by destroying the epithelium and basement membrane integrity and degrading the extracellular matrix of the lamina propria to produce ulcers. Protease-mediated barrier disruption in turn results in increased amounts of antigen crossing into the lamina propria, driving further immune responses and sustaining the inflammatory process.

  9. Hybrid male sterility and genome-wide misexpression of male reproductive proteases.

    PubMed

    Gomes, Suzanne; Civetta, Alberto

    2015-07-06

    Hybrid male sterility is a common barrier to gene flow between species. Previous studies have posited a link between misregulation of spermatogenesis genes in interspecies hybrids and sterility. However, in the absence of fully fertile control hybrids, it is impossible to differentiate between misregulation associated with sterility vs. fast male gene regulatory evolution. Here, we differentiate between these two possibilities using a D. pseudoobscura species pair that experiences unidirectional hybrid sterility. We identify genes uniquely misexpressed in sterile hybrid male reproductive tracts via RNA-seq. The sterile male hybrids had more misregulated and more over or under expressed genes relative to parental species than the fertile male hybrids. Proteases were the only gene ontology class overrepresented among uniquely misexpressed genes, with four located within a previously identified hybrid male sterility locus. This result highlights the potential role of a previously unexplored class of genes in interspecific hybrid male sterility and speciation.

  10. Ovarian Tumor (OTU)-domain Containing Viral Proteases Evade Ubiquitin- and ISG15-dependent Innate Immune Responses

    PubMed Central

    Frias-Staheli, Natalia; Giannakopoulos, Nadia V.; Kikkert, Marjolein; Taylor, Shannon L.; Bridgen, Anne; Paragas, Jason J.; Richt, Juergen A.; Rowland, Raymond R.; Schmaljohn, Connie S.; Lenschow, Deborah J.; Snijder, Eric J.; García-Sastre, Adolfo; Virgin, Herbert Whiting

    2007-01-01

    Summary Ubiquitin (Ub) and interferon stimulated gene product 15 (ISG15) reversibly conjugate to proteins via a conserved LRLRGG C-terminal motif, mediating important innate antiviral responses. The ovarian tumor (OTU) domain represents a superfamily of predicted proteases found in eukaryotic, bacterial and viral proteins, some of which have Ub-deconjugating activity. We show that the OTU domain-containing proteases of nairoviruses and arteriviruses hydrolyze Ub and ISG15 from cellular target proteins. This broad activity contrasts with the target specificity of known mammalian OTU domain-containing proteins. The biological significance of this activity of viral OTU domain-containing proteases was evidenced by their capacity to inhibit NF-κB dependent signaling and to antagonize the antiviral effects of ISG15 during Sindbis virus infection in vivo. The deconjugating activity of viral OTU proteases represents a novel viral immune evasion mechanism that inhibits Ub-and ISG15-dependent antiviral pathways. PMID:18078692

  11. Production, purification and characterization of an aspartic protease from Aspergillus foetidus.

    PubMed

    Souza, Paula Monteiro; Werneck, Gabriela; Aliakbarian, Bahar; Siqueira, Felix; Ferreira Filho, Edivaldo Ximenes; Perego, Patrizia; Converti, Attilio; Magalhães, Pérola Oliveira; Junior, Adalberto Pessoa

    2017-11-01

    An acidic thermostable protease was extracellularly produced either in shake flask or in stirred tank bioreactor by an Aspergillus foetidus strain isolated from the Brazilian savanna soil using different nitrogen sources. Its maximum activity (63.7 U mL -1 ) was obtained in a medium containing 2% (w/v) peptone. A cultivation carried out in a 5.0 L stirred-tank bioreactor provided a maximum protease activity 9% lower than that observed in Erlenmeyer flasks, which was obtained after a significantly shorter (by 16-29%) time. Protease purification by a combination of gel-filtration chromatography resulted in a 16.9-fold increase in specific activity (248.1 U g -1 ). The estimated molecular weight of the purified enzyme was 50.6 kDa, and the optimal pH and temperature were 5.0 and 55 °C, respectively. The enzyme was completely inhibited by pepstatin A, and its activity enhanced by some metals. According to the inhibition profiles, it was confirmed that the purified acid protease belongs to the aspartic protease type. These results are quite promising for future development of large-scale production of such protease, which can be useful in biotechnological applications requiring high enzyme activity and stability under acidic conditions. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Broad spectrum activity of a lectin-like bacterial serine protease family on human leukocytes.

    PubMed

    Ayala-Lujan, Jorge Luis; Vijayakumar, Vidhya; Gong, Mei; Smith, Rachel; Santiago, Araceli E; Ruiz-Perez, Fernando

    2014-01-01

    The serine protease autotransporter from Enterobacteriaceae (SPATE) family, which number more than 25 proteases with apparent diverse functions, have been phylogenetically divided into two distinct classes, designated 1 and 2. We recently demonstrated that Pic and Tsh, two members of the class-2 SPATE family produced by intestinal and extraintestinal pathogenic E. coli, were able to cleave a number of O-glycosylated proteins on neutrophils and lymphocytes resulting in impaired leukocyte functions. Here we show that most members of the class-2 SPATE family have lectin-like properties and exhibit differential protease activity reliant on glycoprotein type and cell lineage. Protease activity was seen in virtually all tested O-glycosylated proteins including CD34, CD55, CD164, TIM1, TIM3, TIM4 and C1-INH. We also show that although SPATE proteins bound and cleaved glycoproteins more efficiently on granulocytes and monocytes, they also targeted glycoproteins on B, T and natural killer lymphocytes. Finally, we found that the characteristic domain-2 of class-2 SPATEs is not required for glycoprotease activity, but single amino acid mutations in Pic domain-1 to those residues naturally occurring in domain-1 of SepA, were sufficient to hamper Pic glycoprotease activity. This study shows that most class-2 SPATEs have redundant activities and suggest that they may function as immunomodulators at several levels of the immune system.

  13. Broad Spectrum Activity of a Lectin-Like Bacterial Serine Protease Family on Human Leukocytes

    PubMed Central

    Ayala-Lujan, Jorge Luis; Vijayakumar, Vidhya; Gong, Mei; Smith, Rachel; Santiago, Araceli E.; Ruiz-Perez, Fernando

    2014-01-01

    The serine protease autotransporter from Enterobacteriaceae (SPATE) family, which number more than 25 proteases with apparent diverse functions, have been phylogenetically divided into two distinct classes, designated 1 and 2. We recently demonstrated that Pic and Tsh, two members of the class-2 SPATE family produced by intestinal and extraintestinal pathogenic E. coli, were able to cleave a number of O-glycosylated proteins on neutrophils and lymphocytes resulting in impaired leukocyte functions. Here we show that most members of the class-2 SPATE family have lectin-like properties and exhibit differential protease activity reliant on glycoprotein type and cell lineage. Protease activity was seen in virtually all tested O-glycosylated proteins including CD34, CD55, CD164, TIM1, TIM3, TIM4 and C1-INH. We also show that although SPATE proteins bound and cleaved glycoproteins more efficiently on granulocytes and monocytes, they also targeted glycoproteins on B, T and natural killer lymphocytes. Finally, we found that the characteristic domain-2 of class-2 SPATEs is not required for glycoprotease activity, but single amino acid mutations in Pic domain-1 to those residues naturally occurring in domain-1 of SepA, were sufficient to hamper Pic glycoprotease activity. This study shows that most class-2 SPATEs have redundant activities and suggest that they may function as immunomodulators at several levels of the immune system. PMID:25251283

  14. Neural ECM proteases in learning and synaptic plasticity.

    PubMed

    Tsilibary, Effie; Tzinia, Athina; Radenovic, Lidija; Stamenkovic, Vera; Lebitko, Tomasz; Mucha, Mariusz; Pawlak, Robert; Frischknecht, Renato; Kaczmarek, Leszek

    2014-01-01

    Recent studies implicate extracellular proteases in synaptic plasticity, learning, and memory. The data are especially strong for such serine proteases as thrombin, tissue plasminogen activator, neurotrypsin, and neuropsin as well as matrix metalloproteinases, MMP-9 in particular. The role of those enzymes in the aforementioned phenomena is supported by the experimental results on the expression patterns (at the gene expression and protein and enzymatic activity levels) and functional studies, including knockout mice, specific inhibitors, etc. Counterintuitively, the studies have shown that the extracellular proteolysis is not responsible mainly for an overall degradation of the extracellular matrix (ECM) and loosening perisynaptic structures, but rather allows for releasing signaling molecules from the ECM, transsynaptic proteins, and latent form of growth factors. Notably, there are also indications implying those enzymes in the major neuropsychiatric disorders, probably by contributing to synaptic aberrations underlying such diseases as schizophrenia, bipolar, autism spectrum disorders, and drug addiction.

  15. Production of Alkaline Protease by Solvent-Tolerant Alkaliphilic Bacillus circulans MTCC 7942 Isolated from Hydrocarbon Contaminated Habitat: Process Parameters Optimization

    PubMed Central

    Patil, Ulhas; Chaudhari, Ambalal

    2013-01-01

    In the present investigation, a newly isolated organic solvent-tolerant and alkaliphilic bacterial strain was reported from a hydrocarbon (gasoline and diesel) contaminated soil collected from the petrol station, Shirpur (India). The strain was identified as Bacillus circulans MTCC 7942, based on phenotype, biochemical, and phylogenetic analysis of 16S rRNA gene sequence. The capability of Bacillus circulans to secrete an extracellular, thermostable, alkaline protease and grow in the presence of organic solvents was explored. Bacillus circulans produced maximum alkaline protease (412 U/mL) in optimized medium (g/L): soybean meal, 15; starch, 10; KH2PO4, 1; MgSO4·7H2O, 0.05; CaCl2, 1; Na2CO3, 8; pH 10.0 at 37°C and 100 rpm. The competence of strain to grow in various organic solvents—n-octane, dodecane, n-decane, N,N-dimethylformamide, n-hexane, and dimethyl sulfoxide, establishes its potential as solvent-stable protease source for the possible applications in nonaqueous reactions and fine chemical synthesis. PMID:25937965

  16. An oxidant- and solvent-stable protease produced by Bacillus cereus SV1: application in the deproteinization of shrimp wastes and as a laundry detergent additive.

    PubMed

    Manni, Laila; Jellouli, Kemel; Ghorbel-Bellaaj, Olfa; Agrebi, Rym; Haddar, Anissa; Sellami-Kamoun, Alya; Nasri, Moncef

    2010-04-01

    The current increase in amount of shrimp wastes produced by the shrimp industry has led to the need in finding new methods for shrimp wastes disposal. In this study, an extracellular organic solvent- and oxidant-stable metalloprotease was produced by Bacillus cereus SV1. Maximum protease activity (5,900 U/mL) was obtained when the strain was grown in medium containing 40 g/L shrimp wastes powder as a sole carbon source. The optimum pH, optimum temperature, pH stability, and thermal stability of the crude enzyme preparation were pH 8.0, 60 degrees C, pH 6-9.5, and <55 degrees C, respectively. The crude protease was extremely stable toward several organic solvents. No loss of activity was observed even after 60 days of incubation at 30 degrees C in the presence of 50% (v/v) dimethyl sulfoxide and ethyl ether; the enzyme retained more than 70% of its original activity in the presence of ethanol and N,N-dimethylformamide. The protease showed high stability toward anionic (SDS) and non-ionic (Tween 80, Tween 20, and Triton X-100) surfactants. Interestingly, the activity of the enzyme was significantly enhanced by oxidizing agents. In addition, the enzyme showed excellent compatibility with some commercial liquid detergents. The protease of B. cereus SV1, produced under the optimal culture conditions, was tested for shrimp waste deproteinization in the preparation of chitin. The protein removal with a ratio E/S of 20 was about 88%. The novelties of the SV1 protease include its high stability to organic solvents and surfactants. These unique properties make it an ideal choice for application in detergent formulations and enzymatic peptide synthesis. In addition, the enzyme may find potential applications in the deproteinization of shrimp wastes to produce chitin.

  17. Cowpea bruchid Callosobruchus maculatus counteracts dietary protease inhibitors by modulating propeptides of major digestive enzymes.

    PubMed

    Ahn, J-E; Lovingshimer, M R; Salzman, R A; Presnail, J K; Lu, A L; Koiwa, H; Zhu-Salzman, K

    2007-06-01

    Cowpea bruchids, when challenged by consumption of the soybean cysteine protease inhibitor scN, reconfigure expression of their major CmCP digestive proteases and resume normal feeding and development. Previous evidence indicated that insects selectively induced CmCPs from subfamily B, that were more efficient in autoprocessing and possessed not only higher proteolytic, but also scN-degrading activities. In contrast, dietary scN only marginally up-regulated genes from the more predominant CmCP subfamily A that were inferior to subfamily B. To gain further molecular insight into this adaptive adjustment, we performed domain swapping between the two respective subfamily members B1 and A16, the latter unable to autoprocess or degrade scN even after intermolecular processing. Swapping the propeptides did not qualitatively alter autoprocessing in either protease isoform. Incorporation of either the N- (pAmBA) or C-terminal (pAmAB) mature B1 segment into A16, however, was sufficient to prime autoprocessing of A16 to its mature form. Further, the swap at the N-terminal mature A16 protein region (pAmBA) resulted in four amino acid changes. Replacement of these amino acid residues by the corresponding B1 residues, singly and pair-wise, revealed that autoprocessing activation in pAmBA resulted from cumulative and/or coordinated individual effects. Bacterially expressed isolated propeptides (pA16 and pB1) differed in their ability to inhibit mature B1 enzyme. Lower inhibitory activity in pB1 is likely attributable to its lack of protein stability. This instability in the cleaved propeptide is necessary, although insufficient by itself, for scN-degradation by the mature B1 enzyme. Taken together, cowpea bruchids modulate proteolysis of their digestive enzymes by controlling proCmCP cleavage and propeptide stability, which explains at least in part the plasticity cowpea bruchids demonstrate in response to protease inhibitors.

  18. Cathepsin K in Lymphangioleiomyomatosis: LAM Cell-Fibroblast Interactions Enhance Protease Activity by Extracellular Acidification.

    PubMed

    Dongre, Arundhati; Clements, Debbie; Fisher, Andrew J; Johnson, Simon R

    2017-08-01

    Lymphangioleiomyomatosis (LAM) is a rare disease in which LAM cells and fibroblasts form lung nodules and it is hypothesized that LAM nodule-derived proteases cause cyst formation and tissue damage. On protease gene expression profiling in whole lung tissue, cathepsin K gene expression was 40-fold overexpressed in LAM compared with control lung tissue (P ≤ 0.0001). Immunohistochemistry confirmed cathepsin K protein was expressed in LAM but not control lungs. Cathepsin K gene expression and protein and protease activity were detected in LAM-associated fibroblasts but not the LAM cell line 621-101. In lung nodules, cathepsin K immunoreactivity predominantly co-localized with LAM-associated fibroblasts. In vitro, fibroblast extracellular cathepsin K activity was minimal at pH 7.5 but significantly enhanced at pH 7 and 6. 621-101 cells reduced extracellular pH with acidification dependent on 621-101 mechanistic target of rapamycin activity and net hydrogen ion exporters, particularly sodium bicarbonate co-transporters and carbonic anhydrases, which were also expressed in LAM lung tissue. In LAM cell-fibroblast co-cultures, acidification paralleled cathepsin K activity, and both were reduced by sodium bicarbonate co-transporter (P ≤ 0.0001) and carbonic anhydrase inhibitors (P = 0.0021). Our findings suggest that cathepsin K activity is dependent on LAM cell-fibroblast interactions, and inhibitors of extracellular acidification may be potential therapies for LAM. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  19. The novel monoclonal antibody 9F5 reveals expression of a fragment of GPNMB/osteoactivin processed by furin‐like protease(s) in a subpopulation of microglia in neonatal rat brain

    PubMed Central

    Hirata, Hiroshi; Ohbuchi, Kengo; Nishi, Kentaro; Maeda, Akira; Kuniyasu, Akihiko; Yamada, Daisuke; Maeda, Takehiko; Tsuji, Akihiko; Sawada, Makoto

    2016-01-01

    To differentiate subtypes of microglia (MG), we developed a novel monoclonal antibody, 9F5, against one subtype (type 1) of rat primary MG. The 9F5 showed high selectivity for this cell type in Western blot and immunocytochemical analyses and no cross‐reaction with rat peritoneal macrophages (Mφ). We identified the antigen molecule for 9F5: the 50‐ to 70‐kDa fragments of rat glycoprotein nonmetastatic melanoma protein B (GPNMB)/osteoactivin, which started at Lys170. In addition, 9F5 immunoreactivity with GPNMB depended on the activity of furin‐like protease(s). More important, rat type 1 MG expressed the GPNMB fragments, but type 2 MG and Mφ did not, although all these cells expressed mRNA and the full‐length protein for GPNMB. These results suggest that 9F5 reactivity with MG depends greatly on cleavage of GPNMB and that type 1 MG, in contrast to type 2 MG and Mφ, may have furin‐like protease(s) for GPNMB cleavage. In neonatal rat brain, amoeboid 9F5+ MG were observed in specific brain areas including forebrain subventricular zone, corpus callosum, and retina. Double‐immunοstaining with 9F5 antibody and anti‐Iba1 antibody, which reacts with MG throughout the CNS, revealed that 9F5+ MG were a portion of Iba1+ MG, suggesting that MG subtype(s) exist in vivo. We propose that 9F5 is a useful tool to discriminate between rat type 1 MG and other subtypes of MG/Mφ and to reveal the role of the GPNMB fragments during developing brain. GLIA 2016;64:1938–1961 PMID:27464357

  20. Optimum production and characterization of an acid protease from marine yeast Metschnikowia reukaufii W6b

    NASA Astrophysics Data System (ADS)

    Li, Jing; Peng, Ying; Wang, Xianghong; Chi, Zhenming

    2010-12-01

    The marine yeast strain W6b isolated from sediment of the South China Sea was found to produce a cell-bound acid protease. The crude acid protease produced by this marine yeast showed the highest activity at pH 3.5 and 40 °C. The optimal pH and temperature for the crude acid protease were in agreement with those for acid protease produced by the terrestrial yeasts. The optimal medium of the acid protease production was seawater containing 1.0% glucose, 1.5% casein, and 0.5% yeast extract, and the optimal cultivation conditions of the acid protease production were pH 4.0, a temperature of 25 °C and a shaking speed of 140 rmin-1. Under the optimal conditions, 72.5 UmL-1 of acid protease activity could be obtained in cell suspension within 48 h of fermentation at shake flask level. The acid protease production was induced by high-molecular-weight nitrogen sources and repressed by low-molecular-weight nitrogen sources. Skimmed-milk-clotting test showed that the crude acid protease from the cell suspension of the yeast W6b had high skimmed milk coagulability. The acid protease produced by M. reukaufii W6b may have highly potential applications in cheese, food and fermentation industries.

  1. Protease inhibitor in scorpion (Mesobuthus eupeus) venom prolongs the biological activities of the crude venom.

    PubMed

    Ma, Hakim; Xiao-Peng, Tang; Yang, Shi-Long; Lu, Qiu-Min; Lai, Ren

    2016-08-01

    It is hypothesized that protease inhibitors play an essential role in survival of venomous animals through protecting peptide/protein toxins from degradation by proteases in their prey or predators. However, the biological function of protease inhibitors in scorpion venoms remains unknown. In the present study, a trypsin inhibitor was purified and characterized from the venom of scorpion Mesobuthus eupeus, which enhanced the biological activities of crude venom components in mice when injected in combination with crude venom. This protease inhibitor, named MeKTT-1, belonged to Kunitz-type toxins subfamily. Native MeKTT-1 selectively inhibited trypsin with a Kivalue of 130 nmol·L(-1). Furthermore, MeKTT-1 was shown to be a thermo-stable peptide. In animal behavioral tests, MeKTT-1 prolonged the pain behavior induced by scorpion crude venom, suggesting that protease inhibitors in scorpion venom inhibited proteases and protect the functionally important peptide/protein toxins from degradation, consequently keeping them active longer. In conclusion, this was the first experimental evidence about the natural existence of serine protease inhibitor in the venom of scorpion Mesobuthus eupeus, which preserved the activity of venom components, suggests that scorpions may use protease inhibitors for survival. Copyright © 2016 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

  2. Ubiquitin-Specific Protease 2 Regulates Hepatic Gluconeogenesis and Diurnal Glucose Metabolism Through 11β-Hydroxysteroid Dehydrogenase 1

    PubMed Central

    Molusky, Matthew M.; Li, Siming; Ma, Di; Yu, Lei; Lin, Jiandie D.

    2012-01-01

    Hepatic gluconeogenesis is important for maintaining steady blood glucose levels during starvation and through light/dark cycles. The regulatory network that transduces hormonal and circadian signals serves to integrate these physiological cues and adjust glucose synthesis and secretion by the liver. In this study, we identified ubiquitin-specific protease 2 (USP2) as an inducible regulator of hepatic gluconeogenesis that responds to nutritional status and clock. Adenoviral-mediated expression of USP2 in the liver promotes hepatic glucose production and exacerbates glucose intolerance in diet-induced obese mice. In contrast, in vivo RNA interference (RNAi) knockdown of this factor improves systemic glycemic control. USP2 is a target gene of peroxisome proliferator–activated receptor γ coactivator-1α (PGC-1α), a coactivator that integrates clock and energy metabolism, and is required for maintaining diurnal glucose homeostasis during restricted feeding. At the mechanistic level, USP2 regulates hepatic glucose metabolism through its induction of 11β-hydroxysteroid dehydrogenase 1 (HSD1) and glucocorticoid signaling in the liver. Pharmacological inhibition and liver-specific RNAi knockdown of HSD1 significantly impair the stimulation of hepatic gluconeogenesis by USP2. Together, these studies delineate a novel pathway that links hormonal and circadian signals to gluconeogenesis and glucose homeostasis. PMID:22447855

  3. CYP1A1, GCLC, AGT, AGTR1 gene-gene interactions in community-acquired pneumonia pulmonary complications.

    PubMed

    Salnikova, Lyubov E; Smelaya, Tamara V; Golubev, Arkadiy M; Rubanovich, Alexander V; Moroz, Viktor V

    2013-11-01

    This study was conducted to establish the possible contribution of functional gene polymorphisms in detoxification/oxidative stress and vascular remodeling pathways to community-acquired pneumonia (CAP) susceptibility in the case-control study (350 CAP patients, 432 control subjects) and to predisposition to the development of CAP complications in the prospective study. All subjects were genotyped for 16 polymorphic variants in the 14 genes of xenobiotics detoxification CYP1A1, AhR, GSTM1, GSTT1, ABCB1, redox-status SOD2, CAT, GCLC, and vascular homeostasis ACE, AGT, AGTR1, NOS3, MTHFR, VEGFα. Risk of pulmonary complications (PC) in the single locus analysis was associated with CYP1A1, GCLC and AGTR1 genes. Extra PC (toxic shock syndrome and myocarditis) were not associated with these genes. We evaluated gene-gene interactions using multi-factor dimensionality reduction, and cumulative gene risk score approaches. The final model which included >5 risk alleles in the CYP1A1 (rs2606345, rs4646903, rs1048943), GCLC, AGT, and AGTR1 genes was associated with pleuritis, empyema, acute respiratory distress syndrome, all PC and acute respiratory failure (ARF). We considered CYP1A1, GCLC, AGT, AGTR1 gene set using Set Distiller mode implemented in GeneDecks for discovering gene-set relations via the degree of sharing descriptors within a given gene set. N-acetylcysteine and oxygen were defined by Set Distiller as the best descriptors for the gene set associated in the present study with PC and ARF. Results of the study are in line with literature data and suggest that genetically determined oxidative stress exacerbation may contribute to the progression of lung inflammation.

  4. Potential Roles of Protease Inhibitors in Cancer Progression.

    PubMed

    Yang, Peng; Li, Zhuo-Yu; Li, Han-Qing

    2015-01-01

    Proteases are important molecules that are involved in many key physiological processes. Protease signaling pathways are strictly controlled, and disorders in protease activity can result in pathological changes such as cardiovascular and inflammatory diseases, cancer and neurological disorders. Many proteases have been associated with increasing tumor metastasis in various human cancers, suggesting important functional roles in the metastatic process because of their ability to degrade the extracellular matrix barrier. Proteases are also capable of cleaving non-extracellular matrix molecules. Inhibitors of proteases to some extent can reduce invasion and metastasis of cancer cells, and slow down cancer progression. In this review, we focus on the role of a few proteases and their inhibitors in tumors as a basis for cancer prognostication and therapy.

  5. Emerging principles in protease-based drug discovery

    PubMed Central

    Drag, Marcin; Salvesen, Guy S.

    2010-01-01

    Proteases have an important role in many signalling pathways, and represent potential drug targets for diseases ranging from cardiovascular disorders to cancer, as well as for combating many parasites and viruses. Although inhibitors of well-established protease targets such as angiotensin-converting enzyme and HIV protease have shown substantial therapeutic success, developing drugs for new protease targets has proved challenging in recent years. This in part could be due to issues such as the difficulty of achieving selectivity when targeting protease active sites. This Perspective discusses the general principles in protease-based drug discovery, highlighting the lessons learned and the emerging strategies, such as targeting allosteric sites, which could help harness the therapeutic potential of new protease targets. PMID:20811381

  6. Regulation of intestinal permeability: The role of proteases

    PubMed Central

    Van Spaendonk, Hanne; Ceuleers, Hannah; Witters, Leonie; Patteet, Eveline; Joossens, Jurgen; Augustyns, Koen; Lambeir, Anne-Marie; De Meester, Ingrid; De Man, Joris G; De Winter, Benedicte Y

    2017-01-01

    The gastrointestinal barrier is - with approximately 400 m2 - the human body’s largest surface separating the external environment from the internal milieu. This barrier serves a dual function: permitting the absorption of nutrients, water and electrolytes on the one hand, while limiting host contact with noxious luminal antigens on the other hand. To maintain this selective barrier, junction protein complexes seal the intercellular space between adjacent epithelial cells and regulate the paracellular transport. Increased intestinal permeability is associated with and suggested as a player in the pathophysiology of various gastrointestinal and extra-intestinal diseases such as inflammatory bowel disease, celiac disease and type 1 diabetes. The gastrointestinal tract is exposed to high levels of endogenous and exogenous proteases, both in the lumen and in the mucosa. There is increasing evidence to suggest that a dysregulation of the protease/antiprotease balance in the gut contributes to epithelial damage and increased permeability. Excessive proteolysis leads to direct cleavage of intercellular junction proteins, or to opening of the junction proteins via activation of protease activated receptors. In addition, proteases regulate the activity and availability of cytokines and growth factors, which are also known modulators of intestinal permeability. This review aims at outlining the mechanisms by which proteases alter the intestinal permeability. More knowledge on the role of proteases in mucosal homeostasis and gastrointestinal barrier function will definitely contribute to the identification of new therapeutic targets for permeability-related diseases. PMID:28405139

  7. A vitamin-B2-sensing mechanism that regulates gut protease activity to impact animal’s food behavior and growth

    PubMed Central

    Qi, Bin; Kniazeva, Marina; Han, Min

    2017-01-01

    To survive challenging environments, animals acquired the ability to evaluate food quality in the intestine and respond to nutrient deficiencies with changes in food-response behavior, metabolism and development. However, the regulatory mechanisms underlying intestinal sensing of specific nutrients, especially micronutrients such as vitamins, and the connections to downstream physiological responses in animals remain underexplored. We have established a system to analyze the intestinal response to vitamin B2 (VB2) deficiency in Caenorhabditis elegans, and demonstrated that VB2 level critically impacts food uptake and foraging behavior by regulating specific protease gene expression and intestinal protease activity. We show that this impact is mediated by TORC1 signaling through reading the FAD-dependent ATP level. Thus, our study in live animals uncovers a VB2-sensing/response pathway that regulates food-uptake, a mechanism by which a common signaling pathway translates a specific nutrient signal into physiological activities, and the importance of gut microbiota in supplying micronutrients to animals. DOI: http://dx.doi.org/10.7554/eLife.26243.001 PMID:28569665

  8. Diversity and transcription of proteases involved in the maturation of hydrogenases in Nostoc punctiforme ATCC 29133 and Nostoc sp. strain PCC 7120

    PubMed Central

    2009-01-01

    Background The last step in the maturation process of the large subunit of [NiFe]-hydrogenases is a proteolytic cleavage of the C-terminal by a hydrogenase specific protease. Contrary to other accessory proteins these hydrogenase proteases are believed to be specific whereby one type of hydrogenases specific protease only cleaves one type of hydrogenase. In cyanobacteria this is achieved by the gene product of either hupW or hoxW, specific for the uptake or the bidirectional hydrogenase respectively. The filamentous cyanobacteria Nostoc punctiforme ATCC 29133 and Nostoc sp strain PCC 7120 may contain a single uptake hydrogenase or both an uptake and a bidirectional hydrogenase respectively. Results In order to examine these proteases in cyanobacteria, transcriptional analyses were performed of hupW in Nostoc punctiforme ATCC 29133 and hupW and hoxW in Nostoc sp. strain PCC 7120. These studies revealed numerous transcriptional start points together with putative binding sites for NtcA (hupW) and LexA (hoxW). In order to investigate the diversity and specificity among hydrogeanse specific proteases we constructed a phylogenetic tree which revealed several subgroups that showed a striking resemblance to the subgroups previously described for [NiFe]-hydrogenases. Additionally the proteases specificity was also addressed by amino acid sequence analysis and protein-protein docking experiments with 3D-models derived from bioinformatic studies. These studies revealed a so called "HOXBOX"; an amino acid sequence specific for protease of Hox-type which might be involved in docking with the large subunit of the hydrogenase. Conclusion Our findings suggest that the hydrogenase specific proteases are under similar regulatory control as the hydrogenases they cleave. The result from the phylogenetic study also indicates that the hydrogenase and the protease have co-evolved since ancient time and suggests that at least one major horizontal gene transfer has occurred. This co

  9. A sensitive assay using a native protein substrate for screening HIV-1 maturation inhibitors targeting the protease cleavage site between the matrix and capsid.

    PubMed

    Lee, Sook-Kyung; Cheng, Nancy; Hull-Ryde, Emily; Potempa, Marc; Schiffer, Celia A; Janzen, William; Swanstrom, Ronald

    2013-07-23

    The matrix/capsid processing site in the HIV-1 Gag precursor is likely the most sensitive target to inhibit HIV-1 replication. We have previously shown that modest incomplete processing at the site leads to a complete loss of virion infectivity. In the study presented here, a sensitive assay based on fluorescence polarization that can monitor cleavage at the MA/CA site in the context of the folded protein substrate is described. The substrate, an MA/CA fusion protein, was labeled with the fluorescein-based FlAsH (fluorescein arsenical hairpin) reagent that binds to a tetracysteine motif (CCGPCC) that was introduced within the N-terminal domain of CA. By limiting the size of CA and increasing the size of MA (with an N-terminal GST fusion), we were able to measure significant differences in polarization values as a function of HIV-1 protease cleavage. The sensitivity of the assay was tested in the presence of increasing amounts of an HIV-1 protease inhibitor, which resulted in a gradual decrease in the fluorescence polarization values demonstrating that the assay is sensitive in discerning changes in protease processing. The high-throughput screening assay validation in 384-well plates showed that the assay is reproducible and robust with an average Z' value of 0.79 and average coefficient of variation values of <3%. The robustness and reproducibility of the assay were further validated using the LOPAC(1280) compound library, demonstrating that the assay provides a sensitive high-throughput screening platform that can be used with large compound libraries for identifying novel maturation inhibitors targeting the MA/CA site of the HIV-1 Gag polyprotein.

  10. Significance of Cuscutain, a cysteine protease from Cuscuta reflexa, in host-parasite interactions

    PubMed Central

    2010-01-01

    Background Plant infestation with parasitic weeds like Cuscuta reflexa induces morphological as well as biochemical changes in the host and the parasite. These modifications could be caused by a change in protein or gene activity. Using a comparative macroarray approach Cuscuta genes specifically upregulated at the host attachment site were identified. Results One of the infestation specific Cuscuta genes encodes a cysteine protease. The protein and its intrinsic inhibitory peptide were heterologously expressed, purified and biochemically characterized. The haustoria specific enzyme was named cuscutain in accordance with similar proteins from other plants, e.g. papaya. The role of cuscutain and its inhibitor during the host parasite interaction was studied by external application of an inhibitor suspension, which induced a significant reduction of successful infection events. Conclusions The study provides new information about molecular events during the parasitic plant - host interaction. Inhibition of cuscutain cysteine proteinase could provide means for antagonizing parasitic plants. PMID:20964874

  11. Structural and Functional Characterization of Cleavage and Inactivation of Human Serine Protease Inhibitors by the Bacterial SPATE Protease EspPα from Enterohemorrhagic E. coli

    PubMed Central

    Weiss, André; Joerss, Hanna; Brockmeyer, Jens

    2014-01-01

    EspPα and EspI are serine protease autotransporters found in enterohemorrhagic Escherichia coli. They both belong to the SPATE autotransporter family and are believed to contribute to pathogenicity via proteolytic cleavage and inactivation of different key host proteins during infection. Here, we describe the specific cleavage and functional inactivation of serine protease inhibitors (serpins) by EspPα and compare this activity with the related SPATE EspI. Serpins are structurally related proteins that regulate vital protease cascades, such as blood coagulation and inflammatory host response. For the rapid determination of serpin cleavage sites, we applied direct MALDI-TOF-MS or ESI-FTMS analysis of coincubations of serpins and SPATE proteases and confirmed observed cleavage positions using in-gel-digest of SDS-PAGE-separated degradation products. Activities of both serpin and SPATE protease were assessed in a newly developed photometrical assay using chromogenic peptide substrates. EspPα cleaved the serpins α1-protease inhibitor (α1-PI), α1-antichymotrypsin, angiotensinogen, and α2-antiplasmin. Serpin cleavage led to loss of inhibitory function as demonstrated for α1-PI while EspPα activity was not affected. Notably, EspPα showed pronounced specificity and cleaved procoagulatory serpins such as α2-antiplasmin while the anticoagulatory antithrombin III was not affected. Together with recently published research, this underlines the interference of EspPα with hemostasis or inflammatory responses during infection, while the observed interaction of EspI with serpins is likely to be not physiologically relevant. EspPα-mediated serpin cleavage occurred always in flexible loops, indicating that this structural motif might be required for substrate recognition. PMID:25347319

  12. Functional Analysis of the Trichoderma harzianum nox1 Gene, Encoding an NADPH Oxidase, Relates Production of Reactive Oxygen Species to Specific Biocontrol Activity against Pythium ultimum▿†

    PubMed Central

    Montero-Barrientos, M.; Hermosa, R.; Cardoza, R. E.; Gutiérrez, S.; Monte, E.

    2011-01-01

    The synthesis of reactive oxygen species (ROS) is one of the first events following pathogenic interactions in eukaryotic cells, and NADPH oxidases are involved in the formation of such ROS. The nox1 gene of Trichoderma harzianum was cloned, and its role in antagonism against phytopathogens was analyzed in nox1-overexpressed transformants. The increased levels of nox1 expression in these transformants were accompanied by an increase in ROS production during their direct confrontation with Pythium ultimum. The transformants displayed an increased hydrolytic pattern, as determined by comparing protease, cellulase, and chitinase activities with those for the wild type. In confrontation assays against P. ultimum the nox1-overexpressed transformants were more effective than the wild type, but not in assays against Botrytis cinerea or Rhizoctonia solani. A transcriptomic analysis using a Trichoderma high-density oligonucleotide (HDO) microarray also showed that, compared to gene expression for the interaction of wild-type T. harzianum and P. ultimum, genes related to protease, cellulase, and chitinase activities were differentially upregulated in the interaction of a nox1-overexpressed transformant with this pathogen. Our results show that nox1 is involved in T. harzianum ROS production and antagonism against P. ultimum. PMID:21421791

  13. Cysteine desulfurase Nfs1 and Pim1 protease control levels of Isu, the Fe-S cluster biogenesis scaffold.

    PubMed

    Song, Ji-Yoon; Marszalek, Jaroslaw; Craig, Elizabeth Anne

    2012-06-26

    Fe-S clusters are critical prosthetic groups for proteins involved in various critical biological processes. Before being transferred to recipient apo-proteins, Fe-S clusters are assembled on the highly conserved scaffold protein Isu, the abundance of which is regulated posttranslationally on disruption of the cluster biogenesis system. Here we report that Isu is degraded by the Lon-type AAA+ ATPase protease of the mitochondrial matrix, Pim1. Nfs1, the cysteine desulfurase responsible for providing sulfur for cluster formation, is required for the increased Isu stability occurring after disruption of cluster formation on or transfer from Isu. Physical interaction between the Isu and Nfs1 proteins, not the enzymatic activity of Nfs1, is the important factor in increased stability. Analysis of several conditions revealed that high Isu levels can be advantageous or disadvantageous, depending on the physiological condition. During the stationary phase, elevated Isu levels were advantageous, resulting in prolonged chronological lifespan. On the other hand, under iron-limiting conditions, high Isu levels were deleterious. Compared with cells expressing normal levels of Isu, such cells grew poorly and exhibited reduced activity of the heme-containing enzyme ferric reductase. Our results suggest that modulation of the degradation of Isu by the Pim1 protease is a regulatory mechanism serving to rapidly help balance the cell's need for critical iron-requiring processes under changing environmental conditions.

  14. Advances in protease engineering for laundry detergents.

    PubMed

    Vojcic, Ljubica; Pitzler, Christian; Körfer, Georgette; Jakob, Felix; Ronny Martinez; Maurer, Karl-Heinz; Schwaneberg, Ulrich

    2015-12-25

    Proteases are essential ingredients in modern laundry detergents. Over the past 30 years, subtilisin proteases employed in the laundry detergent industry have been engineered by directed evolution and rational design to tailor their properties towards industrial demands. This comprehensive review discusses recent success stories in subtilisin protease engineering. Advances in protease engineering for laundry detergents comprise simultaneous improvement of thermal resistance and activity at low temperatures, a rational strategy to modulate pH profiles, and a general hypothesis for how to increase promiscuous activity towards the production of peroxycarboxylic acids as mild bleaching agents. The three protease engineering campaigns presented provide in-depth analysis of protease properties and have identified principles that can be applied to improve or generate enzyme variants for industrial applications beyond laundry detergents. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Extracellular proteases as targets for drug development

    PubMed Central

    Cudic, Mare

    2015-01-01

    Proteases constitute one of the primary targets in drug discovery. In the present review, we focus on extracellular proteases (ECPs) because of their differential expression in many pathophysiological processes, including cancer, cardiovascular conditions, and inflammatory, pulmonary, and periodontal diseases. Many new ECP inhibitors are currently under clinical investigation and a significant increase in new therapies based on protease inhibition can be expected in the coming years. In addition to directly blocking the activity of a targeted protease, one can take advantage of differential expression in disease states to selectively deliver therapeutic or imaging agents. Recent studies in targeted drug development for the metalloproteases (matrix metalloproteinases, adamalysins, pappalysins, neprilysin, angiotensin-converting enzyme, metallocarboxypeptidases, and glutamate carboxypeptidase II), serine proteases (elastase, coagulation factors, tissue/urokinase plasminogen activator system, kallikreins, tryptase, dipeptidyl peptidase IV), cysteine proteases (cathepsin B), and renin system are discussed herein. PMID:19689354

  16. A Molecular Sensor To Characterize Arenavirus Envelope Glycoprotein Cleavage by Subtilisin Kexin Isozyme 1/Site 1 Protease.

    PubMed

    Oppliger, Joel; da Palma, Joel Ramos; Burri, Dominique J; Bergeron, Eric; Khatib, Abdel-Majid; Spiropoulou, Christina F; Pasquato, Antonella; Kunz, Stefan

    2016-01-15

    Arenaviruses are emerging viruses including several causative agents of severe hemorrhagic fevers in humans. The advent of next-generation sequencing technology has greatly accelerated the discovery of novel arenavirus species. However, for many of these viruses, only genetic information is available, and their zoonotic disease potential remains unknown. During the arenavirus life cycle, processing of the viral envelope glycoprotein precursor (GPC) by the cellular subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P) is crucial for productive infection. The ability of newly emerging arenaviruses to hijack human SKI-1/S1P appears, therefore, to be a requirement for efficient zoonotic transmission and human disease potential. Here we implement a newly developed cell-based molecular sensor for SKI-1/S1P to characterize the processing of arenavirus GPC-derived target sequences by human SKI-1/S1P in a quantitative manner. We show that only nine amino acids flanking the putative cleavage site are necessary and sufficient to accurately recapitulate the efficiency and subcellular location of arenavirus GPC processing. In a proof of concept, our sensor correctly predicts efficient processing of the GPC of the newly emergent pathogenic Lujo virus by human SKI-1/S1P and defines the exact cleavage site. Lastly, we employed our sensor to show efficient GPC processing of a panel of pathogenic and nonpathogenic New World arenaviruses, suggesting that GPC cleavage represents no barrier for zoonotic transmission of these pathogens. Our SKI-1/S1P sensor thus represents a rapid and robust test system for assessment of the processing of putative cleavage sites derived from the GPCs of newly discovered arenavirus by the SKI-1/S1P of humans or any other species, based solely on sequence information. Arenaviruses are important emerging human pathogens that can cause severe hemorrhagic fevers with high mortality in humans. A crucial step in productive arenavirus infection of human

  17. Hybrid male sterility and genome-wide misexpression of male reproductive proteases

    PubMed Central

    Gomes, Suzanne; Civetta, Alberto

    2015-01-01

    Hybrid male sterility is a common barrier to gene flow between species. Previous studies have posited a link between misregulation of spermatogenesis genes in interspecies hybrids and sterility. However, in the absence of fully fertile control hybrids, it is impossible to differentiate between misregulation associated with sterility vs. fast male gene regulatory evolution. Here, we differentiate between these two possibilities using a D. pseudoobscura species pair that experiences unidirectional hybrid sterility. We identify genes uniquely misexpressed in sterile hybrid male reproductive tracts via RNA-seq. The sterile male hybrids had more misregulated and more over or under expressed genes relative to parental species than the fertile male hybrids. Proteases were the only gene ontology class overrepresented among uniquely misexpressed genes, with four located within a previously identified hybrid male sterility locus. This result highlights the potential role of a previously unexplored class of genes in interspecific hybrid male sterility and speciation. PMID:26146165

  18. Protection of scaffold protein Isu from degradation by the Lon protease Pim1 as a component of Fe-S cluster biogenesis regulation.

    PubMed

    Ciesielski, Szymon J; Schilke, Brenda; Marszalek, Jaroslaw; Craig, Elizabeth A

    2016-04-01

    Iron-sulfur (Fe-S) clusters, essential protein cofactors, are assembled on the mitochondrial scaffold protein Isu and then transferred to recipient proteins via a multistep process in which Isu interacts sequentially with multiple protein factors. This pathway is in part regulated posttranslationally by modulation of the degradation of Isu, whose abundance increases >10-fold upon perturbation of the biogenesis process. We tested a model in which direct interaction with protein partners protects Isu from degradation by the mitochondrial Lon-type protease. Using purified components, we demonstrated that Isu is indeed a substrate of the Lon-type protease and that it is protected from degradation by Nfs1, the sulfur donor for Fe-S cluster assembly, as well as by Jac1, the J-protein Hsp70 cochaperone that functions in cluster transfer from Isu. Nfs1 and Jac1 variants known to be defective in interaction with Isu were also defective in protecting Isu from degradation. Furthermore, overproduction of Jac1 protected Isu from degradation in vivo, as did Nfs1. Taken together, our results lead to a model of dynamic interplay between a protease and protein factors throughout the Fe-S cluster assembly and transfer process, leading to up-regulation of Isu levels under conditions when Fe-S cluster biogenesis does not meet cellular demands. © 2016 Ciesielski et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  19. Enhanced enteroviral infectivity via viral protease-mediated cleavage of Grb2-associated binder 1

    PubMed Central

    Deng, Haoyu; Fung, Gabriel; Shi, Junyan; Xu, Suowen; Wang, Chen; Yin, Meimei; Hou, Jun; Zhang, Jingchun; Jin, Zheng-Gen; Luo, Honglin

    2015-01-01

    Coxsackievirus B3 (CVB3), an important human causative pathogen for viral myocarditis, pancreatitis, and meningitis, has evolved different strategies to manipulate the host signaling machinery to ensure successful viral infection. We previously revealed a crucial role for the ERK1/2 signaling pathway in regulating viral infectivity. However, the detail mechanism remains largely unknown. Grb2-associated binder 1 (GAB1) is an important docking protein responsible for intracellular signaling assembly and transduction. In this study, we demonstrated that GAB1 was proteolytically cleaved after CVB3 infection at G175 and G436 by virus-encoded protease 2Apro, independent of caspase activation. Knockdown of GAB1 resulted in a significant reduction of viral protein expression and virus titers. Moreover, we showed that virus-induced cleavage of GAB1 is beneficial to viral growth as the N-terminal proteolytic product of GAB1 (GAB1-N1–174) further enhances ERK1/2 activation and promotes viral replication. Our results collectively suggest that CVB3 targets host GAB1 to generate a GAB1-N1–174 fragment that enhances viral infectivity, at least in part, via activation of the ERK pathway. The findings in this study suggest a novel mechanism that CVB3 employs to subvert the host signaling and facilitate consequent viral replication.—Deng, H., Fung, G., Shi, J., Xu, S., Wang, C., Yin, M., Hou, J., Zhang, J., Jin, Z.-G., Luo, H. Enhanced enteroviral infectivity via viral protease-mediated cleavage of Grb2-associated binder 1. PMID:26183772

  20. Discovery of potent peptide-mimetic antagonists for the human thrombin receptor, protease-activated receptor-1 (PAR-1).

    PubMed

    Maryanoff, Bruce E; Zhang, Han-Cheng; Andrade-Gordon, Patricia; Derian, Claudia K

    2003-03-01

    Protease-activated receptors (PARs) represent a unique family of seven-transmembrane G-protein-coupled receptors, which are enzymatically cleaved to expose a new extracellular N-terminus that acts as a tethered activating ligand. PAR-1 is cleaved and activated by the serine protease alpha-thrombin, is expressed in various tissues (e.g. platelets and vascular cells), and is involved in cellular responses associated with hemostasis, proliferation, and tissue injury. By using a de novo design approach, we have discovered a series of potent heterocycle-based peptide-miimetic antagonists of PAR-1, exemplified by advanced leads RWJ-56110 (22) and RWJ-58259 (32). These compounds are potent, selective PAR-1 antagonists, devoid of PAR-1 agonist and thrombin inhibitory activity: they bind to PAR-1, interfere with calcium mobilization and cellular functions associated with PAR-1, and do not affect PAR-2, PAR-3, or PAR-4. RWJ-56110 was determined to be a direct inhibitor of PAR-1 activation and internalization, without affecting PAR-1 N-terminal cleavage. At high concentrations of alpha-thrombin, RWJ-56110 fully blocked activation responses in human vascular cells, but not in human platelets; whereas, at high concentrations of TRAP-6, RWJ-56110 blocked activation responses in both cell types. This result is consistent with the presence of another thrombin receptor on human platelets, namely PAR-4. RWJ-56110 and RWJ-58259 clearly interrupt the binding of a tethered ligand to its receptor. RWJ-58259 demonstrated antirestenotic activity in a rat balloon angioplasty model and antithrombotic activity in a cynomolgus monkey arterial injury model. Such PAR-1 antagonists should not only serve as useful tools to delineate the physiological and pathophysiological roles of PAR-1, but also may have therapeutic potential for treating thrombosis and restenosis in humans.

  1. Novel IgG-Degrading Enzymes of the IgdE Protease Family Link Substrate Specificity to Host Tropism of Streptococcus Species

    PubMed Central

    Spoerry, Christian; Hessle, Pontus; Lewis, Melanie J.; Paton, Lois; Woof, Jenny M.

    2016-01-01

    Recently we have discovered an IgG degrading enzyme of the endemic pig pathogen S. suis designated IgdE that is highly specific for porcine IgG. This protease is the founding member of a novel cysteine protease family assigned C113 in the MEROPS peptidase database. Bioinformatical analyses revealed putative members of the IgdE protease family in eight other Streptococcus species. The genes of the putative IgdE family proteases of S. agalactiae, S. porcinus, S. pseudoporcinus and S. equi subsp. zooepidemicus were cloned for production of recombinant protein into expression vectors. Recombinant proteins of all four IgdE family proteases were proteolytically active against IgG of the respective Streptococcus species hosts, but not against IgG from other tested species or other classes of immunoglobulins, thereby linking the substrate specificity to the known host tropism. The novel IgdE family proteases of S. agalactiae, S. pseudoporcinus and S. equi showed IgG subtype specificity, i.e. IgdE from S. agalactiae and S. pseudoporcinus cleaved human IgG1, while IgdE from S. equi was subtype specific for equine IgG7. Porcine IgG subtype specificities of the IgdE family proteases of S. porcinus and S. pseudoporcinus remain to be determined. Cleavage of porcine IgG by IgdE of S. pseudoporcinus is suggested to be an evolutionary remaining activity reflecting ancestry of the human pathogen to the porcine pathogen S. porcinus. The IgG subtype specificity of bacterial proteases indicates the special importance of these IgG subtypes in counteracting infection or colonization and opportunistic streptococci neutralize such antibodies through expression of IgdE family proteases as putative immune evasion factors. We suggest that IgdE family proteases might be valid vaccine targets against streptococci of both human and veterinary medical concerns and could also be of therapeutic as well as biotechnological use. PMID:27749921

  2. Involvement of an Extracellular Protease in Algicidal Activity of the Marine Bacterium Pseudoalteromonas sp. Strain A28

    PubMed Central

    Lee, Sun-og; Kato, Junichi; Takiguchi, Noboru; Kuroda, Akio; Ikeda, Tsukasa; Mitsutani, Atsushi; Ohtake, Hisao

    2000-01-01

    The marine bacterium Pseudoalteromonas sp. strain A28 was able to kill the diatom Skeletonema costatum strain NIES-324. The culture supernatant of strain A28 showed potent algicidal activity when it was applied to a paper disk placed on a lawn of S. costatum NIES-324. The condensed supernatant, which was prepared by subjecting the A28 culture supernatant to ultrafiltration with a 10,000-Mw-cutoff membrane, showed algicidal activity, suggesting that strain A28 produced extracellular substances capable of killing S. costatum cells. The condensed supernatant was then found to have protease and DNase activities. Two Pseudoalteromonas mutants lacking algicidal activity, designated NH1 and NH2, were selected after N-methyl-N′-nitrosoguanidine mutagenesis. The culture supernatants of NH1 and NH2 showed less than 15% of the protease activity detected with the parental strain, A28. The protease was purified to homogeneity from A28 culture supernatants by using ion-exchange chromatography followed by preparative gel electrophoresis. Paper-disk assays revealed that the purified protease had potent algicidal activity. The purified protease had a molecular mass for 50 kDa, and the N-terminal amino acid sequence was determined to be Ala-Thr-Pro-Asn-Asp-Pro. The optimum pH and temperature of the protease were found to be 8.8 and 30°C, respectively, by using succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as a substrate. The protease activity was strongly inhibited by phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate, antipain, chymostatin, and leupeptin. No significant inhibition was detected with EDTA, EGTA, phenanthroline or tetraethylenepentamine. These results suggest that Pseudoalteromonas sp. strain A28 produced an extracellular serine protease which was responsible for the algicidal activity of this marine bacterium. PMID:11010878

  3. Cloning and characterization of Bacillus subtilis iep, which has positive and negative effects on production of extracellular proteases.

    PubMed Central

    Tanaka, T; Kawata, M

    1988-01-01

    We have isolated a DNA fragment from Bacillus subtilis 168 which, when present in a high-copy plasmid, inhibited production of extracellular alkaline and neutral proteases. The gene responsible for this activity was referred to as iep. The open reading frame of iep was found to be incomplete in the cloned DNA fragment. When the intact iep gene was reconstructed after the missing part of the iep gene had been cloned, it showed an enhancing effect on the production of the extracellular proteases. The open reading frame encodes a polypeptide of 229 amino acids with a molecular weight of ca. 25,866. Deletion of two amino acids from the N-terminal half of the putative iep protein resulted in dual effects, i.e., a decrease in the inhibitory activity shown by the incomplete iep gene and a slight increase in the enhancing activity shown by the complete iep gene. These results show that the iep gene product is a bifunctional protein, containing inhibitory and enhancing activities for the exoprotease production in the N-terminal and C-terminal regions, respectively. It was found by genetic and functional analyses that iep lies very close to sacU. Images PMID:3136143

  4. Cloning and characterization of Bacillus subtilis iep, which has positive and negative effects on production of extracellular proteases.

    PubMed

    Tanaka, T; Kawata, M

    1988-08-01

    We have isolated a DNA fragment from Bacillus subtilis 168 which, when present in a high-copy plasmid, inhibited production of extracellular alkaline and neutral proteases. The gene responsible for this activity was referred to as iep. The open reading frame of iep was found to be incomplete in the cloned DNA fragment. When the intact iep gene was reconstructed after the missing part of the iep gene had been cloned, it showed an enhancing effect on the production of the extracellular proteases. The open reading frame encodes a polypeptide of 229 amino acids with a molecular weight of ca. 25,866. Deletion of two amino acids from the N-terminal half of the putative iep protein resulted in dual effects, i.e., a decrease in the inhibitory activity shown by the incomplete iep gene and a slight increase in the enhancing activity shown by the complete iep gene. These results show that the iep gene product is a bifunctional protein, containing inhibitory and enhancing activities for the exoprotease production in the N-terminal and C-terminal regions, respectively. It was found by genetic and functional analyses that iep lies very close to sacU.

  5. Singlet Oxygen-Induced Membrane Disruption and Serpin-Protease Balance in Vacuolar-Driven Cell Death.

    PubMed

    Koh, Eugene; Carmieli, Raanan; Mor, Avishai; Fluhr, Robert

    2016-07-01

    Singlet oxygen plays a role in cellular stress either by providing direct toxicity or through signaling to initiate death programs. It was therefore of interest to examine cell death, as occurs in Arabidopsis, due to differentially localized singlet oxygen photosensitizers. The photosensitizers rose bengal (RB) and acridine orange (AO) were localized to the plasmalemma and vacuole, respectively. Their photoactivation led to cell death as measured by ion leakage. Cell death could be inhibited by the singlet oxygen scavenger histidine in treatments with AO but not with RB In the case of AO treatment, the vacuolar membrane was observed to disintegrate. Concomitantly, a complex was formed between a vacuolar cell-death protease, RESPONSIVE TO DESSICATION-21 and its cognate cytoplasmic protease inhibitor ATSERPIN1. In the case of RB treatment, the tonoplast remained intact and no complex was formed. Over-expression of AtSerpin1 repressed cell death, only under AO photodynamic treatment. Interestingly, acute water stress showed accumulation of singlet oxygen as determined by fluorescence of Singlet Oxygen Sensor Green, by electron paramagnetic resonance spectroscopy and the induction of singlet oxygen marker genes. Cell death by acute water stress was inhibited by the singlet oxygen scavenger histidine and was accompanied by vacuolar collapse and the appearance of serpin-protease complex. Over-expression of AtSerpin1 also attenuated cell death under this mode of cell stress. Thus, acute water stress damage shows parallels to vacuole-mediated cell death where the generation of singlet oxygen may play a role. © 2016 American Society of Plant Biologists. All Rights Reserved.

  6. Characterizing Protease Specificity: How Many Substrates Do We Need?

    PubMed Central

    Schauperl, Michael; Fuchs, Julian E.; Waldner, Birgit J.; Huber, Roland G.; Kramer, Christian; Liedl, Klaus R.

    2015-01-01

    Calculation of cleavage entropies allows to quantify, map and compare protease substrate specificity by an information entropy based approach. The metric intrinsically depends on the number of experimentally determined substrates (data points). Thus a statistical analysis of its numerical stability is crucial to estimate the systematic error made by estimating specificity based on a limited number of substrates. In this contribution, we show the mathematical basis for estimating the uncertainty in cleavage entropies. Sets of cleavage entropies are calculated using experimental cleavage data and modeled extreme cases. By analyzing the underlying mathematics and applying statistical tools, a linear dependence of the metric in respect to 1/n was found. This allows us to extrapolate the values to an infinite number of samples and to estimate the errors. Analyzing the errors, a minimum number of 30 substrates was found to be necessary to characterize substrate specificity, in terms of amino acid variability, for a protease (S4-S4’) with an uncertainty of 5 percent. Therefore, we encourage experimental researchers in the protease field to record specificity profiles of novel proteases aiming to identify at least 30 peptide substrates of maximum sequence diversity. We expect a full characterization of protease specificity helpful to rationalize biological functions of proteases and to assist rational drug design. PMID:26559682

  7. The herpesvirus proteases as targets for antiviral chemotherapy.

    PubMed

    Waxman, L; Darke, P L

    2000-01-01

    Viruses of the family Herpesviridae are responsible for a diverse set of human diseases. The available treatments are largely ineffective, with the exception of a few drugs for treatment of herpes simplex virus (HSV) infections. For several members of this DNA virus family, advances have been made recently in the biochemistry and structural biology of the essential viral protease, revealing common features that may be possible to exploit in the development of a new class of anti-herpesvirus agents. The herpesvirus proteases have been identified as belonging to a unique class of serine protease, with a Ser-His-His catalytic triad. A new, single domain protein fold has been determined by X-ray crystallography for the proteases of at least three different herpesviruses. Also unique for serine proteases, dimerization has been shown to be required for activity of the cytomegalovirus and HSV proteases. The dimerization requirement seriously impacts methods needed for productive, functional analysis and inhibitor discovery. The conserved functional and catalytic properties of the herpesvirus proteases lead to common considerations for this group of proteases in the early phases of inhibitor discovery. In general, classical serine protease inhibitors that react with active site residues do not readily inactivate the herpesvirus proteases. There has been progress however, with activated carbonyls that exploit the selective nucleophilicity of the active site serine. In addition, screening of chemical libraries has yielded novel structures as starting points for drug development. Recent crystal structures of the herpesvirus proteases now allow more direct interpretation of ligand structure-activity relationships. This review first describes basic functional aspects of herpesvirus protease biology and enzymology. Then we discuss inhibitors identified to date and the prospects for their future development.

  8. The novel monoclonal antibody 9F5 reveals expression of a fragment of GPNMB/osteoactivin processed by furin-like protease(s) in a subpopulation of microglia in neonatal rat brain.

    PubMed

    Kawahara, Kohichi; Hirata, Hiroshi; Ohbuchi, Kengo; Nishi, Kentaro; Maeda, Akira; Kuniyasu, Akihiko; Yamada, Daisuke; Maeda, Takehiko; Tsuji, Akihiko; Sawada, Makoto; Nakayama, Hitoshi

    2016-11-01

    To differentiate subtypes of microglia (MG), we developed a novel monoclonal antibody, 9F5, against one subtype (type 1) of rat primary MG. The 9F5 showed high selectivity for this cell type in Western blot and immunocytochemical analyses and no cross-reaction with rat peritoneal macrophages (Mφ). We identified the antigen molecule for 9F5: the 50- to 70-kDa fragments of rat glycoprotein nonmetastatic melanoma protein B (GPNMB)/osteoactivin, which started at Lys(170) . In addition, 9F5 immunoreactivity with GPNMB depended on the activity of furin-like protease(s). More important, rat type 1 MG expressed the GPNMB fragments, but type 2 MG and Mφ did not, although all these cells expressed mRNA and the full-length protein for GPNMB. These results suggest that 9F5 reactivity with MG depends greatly on cleavage of GPNMB and that type 1 MG, in contrast to type 2 MG and Mφ, may have furin-like protease(s) for GPNMB cleavage. In neonatal rat brain, amoeboid 9F5+ MG were observed in specific brain areas including forebrain subventricular zone, corpus callosum, and retina. Double-immunοstaining with 9F5 antibody and anti-Iba1 antibody, which reacts with MG throughout the CNS, revealed that 9F5+ MG were a portion of Iba1+ MG, suggesting that MG subtype(s) exist in vivo. We propose that 9F5 is a useful tool to discriminate between rat type 1 MG and other subtypes of MG/Mφ and to reveal the role of the GPNMB fragments during developing brain. GLIA 2016;64:1938-1961. © 2016 The Authors. Glia Published by Wiley Periodicals, Inc.

  9. Identification and biochemical characterization of small-molecule inhibitors of west nile virus serine protease by a high-throughput screen.

    PubMed

    Mueller, Niklaus H; Pattabiraman, Nagarajan; Ansarah-Sobrinho, Camilo; Viswanathan, Prasanth; Pierson, Theodore C; Padmanabhan, R

    2008-09-01

    West Nile virus and dengue virus are mosquito-borne flaviviruses that cause a large number of human infections each year. No vaccines or chemotherapeutics are currently available. These viruses encode a serine protease that is essential for polyprotein processing, a required step in the viral replication cycle. In this study, a high-throughput screening assay for the West Nile virus protease was employed to screen approximately 32,000 small-molecule compounds for identification of inhibitors. Lead inhibitor compounds with three distinct core chemical structures (1 to 3) were identified. In a secondary screening of selected compounds, two compounds, belonging to the 8-hydroxyquinoline family (compounds A and B) and containing core structure 1, were identified as potent inhibitors of the West Nile virus protease, with K(i) values of 3.2 +/- 0.3 microM and 3.4 +/- 0.6 microM, respectively. These compounds inhibited the dengue virus type 2 protease with K(i) values of 28.6 +/- 5.1 microM and 30.2 +/- 8.6 microM, respectively, showing some selectivity in the inhibition of these viral proteases. However, the compounds show no inhibition of cellular serine proteases, trypsin, or factor Xa. Kinetic analysis and molecular docking of compound B onto the known crystal structure of the West Nile virus protease indicate that the inhibitor binds in the substrate-binding cleft. Furthermore, compound B was capable of inhibiting West Nile virus RNA replication in cultured Vero cells (50% effective concentration, 1.4 +/- 0.4 microM; selectivity index, 100), presumably by inhibition of polyprotein processing.

  10. Human Cathepsin V Protease Participates in Production of Enkephalin and NPY Neuropeptide Neurotransmitters*

    PubMed Central

    Funkelstein, Lydiane; Lu, W. Douglas; Koch, Britta; Mosier, Charles; Toneff, Thomas; Taupenot, Laurent; O'Connor, Daniel T.; Reinheckel, Thomas; Peters, Christoph; Hook, Vivian

    2012-01-01

    Proteases are required for processing precursors into active neuropeptides that function as neurotransmitters for cell-cell communication. This study demonstrates the novel function of human cathepsin V protease for producing the neuropeptides enkephalin and neuropeptide Y (NPY). Cathepsin V is a human-specific cysteine protease gene. Findings here show that expression of cathepsin V in neuroendocrine PC12 cells and human neuronal SK-N-MC cells results in production of (Met)enkephalin from proenkephalin. Gene silencing of cathepsin V by siRNA in human SK-N-MC cells results in reduction of (Met)enkephalin by more than 80%, illustrating the prominent role of cathepsin V for neuropeptide production. In vitro processing of proenkephalin by cathepsin V occurs at dibasic residue sites to generate enkephalin-containing peptides and an ∼24-kDa intermediate present in human brain. Cathepsin V is present in human brain cortex and hippocampus where enkephalin and NPY are produced and is present in purified human neuropeptide secretory vesicles. Colocalization of cathepsin V with enkephalin and NPY in secretory vesicles of human neuroblastoma cells was illustrated by confocal microscopy. Furthermore, expression of cathepsin V with proNPY results in NPY production. These findings indicate the unique function of human cathepsin V for producing enkephalin and NPY neuropeptides required for neurotransmission in health and neurological diseases. PMID:22393040

  11. The m-AAA Protease Associated with Neurodegeneration Limits MCU Activity in Mitochondria.

    PubMed

    König, Tim; Tröder, Simon E; Bakka, Kavya; Korwitz, Anne; Richter-Dennerlein, Ricarda; Lampe, Philipp A; Patron, Maria; Mühlmeister, Mareike; Guerrero-Castillo, Sergio; Brandt, Ulrich; Decker, Thorsten; Lauria, Ines; Paggio, Angela; Rizzuto, Rosario; Rugarli, Elena I; De Stefani, Diego; Langer, Thomas

    2016-10-06

    Mutations in subunits of mitochondrial m-AAA proteases in the inner membrane cause neurodegeneration in spinocerebellar ataxia (SCA28) and hereditary spastic paraplegia (HSP7). m-AAA proteases preserve mitochondrial proteostasis, mitochondrial morphology, and efficient OXPHOS activity, but the cause for neuronal loss in disease is unknown. We have determined the neuronal interactome of m-AAA proteases in mice and identified a complex with C2ORF47 (termed MAIP1), which counteracts cell death by regulating the assembly of the mitochondrial Ca 2+ uniporter MCU. While MAIP1 assists biogenesis of the MCU subunit EMRE, the m-AAA protease degrades non-assembled EMRE and ensures efficient assembly of gatekeeper subunits with MCU. Loss of the m-AAA protease results in accumulation of constitutively active MCU-EMRE channels lacking gatekeeper subunits in neuronal mitochondria and facilitates mitochondrial Ca 2+ overload, mitochondrial permeability transition pore opening, and neuronal death. Together, our results explain neuronal loss in m-AAA protease deficiency by deregulated mitochondrial Ca 2+ homeostasis. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Insecticidal activity of two proteases against Spodoptera frugiperda larvae infected with recombinant baculoviruses

    PubMed Central

    2010-01-01

    Background Baculovirus comprise the largest group of insect viruses most studied worldwide, mainly because they efficiently kill agricutural insect pests. In this study, two recombinant baculoviruses containing the ScathL gene from Sarcophaga peregrina (vSynScathL), and the Keratinase gene from the fungus Aspergillus fumigatus (vSynKerat), were constructed. and their insecticidal properties analysed against Spodoptera frugiperda larvae. Results Bioassays of third-instar and neonate S. frugiperda larvae with vSynScathL and vSynKerat showed a decrease in the time needed to kill the infected insects when compared to the wild type virus. We have also shown that both recombinants were able to increase phenoloxidase activity in the hemolymph of S. frugiperda larvae. The expression of proteases in infected larvae resulted in destruction of internal tissues late in infection, which could be the reason for the increased viral speed of kill. Conclusions Baculoviruses and their recombinant forms constitute viable alternatives to chemical insecticides. Recombinant baculoviruses containing protease genes can be added to the list of engineered baculoviruses with great potential to be used in integrated pest management programs. PMID:20587066

  13. Dynamics of Preferential Substrate Recognition in HIV-1 Protease: Redefining the Substrate Envelope

    PubMed Central

    Özen, Ayşegül; Haliloğlu, Türkan; Schiffer, Celia A.

    2011-01-01

    HIV-1 protease (PR) permits viral maturation by processing the Gag and Gag-Pro-Pol polyproteins. Though HIV-1 PR inhibitors (PIs) are used in combination antiviral therapy, the emergence of drug resistance has limited their efficacy. The rapid evolution of HIV-1 necessitates the consideration of drug resistance in novel drug-design strategies. Drug-resistant HIV-1 PR variants, while no longer efficiently inhibited, continue to efficiently hydrolyze the natural viral substrates. Though highly diverse in sequence, the HIV-1 PR substrates bind in a conserved three-dimensional shape we defined as the “substrate envelope”. We previously showed that resistance mutations arise where PIs protrude beyond the substrate envelope, as these regions are crucial for drug binding but not for substrate recognition. Here, we extend this model by considering the role of protein dynamics in the interaction of HIV-1 PR with its substrates. Seven molecular dynamics simulations of PR-substrate complexes were performed to estimate the conformational flexibility of substrates in their complexes. Interdependency of the substrate-protease interactions may compensate for the variations in cleavage-site sequences, and explain how a diverse set of sequences can be recognized as substrates by the same enzyme. This diversity may be essential for regulating sequential processing of substrates. We also define a dynamic substrate envelope as a more accurate representation of PR-substrate interactions. This dynamic substrate envelope, described by a probability distribution function, is a powerful tool for drug design efforts targeting ensembles of resistant HIV-1 PR variants with the aim of developing drugs that are less susceptible to resistance. PMID:21762811

  14. Characterization of detergent compatible protease of a halophilic Bacillus sp. EMB9: differential role of metal ions in stability and activity.

    PubMed

    Sinha, Rajeshwari; Khare, S K

    2013-10-01

    A moderately halophilic protease producer, Bacillus sp. strain isolated from sea water is described. The protease is purified to homogeneity by ammonium sulphate precipitation and CM cellulose chromatography. The serine protease has a molecular mass of 29 kDa. Enzymatic characterization of protease revealed K(m) 2.22 mg mL(-1), Vmax 1111.11 U mL(-1), pH optimum 9.0, t1/2 190 min at 60°C and salt optima 1% (w/v) NaCl. The protease is remarkably stable in hydrophilic and hydrophobic solvents at high concentrations. The purified preparation is unstable at room temperature. Ca(2+) ions are required for preventing this loss of activity. Interestingly, the activity and stability are modulated differentially. Whereas, divalent cation Ca(2+) are involved in maintaining stability in solution at room temperature by preventing unfolding, monovalent Na(+) and K(+) ions participate in regulating the activity and assist in refolding of the enzyme. Application of the protease is shown in efficient removal of blood stain. Copyright © 2012 Elsevier Ltd. All rights reserved.

  15. Mapping, Complementation, and Targets of the Cysteine Protease Actinidin in Kiwifruit1[C][W][OA

    PubMed Central

    Nieuwenhuizen, Niels J.; Maddumage, Ratnasiri; Tsang, Gianna K.; Fraser, Lena G.; Cooney, Janine M.; De Silva, H. Nihal; Green, Sol; Richardson, Kim A.; Atkinson, Ross G.

    2012-01-01

    Cysteine proteases (CPs) accumulate to high concentration in many fruit, where they are believed to play a role in fungal and insect defense. The fruit of Actinidia species (kiwifruit) exhibit a range of CP activities (e.g. the Actinidia chinensis variety YellowA shows less than 2% of the activity of Actinidia deliciosa variety Hayward). A major quantitative trait locus for CP activity was mapped to linkage group 16 in a segregating population of A. chinensis. This quantitative trait locus colocated with the gene encoding actinidin, the major acidic CP in ripe Hayward fruit encoded by the ACT1A-1 allele. Sequence analysis indicated that the ACT1A locus in the segregating A. chinensis population contained one functional allele (A-2) and three nonfunctional alleles (a-3, a-4, and a-5) each containing a unique frameshift mutation. YellowA kiwifruit contained two further alleles: a-6, which was nonfunctional because of a large insertion, and a-7, which produced an inactive enzyme. Site-directed mutagenesis of the act1a-7 protein revealed a residue that restored CP activity. Expression of the functional ACT1A-1 cDNA in transgenic plants complemented the natural YellowA mutations and partially restored CP activity in fruit. Two consequences of the increase in CP activity were enhanced degradation of gelatin-based jellies in vitro and an increase in the processing of a class IV chitinase in planta. These results provide new insight into key residues required for CP activity and the in vivo protein targets of actinidin. PMID:22039217

  16. Identification of a serine protease as a major allergen (Per a 10) of Periplaneta americana.

    PubMed

    Sudha, V T; Arora, N; Gaur, S N; Pasha, S; Singh, B P

    2008-06-01

    Cockroach allergens are associated with the development of asthma, but none of these has been characterized for proteolytic activity. This study was undertaken to isolate and characterize a protease from Periplaneta americana and determine its allergenicity. A serine protease was isolated from P. americana extract using benzamidine sepharose column and characterized by immunobiochemical methods. Allergenicity of the protease was assessed by enzyme-linked immunosorbent assay, immunoblot, intradermal testing, histamine release and peripheral blood mononuclear cells (PBMCs) proliferation. Affinity purified protein of approximately 28 kDa (Per a 10) showed a single band of activity in gelatin zymogram and agarose plate assay. N-terminal sequence (IVGGRPAQI) revealed similarity with mite serine protease allergens and insect trypsins. It demonstrated proteolytic activity with azocollagen > gelatin > defatted-milk > casein including serine protease specific substrate, N-benzoyl-arginine-ethyl-ester-hydrochloride. It was inhibited by serine protease inhibitors, namely aprotinin > pefabloc > AEBSF > PMSF > benzamidine > antipain > leupeptin and trypsin-specific inhibitor (tosyl-lysyl-chloromethyl-ketone) suggesting it to be a trypsin-like serine protease. Per a 10 was recognized as a major allergen, showing IgE reactivity with >80% of cockroach sensitized patients by skin tests and immunoblot. It could induce significant histamine release (P < 0.05) in blood and secretion of interleukin-4 (IL-4) (P < 0.05) and IL-5 (P < 0.05) in culture supernatant of PBMCs from cockroach hypersensitive patients, suggesting a strong allergenic potency. A serine protease isolated from P. americana was demonstrated to be a major allergen (Per a 10). It has a potential for component-based diagnosis of allergy and will be useful in elucidating the mechanism of allergy.

  17. Nematicidal activity of three novel extracellular proteases of the nematophagous fungus Monacrosporium sinense.

    PubMed

    Soares, Filippe E F; Braga, Fabio R; Araújo, Jackson V; Geniêr, Hugo L A; Gouveia, Angélica S; Queiroz, José H

    2013-04-01

    Extracellular proteases are an important virulence factor for the nematophagous fungi Monacrosporium. The objective of this study was to optimize, purify, partially characterize, and to evaluate the nematicidal activity of the proteases produced by the nematophagous fungus Monacrosporium sinense (SF53) by solid-state fermentation. Wheat bran was used as substrate for protease production. The variables moisture, pH, incubation time, temperature, glucose, yeast extract, and the number of conidia were tested for their influences on protease production by SF53. To determine the optimal level of the selected variables the central composite design was applied. The crude extract obtained was purified in two steps, an ion exchange chromatography and a gel excision. SDS-PAGE and zymogram were performed for analysis of the purification process. Proteolytic activity was also tested at different pHs and temperatures. In the in vitro assay, the nematicidal activity of the three proteases was evaluated. pH and incubation time showed a significant effect (p<0.05) on production of protease. The highest value of activity was 38.0 (U/ml) under the conditions of pH 5.0 and incubation time of 211 h. SF53 produced three different proteases (Ms1, Ms2, and Ms3) which were directly purified from the zymogram. Ms1, Ms2, and Ms3 showed the following percentage of reduction (p<0.05) on the number of Panagrellus redivivus compared to control after 24 h: 76.8, 68.1, and 92.1%. This is the first report of the use of proteases of the isolate SF53 on a phytonematode, which may be a research tool in future works.

  18. Improvement of shelf life of soymilk using immobilized protease of Oerskovia xanthineolytica NCIM 2839.

    PubMed

    Sahoo, A K; Gaikwad, V S; Ranveer, R C; Dandge, P B; Waghmare, S R

    2016-12-01

    Protease enzyme has lot of commercial applications, so the cost-effective production of protease using sunflower oil seed waste was carried out from Oerskovia xanthineolyitca NCIM 2839. The maximum protease production was after 24 h of incubation with 2.5 % oil seed waste concentration. O. xanthineolytica was found to produce two proteases-P1 and P2. The proteases were purified using 60 % cold acetone precipitation and DEAE-cellulose ion exchange chromatography. SDS-PAGE revealed molecular weight of P1 and P2 was 36 and 24 kDa, respectively. P1 and P2 were optimally active at pH 7.0 and pH 7.5 at temperature 35 and 40 °C, respectively. Analysis of hydrolyzed product of P1 and P2 by HPLC reveals that the P1 has endoprotease and P2 has exoprotease activity. The treated soy milk with immobilized proteases showed increased shelf life and removal of off flavor.

  19. Crystallization of a non-B and a B mutant HIV protease.

    PubMed

    Sanches, Mario; Martins, Nádia Helena; Calazans, Alexandre; Brindeiro, Rodrigo de Moraes; Tanuri, Amilcar; Antunes, Octavio Augusto Ceva; Polikarpov, Igor

    2004-09-01

    HIV polymorphism is responsible for the selection of variant viruses resistant to inhibitors used in AIDS treatment. Knowledge of the mechanism of resistance of those viruses is determinant to the development of new inhibitors able to stop, or at least slow down, the disease's progress caused by new mutations. In this paper, the crystallization and preliminary crystallographic structure solution for two multi-resistant 99 amino acid HIV proteases, both isolated from Brazilian patients failing intensive anti-AIDS therapy are presented, viz. the subtype B mutant, with mutations Q7K, S37N, R41K, K45R, I54V, L63P, A71V, V82A and L90M, and the subtype F (wild type), naturally carrying mutations Q7K, I15V, E35D, M36I, S37N, R41K, R57K, D60E, Q61N, I62V, L63S, I64L and L89M, with respect to the B consensus sequence. Both proteins crystallized as a complex with the inhibitor TL-3 in space group P6(1)22. X-ray diffraction data were collected from these crystals to resolutions of 2.1 and 2.6 A for the subtype B mutant and subtype F wild type, respectively, and the enzyme structures were solved by molecular replacement. The crystals of subtype F HIV protease are, to the best of the authors' knowledge, the first protein crystals obtained for a non-B HIV protease.

  20. Serine Proteases of Parasitic Helminths

    PubMed Central

    Yang, Yong; Wen, Yun jun; Cai, Ya Nan; Vallée, Isabelle; Boireau, Pascal; Liu, Ming Yuan; Cheng, Shi Peng

    2015-01-01

    Serine proteases form one of the most important families of enzymes and perform significant functions in a broad range of biological processes, such as intra- and extracellular protein metabolism, digestion, blood coagulation, regulation of development, and fertilization. A number of serine proteases have been identified in parasitic helminths that have putative roles in parasite development and nutrition, host tissues and cell invasion, anticoagulation, and immune evasion. In this review, we described the serine proteases that have been identified in parasitic helminths, including nematodes (Trichinella spiralis, T. pseudospiralis, Trichuris muris, Anisakis simplex, Ascaris suum, Onchocerca volvulus, O. lienalis, Brugia malayi, Ancylostoma caninum, and Steinernema carpocapsae), cestodes (Spirometra mansoni, Echinococcus granulosus, and Schistocephalus solidus), and trematodes (Fasciola hepatica, F. gigantica, and Schistosoma mansoni). Moreover, the possible biological functions of these serine proteases in the endogenous biological phenomena of these parasites and in the host-parasite interaction were also discussed. PMID:25748703

  1. Mosaic serine proteases in the mammalian central nervous system.

    PubMed

    Mitsui, Shinichi; Watanabe, Yoshihisa; Yamaguchi, Tatsuyuki; Yamaguchi, Nozomi

    2008-01-01

    We review the structure and function of three kinds of mosaic serine proteases expressed in the mammalian central nervous system (CNS). Mosaic serine proteases have several domains in the proenzyme fragment, which modulate proteolytic function, and a protease domain at the C-terminus. Spinesin/TMPRSS5 is a transmembrane serine protease whose presynaptic distribution on motor neurons in the spinal cord suggests that it is significant for neuronal plasticity. Cell type-specific alternative splicing gives this protease diverse functions by modulating its intracellular localization. Motopsin/PRSS12 is a mosaic protease, and loss of its function causes mental retardation. Recent reports indicate the significance of this protease for cognitive function. We mention the fibrinolytic protease, tissue plasminogen activator (tPA), which has physiological and pathological functions in the CNS.

  2. Using Hierarchical Virtual Screening To Combat Drug Resistance of the HIV-1 Protease.

    PubMed

    Li, Nan; Ainsworth, Richard I; Ding, Bo; Hou, Tingjun; Wang, Wei

    2015-07-27

    Human immunodeficiency virus (HIV) protease inhibitors (PIs) are important components of highly active anti-retroviral therapy (HAART) that block the catalytic site of HIV protease, thus preventing maturation of the HIV virion. However, with two decades of PI prescriptions in clinical practice, drug-resistant HIV mutants have now been found for all of the PI drugs. Therefore, the continuous development of new PI drugs is crucial both to combat the existing drug-resistant HIV strains and to provide treatments for future patients. Here we purpose an HIV PI drug design strategy to select candidate PIs with binding energy distributions dominated by interactions with conserved protease residues in both wild-type and various drug-resistant mutants. On the basis of this strategy, we have constructed a virtual screening pipeline including combinatorial library construction, combinatorial docking, MM/GBSA-based rescoring, and reranking on the basis of the binding energy distribution. We have tested our strategy on lopinavir by modifying its two functional groups. From an initial 751 689 candidate molecules, 18 candidate inhibitors were selected using the pipeline for experimental validation. IC50 measurements and drug resistance predictions successfully identified two ligands with both HIV protease inhibitor activity and an improved drug resistance profile on 2382 HIV mutants. This study provides a proof of concept for the integration of MM/GBSA energy analysis and drug resistance information at the stage of virtual screening and sheds light on future HIV drug design and the use of virtual screening to combat drug resistance.

  3. Monitoring the impact of an aspartic protease (MpAPr1) on grape proteins and wine properties.

    PubMed

    Theron, Louwrens Wiid; Bely, Marina; Divol, Benoit

    2018-04-23

    The perception of haze in wine is brought about when pathogenesis-related proteins become unstable and aggregate, subsequently resulting in crosslinking until it develops into light-dispersing particles. Elimination of these proteins is usually achieved via bentonite fining, which, although effective, suffers from several drawbacks. The utilization of proteases has been proposed as an ideal alternative. In a previous study, an aspartic protease (MpAPr1) from the yeast Metschnikowia pulcherrima was purified and shown to be partially active against grape proteins in synthetic medium. In this study, the effects of pure MpAPr1 supplemented to Sauvignon Blanc juice on subsequent fermentation were investigated. The juice was incubated for 48 h and thereafter inoculated with Saccharomyces cerevisiae. Results revealed that the enzyme had no observable effects on fermentation performance and retained activity throughout. Protein degradation could be detected and resulted in a significant modification of the wine composition and an increase in the presence of certain volatile compounds, especially those linked to amino acid metabolism.

  4. Structural and functional analysis of human HtrA3 protease and its subdomains

    DOE PAGES

    Glaza, Przemyslaw; Osipiuk, Jerzy; Wenta, Tomasz; ...

    2015-06-25

    Human HtrA3 protease, which induces mitochondria-mediated apoptosis, can be a tumor suppressor and a potential therapeutic target in the treatment of cancer. However, there is little information about its structure and biochemical properties. HtrA3 is composed of an N-terminal domain not required for proteolytic activity, a central serine protease domain and a C-terminal PDZ domain. HtrA3S, its short natural isoform, lacks the PDZ domain which is substituted by a stretch of 7 C-terminal amino acid residues, unique for this isoform. This paper presents the crystal structure of the HtrA3 protease domain together with the PDZ domain (ΔN-HtrA3), showing that themore » protein forms a trimer whose protease domains are similar to those of human HtrA1 and HtrA2. The ΔN-HtrA3 PDZ domains are placed in a position intermediate between that in the flat saucer-like HtrA1 SAXS structure and the compact pyramidal HtrA2 X-ray structure. The PDZ domain interacts closely with the LB loop of the protease domain in a way not found in other human HtrAs. ΔN-HtrA3 with the PDZ removed (ΔN-HtrA3-ΔPDZ) and an N-terminally truncated HtrA3S (ΔN-HtrA3S) were fully active at a wide range of temperatures and their substrate affinity was not impaired. This indicates that the PDZ domain is dispensable for HtrA3 activity. As determined by size exclusion chromatography, ΔN-HtrA3 formed stable trimers while both ΔN-HtrA3-ΔPDZ and ΔN-HtrA3S were monomeric. This suggests that the presence of the PDZ domain, unlike in HtrA1 and HtrA2, influences HtrA3 trimer formation. The unique C-terminal sequence of ΔN-HtrA3S appeared to have little effect on activity and oligomerization. Additionally, we examined the cleavage specificity of ΔN-HtrA3. Results reported in this paper provide new insights into the structure and function of ΔN-HtrA3, which seems to have a unique combination of features among human HtrA proteases.« less

  5. Structural and Functional Analysis of Human HtrA3 Protease and Its Subdomains

    PubMed Central

    Glaza, Przemyslaw; Osipiuk, Jerzy; Wenta, Tomasz; Zurawa-Janicka, Dorota; Jarzab, Miroslaw; Lesner, Adam; Banecki, Bogdan; Skorko-Glonek, Joanna; Joachimiak, Andrzej; Lipinska, Barbara

    2015-01-01

    Human HtrA3 protease, which induces mitochondria-mediated apoptosis, can be a tumor suppressor and a potential therapeutic target in the treatment of cancer. However, there is little information about its structure and biochemical properties. HtrA3 is composed of an N-terminal domain not required for proteolytic activity, a central serine protease domain and a C-terminal PDZ domain. HtrA3S, its short natural isoform, lacks the PDZ domain which is substituted by a stretch of 7 C-terminal amino acid residues, unique for this isoform. This paper presents the crystal structure of the HtrA3 protease domain together with the PDZ domain (ΔN-HtrA3), showing that the protein forms a trimer whose protease domains are similar to those of human HtrA1 and HtrA2. The ΔN-HtrA3 PDZ domains are placed in a position intermediate between that in the flat saucer-like HtrA1 SAXS structure and the compact pyramidal HtrA2 X-ray structure. The PDZ domain interacts closely with the LB loop of the protease domain in a way not found in other human HtrAs. ΔN-HtrA3 with the PDZ removed (ΔN-HtrA3-ΔPDZ) and an N-terminally truncated HtrA3S (ΔN-HtrA3S) were fully active at a wide range of temperatures and their substrate affinity was not impaired. This indicates that the PDZ domain is dispensable for HtrA3 activity. As determined by size exclusion chromatography, ΔN-HtrA3 formed stable trimers while both ΔN-HtrA3-ΔPDZ and ΔN-HtrA3S were monomeric. This suggests that the presence of the PDZ domain, unlike in HtrA1 and HtrA2, influences HtrA3 trimer formation. The unique C-terminal sequence of ΔN-HtrA3S appeared to have little effect on activity and oligomerization. Additionally, we examined the cleavage specificity of ΔN-HtrA3. Results reported in this paper provide new insights into the structure and function of ΔN-HtrA3, which seems to have a unique combination of features among human HtrA proteases. PMID:26110759

  6. Structural and Functional Analysis of Human HtrA3 Protease and Its Subdomains.

    PubMed

    Glaza, Przemyslaw; Osipiuk, Jerzy; Wenta, Tomasz; Zurawa-Janicka, Dorota; Jarzab, Miroslaw; Lesner, Adam; Banecki, Bogdan; Skorko-Glonek, Joanna; Joachimiak, Andrzej; Lipinska, Barbara

    2015-01-01

    Human HtrA3 protease, which induces mitochondria-mediated apoptosis, can be a tumor suppressor and a potential therapeutic target in the treatment of cancer. However, there is little information about its structure and biochemical properties. HtrA3 is composed of an N-terminal domain not required for proteolytic activity, a central serine protease domain and a C-terminal PDZ domain. HtrA3S, its short natural isoform, lacks the PDZ domain which is substituted by a stretch of 7 C-terminal amino acid residues, unique for this isoform. This paper presents the crystal structure of the HtrA3 protease domain together with the PDZ domain (ΔN-HtrA3), showing that the protein forms a trimer whose protease domains are similar to those of human HtrA1 and HtrA2. The ΔN-HtrA3 PDZ domains are placed in a position intermediate between that in the flat saucer-like HtrA1 SAXS structure and the compact pyramidal HtrA2 X-ray structure. The PDZ domain interacts closely with the LB loop of the protease domain in a way not found in other human HtrAs. ΔN-HtrA3 with the PDZ removed (ΔN-HtrA3-ΔPDZ) and an N-terminally truncated HtrA3S (ΔN-HtrA3S) were fully active at a wide range of temperatures and their substrate affinity was not impaired. This indicates that the PDZ domain is dispensable for HtrA3 activity. As determined by size exclusion chromatography, ΔN-HtrA3 formed stable trimers while both ΔN-HtrA3-ΔPDZ and ΔN-HtrA3S were monomeric. This suggests that the presence of the PDZ domain, unlike in HtrA1 and HtrA2, influences HtrA3 trimer formation. The unique C-terminal sequence of ΔN-HtrA3S appeared to have little effect on activity and oligomerization. Additionally, we examined the cleavage specificity of ΔN-HtrA3. Results reported in this paper provide new insights into the structure and function of ΔN-HtrA3, which seems to have a unique combination of features among human HtrA proteases.

  7. The relationship between protease/anti-protease profile, angiogenesis and re-epithelialisation in acute burn wounds.

    PubMed

    Caulfield, Robert H; Tyler, Michael P H; Austyn, Jon M; Dziewulski, Peter; McGrouther, Duncan A

    2008-06-01

    In the management of partial thickness burns, it is difficult to balance between conservative management and surgical intervention. Our hypothesis was that a triangular relationship exists between protease/anti-protease profile at the burn wound surface, angiogenesis and re-epithelialisation. By manipulation of the biochemical profile at the wound level, we determined to affect the nature and extent of angiogenesis and resulting re-epithelialisation. We performed a randomised longitudinal observational study on partial thickness burns in adult patients presenting to two regional burns units. Our results demonstrated that a high-protease wound environment is associated with lower levels of the angiogenic factor VEGF, a lower more uniform change in wound bloodflow and a uniform well healed wound with an architecturally normal epidermis. In addition, we found that a low protease wound environment is associated with higher levels of the angiogenic factor VEGF, a higher wound bloodflow throughout the wound healing period and a more chaotic, hypercellular, overkeratinised, and chaotic thickened epidermis.

  8. A novel subtilase inhibitor in plants shows structural and functional similarities to protease propeptides.

    PubMed

    Hohl, Mathias; Stintzi, Annick; Schaller, Andreas

    2017-04-14

    The propeptides of subtilisin-like serine proteinases (subtilases, SBTs) serve dual functions as intramolecular chaperones that are required for enzyme folding and as inhibitors of the mature proteases. SBT propeptides are homologous to the I9 family of protease inhibitors that have only been described in fungi. Here we report the identification and characterization of subtilisin propeptide-like inhibitor 1 (SPI-1) from Arabidopsis thaliana Sequence similarity and the shared β-α-β-β-α-β core structure identified SPI-1 as a member of the I9 inhibitor family and as the first independent I9 inhibitor in higher eukaryotes. SPI-1 was characterized as a high-affinity, tight-binding inhibitor of Arabidopsis subtilase SBT4.13 with K d and K i values in the picomolar range. SPI-1 acted as a stable inhibitor of SBT4.13 over the physiologically relevant range of pH, and its inhibitory profile included many other SBTs from plants but not bovine chymotrypsin or bacterial subtilisin A. Upon binding to SBT4.13, the C-terminal extension of SPI-1 was proteolytically cleaved. The last four amino acids at the newly formed C terminus of SPI-1 matched both the cleavage specificity of SBT4.13 and the consensus sequence of Arabidopsis SBTs at the junction of the propeptide with the catalytic domain. The data suggest that the C terminus of SPI-1 acts as a competitive inhibitor of target proteases as it remains bound to the active site in a product-like manner. SPI-1 thus resembles SBT propeptides with respect to its mode of protease inhibition. However, in contrast to SBT propeptides, SPI-1 could not substitute as a folding assistant for SBT4.13. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Purification, Cloning and Immuno-Biochemical Characterization of a Fungal Aspartic Protease Allergen Rhi o 1 from the Airborne Mold Rhizopus oryzae

    PubMed Central

    Sircar, Gaurab; Saha, Bodhisattwa; Mandal, Rahul Shubhra; Pandey, Naren; Saha, Sudipto; Gupta Bhattacharya, Swati

    2015-01-01

    Background Fungal allergy is considered as serious health problem worldwide and is increasing at an alarming rate in the industrialized areas. Rhizopus oyzae is a ubiquitously present airborne pathogenic mold and an important source of inhalant allergens for the atopic population of India. Here, we report the biochemical and immunological features of its 44 kDa sero-reactive aspartic protease allergen, which is given the official designation ‘Rhi o 1’. Method The natural Rhi o 1 was purified by sequential column chromatography and its amino acid sequence was determined by mass spectrometry and N-terminal sequencing. Based on its amino acid sequence, the cDNA sequence was identified, cloned and expressed to produce recombinant Rhi o 1. The allergenic activity of rRhi o 1 was assessed by means of its IgE reactivity and histamine release ability. The biochemical property of Rhi o 1 was studied by enzyme assay. IgE-inhibition experiments were performed to identify its cross-reactivity with the German cockroach aspartic protease allergen Bla g 2. For precise characterization of the cross-reactive epitope, we used anti-Bla g 2 monoclonal antibodies for their antigenic specificity towards Rhi o 1. A homology based model of Rhi o 1 was built and mapping of the cross-reactive conformational epitope was done using certain in silico structural studies. Results The purified natural nRhi o 1 was identified as an endopeptidase. The full length allergen cDNA was expressed and purified as recombinant rRhi o 1. Purified rRhi o 1 displayed complete allergenicity similar to the native nRhi o 1. It was recognized by the serum IgE of the selected mold allergy patients and efficiently induced histamine release from the sensitized PBMC cells. This allergen was identified as an active aspartic protease functional in low pH. The Rhi o 1 showed cross reactivity with the cockroach allergen Bla g 2, as it can inhibit IgE binding to rBla g 2 up to certain level. The rBla g 2 was also found

  10. Factors affecting the protease activity of venom from jellyfish Rhopilema esculentum Kishinouye.

    PubMed

    Li, Cuiping; Yu, Huahua; Liu, Song; Xing, Ronge; Guo, Zhanyong; Li, Pengcheng

    2005-12-15

    In this paper, the effects of some chemical and physical factors such as temperature, pH values, glycerol, and divalent metal cations on the protease activity of venom from jellyfish, Rhopilema esculentum Kishinouye, were assayed. Protease activity was dependent on temperature and pH values. Zn(2+), Mg(2+), and Mn(2+) in sodium phosphate buffer (0.02M, pH 8.0) could increase protease activity. Mn(2+) had the best effects among the three metal cations and the effect was about 20 times of that of Zn(2+) or Mg(2+) and its maximal protease activity was 2.3x10(5)U/mL. EDTA could increase protease activity. PMSF had hardly affected protease activity. O-Phenanthroline and glycerol played an important part in inhibiting protease activity and their maximal inhibiting rates were 87.5% and 82.1%, respectively.

  11. Restricting detergent protease action to surface of protein fibres by chemical modification.

    PubMed

    Schroeder, M; Lenting, H B M; Kandelbauer, A; Silva, C J S M; Cavaco-Paulo, A; Gübitz, G M

    2006-10-01

    Due to their excellent properties, such as thermostability, activity over a broad range of pH and efficient stain removal, proteases from Bacillus sp. are commonly used in the textile industry including industrial processes and laundry and represent one of the most important groups of enzymes. However, due to the action of proteases, severe damage on natural protein fibres such as silk and wool result after washing with detergents containing proteases. To include the benefits of proteases in a wool fibre friendly detergent formulation, the soluble polymer polyethylene glycol (PEG) was covalently attached to a protease from Bacillus licheniformis. In contrast to activation of PEG with cyanuric chloride (50%) activation with 1,1'-carbonyldiimidazole (CDI) lead to activity recovery above 90%. With these modified enzymes, hydrolytic attack on wool fibres could be successfully prevented up to 95% compared to the native enzymes. Colour difference (DeltaE) measured in the three dimensional colour space showed good stain removal properties for the modified enzymes. Furthermore, half-life of the modified enzymes in buffers and commercial detergents solutions was nearly twice as high as those of the non-modified enzymes with values of up to 63 min. Out of the different modified proteases especially the B. licheniformis protease with the 2.0-kDa polymer attached both retained stain removal properties and did not hydrolyse/damage wool fibres.

  12. Stability and detergent compatibility of a predominantly β-sheet serine protease from halotolerant B. aquimaris VITP4 strain.

    PubMed

    Thaz, Chittoor Jabeena; Jayaraman, Gurunathan

    2014-01-01

    The present study deals with the characterization of halotolerant protease produced by Bacillus aquimaris VITP4 strain isolated from Kumta coast, Karnataka, India. The studies were performed at 40 °C and pH 8 in Tris buffer. Metal ions such as Mn(2+) and Ca(2+) increased the proteolytic activity of the enzyme by 34 and 30 %, respectively, at 10 mM concentration. Cu(2+) at 1 mM concentration was found to enhance the enzyme activity by 16 %, whereas inhibition was observed at higher concentration (>5 mM). Slight inhibition was observed even with lower (>1 mM) concentrations of Zn(2+), Hg(2+), Fe(3+), Ni(2+), and Co(2+).The activity of protease was completely inhibited by phenylmethylsulfonyl fluoride, indicating that the VITP4 protease is a serine protease. The presence of ethylenediaminetetraacetic acid and 1,10-phenanthroline (>5 mM) moderately inhibited the activity, suggesting that the enzyme is activated by metal ions. The protease was purified to homogeneity with a purification fold of 15.7 with ammonium sulfate precipitation and 46.65 with gel filtration chromatography using Sephadex G-100, resulting in a specific activity of 424 ± 2.6 U mg(-1). The VITP4 protease consists of a single polypeptide chain with a molecular mass of 34.7 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight. Among the different substrates used (casein, egg albumin, gelatin, and bovine serum albumin), the activity was higher with casein with V max, K m, and k cat values of 0.817 mg ml min(-1), 0.472 mg ml(-1), and 2.31 s(-1), respectively. Circular dichroism studies revealed that the VITP4 protease has a predominantly β-sheet structure (51.6 %) with a temperature for half denaturation of 85.8 °C in the presence of 1 mM CaCl2. Additionally, the VITP4 protease was found to retain more than 70 % activity in the presence of 10 mM concentration of different detergents

  13. Production and purification of a protease, a chitosanase, and chitin oligosaccharides by Bacillus cereus TKU022 fermentation.

    PubMed

    Liang, Tzu-Wen; Hsieh, Jia-Lin; Wang, San-Lang

    2012-11-15

    A protease- and chitosanase-producing strain was isolated and identified as Bacillus cereus TKU022. The protease and chitosanase were both produced using 1.5% (w/v) shrimp head powder (SHP) as the sole carbon/nitrogen source, and these enzymes were purified from the culture supernatant. The molecular masses of the TKU022 protease and chitosanase determined using SDS-PAGE were approximately 45 and 44kDa, respectively. The high stability of the TKU022 protease toward surfactants, an optimal pH of 10 and an optimal temperature of 50-60°C suggest that this high-alkaline protease has potential applications for various industrial processes. Concomitant with the production of the TKU022 chitosanase, N-acetyl chitooligosaccharides were also observed in the culture supernatant, including (GlcNAc)(2), (GlcNAc)(4), (GlcNAc)(5), and (GlcNAc)(6) at concentrations of 201.5, 12.4, 0.5, and 0.3μg/mL, respectively, as determined using an HPLC analysis. The chitin oligosaccharides products were also characterized using a MALDI-TOF mass spectrometer. A combination of the HPLC and MALDI-TOF MS results showed that the chitin oligosaccharides of the TKU022 culture supernatant comprise oligomers with degree of polymerization (DP) from 2 to 6. Using this method, the production of a protease, a chitosanase, and chitin oligosaccharides may be useful for various industrial and biological applications. Copyright © 2012 Elsevier Ltd. All rights reserved.

  14. Cloning, characterization, expression and antifungal activity of an alkaline serine protease of Aureobasidium pullulans PL5 involved in the biological control of postharvest pathogens.

    PubMed

    Zhang, Dianpeng; Spadaro, Davide; Valente, Silvia; Garibaldi, Angelo; Gullino, Maria Lodovica

    2012-02-15

    An alkaline protease gene was amplified from genomic DNA and cDNA of the antagonistic yeast-like fungus Aureobasidium pullulans PL5, a biocontrol agent effective against Monilinia laxa on stone fruit and Botrytis cinerea and Penicillium expansum on pome fruits. An open reading frame of 1248 bp encoding a 415-amino acid (aa) protein with a calculated molecular weight (M(r)) of 42.9 kDa and an isoelectric point (pI) of 4.5 was characterized. The cDNAALP5 gene had an 18-amino acid signal peptide, one N-gylcosylation, one histidine active site, and one serine active site. The ALP5 gene with a M(r) of 1351 bp contained two introns. One intron was of 54 bp, while the other was of 50 bp. Protein BLAST and phylogenetic tree analysis of the deduced amino sequences from the cDNAALP5 gene showed that the encoded protein had 100% homology to a protease enzyme (ALP2) of a sea strain of A. pullulans, suggesting that the protein ALP5 was an alkaline serine protease. Expression of ALP5 in Escherichia coli BL21 (DE3), followed by identification with Western-blotting, purification with Ni-NTA and analysis of enzymatic activity, yielded an homogeneous recombinant ALP5 which hydrolysed the substrate casein and inhibited the mycelial growth of the pathogens. At its optimal pH of 10.0 and reaction temperature of 50°C, the recombinant protease exhibited the highest activity towards the substrate casein, though the highest stability was at lower temperatures and pH between 7.0 and 9.0. This study provided the direct evidence that extracellular proteases secreted by the antagonist A. pullulans PL5 played a role in the biocontrol activities against some postharvest pathogens of apple and peach. Copyright © 2011 Elsevier B.V. All rights reserved.

  15. A Bottom-Up Proteomic Approach to Identify Substrate Specificity of Outer-Membrane Protease OmpT.

    PubMed

    Wood, Sarah E; Sinsinbar, Gaurav; Gudlur, Sushanth; Nallani, Madhavan; Huang, Che-Fan; Liedberg, Bo; Mrksich, Milan

    2017-12-22

    Identifying peptide substrates that are efficiently cleaved by proteases gives insights into substrate recognition and specificity, guides development of inhibitors, and improves assay sensitivity. Peptide arrays and SAMDI mass spectrometry were used to identify a tetrapeptide substrate exhibiting high activity for the bacterial outer-membrane protease (OmpT). Analysis of protease activity for the preferred residues at the cleavage site (P1, P1') and nearest-neighbor positions (P2, P2') and their positional interdependence revealed FRRV as the optimal peptide with the highest OmpT activity. Substituting FRRV into a fragment of LL37, a natural substrate of OmpT, led to a greater than 400-fold improvement in OmpT catalytic efficiency, with a k cat /K m value of 6.1×10 6  L mol -1  s -1 . Wild-type and mutant OmpT displayed significant differences in their substrate specificities, demonstrating that even modest mutants may not be suitable substitutes for the native enzyme. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Zebra chip disease decreases tuber (Solanum tuberosum L.) protein content by attenuating protease inhibitor levels and increasing protease activities.

    PubMed

    Kumar, G N Mohan; Knowles, Lisa O; Knowles, N Richard

    2015-11-01

    Zebra chip disease of potato decreases protease inhibitor levels resulting in enhanced serine-type protease activity, decreased protein content and altered protein profiles of fully mature tubers. Zebra-chip (ZC), caused by Candidatus Liberibacter solanacearum (CLso), is a relatively new disease of potato that negatively affects growth, yield, propagation potential, and fresh and process qualities of tubers. Diseased plants produce tubers with characteristic brown discoloration of vascular tissue accompanied by elevated levels of free amino acids and reducing sugars. Here we demonstrate that ZC disease induces selective protein catabolism in tubers through modulating protease inhibitor levels. Soluble protein content of tubers from CLso-infected plants was 33% lower than from non-infected plants and electrophoretic analyses revealed substantial reductions in major tuber proteins. Patatin (~40 kDa) and ser-, asp- (22 kDa) and cys-type (85 kDa) protease inhibitors were either absent or greatly reduced in ZC-afflicted tubers. In contrast to healthy (non-infected) tubers, the proteolytic activity in CLso infected tubers was high and the ability of extracts from infected tubers to inhibit trypsin (ser-type) and papain (cys-type) proteases greatly attenuated. Moreover, extracts from CLso-infected tubers rapidly catabolized proteins purified from healthy tubers (40 kDa patatin, 22 kDa protease inhibitors, 85 kDa potato multicystatin) when subjected to proteolysis individually. In contrast, crude extracts from non-infected tubers effectively inhibited the proteolytic activity from ZC-afflicted tubers. These results suggest that the altered protein profile of ZC afflicted tubers is largely due to loss of ser- and cys-type protease inhibitors. Further analysis revealed a novel PMSF-sensitive (ser) protease (ca. 80-120 kDa) in CLso infected tubers. PMSF abolished the proteolytic activities responsible for degrading patatin, the 22 kDa protease inhibitor(s) and potato

  17. The human CLN2 protein/tripeptidyl-peptidase I is a serine protease that autoactivates at acidic pH.

    PubMed

    Lin, L; Sohar, I; Lackland, H; Lobel, P

    2001-01-19

    The CLN2 gene mutated in the fatal hereditary neurodegenerative disease late infantile neuronal ceroid lipofuscinosis encodes a lysosomal protease with tripeptidyl-peptidase I activity. To understand the enzymological properties of the protein, we purified and characterized C-terminal hexahistidine-tagged human CLN2p/tripeptidyl-peptidase I produced from insect cells transfected with a baculovirus vector. The N terminus of the secreted 66-kDa protein corresponds to residue 20 of the primary CLN2 gene translation product, indicating removal of a 19-residue signal peptide. The purified protein is enzymatically inactive; however, upon acidification, it is proteolytically processed and concomitantly acquires enzymatic activity. The N terminus of the final 46-kDa processed form (Leu196) corresponds to that of mature CLN2p/tripeptidyl-peptidase I purified from human brain. The activity of the mature enzyme is irreversibly inhibited by the serine esterase inhibitor diisopropyl fluorophosphate, which specifically and stoichiometrically reacts with CLN2p/tripeptidyl-peptidase I at Ser475, demonstrating that this residue represents the active site nucleophile. Expression of wild type and mutant proteins in CHO cells indicate that Ser475, Asp360, Asp517, but not His236 are essential for activity. These data indicate that the CLN2 gene product is synthesized as an inactive proenzyme that is autocatalytically converted to an active serine protease.

  18. Sequence analysis and heterologous expression of the wool cuticle-degrading enzyme encoding genes in Fusarium oxysporum 26-1.

    PubMed

    Chaya, Etsushi; Suzuki, Tohru; Karita, Shuichi; Hanya, Akira; Yoshino-Yasuda, Shoko; Kitamoto, Noriyuki

    2014-06-01

    Two protease-like proteins, KrtA and KrtC, were identified in Fusarium oxysporum 26-1. Genes coding these proteins, krtA and krtC, were isolated and characterized. Recombinant KrtA (rKrtA) and KrtC (rKrtC) were successfully expressed in Aspergillus oryzae and secreted. The combination of rKrtA and rKrtC completely removed the cuticle of wool fibers. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  19. Saccharomyces boulardii protease inhibits Clostridium difficile toxin A effects in the rat ileum.

    PubMed Central

    Castagliuolo, I; LaMont, J T; Nikulasson, S T; Pothoulakis, C

    1996-01-01

    Saccharomyces boulardii, a nonpathogenic yeast, is effective in treating some patients with Clostridium difficile diarrhea and colitis. We have previously reported that S. boulardii inhibits rat ileal secretion in response to C. difficile toxin A possibly by releasing a protease that digests the intestinal receptor for this toxin (C. Pothoulakis, C. P. Kelly, M. A. Joshi, N. Gao, C. J. O'Keane, I. Castagliuolo, and J. T. LaMont, Gastroenterology 104: 1108-1115, 1993). The aim of this study was to purify and characterize this protease. S. boulardii protease was partially purified by gel filtration on Sephadex G-50 and octyl-Sepharose. The effect of S. boulardii protease on rat ileal secretion, epithelial permeability, and morphology in response to toxin A was examined in rat ileal loops in vivo. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified S. boulardii protease revealed a major band at 54 kDa. Pretreatment of rat ileal brush border (BB) membranes with partially purified protease reduced specific toxin A receptor binding (by 26%). Partially purified protease digested the toxin A molecule and significantly reduced its binding to BB membranes in vitro (by 42%). Preincubation of toxin A with S. boulardii protease inhibited ileal secretion (46% inhibition, P < 0.01), mannitol permeability (74% inhibition, P < 0.01), and histologic damage caused by toxin A. Thus, S. boulardii protease inhibits the intestinal effects of C. difficile toxin A by proteolysis of the toxin and inhibition of toxin A binding to its BB receptor. Our results may be relevant to the mechanism by which S. boulardii exerts its protective effects in C. difficile infection in humans. PMID:8945570

  20. SERPINE1: A Molecular Switch in the Proliferation-Migration Dichotomy in Wound-“Activated” Keratinocytes

    PubMed Central

    Simone, Tessa M.; Higgins, Craig E.; Czekay, Ralf-Peter; Law, Brian K.; Higgins, Stephen P.; Archambeault, Jaclyn; Kutz, Stacie M.; Higgins, Paul J.

    2014-01-01

    Significance: A highly interactive serine protease/plasmin/matrix metalloproteinase axis regulates stromal remodeling in the wound microenvironment. Current findings highlight the importance of stringent controls on protease expression and their topographic activities in cell proliferation, migration, and tissue homeostasis. Targeting elements in this cascading network may lead to novel therapeutic approaches for fibrotic diseases and chronic wounds. Recent Advances: Matrix-active proteases and their inhibitors orchestrate wound site tissue remodeling, cell migration, and proliferation. Indeed, the serine proteases urokinase plasminogen activator and tissue-type plasminogen activator (uPA/tPA) and their major phsyiological inhibitor, plasminogen activator inhibitor-1 (PAI-1; serine protease inhibitor clade E member 1 [SERPINE1]), are upregulated in several cell types during injury repair. Coordinate expression of proteolytic enzymes and their inhibitors in the wound bed provides a mechanism for fine control of focal proteolysis to facilitate matrix restructuring and cell motility in complex environments. Critical Issues: Cosmetic and tissue functional consequences of wound repair anomalies affect the quality of life of millions of patients in the United States alone. The development of novel therapeutics to manage individuals most affected by healing anomalies will likely derive from the identification of critical, translationally accessible, control elements in the wound site microenvironment. Future Directions: Activation of the PAI-1 gene early after wounding, its prominence in the repair transcriptome and varied functions suggest a key role in the global cutaneous injury response program. Targeting PAI-1 gene expression and/or PAI-1 function with molecular genetic constructs, neutralizing antibodies or small molecule inhibitors may provide a novel, therapeutically relevant approach, to manage the pathophysiology of wound healing disorders associated with