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Sample records for a1 proteases evidence

  1. 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

  2. Pneumococcal IgA1 protease subverts specific protection by human IgA1.

    PubMed

    Janoff, E N; Rubins, J B; Fasching, C; Charboneau, D; Rahkola, J T; Plaut, A G; Weiser, J N

    2014-03-01

    Bacterial immunoglobulin A1 (IgA1) proteases may sabotage the protective effects of IgA. In vitro, both exogenous and endogenously produced IgA1 protease inhibited phagocytic killing of Streptococcus pneumoniae by capsule-specific IgA1 human monoclonal antibodies (hMAbs) but not IgA2. These IgA1 proteases cleaved and reduced binding of the the effector Fcα1 heavy chain but not the antigen-binding F(ab)/light chain to pneumococcal surfaces. In vivo, IgA1 protease-resistant IgA2, but not IgA1 protease-sensitive IgA1, supported 60% survival in mice infected with wild-type S. pneumoniae. IgA1 hMAbs protected mice against IgA1 protease-deficient but not -producing pneumococci. Parallel mouse sera with human IgA2 showed more efficient complement-mediated reductions in pneumococci with neutrophils than did IgA1, particularly with protease-producing organisms. After natural human pneumococcal bacteremia, purified serum IgG inhibited IgA1 protease activity in 7 of 11 patients (64%). These observations provide the first evidence in vivo that IgA1 protease can circumvent killing of S. pneumoniae by human IgA. Acquisition of IgA1 protease-neutralizing IgG after infection directs attention to IgA1 protease both as a determinant of successful colonization and infection and as a potential vaccine candidate. PMID:23820749

  3. Amino acid sequence requirements in the hinge of human immunoglobulin A1 (IgA1) for cleavage by streptococcal IgA1 proteases.

    PubMed

    Batten, Margaret R; Senior, Bernard W; Kilian, Mogens; Woof, Jenny M

    2003-03-01

    The amino acid sequence requirements in the hinge of human immunoglobulin A1 (IgA1) for cleavage by IgA1 proteases of different species of Streptococcus were investigated. Recombinant IgA1 antibodies were generated with point mutations at proline 227 and threonine 228, the residues lying on either side of the peptide bond at which all streptococcal IgA1 proteases cleave wild-type human IgA1. The amino acid substitutions produced no major effect upon the structure of the mutant IgA1 antibodies or their functional ability to bind to Fcalpha receptors. However, the substitutions had a substantial effect upon sensitivity to cleavage with some streptococcal IgA1 proteases, with, in some cases, a single point mutation rendering the antibody resistant to a particular IgA1 protease. This effect was least marked with the IgA1 protease from Streptococcus pneumoniae, which showed no absolute requirement for either proline or threonine at residues 227 to 228. By contrast, the IgA1 proteases of Streptococcus oralis, Streptococcus sanguis, and Streptococcus mitis had an absolute requirement for proline at 227 but not for threonine at 228, which could be replaced by valine. There was evidence in S. mitis that proteases from different strains may have different amino acid requirements for cleavage. Remarkably, some streptococcal proteases appeared able to cleave the hinge at a distant alternative site if substitution prevented efficient cleavage of the original site. Hence, this study has identified key residues required for the recognition of the IgA1 hinge as a substrate by streptococcal IgA1 proteases, and it marks a preliminary step towards development of specific enzyme inhibitors. PMID:12595464

  4. Degradation of phycobilisomes in Synechocystis sp. PCC6803: evidence for essential formation of an NblA1/NblA2 heterodimer and its codegradation by A Clp protease complex.

    PubMed

    Baier, Antje; Winkler, Wiebke; Korte, Thomas; Lockau, Wolfgang; Karradt, Anne

    2014-04-25

    When cyanobacteria acclimate to nitrogen deficiency, they degrade their large (3-5-MDa), light-harvesting complexes, the phycobilisomes. This massive, yet specific, intracellular degradation of the pigmented phycobiliproteins causes a color change of cyanobacterial cultures from blue-green to yellow-green, a process referred to as chlorosis or bleaching. Phycobilisome degradation is induced by expression of the nblA gene, which encodes a protein of ~7 kDa. NblA most likely acts as an adaptor protein that guides a Clp protease to the phycobiliproteins, thereby initiating the degradation process. Most cyanobacteria and red algae possess just one nblA-homologous gene. As an exception, the widely used "model organism" Synechocystis sp. PCC6803 expresses two such genes, nblA16803 and nblA26803, both of whose products are required for phycobilisome degradation. Here, we demonstrate that the two NblA proteins heterodimerize in vitro and in vivo using pull-down assays and a Förster energy-transfer approach, respectively. We further show that the NblA proteins form a ternary complex with ClpC (the HSP100 chaperone partner of Clp proteases) and phycobiliproteins in vitro. This complex is susceptible to ATP-dependent degradation by a Clp protease, a finding that supports a proposed mechanism of the degradation process. Expression of the single nblA gene encoded by the genome of the N2-fixing, filamentous cyanobacterium Nostoc sp. PCC7120 in the nblA1/nblA2 mutant of Synechocystis sp. PCC6803 induced phycobilisome degradation, suggesting that the function of the NblA heterodimer of Synechocystis sp. PCC6803 is combined in the homodimeric protein of Nostoc sp. PCC7120. PMID:24610785

  5. Immunoglobulin A1 Protease, an Exoenzyme of Pathogenic Neisseriae, Is a Potent Inducer of Proinflammatory Cytokines

    PubMed Central

    Lorenzen, Dirk R.; Düx, Frank; Wölk, Uwe; Tsirpouchtsidis, Anastasios; Haas, Gaby; Meyer, Thomas F.

    1999-01-01

    A characteristic of human pathogenic Neisseriae is the production and secretion of an immunoglobulin (Ig)A1-specific serine protease (IgA1 protease) that cleaves preferentially human IgA1 and other target proteins. Here we show a novel function for native IgA1 protease, i.e., the induction of proinflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-8 from peripheral blood mononuclear cells. The capacity of IgA1 protease to elicit such cytokine responses in monocytes was enhanced in the presence of T lymphocytes. IgA1 protease did not induce the regulatory cytokine IL-10, which was, however, found in response to lipopolysaccharide and phytohemagglutinin. The immunomodulatory effects caused by IgA1 protease require a native form of the enzyme, and denaturation abolished cytokine induction. However, the proteolytic activity is not required for the cytokine induction by IgA1 protease. Our results indicate that IgA1 protease exhibits important immunostimulatory properties and may contribute substantially to the pathogenesis of neisserial infections by inducing large amounts of TNF-α and other proinflammatory cytokines. In particular, IgA1 protease may represent a key virulence determinant of bacterial meningitis. PMID:10523603

  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. PMID:9220164

  7. A Neisseria gonorrhoeae Immunoglobulin A1 Protease Mutant Is Infectious in the Human Challenge Model of Urethral Infection

    PubMed Central

    Johannsen, Diana B.; Johnston, David M.; Koymen, Hakan O.; Cohen, Myron S.; Cannon, Janne G.

    1999-01-01

    Many mucosal pathogens, including Neisseria gonorrhoeae, produce proteases that cleave immunoglobulin A (IgA), the predominant immunoglobulin class produced at mucosal surfaces. While considerable circumstantial evidence suggests that IgA1 protease contributes to gonococcal virulence, there is no direct evidence that N. gonorrhoeae requires IgA1 protease activity to infect a human host. We constructed a N. gonorrhoeae iga mutant without introducing new antibiotic resistance markers into the final mutant strain and used human experimental infection to test the ability of the mutant to colonize the male urethra and to cause gonococcal urethritis. Four of the five male volunteers inoculated with the Iga− mutant became infected. In every respect—clinical signs and symptoms, incubation period between inoculation and infection, and the proportion of volunteers infected—the outcome of human experimental infection with FA1090iga was indistinguishable from that previously reported for a variant of parent strain FA1090 matching the mutant in expression of Opa proteins, lipooligosaccharide, and pilin. These results indicate that N. gonorrhoeae does not require IgA1 protease production to cause experimental urethritis in males. PMID:10338512

  8. Detection of immunoglobulin A1 protease-induced Fab alpha fragments on dental plaque bacteria.

    PubMed Central

    Ahl, T; Reinholdt, J

    1991-01-01

    The mechanisms by which immunoglobulin A1 (IgA1) protease activity may enable bacteria to evade the effect of specific secretory IgA (S-IgA) antibodies are not clear. A possibility which has received indirect experimental support is that bacteria, as a consequence of the protease activity, become coated with incompetent Fab alpha fragments instead of with intact antibody molecules. Using a combination of nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting, we detected Fab alpha fragments not only on oral streptococci (Streptococcus sanguis and Streptococcus gordonii) incubated in saliva but also on the bacteria in incipient dental plaque. These results are of relevance to our previous observation that IgA1 protease activity may neutralize the ability of S-IgA antibodies to inhibit the adherence of oral streptococci to saliva-coated hydroxyapatite. Images PMID:1987074

  9. Comparative analysis of immunoglobulin A1 protease activity among bacteria representing different genera, species, and strains.

    PubMed Central

    Reinholdt, J; Kilian, M

    1997-01-01

    Immunoglobulin A1 (IgA1) proteases cleaving human IgA1 in the hinge region are produced constitutively by a number of pathogens, including Haemophilus influenzae, Neisseria meningitidis, Neisseria gonorrhoeae, and Streptococcus pneumoniae, as well as by some members of the resident oropharyngeal flora. Whereas IgA1 proteases have been shown to interfere with the functions of IgA antibodies in vitro, the exact role of these enzymes in the relationship of bacteria to a human host capable of responding with enzyme-neutralizing antibodies is not clear. Conceivably, the role of IgA1 proteases may depend on the quantity of IgA1 protease generated as well as on the balance between secreted and cell-associated forms of the enzyme. Therefore, we have compared levels of IgA1 protease activity in cultures of 38 bacterial strains representing different genera and species as well as strains of different pathogenic potential. Wide variation in activity generation rate was found overall and within some species. High activity was not an exclusive property of bacteria with documented pathogenicity. Almost all activity of H. influenzae, N. meningitidis, and N. gonorrhoeae strains was present in the supernatant. In contrast, large proportions of the activity in Streptococcus, Prevotella, and Capnocytophaga species was cell associated at early stationary phase, suggesting that the enzyme may play the role of a surface antigen. Partial release of cell-associated activity occurred during stationary phase. Within some taxa, the degree of activity variation correlated with degree of antigenic diversity of the enzyme as determined previously. This finding may indicate that the variation observed is of biological significance. PMID:9353019

  10. Evidence for Reduced Drug Susceptibility without Emergence of Major Protease Mutations following Protease Inhibitor Monotherapy Failure in the SARA Trial

    PubMed Central

    Sutherland, Katherine A.; Parry, Chris M.; McCormick, Adele; Kapaata, Anne; Lyagoba, Fred; Kaleebu, Pontiano; Gilks, Charles F.; Goodall, Ruth; Spyer, Moira; Kityo, Cissy; Pillay, Deenan; Gupta, Ravindra K.

    2015-01-01

    Background Major protease mutations are rarely observed following failure with protease inhibitors (PI), and other viral determinants of failure to PI are poorly understood. We therefore characterized Gag-Protease phenotypic susceptibility in subtype A and D viruses circulating in East Africa following viral rebound on PIs. Methods Samples from baseline and treatment failure in patients enrolled in the second line LPV/r trial SARA underwent phenotypic susceptibility testing. Data were expressed as fold-change in susceptibility relative to a LPV-susceptible reference strain. Results We cloned 48 Gag-Protease containing sequences from seven individuals and performed drug resistance phenotyping from pre-PI and treatment failure timepoints in seven patients. For the six patients where major protease inhibitor resistance mutations did not emerge, mean fold-change EC50 to LPV was 4.07 fold (95% CI, 2.08–6.07) at the pre-PI timepoint. Following viral failure the mean fold-change in EC50 to LPV was 4.25 fold (95% CI, 1.39–7.11, p = 0.91). All viruses remained susceptible to DRV. In our assay system, the major PI resistance mutation I84V, which emerged in one individual, conferred a 10.5-fold reduction in LPV susceptibility. One of the six patients exhibited a significant reduction in susceptibility between pre-PI and failure timepoints (from 4.7 fold to 9.6 fold) in the absence of known major mutations in protease, but associated with changes in Gag: V7I, G49D, R69Q, A120D, Q127K, N375S and I462S. Phylogenetic analysis provided evidence of the emergence of genetically distinct viruses at the time of treatment failure, indicating ongoing viral evolution in Gag-protease under PI pressure. Conclusions Here we observe in one patient the development of significantly reduced susceptibility conferred by changes in Gag which may have contributed to treatment failure on a protease inhibitor containing regimen. Further phenotype-genotype studies are required to elucidate genetic

  11. Modulation of the Bacillus anthracis secretome by the immune inhibitor A1 protease.

    PubMed

    Pflughoeft, Kathryn J; Swick, Michelle C; Engler, David A; Yeo, Hye-Jeong; Koehler, Theresa M

    2014-01-01

    The Bacillus anthracis secretome includes protective antigen, lethal factor, and edema factor, which are the components of anthrax toxin, and other proteins with known or potential roles in anthrax disease. Immune inhibitor A1 (InhA1) is a secreted metalloprotease that is unique to pathogenic members of the Bacillus genus and has been associated with cleavage of host proteins during infection. Here, we report the effect of InhA1 on the B. anthracis secretome. Differential in-gel electrophoresis of proteins present in culture supernatants from a parent strain and an isogenic inhA1-null mutant revealed multiple differences. Of the 1,340 protein spots observed, approximately one-third were less abundant and one-third were more abundant in the inhA1 secretome than in the parent strain secretome. Proteases were strongly represented among those proteins exhibiting a 9-fold or greater change. InhA1 purified from a B. anthracis culture supernatant directly cleaved each of the anthrax toxin proteins as well as an additional secreted protease, Npr599. The conserved zinc binding motif HEXXH of InhA1 (HEYGH) was critical for its proteolytic activity. Our data reveal that InhA1 directly and indirectly modulates the form and/or abundance of over half of all the secreted proteins of B. anthracis. The proteolytic activity of InhA1 on established secreted virulence factors, additional proteases, and other secreted proteins suggests that this major protease plays an important role in virulence not only by cleaving mammalian substrates but also by modulating the B. anthracis secretome itself. PMID:24214942

  12. Effect of mutations in the human immunoglobulin A1 (IgA1) hinge on its susceptibility to cleavage by diverse bacterial IgA1 proteases.

    PubMed

    Senior, Bernard W; Woof, Jenny M

    2005-03-01

    Components of the human immunoglobulin A1 (IgA1) hinge governing sensitivity to cleavage by bacterial IgA1 proteases were investigated. Recombinant antibodies with distinct hinge mutations were constructed from a hybrid comprised of human IgA2 bearing half of the human IgA1 hinge region. This hybrid antibody and all the mutant antibodies derived from it were resistant to cleavage by the IgA1 proteases from Streptococcus oralis and Streptococcus mitis biovar 1 strains but were cleaved to various degrees by those of Streptococcus pneumoniae, some Streptococcus sanguis strains, and the type 1 and 2 IgA1 proteases of Haemophilus influenzae, Neisseria meningitidis, and Neisseria gonorrhoeae. Remarkably, those proteases that cleave a Pro-Ser peptide bond in the wild-type IgA1 hinge were able to cleave mutant antibodies lacking a Pro-Ser peptide bond in the hinge, and those that cleave a Pro-Thr peptide bond in the wild-type IgA1 hinge were able to cleave mutant antibodies devoid of a Pro-Thr peptide bond in the hinge. Thus, the enzymes can cleave alternatives to their preferred postproline peptide bond when such a bond is unavailable. Peptide sequence analysis of a representative antibody digestion product confirmed this conclusion. The presence of a cleavable peptide bond near the CH2 end of the hinge appeared to result in greater cleavage than if the scissile bond was at the CH1 end of the hinge. Proline-to-serine substitution at residue 230 in a hinge containing potentially cleavable Pro-Ser and Pro-Thr peptide bonds increased the resistance of the antibody to cleavage by many IgA1 proteases. PMID:15731049

  13. Protoporphyrins Enhance Oligomerization and Enzymatic Activity of HtrA1 Serine Protease

    PubMed Central

    Jo, Hakryul; Patterson, Victoria; Stoessel, Sean; Kuan, Chia-Yi; Hoh, Josephine

    2014-01-01

    High temperature requirement protein A1 (HtrA1), a secreted serine protease of the HtrA family, is associated with a multitude of human diseases. However, the exact functions of HtrA1 in these diseases remain poorly understood. We seek to unravel the mechanisms of HtrA1 by elucidating its interactions with chemical or biological modulators. To this end, we screened a small molecule library of 500 bioactive compounds to identify those that alter the formation of extracellular HtrA1 complexes in the cell culture medium. An initial characterization of two novel hits from this screen showed that protoporphyrin IX (PPP-IX), a precursor in the heme biosynthetic pathway, and its metalloporphyrin (MPP) derivatives fostered the oligomerization of HtrA1 by binding to the protease domain. As a result of the interaction with MPPs, the proteolytic activity of HtrA1 against Fibulin-5, a specific HtrA1 substrate in age-related macular degeneration (AMD), was increased. This physical interaction could be abolished by the missense mutations of HtrA1 found in patients with cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL). Furthermore, knockdown of HtrA1 attenuated apoptosis induced by PPP-IX. These results suggest that PPP-IX, or its derivatives, and HtrA1 may function as co-factors whereby porphyrins enhance oligomerization and the protease activity of HtrA1, while active HtrA1 elevates the pro-apoptotic actions of porphyrin derivatives. Further analysis of this interplay may shed insights into the pathogenesis of diseases such as AMD, CARASIL and protoporphyria, as well as effective therapeutic development. PMID:25506911

  14. Sites in the CH3 domain of human IgA1 that influence sensitivity to bacterial IgA1 proteases.

    PubMed

    Senior, Bernard W; Woof, Jenny M

    2006-09-15

    The influence of regions, other than the hinge, on the susceptibility of human IgA1 to cleavage by diverse bacterial IgA1 proteases, was examined using IgA1 mutants bearing amino acid deletions, substitutions, and domain swaps. IgA1 lacking the tailpiece retained its susceptibility to cleavage by all of the IgA1 proteases. The domain swap molecule alpha1alpha2gamma3, in which the CH3 domain of IgA1 was exchanged for that of human IgG1, was resistant to cleavage with the type 1 and 2 serine IgA1 proteases of Neisseria meningitidis, Neisseria gonorrhoeae, and Haemophilus influenzae, but remained sensitive to cleavage with the metallo-IgA1 proteases of Streptococcus pneumoniae, Streptococcus oralis, Streptococcus sanguis, and Streptococcus mitis. Substitution of the IgA1 Calpha3 domain motif Pro440 -Phe443 into the corresponding position in the Cgamma3 domain of alpha1alpha2gamma3 resulted now in sensitivity to the type 2 IgA1 protease of N. meningitidis, indicating the possible requirement of these amino acids for sensitivity to this protease. For the H. influenzae type 2 protease, resistance of an IgA1 mutant in which the CH3 domain residues 399-409 were exchanged with those from IgG1, but sensitivity of mutant HuBovalpha3 in which the Calpha3 domain of bovine IgA replaces that of human IgA1, suggests that CH3 domain residues Glu403, Gln406, and Thr409 influence sensitivity to this enzyme. Hence, unlike the situation with the metallo-IgA1 proteases of Streptococcus spp., the sensitivity of human IgA1 to cleavage with the serine IgA1 proteases of Neisseria and Haemophilus involves their binding to different sites specifically in the CH3 domain. PMID:16951354

  15. Proresolving Actions of Synthetic and Natural Protease Inhibitors Are Mediated by Annexin A1.

    PubMed

    Vago, Juliana P; Tavares, Luciana P; Sugimoto, Michelle A; Lima, Graziele Letícia N; Galvão, Izabela; de Caux, Thais R; Lima, Kátia M; Ribeiro, Ana Luíza C; Carneiro, Fernanda S; Nunes, Fernanda Freire C; Pinho, Vanessa; Perretti, Mauro; Teixeira, Mauro M; Sousa, Lirlândia P

    2016-02-15

    Annexin A1 (AnxA1) is a glucocorticoid-regulated protein endowed with anti-inflammatory and proresolving properties. Intact AnxA1 is a 37-kDa protein that may be cleaved in vivo at the N-terminal region by neutrophil proteases including elastase and proteinase-3, generating the 33-kDa isoform that is largely inactive. In this study, we investigated the dynamics of AnxA1 expression and the effects of synthetic (sivelestat [SIV]; Eglin) and natural (secretory leukocyte protease inhibitor [SLPI]; Elafin) protease inhibitors on the resolution of LPS-induced inflammation. During the settings of LPS inflammation AnxA1 cleavage associated closely with the peak of neutrophil and elastase expression and activity. SLPI expression increased during resolving phase of the pleurisy. Therapeutic treatment of LPS-challenge mice with recombinant human SLPI or Elafin accelerated resolution, an effect associated with increased numbers of apoptotic neutrophils in the pleural exudates, inhibition of elastase, and modulation of the survival-controlling proteins NF-κB and Mcl-1. Similar effects were observed with SIV, which dose-dependently inhibited neutrophil elastase and shortened resolution intervals. Mechanistically, SIV-induced resolution was caspase-dependent, associated to increased levels of intact AnxA1 and decreased expression of NF-κB and Mcl-1. The proresolving effect of antiproteases was also observed in a model of monosodium urate crystals-induced inflammation. SIV skewed macrophages toward resolving phenotypes and enhanced efferocytosis of apoptotic neutrophils. A neutralizing antiserum against AnxA1 and a nonselective antagonist of AnxA1 receptor abolished the accelerated resolution promoted by SIV. Collectively, these results show that elastase inhibition not only inhibits inflammation but actually promotes resolution, and this response is mediated by protection of endogenous intact AnxA1 with ensuing augmentation of neutrophil apoptosis. PMID:26800869

  16. 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

  17. Comparative characterization of the iga gene encoding IgA1 protease in Neisseria meningitidis, Neisseria gonorrhoeae and Haemophilus influenzae.

    PubMed

    Lomholt, H; Poulsen, K; Kilian, M

    1995-02-01

    Cloning and sequencing of the IgA1 protease gene (iga) from Neisseria meningitidis strain HF13 showed an overall structure equivalent to iga genes from Neisseria gonorrhoeae and Haemophilus influenzae, although no region corresponding to the gonococcal alpha-peptide was evident. An additional 18 N. meningitidis and 3 H. influenzae iga genes were amplified by the polymerase chain reaction technique and sequenced corresponding approximately to the N-terminal half of the mature enzyme. Comparative analyses of a total of 29 iga genes showed that pathogenic Neisseria have iga genes with a significantly lower degree of heterogeneity than H. influenzae iga genes. Recombinational events indicated by mosaic-like structures corresponding to those found among N. gonorrhoeae protease genes were detected among N. meningitidis iga genes. One region showed characteristic differences in sequence and length which correlated with each of the different cleavage specificities. Meningococci were extremely conserved in this region with no evidence of recombination between isolates of different cleavage specificities. Sequences further downstream showed no obvious relationship with enzyme cleavage type. This region consisted of conserved areas interspersed with highly variable areas. Amino acid sequence homologies in the variable regions of meningococci reflected the antigenic types defined by using polyclonal neutralizing antibodies. PMID:7783620

  18. Evidence for possible involvement of an elastolytic serine protease in aspergillosis.

    PubMed Central

    Kolattukudy, P E; Lee, J D; Rogers, L M; Zimmerman, P; Ceselski, S; Fox, B; Stein, B; Copelan, E A

    1993-01-01

    A number of isolates of Aspergillus fumigatus obtained from the hospital environment produced extracellular elastolytic activity. This activity was found to be catalyzed by a single 33-kDa protein which was purified and characterized to be a serine protease. A. fumigatus, when grown on the insoluble structural material obtained from murine and bovine lung, produced the same extracellular 33-kDa elastolytic protease, indicating that this enzyme is likely to be produced when the organism infects the lung. Polymerase chain reaction with an oligonucleotide primer based on the N-terminal amino acid sequence of the elastolytic enzyme yielded a cDNA which was cloned and sequenced. The active serine motif showed more similarity to subtilisin than to mammalian elastase. The amino acid sequence showed 80% identity to the alkaline protease from Aspergillus oryzae. Screening of hospital isolates of Aspergillus flavus showed great variation in the production of elastolytic activity and a much lower level of activity than that produced by A. fumigatus. The elastolytic protease from A. flavus was shown to be a serine protease susceptible to modification and inactivation by active serine and histidine-directed reagents. This protease cross-reacted with the antibodies prepared against the elastolytic protease from A. fumigatus. Immunogold localization of the elastolytic enzyme showed that A. fumigatus germinating and penetrating into the lungs of neutropenic mice secreted the elastolytic protease. An elastase-deficient mutant generated from a highly virulent isolate of A. fumigatus caused drastically reduced mortality when nasally introduced into the lung of neutropenic mice. All of the evidence suggests that extracellular elastolytic protease is a significant virulence factor in invasive aspergillosis. Images PMID:8500876

  19. Proteases in cellular slime mold development: evidence for their involvement.

    PubMed Central

    Fong, D; Bonner, J T

    1979-01-01

    Protein degradation appears to be essential for normal differentiation in the cellular slime mold Dictyostelium discoideum. Several protease inhibitors block normal differentiation, and in most cases this inhibition can be reversed by addition of amino acids. For example, chloroquine, which inhibits slime mold cathepsin B activity, interferred with development by blocking sorocarp formation, and this inhibition was reversed by the addition of amino acids. Tosyllysyl chloromethyl ketone also blocked development, and this inhibition was reversed by simultaneous additions of amino acids and glutathione. Moreover, the addition of antipain and leupeptin delayed sorocarp formation. These results, together with the finding reported earlier that cathepsin B activity is differentially localized in the prestalk-prespore zones of the migrating slugs, suggest that proteolysis might play a regulatory role in cellular slime mold differentiation. Images PMID:293735

  20. Amino acid sequence requirements in the human IgA1 hinge for cleavage by streptococcal IgA1 proteases.

    PubMed

    Senior, B W; Batten, M R; Kilian, M; Woof, J M

    2002-08-01

    All the IgA1 proteases of the different pathogenic species of Streptococcus cleave the hinge of the alpha chain of human IgA1 only at one proline-threonine peptide bond. In order to study the importance of these amino acids for cleavage, several hinge mutant recombinant IgA1 antibodies were constructed. The mutations were found to be without major effect upon the structure or functional abilities of the antibodies. However, they had a major effect upon their sensitivity to cleavage by some of the IgA1 proteases. PMID:12196126

  1. Structural Evidence for Regulation and Specificity of Flaviviral Proteases and Evolution of the Flaviviridae Fold

    SciTech Connect

    Aleshin,A.; Shiryaev, S.; Strongin, A.; Liddington, R.

    2007-01-01

    Pathogenic members of the flavivirus family, including West Nile Virus (WNV) and Dengue Virus (DV), are growing global threats for which there are no specific treatments. The two-component flaviviral enzyme NS2B-NS3 cleaves the viral polyprotein precursor within the host cell, a process that is required for viral replication. Here, we report the crystal structure of WNV NS2B-NS3pro both in a substrate-free form and in complex with the trypsin inhibitor aprotinin/BPTI. We show that aprotinin binds in a substrate-mimetic fashion in which the productive conformation of the protease is fully formed, providing evidence for an 'induced fit' mechanism of catalysis and allowing us to rationalize the distinct substrate specificities of WNV and DV proteases. We also show that the NS2B cofactor of WNV can adopt two very distinct conformations and that this is likely to be a general feature of flaviviral proteases, providing further opportunities for regulation. Finally, by comparing the flaviviral proteases with the more distantly related Hepatitis C virus, we provide insights into the evolution of the Flaviviridae fold. Our work should expedite the design of protease inhibitors to treat a range of flaviviral infections.

  2. 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. PMID:27058767

  3. Cleavage of a Recombinant Human Immunoglobulin A2 (IgA2)-IgA1 Hybrid Antibody by Certain Bacterial IgA1 Proteases

    PubMed Central

    Senior, Bernard W.; Dunlop, James I.; Batten, Margaret R.; Kilian, Mogens; Woof, Jenny M.

    2000-01-01

    To understand more about the factors influencing the cleavage of immunoglobulin A1 (IgA1) by microbial IgA1 proteases, a recombinant human IgA2/IgA1 hybrid molecule was generated. In the hybrid, termed IgA2/A1 half hinge, a seven-amino-acid sequence corresponding to one half of the duplicated sequence making up the IgA1 hinge was incorporated into the equivalent site in IgA2. Insertion of the IgA1 half hinge into IgA2 did not affect antigen binding capacity or the functional activity of the hybrid molecule, as judged by its ability to bind to IgA Fcα receptors and trigger respiratory bursts in neutrophils. Although the IgA2/A1 hybrid contained only half of the IgA1 hinge, it was found to be cleaved by a variety of different bacterial IgA1 proteases, including representatives of those that cleave IgA1 in the different duplicated halves of the hinge, namely, those of Prevotella melaninogenica, Streptococcus pneumoniae, S. sanguis, Neisseria meningitidis types 1 and 2, N. gonorrhoeae types 1 and 2, and Haemophilus influenzae type 2. Thus, for these enzymes the recognition site for IgA1 cleavage is contained within half of the IgA1 hinge region; additional distal elements, if required, are provided by either an IgA1 or an IgA2 framework. In contrast, the IgA2/A1 hybrid appeared to be resistant to cleavage with S. oralis and some H. influenzae type 1 IgA1 proteases, suggesting these enzymes require additional determinants for efficient substrate recognition. PMID:10639405

  4. Cleavage of a recombinant human immunoglobulin A2 (IgA2)-IgA1 hybrid antibody by certain bacterial IgA1 proteases.

    PubMed

    Senior, B W; Dunlop, J I; Batten, M R; Kilian, M; Woof, J M

    2000-02-01

    To understand more about the factors influencing the cleavage of immunoglobulin A1 (IgA1) by microbial IgA1 proteases, a recombinant human IgA2/IgA1 hybrid molecule was generated. In the hybrid, termed IgA2/A1 half hinge, a seven-amino-acid sequence corresponding to one half of the duplicated sequence making up the IgA1 hinge was incorporated into the equivalent site in IgA2. Insertion of the IgA1 half hinge into IgA2 did not affect antigen binding capacity or the functional activity of the hybrid molecule, as judged by its ability to bind to IgA Fcalpha receptors and trigger respiratory bursts in neutrophils. Although the IgA2/A1 hybrid contained only half of the IgA1 hinge, it was found to be cleaved by a variety of different bacterial IgA1 proteases, including representatives of those that cleave IgA1 in the different duplicated halves of the hinge, namely, those of Prevotella melaninogenica, Streptococcus pneumoniae, S. sanguis, Neisseria meningitidis types 1 and 2, N. gonorrhoeae types 1 and 2, and Haemophilus influenzae type 2. Thus, for these enzymes the recognition site for IgA1 cleavage is contained within half of the IgA1 hinge region; additional distal elements, if required, are provided by either an IgA1 or an IgA2 framework. In contrast, the IgA2/A1 hybrid appeared to be resistant to cleavage with S. oralis and some H. influenzae type 1 IgA1 proteases, suggesting these enzymes require additional determinants for efficient substrate recognition. PMID:10639405

  5. 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

  6. Identification of a human immunodominant B-cell epitope within the immunoglobulin A1 protease of Streptococcus pneumoniae

    PubMed Central

    De Paolis, Francesca; Beghetto, Elisa; Spadoni, Andrea; Montagnani, Francesca; Felici, Franco; Oggioni, Marco R; Gargano, Nicola

    2007-01-01

    Background The IgA1 protease of Streptococcus pneumoniae is a proteolytic enzyme that specifically cleaves the hinge regions of human IgA1, which dominates most mucosal surfaces and is the major IgA isotype in serum. This protease is expressed in all of the known pneumococcal strains and plays a major role in pathogen's resistance to the host immune response. The present work was focused at identifying the immunodominant regions of pneumococcal IgA1 protease recognized by the human antibody response. Results An antigenic sequence corresponding to amino acids 420–457 (epiA) of the iga gene product was identified by screening a pneumococcal phage display library with patients' sera. The epiA peptide is conserved in all pneumococci and in two out of three S. mitis strains, while it is not present in other oral streptococci so far sequenced. This epitope was specifically recognized by antibodies present in sera from 90% of healthy adults, thus representing an important target of the humoral response to S. pneumoniae and S. mitis infection. Moreover, sera from 68% of children less than 4 years old reacted with the epiA peptide, indicating that the human immune response against streptococcal antigens occurs during childhood. Conclusion The broad and specific recognition of the epiA polypeptide by human sera demonstrate that the pneumococcal IgA1 protease contains an immunodominant B-cell epitope. The use of phage display libraries to identify microbe or disease-specific antigens recognized by human sera is a valuable approach to epitope discovery. PMID:18088426

  7. The influences of hinge length and composition on the susceptibility of human IgA to cleavage by diverse bacterial IgA1 proteases.

    PubMed

    Senior, Bernard W; Woof, Jenny M

    2005-06-15

    The influences of IgA hinge length and composition on its susceptibility to cleavage by bacterial IgA1 proteases were examined using a panel of IgA hinge mutants. The IgA1 proteases of Streptococcus pneumoniae, Streptococcus sanguis strains SK4 and SK49, Neisseria meningitidis, Neisseria gonorrhoeae, and Haemophilus influenzae cleaved IgA2-IgA1 half hinge, an Ab featuring half of the IgA1 hinge incorporated into the equivalent site in IgA1 protease-resistant IgA2, whereas those of Streptococcus mitis, Streptococcus oralis, and S. sanguis strain SK1 did not. Hinge length reduction by removal of two of the four C-terminal proline residues rendered IgA2-IgA1 half hinge resistant to all streptococcal IgA1 metalloproteinases but it remained sensitive to cleavage by the serine-type IgA1 proteases of Neisseria and Haemophilus spp. The four C-terminal proline residues could be substituted by alanine residues or transferred to the N-terminal extremity of the hinge without affect on the susceptibility of the Ab to cleavage by serine-type IgA1 proteases. However, their removal rendered the Ab resistant to cleavage by all the IgA1 proteases. We conclude that the serine-type IgA1 proteases of Neisseria and Haemophilus require the Fab and Fc regions to be separated by at least ten (or in the case of N. gonorrhoeae type I protease, nine) amino acids between Val(222) and Cys(241) (IgA1 numbering) for efficient access and cleavage. By contrast, the streptococcal IgA1 metalloproteinases require 12 or more appropriate amino acids between the Fab and Fc to maintain a minimum critical distance between the scissile bond and the start of the Fc. PMID:15944283

  8. Studies on the gonococcal IgA1 protease II. Improved methods of enzyme purification and production of monoclonal antibodies to the enzyme.

    PubMed

    Blake, M S; Eastby, C

    1991-11-22

    Two types of extremely active proteases that cleave human IgA1 are produced by pathogenic Neisseria in minute concentrations. To study the antigenicity of these enzymes, a simplified method is described to purify these enzymes from large batch cultures to obtain a sufficient quantity of these IgA1 proteases to study these characteristics. In addition, we describe the production of both rabbit polyclonal and mouse monoclonal antibodies to one of these enzymes. One such monoclonal antibody seemed directed toward the active site of the IgA1 protease and inhibited its enzymatic activity. PMID:1960418

  9. Alpha-1-Antitrypsin: A Novel Human High Temperature Requirement Protease A1 (HTRA1) Substrate in Human Placental Tissue

    PubMed Central

    Frochaux, Violette; Hildebrand, Diana; Talke, Anja; Linscheid, Michael W.; Schlüter, Hartmut

    2014-01-01

    The human serine protease high temperature requirement A1 (HTRA1) is highly expressed in the placental tissue, especially in the last trimester of gestation. This suggests that HTRA1 is involved in placental formation and function. With the aim of a better understanding of the role of HTRA1 in the placenta, candidate substrates were screened in a placenta protein extract using a gel-based mass spectrometric approach. Protease inhibitor alpha-1-antitrypsin, actin cytoplasmic 1, tropomyosin beta chain and ten further proteins were identified as candidate substrates of HTRA1. Among the identified candidate substrates, alpha-1-antitrypsin (A1AT) was considered to be of particular interest because of its important role as protease inhibitor. For investigation of alpha-1-antitrypsin as substrate of HTRA1 synthetic peptides covering parts of the sequence of alpha-1-antitrypsin were incubated with HTRA1. By mass spectrometry a specific cleavage site was identified after met-382 (AIPM382↓383SIPP) within the reactive centre loop of alpha-1-antitrypsin, resulting in a C-terminal peptide comprising 36 amino acids. Proteolytic removal of this peptide from alpha-1-antitrypsin results in a loss of its inhibitor function. Beside placental alpha-1-antitrypsin the circulating form in human plasma was also significantly degraded by HTRA1. Taken together, our data suggest a link between the candidate substrates alpha-1-antitrypsin and the function of HTRA1 in the placenta in the syncytiotrophoblast, the cell layer attending to maternal blood in the villous tree of the human placenta. Data deposition: Mass spectrometry (MS) data have been deposited to the ProteomeXchange with identifier PXD000473. PMID:25329061

  10. Development of a High-Throughput Screen and Its Use In the Discovery of Streptococcus pneumoniae Immunoglobulin A1 Protease (IgA1P) Inhibitors

    PubMed Central

    Garner, Amanda L.; Fullagar, Jessica L.; Day, Joshua A.; Cohen, Seth M.; Janda, Kim D.

    2013-01-01

    Streptococcus pneumoniae relies on a number of virulence factors, including immunoglobulin A1 protease (IgA1P), a Zn2+ metalloprotease produced on the extracellular surface of the bacteria, to promote pathogenic colonization. IgA1P exhibits a unique function, in that it catalyzes the proteolysis of human IgA1 at its hinge region to leave the bacterial cell surface masked by IgA1 Fab, enabling the bacteria to evade the host's immune system and adhere to host epithelial cells to promote colonization. Thus, S. pneumoniae IgA1P has emerged as a promising antibacterial target; however, the lack of an appropriate screening assay has limited the investigation of this metalloprotease virulence factor. Relying on electrostatics-mediated AuNP aggregation, we have designed a promising high-throughput colorimetric assay for IgA1P. By using this assay, we have uncovered inhibitors of the enzyme that should be useful in deciphering its role in pneumococcal colonization and virulence. PMID:23808771

  11. Thermodynamic analysis of unusually thermostable CutA1 protein from human brain and its protease susceptibility.

    PubMed

    Bagautdinov, Bagautdin; Matsuura, Yoshinori; Yamamoto, Hitoshi; Sawano, Masahide; Ogasahara, Kyoko; Takehira, Michiyo; Kunishima, Naoki; Katoh, Etsuko; Yutani, Katsuhide

    2015-03-01

    Unusually stable proteins are a disadvantage for the metabolic turnover of proteins in cells. The CutA1 proteins from Pyrococcus horikoshii and from Oryza sativa (OsCutA1) have unusually high denaturation temperatures (Td) of nearly 150 and 100 °C, respectively, at pH 7.0. It seemed that the CutA1 protein from the human brain (HsCutA1) also has a remarkably high stability. Therefore, the thermodynamic stabilities of HsCutA1 and its protease susceptibility were examined. The Td was remarkably high, being over 95 °C at pH 7.0. The unfolding Gibbs energy (ΔG(0)H2O) was 174 kJ/mol at 37 °C from the denaturant denaturation. The thermodynamic analysis showed that the unfolding enthalpy and entropy values of HsCutA1 were considerably lower than those of OsCutA1 with a similar stability to HsCutA1, which should be related to flexibility of the unstructured properties in both N- and C-terminals of HsCutA1. HsCutA1 was almost completely digested after 1-day incubation at 37 °C by subtilisin, although OsCutA1 was hardly digested at the same conditions. These results indicate that easily available fragmentation of HsCutA1 with remarkably high thermodynamic stability at the body temperature should be important for its protein catabolism in the human cells. PMID:25344844

  12. Protease inhibition by Heterodera glycines cyst content: evidence for effects on the Meloidogyne incognita proteasome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteases from Heterodera glycines and Meloidogyne incognita juveniles were inhibited by heat-stable content of H. glycines female cysts (HglCE), and by the plant polyphenol epigallocatechin gallate (EGCG). General protease activities detected using the nematode peptide KSAYMRFa were inhibited by EG...

  13. Active site gating regulates substrate selectivity in a chymotrypsin-like serine protease. The structure of Haemophilus influenzae IgA1 protease†

    PubMed Central

    Johnson, Troy A.; Qiu, Jiazhou; Plaut, Andrew G.; Holyoak, Todd

    2009-01-01

    We report here the first structure of a member of the IgA protease family at 1.75Å resolution. This protease is a founding member of the Type V (autotransporter) secretion system and is considered a virulence determinant among the bacteria expressing the enzyme. The structure of the enzyme fits that of a classical autotransporter in which several unique domains necessary for protein function are appended to a central, 100 Å long β-helical domain. The N-terminal domain of the IgA protease is found to possess a chymotrypsin-like fold. However, this catalytic domain contains a unique loop D that extends over the active site acting as a lid, gating substrate access. The data presented provide a structural basis for the known ability of IgA proteases to only cleave the P/S/T rich hinge peptide unique to IgA1 in the context of the intact fold of the immunoglobulin. Based upon the structural data as well as molecular modeling, a model is presented that suggests the unique, extended loop D in this IgA protease sterically occludes the active site binding cleft in the absence of immunoglobulin binding. Only in the context of binding of the IgA1 immunoglobulin Fc domain in a valley formed between the N-terminal protease domain and another domain appended to the β-helix spine (domain-2) is the lid stabilized in an open conformation. The stabilization of this open conformation through Fc association subsequently allows access of the hinge peptide to the active site resulting in recognition and cleavage of the substrate. PMID:19393662

  14. Bacterial IgA protease-mediated degradation of agIgA1 and agIgA1 immune complexes as a potential therapy for IgA Nephropathy.

    PubMed

    Wang, Li; Li, Xueying; Shen, Hongchun; Mao, Nan; Wang, Honglian; Cui, Luke; Cheng, Yuan; Fan, Junming

    2016-01-01

    Mesangial deposition of aberrantly glycosylated IgA1 (agIgA1) and its immune complexes is a key pathogenic mechanism of IgA nephropathy (IgAN). However, treatment of IgAN remains ineffective. We report here that bacteria-derived IgA proteases are capable of degrading these pathogenic agIgA1 and derived immune complexes in vitro and in vivo. By screening 14 different bacterial strains (6 species), we found that 4 bacterial IgA proteases from H. influenzae, N. gonorrhoeae and N. meningitidis exhibited high cleaving activities on serum agIgA1 and artificial galactose-depleted IgA1 in vitro and the deposited agIgA1-containing immune complexes in the mesangium of renal biopsy from IgAN patients and in a passive mouse model of IgAN in vitro. In the modified mouse model of passive IgAN with abundant in situ mesangial deposition of the agIgA-IgG immune complexes, a single intravenous delivery of IgA protease from H. influenzae was able to effectively degrade the deposited agIgA-IgG immune complexes within the glomerulus, demonstrating a therapeutic potential for IgAN. In conclusion, the bacteria-derived IgA proteases are biologically active enzymes capable of cleaving the circulating agIgA and the deposited agIgA-IgG immune complexes within the kidney of IgAN. Thus, the use of such IgA proteases may represent a novel therapy for IgAN. PMID:27485391

  15. Bacterial IgA protease-mediated degradation of agIgA1 and agIgA1 immune complexes as a potential therapy for IgA Nephropathy

    PubMed Central

    Wang, Li; Li, Xueying; Shen, Hongchun; Mao, Nan; Wang, Honglian; Cui, Luke; Cheng, Yuan; Fan, Junming

    2016-01-01

    Mesangial deposition of aberrantly glycosylated IgA1 (agIgA1) and its immune complexes is a key pathogenic mechanism of IgA nephropathy (IgAN). However, treatment of IgAN remains ineffective. We report here that bacteria-derived IgA proteases are capable of degrading these pathogenic agIgA1 and derived immune complexes in vitro and in vivo. By screening 14 different bacterial strains (6 species), we found that 4 bacterial IgA proteases from H. influenzae, N. gonorrhoeae and N. meningitidis exhibited high cleaving activities on serum agIgA1 and artificial galactose-depleted IgA1 in vitro and the deposited agIgA1-containing immune complexes in the mesangium of renal biopsy from IgAN patients and in a passive mouse model of IgAN in vitro. In the modified mouse model of passive IgAN with abundant in situ mesangial deposition of the agIgA-IgG immune complexes, a single intravenous delivery of IgA protease from H. influenzae was able to effectively degrade the deposited agIgA-IgG immune complexes within the glomerulus, demonstrating a therapeutic potential for IgAN. In conclusion, the bacteria-derived IgA proteases are biologically active enzymes capable of cleaving the circulating agIgA and the deposited agIgA-IgG immune complexes within the kidney of IgAN. Thus, the use of such IgA proteases may represent a novel therapy for IgAN. PMID:27485391

  16. Antiviral activity of human immunodeficiency virus type 1 protease inhibitors in a single cycle of infection: evidence for a role of protease in the early phase.

    PubMed Central

    Nagy, K; Young, M; Baboonian, C; Merson, J; Whittle, P; Oroszlan, S

    1994-01-01

    The antiviral activities of two substrate-based inhibitors of human immunodeficiency virus type 1 (HIV-1) protease, UK-88,947 and Ro 31-8959, were studied in acute infections. H9 and HeLaCD4-LTR/beta-gal cells were infected either with HIV-1IIIB or a replication-defective virus, HIV-gpt(HXB-2). Both inhibitors were capable of blocking early steps of HIV-1 replication if added to cells prior to infection. Partial inhibition was also obtained by addition of inhibitor at the time of or as late as 15 min after infection. The inhibitors were ineffective if added 30 min postinfection. The inhibitory effects were studied by cDNA analysis with PCR followed by Southern blot hybridization and by infectivity assays allowing quantitation of HIV-1 in a single cycle of replication. When UK-88,947-treated H9 cells were coinfected with HIV-1 and human T-cell leukemia virus type I only the replication of HIV-1 was inhibited, demonstrating viral specificity. Pretreating the infectious virus stocks with the inhibitors also prevented replication, indicating that the inhibitors block the action of the viral protease and not a cellular protease. A panel of primer sets was used to analyze cDNA from cell lysates by PCR amplification at 4 and 18 h postinfection. Four hours after infection, viral specific cDNA was detected with all of the four primer pairs used: R/U5, nef/U3, 5' gag, and long terminal repeat (LTR)/gag. However, after 18 h, only the R/U5 and nef/U3 primer pairs and not the 5' gag or LTR/gag primer pair were able to allow amplification of cDNA. The results suggest a crucial role of HIV-1 protease in the early phase of viral replication. Although it is not clear what early steps are affected by the protease, it is likely that the target is the NC protein, as referred from our previous reports of the in situ cleavage of the nucleocapsid (NC) protein by the viral protease inside lentiviral capsids. The results suggest that it is not the inhibition of initiation and progression

  17. Chymotrypsin protease inhibitor gene family in rice: Genomic organization and evidence for the presence of a bidirectional promoter shared between two chymotrypsin protease inhibitor genes.

    PubMed

    Singh, Amanjot; Sahi, Chandan; Grover, Anil

    2009-01-01

    Protease inhibitors play important roles in stress and developmental responses of plants. Rice genome contains 17 putative members in chymotrypsin protease inhibitor (ranging in size from 7.21 to 11.9 kDa) gene family with different predicted localization sites. Full-length cDNA encoding for a putative subtilisin-chymotrypsin protease inhibitor (OCPI2) was obtained from Pusa basmati 1 (indica) rice seedlings. 620 bp-long OCPI2 cDNA contained 219 bp-long ORF, coding for 72 amino acid-long 7.7 kDa subtilisin-chymotrypsin protease inhibitor (CPI) cytoplasmic protein. Expression analysis by semi-quantitative RT-PCR analysis showed that OCPI2 transcript is induced by varied stresses including salt, ABA, low temperature and mechanical injury in both root and shoot tissues of the seedlings. Transgenic rice plants produced with OCPI2 promoter-gus reporter gene showed that this promoter directs high salt- and ABA-regulated expression of the GUS gene. Another CPI gene (OCPI1) upstream to OCPI2 (with 1126 bp distance between the transcription initiation sites of the two genes; transcription in the reverse orientation) was noted in genome sequence of rice genome. A vector that had GFP and GUS reporter genes in opposite orientations driven by 1881 bp intergenic sequence between the OCPI2 and OCPI1 (encompassing the region between the translation initiation sites of the two genes) was constructed and shot in onion epidermal cells by particle bombardment. Expression of both GFP and GUS from the same epidermal cell showed that this sequence represents a bidirectional promoter. Examples illustrating gene pairs showing co-expression of two divergent neighboring genes sharing a bidirectional promoter have recently been extensively worked out in yeast and human systems. We provide an example of a gene pair constituted of two homologous genes showing co-expression governed by a bidirectional promoter in rice. PMID:18952157

  18. Structural Evidence for Effectiveness of Darunavir and Two Related Antiviral Inhibitors against HIV-2 Protease

    SciTech Connect

    Kovalevsky, Andrey Y.; Louis, John M.; Aniana, Annie; Ghosh, Arun K.; Weber, Irene T.

    2008-12-08

    No drug has been targeted specifically for HIV-2 (human immunodeficiency virus type 2) infection despite its increasing prevalence worldwide. The antiviral HIV-1 (human immunodeficiency virus type 1) protease (PR) inhibitor darunavir and the chemically related GRL98065 and GRL06579A were designed with the same chemical scaffold and different substituents at P2 and P2' to optimize polar interactions for HIV-1 PR (PR1). These inhibitors are also effective antiviral agents for HIV-2-infected cells. Therefore, crystal structures of HIV-2 PR (PR2) complexes with the three inhibitors have been solved at 1.2-{angstrom} resolution to analyze the molecular basis for their antiviral potency. Unusually, the crystals were grown in imidazole and zinc acetate buffer, which formed interactions with the PR2 and the inhibitors. Overall, the structures were very similar to the corresponding inhibitor complexes of PR1 with an RMSD of 1.1 {angstrom} on main-chain atoms. Most hydrogen-bond and weaker C-H...O interactions with inhibitors were conserved in the PR2 and PR1 complexes, except for small changes in interactions with water or disordered side chains. Small differences were observed in the hydrophobic contacts for the darunavir complexes, in agreement with relative inhibition of the two PRs. These near-atomic-resolution crystal structures verify the inhibitor potency for PR1 and PR2 and will provide the basis for the development of antiviral inhibitors targeting PR2.

  19. The Major Phase-Variable Outer Membrane Protein of Escherichia coli Structurally Resembles the Immunoglobulin A1 Protease Class of Exported Protein and Is Regulated by a Novel Mechanism Involving Dam and OxyR

    PubMed Central

    Henderson, Ian R.; Owen, Peter

    1999-01-01

    Here we report the characterization of an Escherichia coli gene (agn43) which encodes the principal phase-variable outer membrane protein termed antigen 43 (Ag43). The agn43 gene encodes a precursor protein of 107 kDa containing a 52-amino-acid signal sequence. Posttranslational processing generates an α43 subunit (predicted Mr of 49,789) and a C-terminal domain (β43) with features typical of a bacterial integral outer membrane protein (predicted Mr of 51,642). Secondary structure analysis predicts that β43 exists as an 18-stranded β barrel and that Ag43 shows structural organization closely resembling that of immunoglobulin A1 protease type of exoprotein produced by pathogenic Neisseria and Haemophilus spp. The correct processing of the polyprotein to α43 and β43 in OmpT, OmpP, and DegP protease-deficient E. coli strains points to an autocatalytic cleavage mechanism, a hypothesis supported by the occurrence of an aspartyl protease active site within α43. Ag43, a species-specific antigen, possesses two RGD motifs of the type implicated in binding to human integrins. The mechanism of reversible phase variation was studied by immunochemical analysis of a panel of well-defined regulatory mutants and by analysis of DNA sequences upstream of agn43. Evidence strongly suggests that phase variation is regulated by both deoxyadenosine methylase (Dam) and by OxyR. Thus, oxyR mutants are locked on for Ag43 expression, whereas dam mutants are locked off for Ag43 expression. We propose a novel mechanism for the regulation of phase switching in which OxyR competes with Dam for unmethylated GATC sites in the regulatory region of the agn43 gene. PMID:10094691

  20. 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

  1. Supermarket Proteases.

    ERIC Educational Resources Information Center

    Hagar, William G.; Bullerwell, Lornie D.

    2003-01-01

    Presents a laboratory activity on enzymes. Uses common items found in the supermarket that contain protease enzymes, such as contact lens cleaner and meat tenderizer. Demonstrates the digestion of gelatin proteins as part of enzymatic reactions. (Author/SOE)

  2. Clinical Evidence for the Role of Trichomonas vaginalis in Regulation of Secretory Leukocyte Protease Inhibitor in the Female Genital Tract

    PubMed Central

    Huppert, Jill S.; Huang, Bin; Chen, Chen; Dawood, Hassan Y.; Fichorova, Raina N.

    2013-01-01

    Background. Secretory leukocyte protease inhibitor (SLPI) is responsible for regulating inflammatory damage to and innate and adaptive immune responses in the vaginal mucosa. Depressed cervicovaginal SLPI levels have been correlated with both Trichomonas vaginalis infection and poor reproductive health outcomes. Methods. We measured levels of SLPI in 215 vaginal specimens collected from adolescent and young adult females aged 14–22 years. Log-transformed SLPI values were compared by analysis of variance or by an unpaired t test before and after adjustment for confounding effects through the propensity score method. Results. Females receiving hormonal contraceptives and those with an abnormal vaginal pH had lower SLPI levels as compared to their peers. After propensity score adjustment for race, behavioral factors, hormonal use, and other sexually transmitted infections (STIs), SLPI levels were lower in females with a positive T. vaginalis antigen test result, a vaginal pH >4.5, vaginal leukocytosis, and recurrent (vs initial) T. vaginalis infection, with the lowest levels observed in those with the highest T. vaginalis loads. Conclusions. The SLPI level was reduced by >50% in a T. vaginalis load–dependent manner. Future research should consider whether identifying and treating females with low levels of T. vaginalis infection (before they become wet mount positive) would prevent the loss of SLPI and impaired vaginal immunity. The SLPI level could be used as a vaginal-health marker to evaluate interventions and vaginal products. PMID:23355743

  3. Evidence supporting the 19 β-strand model for Tom40 from cysteine scanning and protease site accessibility studies.

    PubMed

    Lackey, Sebastian W K; Taylor, Rebecca D; Go, Nancy E; Wong, Annie; Sherman, E Laura; Nargang, Frank E

    2014-08-01

    Most proteins found in mitochondria are translated in the cytosol and enter the organelle via the TOM complex (translocase of the outer mitochondrial membrane). Tom40 is the pore forming component of the complex. Although the three-dimensional structure of Tom40 has not been determined, the structure of porin, a related protein, has been shown to be a β-barrel containing 19 membrane spanning β-strands and an N-terminal α-helical region. The evolutionary relationship between the two proteins has allowed modeling of Tom40 into a similar structure by several laboratories. However, it has been suggested that the 19-strand porin structure does not represent the native form of the protein. If true, modeling of Tom40 based on the porin structure would also be invalid. We have used substituted cysteine accessibility mapping to identify several potential β-strands in the Tom40 protein in isolated mitochondria. These data, together with protease accessibility studies, support the 19 β-strand model for Tom40 with the C-terminal end of the protein localized to the intermembrane space. PMID:24947507

  4. Evidence Supporting the 19 β-Strand Model for Tom40 from Cysteine Scanning and Protease Site Accessibility Studies*

    PubMed Central

    Lackey, Sebastian W. K.; Taylor, Rebecca D.; Go, Nancy E.; Wong, Annie; Sherman, E. Laura; Nargang, Frank E.

    2014-01-01

    Most proteins found in mitochondria are translated in the cytosol and enter the organelle via the TOM complex (translocase of the outer mitochondrial membrane). Tom40 is the pore forming component of the complex. Although the three-dimensional structure of Tom40 has not been determined, the structure of porin, a related protein, has been shown to be a β-barrel containing 19 membrane spanning β-strands and an N-terminal α-helical region. The evolutionary relationship between the two proteins has allowed modeling of Tom40 into a similar structure by several laboratories. However, it has been suggested that the 19-strand porin structure does not represent the native form of the protein. If true, modeling of Tom40 based on the porin structure would also be invalid. We have used substituted cysteine accessibility mapping to identify several potential β-strands in the Tom40 protein in isolated mitochondria. These data, together with protease accessibility studies, support the 19 β-strand model for Tom40 with the C-terminal end of the protein localized to the intermembrane space. PMID:24947507

  5. Evidence for the presence of a protease-activated receptor distinct from the thrombin receptor in human keratinocytes.

    PubMed Central

    Santulli, R J; Derian, C K; Darrow, A L; Tomko, K A; Eckardt, A J; Seiberg, M; Scarborough, R M; Andrade-Gordon, P

    1995-01-01

    Thrombin receptor activation was explored in human epidermal keratinocytes and human dermal fibroblasts, cells that are actively involved in skin tissue repair. The effects of thrombin, trypsin, and the receptor agonist peptides SFLLRN and TFRIFD were assessed in inositolphospholipid hydrolysis and calcium mobilization studies. Thrombin and SFLLRN stimulated fibroblasts in both assays to a similar extent, whereas TFRIFD was less potent. Trypsin demonstrated weak efficacy in these assays in comparison with thrombin. Results in fibroblasts were consistent with human platelet thrombin receptor activation. Keratinocytes, however, exhibited a distinct profile, with trypsin being a far better activator of inositolphospholipid hydrolysis and calcium mobilization than thrombin. Furthermore, SFLLRN was more efficacious than thrombin, whereas no response was observed with TFRIFD. Since our data indicated that keratinocytes possess a trypsin-sensitive receptor, we addressed the possibility that these cells express the human homologue of the newly described murine protease-activated receptor, PAR-2 [Nystedt, S., Emilsson, K., Wahlestedt, C. & Sundelin, J. (1994) Proc. Natl. Acad. Sci. USA 91, 9208-9212]. PAR-2 is activated by nanomolar concentrations of trypsin and possesses the tethered ligand sequence SLIGRL. SLIGRL was found to be equipotent with SFLLRN in activating keratinocyte inositolphospholipid hydrolysis and calcium mobilization. Desensitization studies indicated that SFLLRN, SLIGRL, and trypsin activate a common receptor, PAR-2. Northern blot analyses detected a transcript of PAR-2 in total RNA from keratinocytes but not fibroblasts. Levels of thrombin receptor message were equivalent in the two cell types. Our results indicate that human keratinocytes possess PAR-2, suggesting a potential role for this receptor in tissue repair and/or skin-related disorders. Images Fig. 6 PMID:7568091

  6. Proteases as Insecticidal Agents

    PubMed Central

    Harrison, Robert L.; Bonning, Bryony C.

    2010-01-01

    Proteases from a variety of sources (viruses, bacteria, fungi, plants, and insects) have toxicity towards insects. Some of these insecticidal proteases evolved as venom components, herbivore resistance factors, or microbial pathogenicity factors, while other proteases play roles in insect development or digestion, but exert an insecticidal effect when over-expressed from genetically engineered plants or microbial pathogens. Many of these proteases are cysteine proteases, although insect-toxic metalloproteases and serine proteases have also been examined. The sites of protease toxic activity range from the insect midgut to the hemocoel (body cavity) to the cuticle. This review discusses these insecticidal proteases along with their evaluation and use as potential pesticides. PMID:22069618

  7. Evidence for charged B meson decays to a1+/-(1260)pi0 and a1(0)(1260)pi+/-.

    PubMed

    Aubert, B; Bona, M; Boutigny, D; Karyotakis, Y; Lees, J P; Poireau, V; Prudent, X; Tisserand, V; Zghiche, A; Garra Tico, J; Grauges, E; Lopez, L; Palano, A; Eigen, G; Stugu, B; Sun, L; Abrams, G S; Battaglia, M; Brown, D N; Button-Shafer, J; Cahn, R N; Groysman, Y; Jacobsen, R G; Kadyk, J A; Kerth, L T; Kolomensky, Yu G; Kukartsev, G; Lopes Pegna, D; Lynch, G; Mir, L M; Orimoto, T J; Ronan, M T; Tackmann, K; Wenzel, W A; Del Amo Sanchez, P; Hawkes, C M; Watson, A T; Held, T; Koch, H; Lewandowski, B; Pelizaeus, M; Schroeder, T; Steinke, M; Walker, D; Asgeirsson, D J; Cuhadar-Donszelmann, T; Fulsom, B G; Hearty, C; Mattison, T S; McKenna, J A; Khan, A; Saleem, M; Teodorescu, L; Blinov, V E; Bukin, A D; Druzhinin, V P; Golubev, V B; Onuchin, A P; Serednyakov, S I; Skovpen, Yu I; Solodov, E P; Todyshev, K Yu; Bondioli, M; Curry, S; Eschrich, I; Kirkby, D; Lankford, A J; Lund, P; Mandelkern, M; Martin, E C; Stoker, D P; Abachi, S; Buchanan, C; Foulkes, S D; Gary, J W; Liu, F; Long, O; Shen, B C; Zhang, L; Paar, H P; Rahatlou, S; Sharma, V; Berryhill, J W; Campagnari, C; Cunha, A; Dahmes, B; Hong, T M; Kovalskyi, D; Richman, J D; Beck, T W; Eisner, A M; Flacco, C J; Heusch, C A; Kroseberg, J; Lockman, W S; Schalk, T; Schumm, B A; Seiden, A; Wilson, M G; Winstrom, L O; Chen, E; Cheng, C H; Fang, F; Hitlin, D G; Narsky, I; Piatenko, T; Porter, F C; Andreassen, R; Mancinelli, G; Meadows, B T; Mishra, K; Sokoloff, M D; Blanc, F; Bloom, P C; Chen, S; Ford, W T; Hirschauer, J F; Kreisel, A; Nagel, M; Nauenberg, U; Olivas, A; Smith, J G; Ulmer, K A; Wagner, S R; Zhang, J; Gabareen, A M; Soffer, A; Toki, W H; Wilson, R J; Winklmeier, F; Altenburg, D D; Feltresi, E; Hauke, A; Jasper, H; Merkel, J; Petzold, A; Spaan, B; Wacker, K; Klose, V; Kobel, M J; Lacker, H M; Mader, W F; Nogowski, R; Schubert, J; Schubert, K R; Schwierz, R; Sundermann, J E; Volk, A; Bernard, D; Bonneaud, G R; Latour, E; Lombardo, V; Thiebaux, Ch; Verderi, M; Clark, P J; Gradl, W; Muheim, F; Playfer, S; Robertson, A I; Xie, Y; Andreotti, M; Bettoni, D; Bozzi, C; Calabrese, R; Cecchi, A; Cibinetto, G; Franchini, P; Luppi, E; Negrini, M; Petrella, A; Piemontese, L; Prencipe, E; Santoro, V; Anulli, F; Baldini-Ferroli, R; Calcaterra, A; de Sangro, R; Finocchiaro, G; Pacetti, S; Patteri, P; Peruzzi, I M; Piccolo, M; Rama, M; Zallo, A; Buzzo, A; Contri, R; Lo Vetere, M; Macri, M M; Monge, M R; Passaggio, S; Patrignani, C; Robutti, E; Santroni, A; Tosi, S; Chaisanguanthum, K S; Morii, M; Wu, J; Dubitzky, R S; Marks, J; Schenk, S; Uwer, U; Bard, D J; Dauncey, P D; Flack, R L; Nash, J A; Panduro Vazquez, W; Tibbetts, M; Behera, P K; Chai, X; Charles, M J; Mallik, U; Ziegler, V; Cochran, J; Crawley, H B; Dong, L; Eyges, V; Meyer, W T; Prell, S; Rosenberg, E I; Rubin, A E; Gao, Y Y; Gritsan, A V; Guo, Z J; Lae, C K; Denig, A G; Fritsch, M; Schott, G; Arnaud, N; Béquilleux, J; Davier, M; Grosdidier, G; Höcker, A; Lepeltier, V; Le Diberder, F; Lutz, A M; Pruvot, S; Rodier, S; Roudeau, P; Schune, M H; Serrano, J; Sordini, V; Stocchi, A; Wang, W F; Wormser, G; Lange, D J; Wright, D M; Bingham, I; Chavez, C A; Forster, I J; Fry, J R; Gabathuler, E; Gamet, R; Hutchcroft, D E; Payne, D J; Schofield, K C; Touramanis, C; Bevan, A J; George, K A; Di Lodovico, F; Menges, W; Sacco, R; Cowan, G; Flaecher, H U; Hopkins, D A; Paramesvaran, S; Salvatore, F; Wren, A C; Brown, D N; Davis, C L; Allison, J; Barlow, N R; Barlow, R J; Chia, Y M; Edgar, C L; Lafferty, G D; West, T J; Yi, J I; Anderson, J; Chen, C; Jawahery, A; Roberts, D A; Simi, G; Tuggle, J M; Blaylock, G; Dallapiccola, C; Hertzbach, S S; Li, X; Moore, T B; Salvati, E; Saremi, S; Cowan, R; Dujmic, D; Fisher, P H; Koeneke, K; Sciolla, G; Sekula, S J; Spitznagel, M; Taylor, F; Yamamoto, R K; Zhao, M; Zheng, Y; McLachlin, S E; Patel, P M; Robertson, S H; Lazzaro, A; Palombo, F; Bauer, J M; Cremaldi, L; Eschenburg, V; Godang, R; Kroeger, R; Sanders, D A; Summers, D J; Zhao, H W; Brunet, S; Côté, D; Simard, M; Taras, P; Viaud, F B; Nicholson, H; De Nardo, G; Fabozzi, F; Lista, L; Monorchio, D; Sciacca, C; Baak, M A; Raven, G; Snoek, H L; Jessop, C P; Losecco, J M; Benelli, G; Corwin, L A; Honscheid, K; Kagan, H; Kass, R; Morris, J P; Rahimi, A M; Regensburger, J J; Wong, Q K; Blount, N L; Brau, J; Frey, R; Igonkina, O; Kolb, J A; Lu, M; Rahmat, R; Sinev, N B; Strom, D; Strube, J; Torrence, E; Gagliardi, N; Gaz, A; Margoni, M; Morandin, M; Pompili, A; Posocco, M; Rotondo, M; Simonetto, F; Stroili, R; Voci, C; Ben-Haim, E; Briand, H; Calderini, G; Chauveau, J; David, P; Del Buono, L; de la Vaissière, Ch; Hamon, O; Leruste, Ph; Malclès, J; Ocariz, J; Perez, A; Gladney, L; Biasini, M; Covarelli, R; Manoni, E; Angelini, C; Batignani, G; Bettarini, S; Carpinelli, M; Cenci, R; Cervelli, A; Forti, F; Giorgi, M A; Lusiani, A; Marchiori, G; Mazur, M A; Morganti, M; Neri, N; Paoloni, E; Rizzo, G; Walsh, J J; Haire, M; Biesiada, J; Elmer, P; Lau, Y P; Lu, C; Olsen, J; Smith, A J S; Telnov, A V; Baracchini, E; Bellini, F; Cavoto, G; D'Orazio, A; Del Re, D; Di Marco, E; Faccini, R; Ferrarotto, F; Ferroni, F; Gaspero, M; Jackson, P D; Li Gioi, L; Mazzoni, M A; Morganti, S; Piredda, G; Polci, F; Renga, F; Voena, C; Ebert, M; Hartmann, T; Schröder, H; Waldi, R; Adye, T; Castelli, G; Franek, B; Olaiya, E O; Ricciardi, S; Roethel, W; Wilson, F F; Aleksan, R; Emery, S; Escalier, M; Gaidot, A; Ganzhur, S F; Hamel de Monchenault, G; Kozanecki, W; Vasseur, G; Yèche, Ch; Zito, M; Chen, X R; Liu, H; Park, W; Purohit, M V; Wilson, J R; Allen, M T; Aston, D; Bartoldus, R; Bechtle, P; Berger, N; Claus, R; Coleman, J P; Convery, M R; Dingfelder, J C; Dorfan, J; Dubois-Felsmann, G P; Dunwoodie, W; Field, R C; Glanzman, T; Gowdy, S J; Graham, M T; Grenier, P; Hast, C; Hryn'ova, T; Innes, W R; Kaminski, J; Kelsey, M H; Kim, H; Kim, P; Kocian, M L; Leith, D W G S; Li, S; Luitz, S; Luth, V; Lynch, H L; Macfarlane, D B; Marsiske, H; Messner, R; Muller, D R; O'Grady, C P; Ofte, I; Perazzo, A; Perl, M; Pulliam, T; Ratcliff, B N; Roodman, A; Salnikov, A A; Schindler, R H; Schwiening, J; Snyder, A; Stelzer, J; Su, D; Sullivan, M K; Suzuki, K; Swain, S K; Thompson, J M; Va'vra, J; van Bakel, N; Wagner, A P; Weaver, M; Wisniewski, W J; Wittgen, M; Wright, D H; Yarritu, A K; Yi, K; Young, C C; Burchat, P R; Edwards, A J; Majewski, S A; Petersen, B A; Wilden, L; Ahmed, S; Alam, M S; Bula, R; Ernst, J A; Jain, V; Pan, B; Saeed, M A; Wappler, F R; Zain, S B; Bugg, W; Krishnamurthy, M; Spanier, S M; Eckmann, R; Ritchie, J L; Ruland, A M; Schilling, C J; Schwitters, R F; Izen, J M; Lou, X C; Ye, S; Bianchi, F; Gallo, F; Gamba, D; Pelliccioni, M; Bomben, M; Bosisio, L; Cartaro, C; Cossutti, F; Della Ricca, G; Lanceri, L; Vitale, L; Azzolini, V; Lopez-March, N; Martinez-Vidal, F; Milanes, D A; Oyanguren, A; Albert, J; Banerjee, Sw; Bhuyan, B; Hamano, K; Kowalewski, R; Nugent, I M; Roney, J M; Sobie, R J; Harrison, P F; Ilic, J; Latham, T E; Mohanty, G B; Pappagallo, M; Band, H R; Chen, X; Dasu, S; Flood, K T; Hollar, J J; Kutter, P E; Pan, Y; Pierini, M; Prepost, R; Wu, S L; Neal, H

    2007-12-31

    We present measurements of the branching fractions for the decays B;{+/-}-->a_{1};{+/-}(1260)pi;{0} and B;{+/-}-->a_{1};{0}(1260)pi;{+/-} from a data sample of 232x10;{6} BB[over ] pairs produced in e;{+}e;{-} annihilation through the Upsilon(4S) resonance. We measure the branching fraction B(B;{+/-}-->a_{1};{+/-}(1260)pi;{0})xB(a_{1};{+/-}(1260)-->pi;{-}pi;{+}pi;{+/-})=(13.2+/-2.7+/-2.1)x10;{-6} with a significance of 4.2sigma, and the branching fraction B(B;{+/-}-->a_{1};{0}(1260)pi;{+/-})xB(a_{1};{0}(1260)-->pi;{-}pi;{+}pi;{0})=(20.4+/-4.7+/-3.4)x10;{-6} with a significance of 3.8sigma, where the first error quoted is statistical and the second is systematic. PMID:18233566

  8. Laboratory Detection of IZnCH_{3} (X^{1}A_{1}) : Further Evidence for Zinc Insertion

    NASA Astrophysics Data System (ADS)

    Bucchino, Matthew P.; Young, Justin P.; Sheridan, Phil M.; Ziurys, Lucy M.

    2013-06-01

    Millimeter-wave direct absorption techniques were used to record the pure rotational spectrum of IZnCH_{3} (X^{1}A_{1}). This species was produced by the reaction of zinc vapor with ICH_{3} in the presence of a DC discharge. Rotational transitions ranging from J = 109 {→} 108 to J = 122 {→} 121 were recorded for I^{64}ZnCH_{3} and I^{66}ZnCH_{3} in the frequency range of 250{-290} GHz. The Ka = 0{-4} components were measured for each transition, with the K-ladder structure and nuclear spin statistics indicative of a symmetric top. As with HZnCH_{3} (X^{1}A_{1}), the detection of IZnCH_{3} provides further evidence for a zinc insertion process.

  9. Evidence of association with type 1 diabetes in the SLC11A1 gene region

    PubMed Central

    2011-01-01

    Background Linkage and congenic strain analyses using the nonobese diabetic (NOD) mouse as a model for human type 1 autoimmune diabetes (T1D) have identified several NOD mouse Idd (insulin dependent diabetes) loci, including Slc11a1 (formerly known as Nramp1). Genetic variants in the orthologous region encompassing SLC11A1 in human chromosome 2q35 have been reported to be associated with various immune-related diseases including T1D. Here, we have conducted association analysis of this candidate gene region, and then investigated potential correlations between the most T1D-associated variant and RNA expression of the SLC11A1 gene and its splice isoform. Methods Nine SNPs (rs2276631, rs2279015, rs1809231, rs1059823, rs17235409 (D543N), rs17235416 (3'UTR), rs3731865 (INT4), rs7573065 (-237 C→T) and rs4674297) were genotyped using TaqMan genotyping assays and the polymorphic promoter microsatellite (GT)n was genotyped using PCR and fragment length analysis. A maximum of 8,863 T1D British cases and 10,841 British controls, all of white European descent, were used to test association using logistic regression. A maximum of 5,696 T1D families were also tested for association using the transmission/disequilibrium test (TDT). We considered P ≤ 0.005 as evidence of association given that we tested nine variants in total. Upon identification of the most T1D-associated variant, we investigated the correlation between its genotype and SLC11A1 expression overall or with splice isoform ratio using 42 PAXgene whole blood samples from healthy donors by quantitative PCR (qPCR). Results Using the case-control collection, rs3731865 (INT4) was identified to be the variant most associated with T1D (P = 1.55 × 10-6). There was also some evidence of association at rs4674297 (P = 1.57 × 10-4). No evidence of disease association was obtained at any of the loci using the family collections (PTDT ≥ 0.13). We also did not observe a correlation between rs3731865 genotypes and SLC11A1

  10. Investigations with Protease.

    ERIC Educational Resources Information Center

    Yip, Din Yan

    1997-01-01

    Presents two simple and reliable ways for measuring protease activity that can be used for a variety of investigations in a range of biology class levels. The investigations use protease from a variety of sources. (DDR)

  11. ITPA and SLC29A1 Genotyping for the Prediction of Ribavirin Dose Reduction in Anti-HCV Triple Therapy with Protease Inhibitors.

    PubMed

    Lombardi, Andrea; Landonio, Simona; Magni, Carlo; Cheli, Stefania; Mazzali, Cristina; Mondelli, Mario U; Rizzardini, Giuliano; Clementi, Emilio; Falvella, Felicia Stefania

    2015-01-01

    Chronic hepatitis C is one of the most important causes of liver disease, leading to cirrhosis, hepatic decompensation and hepatocellular carcinoma. Recently some important advances in therapy have been achieved with the introduction of first wave, first generation direct acting antiviral agents (DAAs) such as boceprevir (BOC), in combination with pegylated interferon (Peg-IFN) and ribavirin (RBV). The superior rate of sustained virological response with this treatment is accompanied by an elevated frequency of anaemia. Several studies have evidenced the importance of single nucleotide polymorphisms (SNPs) in inosine triphosphatase (ITPA) and solute carrier family 29, member 1 (SLC29A1) genes in the development of this adverse event. Here, we investigated haemoglobin levels and the best-known functional SNPs in ITPA and SLC29A1 genes in 22 patients treated with triple therapy with BOC/Peg-IFN/RBV. The identification of ITPA protective and SLC29A1 risk genotypes still appears to be a current methodology in RBV dosing during hepatitis C virus therapy with DAAs. PMID:26279293

  12. 15N NMR spectroscopy of hydrogen-bonding interactions in the active site of serine proteases: evidence for a moving histidine mechanism.

    PubMed

    Bachovchin, W W

    1986-11-18

    Nitrogen-15 NMR spectroscopy has been used to study the hydrogen-bonding interactions involving the histidyl residue in the catalytic triad of alpha-lytic protease in the resting enzyme and in the transition-state or tetrahedral intermediate analogue complexes formed with phenylmethanesulfonyl fluoride and diisopropyl fluorophosphate. The 15N shifts indicate that a strong hydrogen bond links the active site histidine and serine residues in the resting enzyme in solution. This result is at odds with interpretations of the X-ray diffraction data of alpha-lytic protease and of other serine proteases, which indicate that the serine and histidine residues are too far apart and not properly aligned for the formation of a hydrogen bond. In addition, the nitrogen-15 shifts demonstrate that protonation of the histidine imidazole ring at low pH in the transition-state or tetrahedral intermediate analogue complexes formed with phenylmethanesulfonyl fluoride and diisopropyl fluorophosphate triggers the disruption of the aspartate-histidine hydrogen bond. These results suggest a catalytic mechanism involving directed movement of the imidazole ring of the active site histidyl residue. PMID:3542033

  13. Evidence for a SULT4A1 haplotype correlating with baseline psychopathology and atypical antipsychotic response

    PubMed Central

    Ramsey, Timothy L; Meltzer, Herbert Y; Brock, Guy N; Mehrotra, Bharat; Jayathilake, Karu; Bobo, William V; Brennan, Mark D

    2011-01-01

    Aim This study evaluated the impact of SULT4A1 gene variation on psychopathology and antipsychotic drug response in Caucasian subjects from the Clinical Antipsychotic Trials of Intervention Effectiveness (CATIE) study and a replication sample. Patients & methods SULT4A1 haplotypes were determined using SNP data. The relationship to baseline psychopathology was evaluated using linear regression of Positive and Negative Syndrome Scale (PANSS) total score. Drug response was evaluated using Mixed Model Repeat Measures (MMRM) for change in PANSS. Results For the CATIE sample, patients carrying a haplotype designated SULT4A1-1(+) displayed higher baseline PANSS (p = 0.03) and, when treated with olanzapine, demonstrated a significant interaction with time (p = 0.009) in the MMRM. SULT4A1-1(+) patients treated with olanzapine displayed improved response compared with SULT4A1-1(−) patients treated with olanzapine (p = 0.008) or to SULT4A1-1(+) patients treated with risperidone (p = 0.006). In the replication sample, SULT4A1-1(+) patients treated with olanzapine demonstrated greater improvement than SULT4A1-1(−) patients treated with olanzapine (p = 0.05) or than SULT4A1-1(+) patients treated with risperidone (p = 0.05). Conclusion If validated, determination of SULT4A1-1 haplotype status might be useful for identifying patients who show an enhanced response to long-term olanzapine treatment. PMID:21521020

  14. Cloning and nucleotide sequence of the Vibrio cholerae hemagglutinin/protease (HA/protease) gene and construction of an HA/protease-negative strain.

    PubMed Central

    Häse, C C; Finkelstein, R A

    1991-01-01

    The structural gene hap for the extracellular hemagglutinin/protease (HA/protease) of Vibrio cholerae was cloned and sequenced. The cloned DNA fragment contained a 1,827-bp open reading frame potentially encoding a 609-amino-acid polypeptide. The deduced protein contains a putative signal sequence followed by a large propeptide. The extracellular HA/protease consists of 414 amino acids with a computed molecular weight of 46,700. In the absence of protease inhibitors, this is processed to the 32-kDa form which is usually isolated. The deduced amino acid sequence of the mature HA/protease showed 61.5% identity with the Pseudomonas aeruginosa elastase. The cloned hap gene was inactivated and introduced into the chromosome of V. cholerae by recombination to construct the HA/protease-negative strain HAP-1. The cloned fragment containing the hap gene was then shown to complement the mutant strain. Images PMID:2045361

  15. Fibrin(ogen)olytic activity of bumblebee venom serine protease

    SciTech Connect

    Qiu Yuling; Choo, Young Moo; Yoon, Hyung Joo; Jia Jingming; Cui Zheng; Wang Dong; Kim, Doh Hoon; Sohn, Hung Dae; Jin, Byung Rae

    2011-09-01

    Bee venom is a rich source of pharmacologically active components; it has been used as an immunotherapy to treat bee venom hypersensitivity, and venom therapy has been applied as an alternative medicine. Here, we present evidence that the serine protease found in bumblebee venom exhibits fibrin(ogen)olytic activity. Compared to honeybee venom, bumblebee venom contains a higher content of serine protease, which is one of its major components. Venom serine proteases from bumblebees did not cross-react with antibodies against the honeybee venom serine protease. We provide functional evidence indicating that bumblebee (Bombus terrestris) venom serine protease (Bt-VSP) acts as a fibrin(ogen)olytic enzyme. Bt-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products. However, Bt-VSP is not a plasminogen activator, and its fibrinolytic activity is less than that of plasmin. Taken together, our results define roles for Bt-VSP as a prothrombin activator, a thrombin-like protease, and a plasmin-like protease. These findings offer significant insight into the allergic reaction sequence that is initiated by bee venom serine protease and its potential usefulness as a clinical agent in the field of hemostasis and thrombosis. - Graphical abstract: Display Omitted Highlights: > Bumblebee venom serine protease (Bt-VSP) is a fibrin(ogen)olytic enzyme. > Bt-VSP activates prothrombin. > Bt-VSP directly degrades fibrinogen into fibrin degradation products. > Bt-VSP is a hemostatically active protein that is a potent clinical agent.

  16. Extracellular acid proteases from Neurospora crassa.

    PubMed Central

    Lindberg, R A; Rhodes, W G; Eirich, L D; Drucker, H

    1982-01-01

    Three electrophoretically distinct acid proteases appear in culture filtrates of Neurospora crassa. Like the previously investigated alkaline and neutral proteases, these enzymes require induction by an exogenous protein. But in contrast to alkaline and neutral proteases, which are synthesized and secreted in response to limitation of any one of three nutrilites (carbon, nitrogen or sulfur), extracellular elaboration of the acidic proteases is more specifically a function of the missing nutrilite. AcP, a pepstatin-inhibitable enzyme similar to other fungal carboxyl proteases, was secreted in large amounts when protein was the sole source of sulfur. Only trace amounts were secreted when nitrogen was the limiting nutrilite, and it was undetectable under carbon limitation. M-1, a chelator-sensitive protease, was secreted when nitrogen or carbon was limiting. M-2, also chelator sensitive, was present only when nitrogen or sulfur was limiting. The evidence presented suggests that the differential regulation of the acidic proteases with respect to nutrilite deprivation may not occur at the level of transcription. AcP and M-2 were partially purified from nitrogen-derepressed cultures by ultrafiltration, cation-exchange chromatography, and gel filtration. AcP has a molecular weight of 66,000, is stable from pH 3.0 to 6.0, and is optimally active toward bovine serum albumin at pH 4.0. M-2 has a molecular weight of 18,000, is stable from pH 1.6 to 5.5, and has optimal activity at pH 4.5. Images PMID:6210687

  17. Extracellular acid proteases from Neurospora crassa.

    PubMed

    Lindberg, R A; Rhodes, W G; Eirich, L D; Drucker, H

    1982-06-01

    Three electrophoretically distinct acid proteases appear in culture filtrates of Neurospora crassa. Like the previously investigated alkaline and neutral proteases, these enzymes require induction by an exogenous protein. But in contrast to alkaline and neutral proteases, which are synthesized and secreted in response to limitation of any one of three nutrilites (carbon, nitrogen or sulfur), extracellular elaboration of the acidic proteases is more specifically a function of the missing nutrilite. AcP, a pepstatin-inhibitable enzyme similar to other fungal carboxyl proteases, was secreted in large amounts when protein was the sole source of sulfur. Only trace amounts were secreted when nitrogen was the limiting nutrilite, and it was undetectable under carbon limitation. M-1, a chelator-sensitive protease, was secreted when nitrogen or carbon was limiting. M-2, also chelator sensitive, was present only when nitrogen or sulfur was limiting. The evidence presented suggests that the differential regulation of the acidic proteases with respect to nutrilite deprivation may not occur at the level of transcription. AcP and M-2 were partially purified from nitrogen-derepressed cultures by ultrafiltration, cation-exchange chromatography, and gel filtration. AcP has a molecular weight of 66,000, is stable from pH 3.0 to 6.0, and is optimally active toward bovine serum albumin at pH 4.0. M-2 has a molecular weight of 18,000, is stable from pH 1.6 to 5.5, and has optimal activity at pH 4.5. PMID:6210687

  18. Extracellular acid proteases from Neurospora crassa

    SciTech Connect

    Lindberg, R.A.; Rhodes, W.G.; Eirich, L.D.; Drucker, H.

    1982-06-01

    Three electrophoretically distinct acid proteases appear in culture filtrates of Neurospora crassa. Like the previously investigated alkaline and neutral proteases, these enzymes require induction by an exogenous protein. But in contrast to alkaline and neutral proteases, which are synthesized and secreted in response to limitation of any one of three nutrilites (carbon, nitrogen or sulfur), extracellular elaboration of the acidic proteases is more specifically a function of the missing nutrilite. AcP, a pepstatin-inhibitable enzyme similar to other fungal carboxyl proteases, was secreted in large amounts when protein was the sole source of sulfur. Only trace amounts were secreted when nitrogen was the limiting nutrilite, and it was undetectable under carbon limitation. M-1, a chelator-sensitive protease, was secreted when nitrogen or carbon was limiting. M-2, also chelator sensitive, was present only when nitrogen or sulfur was limiting. The evidence presented suggests that the differential regulation of the acidic proteases with respect to nutrilite deprivation may not occur at the level of transcription. AcP and M-2 were partially purified from nitrogen-derepressed cultures by ultrafiltration, cation-exchange chromatography, and gel filtration. AcP has a molecular weight of 66,000, is stable from pH 3.0 to 6.0, and is optimally active toward bovine serum albumin at pH 4.0. M-2 has a molecular weight of 18,000, is stable from pH 1.6 to 5.5, and has optimal activity at pH 4.5.

  19. Substrate properties of C1 inhibitor Ma (alanine 434----glutamic acid). Genetic and structural evidence suggesting that the P12-region contains critical determinants of serine protease inhibitor/substrate status.

    PubMed

    Skriver, K; Wikoff, W R; Patston, P A; Tausk, F; Schapira, M; Kaplan, A P; Bock, S C

    1991-05-15

    The serine protease inhibitor (serpin) C1 inhibitor inactivates enzymes involved in the regulation of vascular permeability. A patient from the Ma family with the genetic disorder hereditary angioedema inherited a dysfunctional C1 inhibitor allele. Relative to normal plasma, the patients's plasma contained an additional C1 inhibitor immunoreactive band, which comigrated with normal C1 inhibitor cleaved by plasma kallikrein, C1s, or factor XIIa. C1 inhibitor Ma did not react with a monoclonal antibody to a neoepitope that is present in complexed and cleaved normal C1 inhibitor, suggesting conformational differences between cleaved normal C1- inhibitor and cleaved C1 inhibitor Ma. Molecular cloning and sequencing of exon 8 of the C1 inhibitor Ma allele revealed a single C to A mutation, changing alanine 434 to glutamic acid. Ala 434 of C1 inhibitor aligns with the P12 residue of the prototypical serpin alpha 1-antitrypsin. The P12 amino acid of all inhibitory serpins is alanine, and it is present in a highly conserved region on the amino-terminal side of the serpin-reactive center loop. Whereas normal C1 inhibitor expressed by transfected COS-1 cells formed complexes with and was cleaved by kallikrein, fXIIa, and C1s, COS-1-expressed Ala434---Glu C1 inhibitor was cleaved by these enzymes but did not form complexes with them. These results, together with evidence from other studies, suggest that serpin protease inhibitor activity is the result of protein conformational change that occurs when the P12 region of a serpin moves from a surface location, on the reactive site loop of the native molecule, to an internal location within sheet A of the complexed inhibitor. PMID:2026621

  20. Characterization of the immunoglobulin A protease of Ureaplasma urealyticum.

    PubMed Central

    Spooner, R K; Russell, W C; Thirkell, D

    1992-01-01

    Ureaplasma urealyticum strains of all serotypes express a specific human immunoglobulin A1 protease that cleaves immunoglobulin A1 to produce intact Fab and Fc fragments. The use of a variety of inhibitors suggests that the enzyme is a serine protease. N-terminal sequencing of the Fc digestion product showed that the enzyme cleaves between the proline and threonine residues 235 and 236 in the hinge region of the heavy chain of immunoglobulin A1. Images PMID:1587621

  1. Enteroviral proteases: structure, host interactions and pathogenicity.

    PubMed

    Laitinen, Olli H; Svedin, Emma; Kapell, Sebastian; Nurminen, Anssi; Hytönen, Vesa P; Flodström-Tullberg, Malin

    2016-07-01

    Enteroviruses are common human pathogens, and infections are particularly frequent in children. Severe infections can lead to a variety of diseases, including poliomyelitis, aseptic meningitis, myocarditis and neonatal sepsis. Enterovirus infections have also been implicated in asthmatic exacerbations and type 1 diabetes. The large disease spectrum of the closely related enteroviruses may be partially, but not fully, explained by differences in tissue tropism. The molecular mechanisms by which enteroviruses cause disease are poorly understood, but there is increasing evidence that the two enteroviral proteases, 2A(pro) and 3C(pro) , are important mediators of pathology. These proteases perform the post-translational proteolytic processing of the viral polyprotein, but they also cleave several host-cell proteins in order to promote the production of new virus particles, as well as to evade the cellular antiviral immune responses. Enterovirus-associated processing of cellular proteins may also contribute to pathology, as elegantly demonstrated by the 2A(pro) -mediated cleavage of dystrophin in cardiomyocytes contributing to Coxsackievirus-induced cardiomyopathy. It is likely that improved tools to identify targets for these proteases will reveal additional host protein substrates that can be linked to specific enterovirus-associated diseases. Here, we discuss the function of the enteroviral proteases in the virus replication cycle and review the current knowledge regarding how these proteases modulate the infected cell in order to favour virus replication, including ways to avoid detection by the immune system. We also highlight new possibilities for the identification of protease-specific cellular targets and thereby a way to discover novel mechanisms contributing to disease. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27145174

  2. Inhibitors of rhomboid proteases.

    PubMed

    Wolf, Eliane V; Verhelst, Steven H L

    2016-03-01

    Rhomboid proteases form one of the most widespread families of intramembrane proteases. They utilize a catalytic serine-histidine dyad located several Å below the surface of the membrane for substrate hydrolysis. Multiple studies have implicated rhomboid proteases in biologically and medically relevant processes. Several assays have been developed that are able to monitor rhomboid activity. With the aid of these assays, different types of inhibitors have been found, all based on electrophiles that covalently react with the active site machinery. Although the currently available inhibitors have limited selectivity and moderate potency, they can function as research tools and as starting point for the development of activity-based probes, which are reagents that can specifically detect active rhomboid species. Structural studies on complexes of inhibitors with the Escherichia coli rhomboid GlpG have provided insight into how substrate recognition may occur. Future synthetic efforts, aided by high-throughput screening or structure-based design, may lead to more potent and selective inhibitors for this interesting family of proteases. PMID:26166068

  3. Evidence that cytochrome b5 acts as a redox donor in CYP17A1 mediated androgen synthesis.

    PubMed

    Duggal, Ruchia; Liu, Yilin; Gregory, Michael C; Denisov, Ilia G; Kincaid, James R; Sligar, Stephen G

    2016-08-19

    Cytochrome P450 17A1 (CYP17A1) is an important drug target for castration resistant prostate cancer. It is a bi-functional enzyme, catalyzing production of glucocorticoid precursors by hydroxylation of pregnene-nucleus, and androgen biosynthesis by a second CC lyase step, at the expense of glucocorticoid production. Cytochrome b5 (cyt b5) is known to be a key regulator of the androgen synthesis reaction in vivo, by a mechanism that is not well understood. Two hypotheses have been proposed for the mechanism by which cyt b5 increases androgen biosynthesis. Cyt b5 could act as an allosteric effector, binding to CYP17A1 and either changing its selective substrate affinity or altering the conformation of the P450 to increase the catalytic rate or decrease unproductive uncoupling channels. Alternatively, cyt b5 could act as a redox donor for supply of the second electron in the P450 cycle, reducing the oxyferrous complex to form the reactive peroxo-intermediate. To understand the mechanism of lyase enhancement by cyt b5, we generated a redox-inactive form of cyt b5, in which the heme is replaced with a Manganese-protoporphyrin IX (Mn-b5), and investigated enhancement of androgen producing lyase reaction by CYP17A1. Given the critical significance of a stable membrane anchor for all of the proteins involved and the need for controlled stoichiometric ratios, we employed the Nanodisc system for this study. The redox inactive form was observed to have no effect on the lyase reaction, while reactions with the normal heme-iron containing cyt b5 were enhanced ∼5 fold as compared to reactions in the absence of cyt b5. We also performed resonance Raman measurements on ferric CYP17A1 bound to Mn-b5. Upon addition of Mn-b5 to Nanodisc reconstituted CYP17A1, we observed clear evidence for the formation of a b5-CYP17A1 complex, as noted by changes in the porphyrin modes and alteration in the proximal FeS vibrational frequency. Thus, although Mn-b5 binds to CYP17A1, it is unable to

  4. Protease inhibition as new therapeutic strategy for GI diseases

    PubMed Central

    Vergnolle, Nathalie

    2016-01-01

    The GI tract is the most exposed organ to proteases, both in physiological and pathophysiological conditions. For digestive purposes, the lumen of the upper GI tract contains large amounts of pancreatic proteases, but studies have also demonstrated increased proteolytic activity into mucosal tissues (both in the upper and lower GI tract), associated with pathological conditions. This review aims at outlining the evidences for dysregulated proteolytic homeostasis in GI diseases and the pathogenic mechanisms of increased proteolytic activity. The therapeutic potential of protease inhibition in GI diseases is discussed, with a particular focus on IBDs, functional GI disorders and colorectal cancer. PMID:27196587

  5. Protease inhibition as new therapeutic strategy for GI diseases.

    PubMed

    Vergnolle, Nathalie

    2016-07-01

    The GI tract is the most exposed organ to proteases, both in physiological and pathophysiological conditions. For digestive purposes, the lumen of the upper GI tract contains large amounts of pancreatic proteases, but studies have also demonstrated increased proteolytic activity into mucosal tissues (both in the upper and lower GI tract), associated with pathological conditions. This review aims at outlining the evidences for dysregulated proteolytic homeostasis in GI diseases and the pathogenic mechanisms of increased proteolytic activity. The therapeutic potential of protease inhibition in GI diseases is discussed, with a particular focus on IBDs, functional GI disorders and colorectal cancer. PMID:27196587

  6. Proteases and Peptidases of Castor Bean Endosperm

    PubMed Central

    Tully, Raymond E.; Beevers, Harry

    1978-01-01

    The endosperm of castor bean seeds (Ricinus communis L.) contains two —SH-dependent aminopeptidases, one hydrolyzing l-leucine-β-naphthylamide optimally at pH 7.0, and the other hydrolyzing l-proline-β-naphthylamide optimally at pH 7.5. After germination the endosperm contains in addition an —SH-dependent hemoglobin protease, a serine-dependent carboxypeptidase, and at least two —SH-dependent enzymes hydrolyzing the model substrate α-N-benzoyl-dl-arginine-β-naphthylamide (BANA). The carboxypeptidase is active on a variety of N-carbobenzoxy dipeptides, especially N-carbobenzoxy-L-phenylalanine-l-alanine and N-carbobenzoxy-l-tyrosine-l-leucine. The pH optima for the protease, carboxypeptidase, and BANAase acivities are 3.5 to 4.0, 5.0 to 5.5, and 6 to 8, respectively. The two aminopeptidases increased about 4-fold in activity during the first 4 days of growth, concurrent with the period of rapid depletion of storage protein. Activities then declined as the endosperm senesced, but were still evident after 6 days. Senescence was complete by day 7 to 8. Hemoglobin protease, carboxypeptidase, and BANAase activities appeared in the endosperm at day 2 to 3, and reached peak activity at day 5 to 6. The data indicate that the aminopeptidases are involved in the early mobilization of endosperm storage protein, whereas protease, carboxypeptidase, and BANAase may take part in later turnover and/or senescence. PMID:16660598

  7. From proteases to proteomics

    PubMed Central

    Neurath, Hans

    2001-01-01

    This personal and professional autobiography covers the 50-yr period of 1950–2000 and includes the following topics: History of the University of Washington School of Medicine and its Department of Biochemistry (Mount Rainier and the University of Washington, recruiting faculty, biology, research programs); scientific editing (publication, Biochemistry, Protein Science, electronic publication); Europe revisited (Heidelberg, approaching retirement, the German Research Center, reunion in Vienna); and 50 yr of research on proteolytic enzymes (trypsin, carboxypeptidases, mast cell proteases, future developments). PMID:11274481

  8. Evidence for deactivation of both ectosolic and cytosolic 5'-nucleotidase by adenosine A1 receptor activation in the rat cardiomyocytes.

    PubMed Central

    Kitakaze, M; Hori, M; Minamino, T; Takashima, S; Komamura, K; Node, K; Kurihara, T; Morioka, T; Sato, H; Inoue, M

    1994-01-01

    Adenosine, an important regulator of many cardiac functions, is produced by ectosolic and cytosolic 5'-nucleotidase. The activity of these enzymes is influenced by several ischemia-sensitive metabolic factors, e.g., ATP, ADP, H+, and inorganic phosphate. However, there is no clear evidence that adenosine itself affects 5'-nucleotidase activity. This study tested whether adenosine decreases the activity of ectosolic and cytosolic 5'-nucleotidase. Cardiomyocytes were isolated from adult male Wistar rats and suspended in the modified Hepes-Tyrode buffer solution. After stabilization, isolated cardiomyocytes were incubated with and without adenosine (10(-9) - 10(-4) M). Ectosolic and cytosolic 5'-nucleotidase activity was decreased by exogenous adenosine (ectosolic 5'-nucleotidase activity, 20.6 +/- 2.3 vs. 8.6 +/- 1.6 mumol/min per 10(6) cells [P < 0.05]; cytosolic 5'-nucleotidase activity, 2.47 +/- 0.58 vs. 1.61 +/- 0.54 mumol/min per 10(6) cells [P < 0.05] at 10(-6) M adenosine) after 30 min. The decrease in ectosolic and cytosolic 5'-nucleotidase activity was inhibited by 8-phenyltheophylline and pertussis toxin, and was mimicked by N6-cyclohexyladenosine, an adenosine A1 receptor agonist. Neither CGS21680C, and A2 receptor agonist, nor cycloheximide deactivated ectosolic and cytosolic 5'-nucleotidase. Thus, we conclude that activation of adenosine A1 receptors is coupled to Gi proteins and attenuates ectosolic and cytosolic 5'-nucleotidase activity in rat cardiomyocytes. Images PMID:7989602

  9. Induction of CYP26A1 by Metabolites of Retinoic Acid: Evidence That CYP26A1 Is an Important Enzyme in the Elimination of Active Retinoids

    PubMed Central

    Topletz, Ariel R.; Tripathy, Sasmita; Foti, Robert S.; Shimshoni, Jakob A.; Nelson, Wendel L.

    2015-01-01

    All-trans-retinoic acid (atRA), the active metabolite of vitamin A, induces gene transcription via binding to nuclear retinoic acid receptors (RARs). The primary hydroxylated metabolites formed from atRA by CYP26A1, and the subsequent metabolite 4-oxo-atRA, bind to RARs and potentially have biologic activity. Hence, CYP26A1, the main atRA hydroxylase, may function either to deplete bioactive retinoids or to form active metabolites. This study aimed to determine the role of CYP26A1 in modulating RAR activation via formation and elimination of active retinoids. After treatment of HepG2 cells with atRA, (4S)-OH-atRA, (4R)-OH-atRA, 4-oxo-atRA, and 18-OH-atRA, mRNAs of CYP26A1 and RARβ were increased 300- to 3000-fold, with 4-oxo-atRA and atRA being the most potent inducers. However, >60% of the 4-OH-atRA enantiomers were converted to 4-oxo-atRA in the first 12 hours of treatment, suggesting that the activity of the 4-OH-atRA was due to 4-oxo-atRA. In human hepatocytes, atRA, 4-OH-atRA, and 4-oxo-atRA induced CYP26A1 and 4-oxo-atRA formation was observed from 4-OH-atRA. In HepG2 cells, 4-oxo-atRA formation was observed even in the absence of CYP26A1 activity and this formation was not inhibited by ketoconazole. In human liver microsomes, 4-oxo-atRA formation was supported by NAD+, suggesting that 4-oxo-atRA formation is mediated by a microsomal alcohol dehydrogenase. Although 4-oxo-atRA was not formed by CYP26A1, it was depleted by CYP26A1 (Km = 63 nM and intrinsic clearance = 90 μl/min per pmol). Similarly, CYP26A1 depleted 18-OH-atRA and the 4-OH-atRA enantiomers. These data support the role of CYP26A1 to clear bioactive retinoids, and suggest that the enzyme forming active 4-oxo-atRA may be important in modulating retinoid action. PMID:25492813

  10. Novel proteases: common themes and surprising features.

    PubMed

    Vandeputte-Rutten, Lucy; Gros, Piet

    2002-12-01

    Proteases perform a wide variety of functions, inside and outside cells, regulating many biological processes. Recent years have witnessed a number of significant advances in the structural biology of proteases, including aspects of intracellular protein and peptide degradation by self-compartmentalizing proteases, activation of proteases in proteolytic cascades of regulatory pathways, and mechanisms of microbial proteases in pathogenicity. PMID:12504673

  11. 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.

  12. Alpha-1 Antitrypsin Deficiency: Beyond the Protease/Antiprotease Paradigm.

    PubMed

    Cosio, Manuel G; Bazzan, Erica; Rigobello, Chiara; Tinè, Mariaenrica; Turato, Graziella; Baraldo, Simonetta; Saetta, Marina

    2016-08-01

    From the discovery that alpha-1 antitrypsin (AAT) was an effective inhibitor of neutrophil elastase originated the classic paradigm of protease/antiprotease imbalance, linking lung destruction to the unopposed effect of proteases in patients with the deficiency. Notwithstanding its importance as an antiprotease, it has become evident that alpha-1 antitrypsin has important antiinflammatory and immune-regulatory activities, which may be critically involved in lung destruction. We review here recent evidence showing that, indeed, an important adaptive immune reaction is present in lungs with AAT deficiency, similar to the one seen in severe chronic obstructive pulmonary disease with normal AAT. On the basis of recent evidence from epidemiological, clinical, and pathogenetic studies, it is likely time to move on from the original protease/antiprotease hypothesis for the production of emphysema toward a more complex paradigm, involving the antiinflammatory and immune modulating functions of AAT. PMID:27564665

  13. Feces Derived Allergens of Tyrophagus putrescentiae Reared on Dried Dog Food and Evidence of the Strong Nutritional Interaction between the Mite and Bacillus cereus Producing Protease Bacillolysins and Exo-chitinases

    PubMed Central

    Erban, Tomas; Rybanska, Dagmar; Harant, Karel; Hortova, Bronislava; Hubert, Jan

    2016-01-01

    Tyrophagus putrescentiae (Schrank, 1781) is an emerging source of allergens in stored products and homes. Feces proteases are the major allergens of astigmatid mites (Acari: Acaridida). In addition, the mites are carriers of microorganisms and microbial adjuvant compounds that stimulate innate signaling pathways. We sought to analyze the mite feces proteome, proteolytic activities, and mite-bacterial interaction in dry dog food (DDF). Proteomic methods comprising enzymatic and zymographic analysis of proteases and 2D-E-MS/MS were performed. The highest protease activity was assigned to trypsin-like proteases; lower activity was assigned to chymotrypsin-like proteases, and the cysteine protease cathepsin B-like had very low activity. The 2D-E-MS/MS proteomic analysis identified mite trypsin allergen Tyr p3, fatty acid-binding protein Tyr p13 and putative mite allergens ferritin (Grp 30) and (poly)ubiquitins. Tyr p3 was detected at different positions of the 2D-E. It indicates presence of zymogen at basic pI, and mature-enzyme form and enzyme fragment at acidic pI. Bacillolysins (neutral and alkaline proteases) of Bacillus cereus symbiont can contribute to the protease activity of the mite extract. The bacterial exo-chitinases likely contribute to degradation of mite exuviae, mite bodies or food boluses consisting of chitin, including the peritrophic membrane. Thus, the chitinases disrupt the feces and facilitate release of the allergens. B. cereus was isolated and identified based on amplification and sequencing of 16S rRNA and motB genes. B. cereus was added into high-fat, high-protein (DDF) and low-fat, low-protein (flour) diets to 1 and 5% (w/w), and the diets palatability was evaluated in 21-day population growth test. The supplementation of diet with B. cereus significantly suppressed population growth and the suppressive effect was higher in the high-fat, high-protein diet than in the low-fat, low-protein food. Thus, B. cereus has to coexist with the mite in

  14. Feces Derived Allergens of Tyrophagus putrescentiae Reared on Dried Dog Food and Evidence of the Strong Nutritional Interaction between the Mite and Bacillus cereus Producing Protease Bacillolysins and Exo-chitinases.

    PubMed

    Erban, Tomas; Rybanska, Dagmar; Harant, Karel; Hortova, Bronislava; Hubert, Jan

    2016-01-01

    Tyrophagus putrescentiae (Schrank, 1781) is an emerging source of allergens in stored products and homes. Feces proteases are the major allergens of astigmatid mites (Acari: Acaridida). In addition, the mites are carriers of microorganisms and microbial adjuvant compounds that stimulate innate signaling pathways. We sought to analyze the mite feces proteome, proteolytic activities, and mite-bacterial interaction in dry dog food (DDF). Proteomic methods comprising enzymatic and zymographic analysis of proteases and 2D-E-MS/MS were performed. The highest protease activity was assigned to trypsin-like proteases; lower activity was assigned to chymotrypsin-like proteases, and the cysteine protease cathepsin B-like had very low activity. The 2D-E-MS/MS proteomic analysis identified mite trypsin allergen Tyr p3, fatty acid-binding protein Tyr p13 and putative mite allergens ferritin (Grp 30) and (poly)ubiquitins. Tyr p3 was detected at different positions of the 2D-E. It indicates presence of zymogen at basic pI, and mature-enzyme form and enzyme fragment at acidic pI. Bacillolysins (neutral and alkaline proteases) of Bacillus cereus symbiont can contribute to the protease activity of the mite extract. The bacterial exo-chitinases likely contribute to degradation of mite exuviae, mite bodies or food boluses consisting of chitin, including the peritrophic membrane. Thus, the chitinases disrupt the feces and facilitate release of the allergens. B. cereus was isolated and identified based on amplification and sequencing of 16S rRNA and motB genes. B. cereus was added into high-fat, high-protein (DDF) and low-fat, low-protein (flour) diets to 1 and 5% (w/w), and the diets palatability was evaluated in 21-day population growth test. The supplementation of diet with B. cereus significantly suppressed population growth and the suppressive effect was higher in the high-fat, high-protein diet than in the low-fat, low-protein food. Thus, B. cereus has to coexist with the mite in

  15. New insights into the evolution of subtilisin-like serine protease genes in Pezizomycotina

    PubMed Central

    2010-01-01

    Background Subtilisin-like serine proteases play an important role in pathogenic fungi during the penetration and colonization of their hosts. In this study, we perform an evolutionary analysis of the subtilisin-like serine protease genes of subphylum Pezizomycotina to find if there are similar pathogenic mechanisms among the pathogenic fungi with different life styles, which utilize subtilisin-like serine proteases as virulence factors. Within Pezizomycotina, nematode-trapping fungi are unique because they capture soil nematodes using specialized trapping devices. Increasing evidence suggests subtilisin-like serine proteases from nematode-trapping fungi are involved in the penetration and digestion of nematode cuticles. Here we also conduct positive selection analysis on the subtilisin-like serine protease genes from nematode-trapping fungi. Results Phylogenetic analysis of 189 subtilisin-like serine protease genes from Pezizomycotina suggests five strongly-supported monophyletic clades. The subtilisin-like serine protease genes previously identified or presumed as endocellular proteases were clustered into one clade and diverged the earliest in the phylogeny. In addition, the cuticle-degrading protease genes from entomopathogenic and nematode-parasitic fungi were clustered together, indicating that they might have overlapping pathogenic mechanisms against insects and nematodes. Our experimental bioassays supported this conclusion. Interestingly, although they both function as cuticle-degrading proteases, the subtilisin-like serine protease genes from nematode-trapping fungi and nematode-parasitic fungi were not grouped together in the phylogenetic tree. Our evolutionary analysis revealed evidence for positive selection on the subtilisin-like serine protease genes of the nematode-trapping fungi. Conclusions Our study provides new insights into the evolution of subtilisin-like serine protease genes in Pezizomycotina. Pezizomycotina subtilisins most likely evolved

  16. Protease degradable electrospun fibrous hydrogels

    PubMed Central

    Wade, Ryan J.; Bassin, Ethan J.; Rodell, Christopher B.; Burdick, Jason A.

    2015-01-01

    Electrospun nanofibers are promising in biomedical applications to replicate features of the natural extracellular matrix (ECM). However, nearly all electrospun scaffolds are either non-degradable or degrade hydrolytically, whereas natural ECM degrades proteolytically, often through matrix metalloproteinases (MMPs). Here, we synthesize reactive macromers that contain protease-cleavable and fluorescent peptides and are able to form both isotropic hydrogels and electrospun fibrous hydrogels through a photoinitiated polymerization. These biomimetic scaffolds are susceptible to protease-mediated cleavage in vitro in a protease dose dependent manner and in vivo in a subcutaneous mouse model using transdermal fluorescent imaging to monitor degradation. Importantly, materials containing an alternate and non-protease-cleavable peptide sequence are stable in both in vitro and in vivo settings. To illustrate the specificity in degradation, scaffolds with mixed fiber populations support selective fiber degradation based on individual fiber degradability. Overall, this represents a novel biomimetic approach to generate protease-sensitive fibrous scaffolds for biomedical applications. PMID:25799370

  17. 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

  18. A bead-based cleavage method for large-scale identification of protease substrates

    PubMed Central

    Wang, Chunli; Ye, Mingliang; Wei, Xiaoluan; Bian, Yangyang; Cheng, Kai; Zou, Hanfa

    2016-01-01

    Proteolysis is a major form of post translational modification which occurs when a protease cleaves peptide bonds in a target protein to modify its activity. Tracking protease substrates is indispensable for understanding its cellular functions. However, it is difficult to directly identify protease substrates because the end products of proteolysis, the cleaved protein fragments, must be identified among the pool of cellular proteins. Here we present a bead-based cleavage approach using immobilized proteome as the screening library to identify protease substrates. This method enables efficient separation of proteolyzed proteins from background protein mixture. Using caspase-3 as the model protease, we have identified 1159 high confident substrates, among which, strikingly, 43.9% of substrates undergo degradation during apoptosis. The huge number of substrates and positive support of in vivo evidence indicate that the BBC method is a powerful tool for protease substrates identification. PMID:26935269

  19. The salt-sensitive structure and zinc inhibition of Borrelia burgdorferi protease BbHtrA.

    PubMed

    Russell, Theresa M; Tang, Xiaoling; Goldstein, Jason M; Bagarozzi, Dennis; Johnson, Barbara J B

    2016-02-01

    HtrA serine proteases are highly conserved and essential ATP-independent proteases with chaperone activity. Bacteria express a variable number of HtrA homologues that contribute to the virulence and pathogenicity of bacterial pathogens. Lyme disease spirochetes possess a single HtrA protease homologue, Borrelia burgdorferi HtrA (BbHtrA). Previous studies established that, like the human orthologue HtrA1, BbHtrA is proteolytically active against numerous extracellular proteins in vitro. In this study, we utilized size exclusion chromatography and blue native polyacrylamide gel electrophoresis (BN-PAGE) to demonstrate BbHtrA oligomeric structures that were substrate independent and salt sensitive. Examination of the influence of transition metals on the activity of BbHtrA revealed that this protease is inhibited by Zn(2+) > Cu(2+) > Mn(2+). Extending this analysis to two other HtrA proteases, E. coli DegP and HtrA1, revealed that all three HtrA proteases were reversibly inhibited by ZnCl2 at all micro molar concentrations examined. Commercial inhibitors for HtrA proteases are not available and physiologic HtrA inhibitors are unknown. Our observation of conserved zinc inhibition of HtrA proteases will facilitate structural and functional studies of additional members of this important class of proteases. PMID:26480895

  20. Serine proteases of parasitic helminths.

    PubMed

    Yang, Yong; Wen, Yun jun; Cai, Ya Nan; Vallée, Isabelle; Boireau, Pascal; Liu, Ming Yuan; Cheng, Shi Peng

    2015-02-01

    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. 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

  2. 11B NMR spectroscopy of peptide boronic acid inhibitor complexes of alpha-lytic protease. Direct evidence for tetrahedral boron in both boron-histidine and boron-serine adduct complexes.

    PubMed

    Tsilikounas, E; Kettner, C A; Bachovchin, W W

    1993-11-30

    We have previously shown, using 15N and 1H NMR spectroscopy, that MeOSuc-Ala-Ala-Pro-boroPhe and certain other boronic acid inhibitors form boron-histidine adducts with alpha-lytic protease instead of transition-state-like tetrahedral boron-serine adducts as is generally supposed [Bachovchin, W. W., Wong, W. Y. L., Farr-Jones, S., Shenvi, A. B., & Kettner, C. (1988) Biochemistry 27, 7689-7697]. An X-ray crystallographic study of the MeOSuc-Ala-Ala-Pro-boroPhe complex with alpha-lytic protease [Bone, R., Frank, D., Kettner, C. A., & Agard, D. A. (1989) Biochemistry 28, 7600-7609] has confirmed the existence of the boron-histidine bond but has concluded that the boron atom is trigonal rather than tetrahedral. Here we report a 11B NMR study at 160.46 MHz of this histidine adduct complex and of two other complexes known to be serine adducts: alpha-lytic protease with MeOSuc-Ala-Ala-Pro-boroVal and chymotrypsin with MeOSucAla-Ala-Pro-boroPhe. The 11B NMR chemical shifts demonstrate that the boron atom is tetrahedral in both the histidine and serine adduct complexes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8251483

  3. Association between CYP1A1 Ile462Val Polymorphism and Oral Squamous Cell Carcinoma Susceptibility: Evidence from 13 Investigations

    PubMed Central

    Yang, Xiao-Lei; Xie, Shang; Jiang, Yi-Yan; Shi, Chang; Cai, Zhi-Gang; Chen, Su-Xiu

    2015-01-01

    CYP1A1 Ile462Val polymorphism might play a key role in pathogenesis of oral squamous cell carcinoma (OSCC). Many case-control studies have investigated the association between CYP1A1 Ile462Val polymorphism and OSCC susceptibility. However, the conclusions are inconsistent. To aim a convincible conclusion, we carried out a meta-analysis to systematically evaluate the association of CYP1A1 Ile462Val polymorphism with OSCC susceptibility. We searched Pubmed, Web of Science, Ovid and Embase databases for available publications. The odds ratio (OR) with the corresponding 95% confidence interval (95% CI) was carried out to estimate the association. A total of 13 papers including 1468 cases and 2183 controls were included, a significant increased OSCC risk was observed in recessive model (OR=1.64, 95% CI=1.08-2.49), but not other genetic models. Our results suggest that the homozygous variant of CYP1A1 Ile462Val might be a risk factor of OSCC. PMID:25767599

  4. Homology modelling and structural analysis of human arylamine N-acetyltransferase NAT1: evidence for the conservation of a cysteine protease catalytic domain and an active-site loop.

    PubMed Central

    Rodrigues-Lima, F; Deloménie, C; Goodfellow, G H; Grant, D M; Dupret, J M

    2001-01-01

    Arylamine N-acetyltransferases (EC 2.3.1.5) (NATs) catalyse the biotransformation of many primary arylamines, hydrazines and their N-hydroxylated metabolites, thereby playing an important role in both the detoxification and metabolic activation of numerous xenobiotics. The recently published crystal structure of the Salmonella typhimurium NAT (StNAT) revealed the existence of a cysteine protease-like (Cys-His-Asp) catalytic triad. In the present study, a three-dimensional homology model of human NAT1, based upon the crystal structure of StNAT [Sinclair, Sandy, Delgoda, Sim and Noble (2000) Nat. Struct. Biol. 7, 560-564], is demonstrated. Alignment of StNAT and NAT1, together with secondary structure predictions, have defined a consensus region (residues 29-131) in which 37% of the residues are conserved. Homology modelling provided a good quality model of the corresponding region in human NAT1. The location of the catalytic triad was found to be identical in StNAT and NAT1. Comparison of active-site structural elements revealed that a similar length loop is conserved in both species (residues 122-131 in NAT1 model and residues 122-133 in StNAT). This observation may explain the involvement of residues 125, 127 and 129 in human NAT substrate selectivity. Our model, and the fact that cysteine protease inhibitors do not affect the activity of NAT1, suggests that human NATs may have adapted a common catalytic mechanism from cysteine proteases to accommodate it for acetyl-transfer reactions. PMID:11368758

  5. Microbial inhibitors of cysteine proteases.

    PubMed

    Kędzior, Mateusz; Seredyński, Rafał; Gutowicz, Jan

    2016-08-01

    Cysteine proteases are one of the major classes of proteolytic enzymes involved in a number of physiological and pathological processes in plants, animals and microorganisms. When their synthesis, activity and localization in mammalian cells are altered, they may contribute to the development of many diseases, including rheumatoid arthritis, osteoporosis and cancer. Therefore, cysteine proteases have become promising drug targets for the medical treatment of these disorders. Inhibitors of cysteine proteases are also produced by almost every group of living organisms, being responsible for the control of intracellular proteolytic activity. Microorganisms synthesize cysteine protease inhibitors not only to regulate the activity of endogenous, often virulent enzymes, but also to hinder the host's proteolytic defense system and evade its immune responses against infections. Present work describes known to date microbial inhibitors of cysteine proteases in terms of their structure, enzyme binding mechanism, specificity and pathophysiological roles. The overview of both proteinaceous and small-molecule inhibitors produced by all groups of microorganisms (bacteria, archaea, fungi, protists) and viruses is provided. Subsequently, possible applications of microbial inhibitors in science, medicine and biotechnology are also highlighted. PMID:27048482

  6. Unraveling the roles of Atg4 proteases from autophagy modulation to targeted cancer therapy.

    PubMed

    Zhang, Lan; Li, Jingjing; Ouyang, Liang; Liu, Bo; Cheng, Yan

    2016-04-01

    Atg4 proteases are cysteine proteases, which are well known for their crucial roles in the lipidation and delipidation of LC3 during the autophagy process. At least four human Atg4 homologs have been reported according to their sequence homology to the yeast Saccharomyces cerevisiae (Sc) Atg4. Accumulating evidence has recently indicated that abnormal expression levels of some human Atg4 proteins occur in several types of cancer cells, which may be closely related to tumor progression, tumor suppression and cancer therapy resistance. In this review, we focus on highlighting the pivotal roles of the human Atg4 proteases in the LC3 conjugation system and their major function in autophagy. Moreover, we further explore the roles of human Atg4 proteases as potential novel targets for cancer therapy. Taken together, these findings would shed light on elucidating the key roles of Atg4 proteases from autophagy modulation to targeted cancer therapy. PMID:26805760

  7. Mitochondrial AAA proteases--towards a molecular understanding of membrane-bound proteolytic machines.

    PubMed

    Gerdes, Florian; Tatsuta, Takashi; Langer, Thomas

    2012-01-01

    Mitochondrial AAA proteases play an important role in the maintenance of mitochondrial proteostasis. They regulate and promote biogenesis of mitochondrial proteins by acting as processing enzymes and ensuring the selective turnover of misfolded proteins. Impairment of AAA proteases causes pleiotropic defects in various organisms including neurodegeneration in humans. AAA proteases comprise ring-like hexameric complexes in the mitochondrial inner membrane and are functionally conserved from yeast to man, but variations are evident in the subunit composition of orthologous enzymes. Recent structural and biochemical studies revealed how AAA proteases degrade their substrates in an ATP dependent manner. Intersubunit coordination of the ATP hydrolysis leads to an ordered ATP hydrolysis within the AAA ring, which ensures efficient substrate dislocation from the membrane and translocation to the proteolytic chamber. In this review, we summarize recent findings on the molecular mechanisms underlying the versatile functions of mitochondrial AAA proteases and their relevance to those of the other AAA+ machines. PMID:22001671

  8. 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

  9. 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. PMID:27608950

  10. Analysis of the immunoglobulin A protease gene of Streptococcus sanguis.

    PubMed Central

    Gilbert, J V; Plaut, A G; Wright, A

    1991-01-01

    The amino acid sequence T-P-P-T-P-S-P-S is tandemly duplicated in the heavy chain of human immunoglobulin A1 (IgA1), the major antibody in secretions. The bacterial pathogen Streptococcus sanguis, a precursor to dental caries and a cause of bacterial endocarditis, yields IgA protease that cleaves only the Pro-Thr peptide bond in the left duplication, while the type 2 IgA proteases of the genital pathogen Neisseria gonorrhoeae and the respiratory pathogen Haemophilus influenzae cleave only the P-T bond in the right half. We have sequenced the entire S. sanguis iga gene cloned into Escherichia coli. A segment consisting of 20 amino acids tandemly repeated 10 times, of unknown function, occurs near the amino-terminal end of the enzyme encoded in E. coli. Identification of a predicted zinc-binding region in the S. sanguis enzyme and the demonstration that mutations in this region result in production of a catalytically inactive protein support the idea that the enzyme is a metalloprotease. The N. gonorrhoeae and H. influenzae enzymes were earlier shown to be serine-type proteases, while the Bacteroides melaninogenicus IgA protease was shown to be a cysteine-type enzyme. The streptococcal IgA protease amino acid sequence has no significant homology with either of the two previously determined IgA protease sequences, that of type 2 N. gonorrhoeae and type 1 H. influenzae. The differences in both structure and mechanism among these functionally analogous enzymes underscore their role in the infectious process and offer some prospect of therapeutic intervention. Images PMID:1987065

  11. Determination of the protease cleavage site repertoire—The RNase H but not the RT domain is essential for foamy viral protease activity

    SciTech Connect

    Spannaus, Ralf; Bodem, Jochen

    2014-04-15

    In contrast to orthoretroviruses, the foamy virus protease is only active as a protease-reverse transcriptase fusion protein and requires viral RNA for activation. Maturation of foamy viral proteins seems to be restricted to a single cleavage site in Gag and Pol. We provide evidence that unprocessed Gag is required for optimal infectivity, which is unique among retroviruses. Analyses of the cleavage site sequences of the Gag and Pol cleavage sites revealed a high similarity compared to those of Lentiviruses. We show that positions P2' and P2 are invariant and that Gag and Pol cleavage sites are processed with similar efficiencies. The RNase H domain is essential for protease activity, but can functionally be substituted by RNase H domains of other retroviruses. Thus, the RNase H domain might be involved in the stabilization of the protease dimer, while the RT domain is essential for RNA dependent protease activation. - Highlights: • Unprocessed Gag is required for optimal infectivity of foamy viruses. • Positions P2 and P2' are invariant in the foamy viral cleavage sites. • The RNaseH domain is essential for protease activity. • The RNaseH domains of other retroviruses support foamy viral protease activity.

  12. Taspase1: a 'misunderstood' protease with translational cancer relevance.

    PubMed

    Wünsch, D; Hahlbrock, A; Jung, S; Schirmeister, T; van den Boom, J; Schilling, O; Knauer, S K; Stauber, R H

    2016-06-30

    Proteolysis is not only a critical requirement for life, but the executing enzymes also play important roles in numerous pathological conditions, including cancer. Therefore, targeting proteases is clearly relevant for improving cancer patient care. However, to effectively control proteases, a profound knowledge of their mechanistic function as well as their regulation and downstream signalling in health and disease is required. The highly conserved protease Threonine Aspartase1 (Taspase1) is overexpressed in numerous liquid and solid malignancies and was characterized as a 'non-oncogene addiction' protease. Although Taspase1 was shown to cleave various regulatory proteins in humans as well as leukaemia provoking mixed lineage leukaemia fusions, our knowledge on its detailed functions and the underlying mechanisms contributing to cancer is still incomplete. Despite superficial similarity to type 2 asparaginases as well as Ntn proteases, such as the proteasome, Taspase1-related research so far gives us the picture of a unique protease exhibiting special features. Moreover, neither effective genetic nor chemical inhibitors for this enzyme are available so far, thus hampering not only to further dissect Taspase1's pathobiological functions but also precluding the assessment of its clinical impact. Based on recent insights, we here critically review the current knowledge of Taspase1's structure-function relationship and its mechanistic relevance for tumorigenesis obtained from in vitro and in vivo cancer models. We provide a comprehensive overview of tumour entities for which Taspase1 might be of predictive and therapeutic value, and present the respective experimental evidence. To stimulate progress in the field, a comprehensive overview of Taspase1 targeting approaches is presented, including coverage of Taspase1-related patents. We conclude by discussing future inhibition strategies and relevant challenges, which need to be resolved by the field. PMID:26657154

  13. Laboratory Detection of ClZnCH3 (X1A1): Further Evidence for Zinc Insertion

    NASA Astrophysics Data System (ADS)

    Bucchino, Matthew; Ziurys, Lucy

    2014-06-01

    The pure rotational spectrum of methylzinc chloride, ClZnCH3 (X1A1), has been recorded in the gas phase using direct absorption spectroscopic techniques. ClZnCH3 was synthesized by the reaction of zinc vapor, generated in a Broida-type oven, with chloromethane in the presence of a DC discharge. Rotational transitions of the main isotopologue, 35Cl64ZnCH3, were measured in the frequency range of 260{-297} GHz. The presence of clear K-ladder structure (K = 0{-6}) indicates that the species is a symmetric top with C3V symmetry. ClZnCH3 appears to be formed by the oxidative addition of atomic zinc to chloromethane. Searches for the 37Cl, 66Zn, 13C, and D substituted isotopologues are currently in progress. The geometries of ClZnCH3 and IZnCH3 will be compared with reactivity trends for halogen-substituted organometallic reagents.

  14. Exogenous proteases for meat tenderization.

    PubMed

    Bekhit, Alaa A; Hopkins, David L; Geesink, Geert; Bekhit, Adnan A; Franks, Philip

    2014-01-01

    The use of exogenous proteases to improve meat tenderness has attracted much interest recently, with a view to consistent production of tender meat and added value to lower grade meat cuts. This review discusses the sources, characteristics, and use of exogenous proteases in meat tenderization to highlight the specificity of the proteases toward meat proteins and their impact on meat quality. Plant enzymes (such as papain, bromelain, and ficin) have been extensively investigated as meat tenderizers. New plant proteases (actinidin and zingibain) and microbial enzyme preparations have been of recent interest due to controlled meat tenderization and other advantages. Successful use of these enzymes in fresh meat requires their enzymatic kinetics and characteristics to be determined, together with an understanding of the impact of the surrounding environmental conditions of the meat (pH, temperature) on enzyme function. This enables the optimal conditions for tenderizing fresh meat to be established, and the elimination or reduction of any negative impacts on other quality attributes. PMID:24499119

  15. Characterization of 18 new mutations in COL7A1 in recessive dystrophic epidermolysis bullosa provides evidence for distinct molecular mechanisms underlying defective anchoring fibril formation.

    PubMed Central

    Hovnanian, A; Rochat, A; Bodemer, C; Petit, E; Rivers, C A; Prost, C; Fraitag, S; Christiano, A M; Uitto, J; Lathrop, M; Barrandon, Y; de Prost, Y

    1997-01-01

    We have characterized 21 mutations in the type VII collagen gene (COL7A1) encoding the anchoring fibrils, 18 of which were not previously reported, in patients from 15 unrelated families with recessive dystrophic epidermolysis bullosa (RDEB). COL7A1 mutations in both alleles were identified by screening the 118 exons of COL7A1 and flanking intron regions. Fourteen mutations created premature termination codons (PTCs) and consisted of nonsense mutations, small insertions, deletions, and splice-site mutations. A further seven mutations predicted glycine or arginine substitutions in the collagenous domain of the molecule. Two mutations were found in more than one family reported in this study, and six of the seven missense mutations showed clustering within exons 72-74 next to the hinge region of the protein. Patients who were homozygous or compound heterozygotes for mutations leading to PTCs displayed both absence or drastic reduction of COL7A1 transcripts and undetectable type VII collagen protein in skin. In contrast, missense mutations were associated with clearly detectable COL7A1 transcripts and with normal or reduced expression of type VII collagen protein at the dermo/epidermal junction. Our results provide evidence for at least two distinct molecular mechanisms underlying defective anchoring fibril formation in RDEB: one involving PTCs leading to mRNA instability and absence of protein synthesis, the other implicating missense mutations resulting in the synthesis of type VII collagen polypeptide with decreased stability and/or altered function. Genotype-phenotype correlations suggested that the nature and location of these mutations are important determinants of the disease phenotype and showed evidence for interfamilial phenotypic variability. Images Figure 1 Figure 2 Figure 4 Figure 5 Figure 6 PMID:9326325

  16. Biotechnology of Cold-Active Proteases

    PubMed Central

    Joshi, Swati; Satyanarayana, Tulasi

    2013-01-01

    The bulk of Earth’s biosphere is cold (<5 °C) and inhabited by psychrophiles. Biocatalysts from psychrophilic organisms (psychrozymes) have attracted attention because of their application in the ongoing efforts to decrease energy consumption. Proteinases as a class represent the largest category of industrial enzymes. There has been an emphasis on employing cold-active proteases in detergents because this allows laundry operations at ambient temperatures. Proteases have been used in environmental bioremediation, food industry and molecular biology. In view of the present limited understanding and availability of cold-active proteases with diverse characteristics, it is essential to explore Earth’s surface more in search of an ideal cold-active protease. The understanding of molecular and mechanistic details of these proteases will open up new avenues to tailor proteases with the desired properties. A detailed account of the developments in the production and applications of cold-active proteases is presented in this review. PMID:24832807

  17. A Role in Immunity for Arabidopsis Cysteine Protease RD21, the Ortholog of the Tomato Immune Protease C14

    PubMed Central

    Shindo, Takayuki; Misas-Villamil, Johana C.; Hörger, Anja C.; Song, Jing; van der Hoorn, Renier A. L.

    2012-01-01

    Secreted papain-like Cys proteases are important players in plant immunity. We previously reported that the C14 protease of tomato is targeted by cystatin-like EPIC proteins that are secreted by the oomycete pathogen Phytophthora infestans (Pinf) during infection. C14 has been under diversifying selection in wild potato species coevolving with Pinf and reduced C14 levels result in enhanced susceptibility for Pinf. Here, we investigated the role C14-EPIC-like interactions in the natural pathosystem of Arabidopsis with the oomycete pathogen Hyaloperonospora arabidopsidis (Hpa). In contrast to the Pinf-solanaceae pathosystem, the C14 orthologous protease of Arabidopsis, RD21, does not evolve under diversifying selection in Arabidopsis, and rd21 null mutants do not show phenotypes upon compatible and incompatible Hpa interactions, despite the evident lack of a major leaf protease. Hpa isolates express highly conserved EPIC-like proteins during infections, but it is unknown if these HpaEPICs can inhibit RD21 and one of these HpaEPICs even lacks the canonical cystatin motifs. The rd21 mutants are unaffected in compatible and incompatible interactions with Pseudomonas syringae pv. tomato, but are significantly more susceptible for the necrotrophic fungal pathogen Botrytis cinerea, demonstrating that RD21 provides immunity to a necrotrophic pathogen. PMID:22238602

  18. Molecular Imaging of Proteases in Cancer

    PubMed Central

    Yang, Yunan; Hong, Hao; Zhang, Yin; Cai, Weibo

    2010-01-01

    Proteases play important roles during tumor angiogenesis, invasion, and metastasis. Various molecular imaging techniques have been employed for protease imaging: optical (both fluorescence and bioluminescence), magnetic resonance imaging (MRI), single-photon emission computed tomography (SPECT), and positron emission tomography (PET). In this review, we will summarize the current status of imaging proteases in cancer with these techniques. Optical imaging of proteases, in particular with fluorescence, is the most intensively validated and many of the imaging probes are already commercially available. It is generally agreed that the use of activatable probes is the most accurate and appropriate means for measuring protease activity. Molecular imaging of proteases with other techniques (i.e. MRI, SPECT, and PET) has not been well-documented in the literature which certainly deserves much future effort. Optical imaging and molecular MRI of protease activity has very limited potential for clinical investigation. PET/SPECT imaging is suitable for clinical investigation; however the optimal probes for PET/SPECT imaging of proteases in cancer have yet to be developed. Successful development of protease imaging probes with optimal in vivo stability, tumor targeting efficacy, and desirable pharmacokinetics for clinical translation will eventually improve cancer patient management. Not limited to cancer, these protease-targeted imaging probes will also have broad applications in other diseases such as arthritis, atherosclerosis, and myocardial infarction. PMID:20234801

  19. Antimicrobial proteins and peptides in human lung diseases: A friend and foe partnership with host proteases.

    PubMed

    Lecaille, Fabien; Lalmanach, Gilles; Andrault, Pierre-Marie

    2016-03-01

    Lung antimicrobial proteins and peptides (AMPs) are major sentinels of innate immunity by preventing microbial colonization and infection. Nevertheless bactericidal activity of AMPs against Gram-positive and Gram-negative bacteria is compromised in patients with chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF) and asthma. Evidence is accumulating that expression of harmful human serine proteases, matrix metalloproteases and cysteine cathepsins is markedely increased in these chronic lung diseases. The local imbalance between proteases and protease inhibitors compromises lung tissue integrity and function, by not only degrading extracellular matrix components, but also non-matrix proteins. Despite the fact that AMPs are somewhat resistant to proteolytic degradation, some human proteases cleave them efficiently and impair their antimicrobial potency. By contrast, certain AMPs may be effective as antiproteases. Host proteases participate in concert with bacterial proteases in the degradation of key innate immunity peptides/proteins and thus may play immunomodulatory activities during chronic lung diseases. In this context, the present review highlights the current knowledge and recent discoveries on the ability of host enzymes to interact with AMPs, providing a better understanding of the role of human proteases in innate host defense. PMID:26341472

  20. 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. PMID:25579194

  1. Extracellular proteases as targets for drug development.

    PubMed

    Cudic, Mare; Fields, Gregg B

    2009-08-01

    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) and cysteine proteases (cathepsin B) are discussed herein. PMID:19689354

  2. Binding modes of a new epoxysuccinyl-peptide inhibitor of cysteine proteases. Where and how do cysteine proteases express their selectivity?

    PubMed

    Czaplewski, C; Grzonka, Z; Jaskólski, M; Kasprzykowski, F; Kozak, M; Politowska, E; Ciarkowski, J

    1999-05-18

    Papain from Carica papaya, an easily available cysteine protease, is the best-studied representative of this family of enzymes. The three dimensional structure of papain is very similar to that of other cysteine proteases of either plant (actinidin, caricain, papaya protease IV) or animal (cathepsins B, K, L, H) origin. As abnormalities in the activities of mammalian cysteine proteases accompany a variety of diseases, there has been a long-lasting interest in the development of potent and selective inhibitors for these enzymes. A covalent inhibitor of cysteine proteases, designed as a combination of epoxysuccinyl and peptide moieties, has been modeled in the catalytic pocket of papain. A number of its configurations have been generated and relaxed by constrained simulated annealing-molecular dynamics in water. A clear conformational variability of this inhibitor is discussed in the context of a conspicuous conformational diversity observed earlier in several solid-state structures of other complexes between cysteine proteases and covalent inhibitors. The catalytic pockets S2 and even more so S3, as defined by the pioneering studies on the papain-ZPACK, papain-E64c and papain-leupeptin complexes, appear elusive in view of the evident flexibility of the present inhibitor and in confrontation with the obvious conformational scatter seen in other examples. This predicts limited chances for the development of selective structure-based inhibitors of thiol proteases, designed to exploit the minute differences in the catalytic pockets of various members of this family. A simultaneous comparison of the three published proenzyme structures suggests the enzyme's prosegment binding loop-prosegment interface as a new potential target for selective inhibitors of papain-related thiol proteases. PMID:10350606

  3. Proteases in biological control and biotechnology

    SciTech Connect

    Cunningham, D.D.; Long, G.L.

    1987-01-01

    This book explores the role of proteases in biological control systems and diseases, examines their structures and evolution, and reviews the methods by which proteases and protease inhibitors are engineered. In addition, the use of recombinant DNA technology is explained throughout the volume. Specific topics examined include: the versatility of proteolytic enzymes, the intricate proteolytic control mechanisms in hemostasis and their application to thrombolytic therapy, the evolution of proteolytic enzymes, and the role of limited proteolytic processing in several biological control processes.

  4. 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.

  5. 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.

  6. Approaches for Analyzing the Roles of Mast Cells and Their Proteases In Vivo

    PubMed Central

    Galli, Stephen J.; Tsai, Mindy; Marichal, Thomas; Tchougounova, Elena; Reber, Laurent L.; Pejler, Gunnar

    2016-01-01

    The roles of mast cells in health and disease remain incompletely understood. While the evidence that mast cells are critical effector cells in IgE-dependent anaphylaxis and other acute IgE-mediated allergic reactions seems unassailable, studies employing various mice deficient in mast cells or mast cell-associated proteases have yielded divergent conclusions about the roles of mast cells or their proteases in certain other immunological responses. Such “controversial” results call into question the relative utility of various older versus newer approaches to ascertain the roles of mast cells and mast cell proteases in vivo. This review discusses how both older and more recent mouse models have been used to investigate the functions of mast cells and their proteases in health and disease. We particularly focus on settings in which divergent conclusions about the importance of mast cells and their proteases have been supported by studies that employed different models of mast cell or mast cell protease deficiency. We think that two major conclusions can be drawn from such findings: (1) no matter which models of mast cell or mast cell protease deficiency one employs, the conclusions drawn from the experiments always should take into account the potential limitations of the models (particularly abnormalities affecting cell types other than mast cells) and (2) even when analyzing a biological response using a single model of mast cell or mast cell protease deficiency, details of experimental design are critical in efforts to define those conditions under which important contributions of mast cells or their proteases can be identified. PMID:25727288

  7. 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.

  8. Proteolytic crosstalk in multi-protease networks.

    PubMed

    Ogle, Curtis T; Mather, William H

    2016-01-01

    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. PMID:27042892

  9. Evidence for defect-induced superconductivity up to 49 K in (C a1 -xRx) F e2A s2

    NASA Astrophysics Data System (ADS)

    Deng, L. Z.; Lv, B.; Zhao, K.; Wei, F. Y.; Xue, Y. Y.; Wu, Z.; Chu, C. W.

    2016-02-01

    To explore the origin of the unusual nonbulk superconductivity with a Tc up to 49 K reported in the rare-earth-doped CaF e2A s2 , the chemical composition, magnetization, specific heat, resistivity, and annealing effect are systematically investigated on nominal (C a1 -xRx) F e2A s2 single crystals with different x and R =La , Ce, Pr, and Nd. All display a doping-independent Tc once superconductivity is induced, a doping-dependent low field superconducting volume fraction f , and a large magnetic anisotropy η in the superconducting state, suggesting a rather inhomogeneous superconducting state in an otherwise microscale homogenous superconductor. The wavelength dispersive spectroscopy and specific heat show the presence of defects that are closely related to f , regardless of the R involved. The magnetism further reveals that the defects are mainly superparamagnetic clusters for R =Ce , Pr, and Nd with strong intercluster interactions, implying that defects are locally self-organized. Annealing at 500 °C, without varying the doping level x , suppresses f profoundly but not the Tc. The above observations provide evidence for the crucial role of defects in the occurrence of the unusually high Tc˜49 K in (C a1 -xRx) F e2A s2 and are consistent with the interface-enhanced superconductivity recently proposed.

  10. Millimeter-Wave Studies of the Isotopologues of IZnCH3(X1A1): Geometric Parameters and Evidence for Zinc Insertion

    NASA Astrophysics Data System (ADS)

    Bucchino, Matthew; Young, Justin; Sheridan, Phillip; Ziurys, Lucy

    2014-06-01

    The laboratory detection of gas-phase IZnCH3 (X1A1), using millimeter-wave direct absorption methods, was reported previously. This work has been extended by the measurement of the pure rotational spectrum of several isotopolgues: I64ZnCH3, I66ZnCH3, I64ZnCD3, and I64Zn13CH3. These species were all created by the reaction of zinc vapor with CH3I, CD3I, or 13CH3I in the presence of a DC discharge. The zinc isotopolgues were observed in natural abundance. Rotational transitions in the range 256{-293} GHz (J = 109 {←} 108 to J = 132 {←} 131, for K = 0 to 6) have been recorded for each species. From these measurements, an r0 structure has been determined. This structure was found to be in good agreement with previous DFT calculations. Interestingly, the 110.2° Zn - C - H bond angle of IZnCH3 is identical to that of the hydrogen substituted zinc insertion complex HZnCH3 (X1A1). These data are further evidence that IZnCH3 is not created by the generation of free radical fragments, but by the direct insertion of atomic zinc into the C - I bond of iodomethane.

  11. Complementary approaches to understanding the role of proteases and their natural inhibitors in neoplastic development: retrospect and prospect.

    PubMed

    Rubin, Harry

    2003-05-01

    A great deal of evidence has accumulated in recent years for an important but complex role for proteases in tumor development. However, attempts to treat cancer in humans with anti-proteases have been disappointing, and it has been suggested that more basic groundwork is needed before anti-proteases can be effectively applied. Considerable basic information comes from the recognition that earlier results on transformation of chicken embryo fibroblasts (CEF) by the Bryan strain of Rous sarcoma virus (B-RSV) can be explained in terms of proteases and their inhibitors. In particular, the full but reversible normalization of discrete transformed foci by appropriate concentrations of fetal bovine or of calf serum implies a causal role for multiple proteases in transformation, and the efficacy of treatment with a physiological balance of their natural inhibitors. Addition of certain proteases to contact-inhibited normal cultures was then found to stimulate their proliferation. The toxicity of medium produced by CEF heavily transformed with B-RSV suggests that cachexia and other systemic effects of human cancer may result from vascular dissemination of peptides from pericellular proteolysis within tumors. Comprehensive studies revealed significant increases of plasminogen activator and matrix metalloproteinases (MMPs) after infection of CEF with other strains of RSV, and correlation of the proteases with aspects of transformation. A similar role for proteases is indicated in the transformation of mammalian cells by chemical and physical agents. The information gained from functional experiments on cell transformation in culture is complementary to that obtained from the molecular identification of proteases and their inhibitors in all stages of tumor development. The speed, quantification and easy manipulation of the RSV-CEF transformation assay can be combined with current methods of characterizing proteases and anti-proteases to further enrich our basic knowledge of

  12. Chemical modification of A1 adenosine receptors in rat brain membranes. Evidence for histidine in different domains of the ligand binding site.

    PubMed

    Klotz, K N; Lohse, M J; Schwabe, U

    1988-11-25

    Chemical modification of amino acid residues was used to probe the ligand recognition site of A1 adenosine receptors from rat brain membranes. The effect of treatment with group-specific reagents on agonist and antagonist radioligand binding was investigated. The histidine-specific reagent diethylpyrocarbonate (DEP) induced a loss of binding of the agonist R-N6-[3H] phenylisopropyladenosine ([3H]PIA), which could be prevented in part by agonists, but not by antagonists. DEP treatment induced also a loss of binding of the antagonist [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX). Antagonists protected A1 receptors from this inactivation while agonists did not. This result provided evidence for the existence of at least 2 different histidine residues involved in ligand binding. Consistent with a modification of the binding site, DEP did not alter the affinity of [3H]DPCPX, but reduced receptor number. From the selective protection of [3H] PIA and [3H]DPCPX binding from inactivation, it is concluded that agonists and antagonists occupy different domains at the binding site. Sulfhydryl modifying reagents did not influence antagonist binding, but inhibited agonist binding. This effect is explained by modification of the inhibitory guanine nucleotide binding protein. Pyridoxal 5-phosphate inactivated both [3H]PIA and [3H]DPCPX binding, but the receptors could not be protected from inactivation by ligands. Therefore, no amino group seems to be located at the ligand binding site. In addition, it was shown that no further amino acids with polar side chains are present. The absence of hydrophilic amino acids from the recognition site of the receptor apart from histidine suggests an explanation for the lack of hydrophilic ligands with high affinity for A1 receptors. PMID:3182861

  13. Tissue dissociation enzyme neutral protease assessment.

    PubMed

    Breite, A G; Dwulet, F E; McCarthy, R C

    2010-01-01

    Neutral proteases, essential components of purified tissue dissociation enzymes required for successful human islet isolation, show variable activities and effects of substrate on their activities. Initially we used a spectrophotometric endpoint assay with azocasein substrate to measure neutral protease activity. After critical review of the results, we observed these data to be inconsistent and not correlating expected differences in specific activities between thermolysin and Bacillus polymyxa proteases. This observation led to the development of a fluorescent microplate assay using fluorescein isothyocyanate-conjugated bovine serum albumin (FITC-BSA) as the substrate. This simpler, more flexible method offered a homogeneous, kinetic enzyme assay allowing determination of steady state reaction rates of sample replicates at various dilutions. The assay had a linear range of 4- to 8-fold and interassay coefficients of variation for B polymyxa protease and thermolysin of <9% and <15%, respectively, which were lower than those using the spectrophotometric endpoint assay, namely, 54% and 36%, respectively. This format allowed for incorporation of enzyme inhibitors, as illustrated by addition of sulfhydryl protease inhibitors, which, consistent with earlier reports, strongly indicated that the main contaminant in purified collagenase preparations was clostripain. Determination of the specific activities for several purified neutral proteases showed that the B polymyxa and Clostridium histolyticum proteases had approximately 40% and 15% specific activities, respectively, of those obtained with purified thermolysin, indicating the different characteristics of neutral protease enzymes for cell isolation procedures. PMID:20692405

  14. Protease-degradable electrospun fibrous hydrogels

    NASA Astrophysics Data System (ADS)

    Wade, Ryan J.; Bassin, Ethan J.; Rodell, Christopher B.; Burdick, Jason A.

    2015-03-01

    Electrospun nanofibres are promising in biomedical applications to replicate features of the natural extracellular matrix (ECM). However, nearly all electrospun scaffolds are either non-degradable or degrade hydrolytically, whereas natural ECM degrades proteolytically, often through matrix metalloproteinases. Here we synthesize reactive macromers that contain protease-cleavable and fluorescent peptides and are able to form both isotropic hydrogels and electrospun fibrous hydrogels through a photoinitiated polymerization. These biomimetic scaffolds are susceptible to protease-mediated cleavage in vitro in a protease dose-dependent manner and in vivo in a subcutaneous mouse model using transdermal fluorescent imaging to monitor degradation. Importantly, materials containing an alternate and non-protease-cleavable peptide sequence are stable in both in vitro and in vivo settings. To illustrate the specificity in degradation, scaffolds with mixed fibre populations support selective fibre degradation based on individual fibre degradability. Overall, this represents a novel biomimetic approach to generate protease-sensitive fibrous scaffolds for biomedical applications.

  15. Progress and prospects on DENV protease inhibitors.

    PubMed

    Timiri, Ajay Kumar; Sinha, Barij Nayan; Jayaprakash, Venkatesan

    2016-07-19

    New treatments are desperately required to combat increasing rate of dengue fever cases reported in tropical and sub-tropical parts of the world. Among the ten proteins (structural and non-structural) encoded by dengue viral genome, NS2B-NS3 protease is an ideal target for drug discovery. It is responsible for the processing of poly protein that is required for genome replication of the virus. Moreover, inhibitors designed against proteases were found successful in Human Immuno-deficiency Virus (HIV) and Hepatitis C Virus (HCV). Complete molecular mechanism and a survey of inhibitors reported against dengue protease will be helpful in designing effective and potent inhibitors. This review provides an insight on molecular mechanism of dengue virus protease and covers up-to-date information on different inhibitors reported against dengue proteases with medicinal chemistry perspective. PMID:27092412

  16. HIV-1 protease mutations and protease inhibitor cross-resistance.

    PubMed

    Rhee, Soo-Yon; Taylor, Jonathan; Fessel, W Jeffrey; Kaufman, David; Towner, William; Troia, Paolo; Ruane, Peter; Hellinger, James; Shirvani, Vivian; Zolopa, Andrew; Shafer, Robert W

    2010-10-01

    The effects of many protease inhibitor (PI)-selected mutations on the susceptibility to individual PIs are unknown. We analyzed in vitro susceptibility test results on 2,725 HIV-1 protease isolates. More than 2,400 isolates had been tested for susceptibility to fosamprenavir, indinavir, nelfinavir, and saquinavir; 2,130 isolates had been tested for susceptibility to lopinavir; 1,644 isolates had been tested for susceptibility to atazanavir; 1,265 isolates had been tested for susceptibility to tipranavir; and 642 isolates had been tested for susceptibility to darunavir. We applied least-angle regression (LARS) to the 200 most common mutations in the data set and identified a set of 46 mutations associated with decreased PI susceptibility of which 40 were not polymorphic in the eight most common HIV-1 group M subtypes. We then used least-squares regression to ascertain the relative contribution of each of these 46 mutations. The median number of mutations associated with decreased susceptibility to each PI was 28 (range, 19 to 32), and the median number of mutations associated with increased susceptibility to each PI was 2.5 (range, 1 to 8). Of the mutations with the greatest effect on PI susceptibility, I84AV was associated with decreased susceptibility to eight PIs; V32I, G48V, I54ALMSTV, V82F, and L90M were associated with decreased susceptibility to six to seven PIs; I47A, G48M, I50V, L76V, V82ST, and N88S were associated with decreased susceptibility to four to five PIs; and D30N, I50L, and V82AL were associated with decreased susceptibility to fewer than four PIs. This study underscores the greater impact of nonpolymorphic mutations compared with polymorphic mutations on decreased PI susceptibility and provides a comprehensive quantitative assessment of the effects of individual mutations on susceptibility to the eight clinically available PIs. PMID:20660676

  17. HIV Protease Inhibitors: Effect on the Opportunistic Protozoan Parasites

    PubMed Central

    Alfonso, Yenisey; Monzote, Lianet

    2011-01-01

    The impact of highly active antiretroviral therapy (HAART) in the natural history of AIDS disease has been allowed to prolong the survival of people with HIV infection, particularly whose with increased HIV viral load. Additionally, the antiretroviral therapy could exert a certain degree of protection against parasitic diseases. A number of studies have been evidenced a decrease in the incidence of opportunistic parasitic infections in the era of HAART. Although these changes have been attributed to the restoration of cell-mediated immunity, induced by either non-nucleoside reverse transcriptase inhibitors or HIV protease inhibitors, in combination with at least two nucleoside reverse transcriptase inhibitors included in HAART, there are evidence that the control of these parasitic infections in HIV-positive persons under HAART, is also induced by the inhibition of the proteases of the parasites. This review focuses on the principal available data related with therapeutic HIV-protease inhibitors and their in vitro and in vivo effects on the opportunistic protozoan parasites. PMID:21629510

  18. Proteases at work: cues for understanding neural development and degeneration

    PubMed Central

    Saftig, Paul; Bovolenta, Paola

    2015-01-01

    Proteolytical processing of membrane bound molecules is a fundamental mechanism for the degradation of these proteins as well as for controlling cell-to-cell communication, which is at the basis of tissue development and homeostasis. Members of families of metalloproteinases and intra-membrane proteases are major effectors of these events. A recent workshop in Baeza, Spain, was devoted to discuss how this mechanism coordinates brain development and how its dysfunction leads to brain pathologies. Herein we summarize the findings presented during this workshop, which illuminate the role of metalloproteinases, including matrix metalloproteinase, A Disintegrin and Metalloproteinase-proteases and intra-membrane proteases, in the regulation of neurogenesis, axon guidance, and synaptogenesis as well as in neurodegeneration. Indeed, there is increasing evidence that proteolysis at the membrane is directly linked to neuropathologies such as Alzheimer Disease and autism spectrum or prion disorders. These proteolytic events are tightly regulated and we are just at the beginning of understanding how these processes could be exploited to design therapeutic treatments aimed at alleviating psychiatric and neurodegenerative pathologies. PMID:25999813

  19. Protease proteomics: revealing protease in vivo functions using systems biology approaches.

    PubMed

    Doucet, Alain; Overall, Christopher M

    2008-10-01

    Proteases irreversibly modify proteins by cleaving their amide bonds and are implicated in virtually every important biological process such as immunity, development and tissue repair. Accordingly, it is easy to see that deregulated proteolysis is a pathognomic feature of many diseases. Most of the current information available on proteases was acquired using in vitro methods, which reveals molecular structure, enzyme kinetics and active-site specificity. However, considerably less is known about the relevant biological functions and combined roles of proteases in moulding the proteome. Although models using genetically modified animals are powerful, they are slow to develop, they can be difficult to interpret, and while useful, they remain only models of human disease. Therefore, to understand how proteases accomplish their tasks in organisms and how they participate in pathology, we need to elucidate the protease degradome-the repertoire of proteases expressed by a cell, a tissue or an organism at a particular time-their expression level, activation state, their biological substrates, also known as the substrate degradome-the repertoire of substrates for each protease-and the effect of the activity of each protease on the pathways of the system under study. Achieving this goal is challenging because several proteases might cleave the same protein, and proteases also form pathways and interact to form the protease web [Overall, C.M., Kleifeld, O., 2006. Tumour microenvironment - opinion: validating matrix metalloproteinases as drug targets and anti-targets for cancer therapy. Nat. Rev. Cancer 6 (3), 227-239]. Hence, the net proteolytic potential of the degradome at a particular time on a substrate and pathway must also be understood. Proteomics offers one of the few routes to the understanding of proteolysis in complex in vivo systems and especially in man where genetic manipulations are impossible. The aim of this chapter is to review methods and tools that allow

  20. Epidermal differentiation: the role of proteases and their inhibitors.

    PubMed

    Zeeuwen, Patrick L J M

    2004-12-01

    Dermatological diseases range from minor cosmetic problems to life-threatening conditions, as seen in some severe disorders of keratinization and cornification. These disorders are commonly due to abnormal epidermal differentiation processes, which result in disturbed barrier function of human skin. Elucidation of the cellular differentiation programs that regulate the formation and homeostasis of the epidermis is therefore of great importance for the understanding and therapy of these disorders. Much of the barrier function of human epidermis against the environment is provided by the cornified cell envelope (CE), which is assembled by transglutaminase (TGase)-mediated cross-linking of several structural proteins and lipids during the terminal stages of normal keratinocyte differentiation. The major constituents of the stratum corneum and the current knowledge on the formation of the stratum corneum will be briefly reviewed here. The discovery of mutations that underlie several human diseases caused by genetic defects in the protein or lipid components of the CE, and recent analyses of mouse mutants with defects in the structural components of the CE, catalyzing enzymes, and lipid processing, have highlighted their essential function in establishing the epidermal barrier. In addition, recent findings have provided evidence that a disturbed protease-antiprotease balance could cause faulty differentiation processes in the epidermis and hair follicle. The importance of regulated proteolysis in epithelia is well demonstrated by the recent identification of the SPINK5 serine proteinase inhibitor as the defective gene in Netherton syndrome, cathepsin C mutations in Papillon-Lefevre syndrome, cathepsin L deficiency infurless mice, targeted ablation of the serine protease Matriptase/MTSP1, targeted ablation of the aspartate protease cathepsin D, and the phenotype of targeted epidermal overexpression of stratum corneum chymotryptic enzyme in mice. Notably, our recent

  1. Potential Elucidation of a Novel CTL Epitope in HIV-1 Protease by the Protease Inhibitor Resistance Mutation L90M

    PubMed Central

    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. PMID:24015196

  2. 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. PMID:16003937

  3. Regulation of protease production in Clostridium sporogenes.

    PubMed Central

    Allison, C; Macfarlane, G T

    1990-01-01

    The physiological and nutritional factors that regulate protease synthesis in Clostridium sporogenes C25 were studied in batch and continuous cultures. Formation of extracellular proteases occurred at the end of active growth and during the stationary phase in batch cultures. Protease production was inversely related to growth rate in glucose-excess and glucose-limited chemostats over the range D = 0.05 to 0.70 h-1. In pulse experiments, glucose, ammonia, phosphate, and some amino acids (tryptophan, proline, tyrosine, and isoleucine) strongly repressed protease synthesis. This repression was not relieved by addition of 4 mM cyclic AMP, cyclic GMP, or dibutyryl cyclic AMP. Protease formation was markedly inhibited by 4 mM ATP and ADP, but GTP and GDP had little effect on the process. It is concluded that protease production by C. sporogenes is strongly influenced by the amount of energy available to the cells, with the highest levels of protease synthesis occurring under energy-limiting conditions. PMID:2268158

  4. A biotechnology perspective of fungal proteases

    PubMed Central

    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, Edivaldo Ximenes; Pessoa, Adalberto; Magalhães, Pérola Oliveira

    2015-01-01

    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. PMID:26273247

  5. 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. PMID:26273247

  6. HIV-1 Protease: Structure, Dynamics and Inhibition

    SciTech Connect

    Louis, John M.; Ishima, R.; Torchia, D.A.; Weber, Irene T.

    2008-06-03

    The HIV-1 protease is synthesized as part of a large Gag-Pol precursor protein. It is responsible for its own release from the precursor and the processing of the Gag and Gag-Pol polyproteins into the mature structural and functional proteins required for virus maturation. Because of its indispensable role, the mature HIV-1 protease dimer has proven to be a successful target for the development of antiviral agents. In the last 5 years, a major emphasis in protease research has been to improve inhibitor design and treatment regimens.

  7. Protease and protease inhibitory activity in pregnant and postpartum involuting uterus

    SciTech Connect

    Milwidsky, A.; Beller, U.; Palti, Z.; Mayer, M.

    1982-08-15

    The presence of two distinct proteolytic activities in the rat uterus was confirmed with /sup 14/C-labeled globin used as a sensitive protein substrate and following release of label into the trichloroacetic acid-soluble supernatant fraction. Protease I is a cytoplasmic acid protease while protease II is associated with the pellet fraction, can be extracted by 0.6 M sodium chloride, and is active at pH 7.0. Protease I activity is low during pregnancy and markedly increases at term achieving maximal activity at day 3 post partum with a subsequent decline to preterm activity values. Lactation did not affect the uterine protease I activity. Protease II activity is not significantly different during pregnancy, at term, and post partum. The presence of an inhibitor of protease I was suggested by a decrease in enzyme activity with an increased cytosolic protein concentration. The inhibitor also lessened bovine trypsin activity but had no effect on protease II. Although its inhibitory potency on trypsin fluctuated during the various uterine physiologic stages, these changes appeared to be statistically insignificant. Human uterine samples were also found to contain the two protease activities with similar changes in protease I post partum. It is suggested that, both in the rat and in man, uterine involution post partum is associated with a marked increase in activity of acid cytosolic protease, while a particulate neutral protease and a soluble inhibitor of trypsin, which are also present in uterine cells, do not appear to play a significant role in the dissolution of uterine tissues after parturition.

  8. Lung protease/anti-protease network and modulation of mucus production and surfactant activity.

    PubMed

    Garcia-Verdugo, Ignacio; Descamps, Delphyne; Chignard, Michel; Touqui, Lhousseine; Sallenave, Jean-Michel

    2010-11-01

    Lung epithelium guarantees gas-exchange (performed in the alveoli) and protects from external insults (pathogens, pollutants…) present within inhaled air. Both functions are facilitated by secretions lining airway surface liquid, mucus (in the upper airways) and pulmonary surfactant (in the alveoli). Mucins, the main glycoproteins present within the mucus, are responsible for its rheologic properties and participate in lung defense mechanisms. In parallel, lung collectins are pattern recognition molecules present in pulmonary surfactant that also modulate lung defense. During chronic airways diseases, excessive protease activity can promote mucus hypersecretion and degradation of lung collectins and therefore contribute to the pathophysiology of these diseases. Importantly, secretion of local and systemic anti-proteases might be crucial to equilibrate the protease/anti-protease unbalance and therefore preserve the function of lung host defense compounds and airway surface liquid homeostasis. In this review we will present information relative to proteases able to modulate mucin production and lung collectin integrity, two important compounds of innate immune defense. One strategy to preserve physiological mucus production and collectin integrity during chronic airways diseases might be the over-expression of local 'alarm' anti-proteases such as SLPI and elafin. Interestingly, a cross-talk between lung collectins and anti-protease activity has recently been described, implicating the presence within the lung of a complex network between proteases, anti-proteases and pattern recognition molecules, which aims to keep or restore homeostasis in resting or inflamed lungs. PMID:20493919

  9. Synthesis of amino heterocycle aspartyl protease inhibitors.

    PubMed

    Chambers, Rachel K; Khan, Tanweer A; Olsen, David B; Sleebs, Brad E

    2016-06-14

    Aspartyl proteases are important pharmacological targets. Historically aspartyl proteases have been commonly targeted with transition state derived peptidomimetics. The strategy to develop aspartyl protease inhibitors has undertaken a dramatic paradigm shift in the last 10 years. The pharmaceutical industry in 2005 disclosed several scaffolds or "head groups" that prompted the field to move beyond peptidomimetic derived inhibitors. Since the discovery of the first amino heterocycle aspartyl protease inhibitor, the amino hydantoin, industry and academia have positioned themselves for a foothold on the new molecular space, designing a variety of related "head groups". Both the design and synthetic efforts involved in constructing these scaffolds are varied and complex. Here we highlight the synthetic strategies used to access these amino heterocycle scaffolds. PMID:27143279

  10. A radiometric assay for HIV-1 protease

    SciTech Connect

    Hyland, L.J.; Dayton, B.D.; Moore, M.L.; Shu, A.Y.; Heys, J.R.; Meek, T.D. )

    1990-08-01

    A rapid, high-throughput radiometric assay for HIV-1 protease has been developed using ion-exchange chromatography performed in 96-well filtration plates. The assay monitors the activity of the HIV-1 protease on the radiolabeled form of a heptapeptide substrate, (tyrosyl-3,5-3H)Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2, which is based on the p17-p24 cleavage site found in the viral polyprotein substrate Pr55gag. Specific cleavage of this uncharged heptapeptide substrate by HIV-1 protease releases the anionic product (tyrosyl-3,5-3H)Ac-Ser-Gln-Asn-Tyr, which is retained upon minicolumns of the anion-exchange resin AG1-X8. Protease activity is determined from the recovery of this radiolabeled product following elution with formic acid. This facile and highly sensitive assay may be utilized for steady-state kinetic analysis of the protease, for measurements of enzyme activity during its purification, and as a routine assay for the evaluation of protease inhibitors from natural product or synthetic sources.

  11. ADAM Proteases and Gastrointestinal Function.

    PubMed

    Jones, Jennifer C; Rustagi, Shelly; Dempsey, Peter J

    2016-01-01

    A disintegrin and metalloproteinases (ADAMs) are a family of cell surface proteases that regulate diverse cellular functions, including cell adhesion, migration, cellular signaling, and proteolysis. Proteolytically active ADAMs are responsible for ectodomain shedding of membrane-associated proteins. ADAMs rapidly modulate key cell signaling pathways in response to changes in the extracellular environment (e.g., inflammation) and play a central role in coordinating intercellular communication within the local microenvironment. ADAM10 and ADAM17 are the most studied members of the ADAM family in the gastrointestinal tract. ADAMs regulate many cellular processes associated with intestinal development, cell fate specification, and the maintenance of intestinal stem cell/progenitor populations. Several signaling pathway molecules that undergo ectodomain shedding by ADAMs [e.g., ligands and receptors from epidermal growth factor receptor (EGFR)/ErbB and tumor necrosis factor α (TNFα) receptor (TNFR) families] help drive and control intestinal inflammation and injury/repair responses. Dysregulation of these processes through aberrant ADAM expression or sustained ADAM activity is linked to chronic inflammation, inflammation-associated cancer, and tumorigenesis. PMID:26667078

  12. ADAM Proteases and Gastrointestinal Function

    PubMed Central

    Jones, Jennifer C.; Rustagi, Shelly; Dempsey, Peter J.

    2016-01-01

    A disintegrin and metalloproteinases (ADAMs) are a family of cell surface proteases that regulate diverse cellular functions, including cell adhesion, migration, cellular signaling, and proteolysis. Proteolytically active ADAMs are responsible for ectodomain shedding of membrane-associated proteins. ADAMs rapidly modulate key cell signaling pathways in response to changes in the extracellular environment (e.g., inflammation) and play a central role in coordinating intercellular communication within the local microenvironment. ADAM10 and ADAM17 are the most studied members of the ADAM family in the gastrointestinal tract. ADAMs regulate many cellular processes associated with intestinal development, cell fate specification, and the maintenance of intestinal stem cell/progenitor populations. Several signaling pathway molecules that undergo ectodomain shedding by ADAMs [e.g., ligands and receptors from epidermal growth factor receptor (EGFR)/ErbB and tumor necrosis factor α (TNFα) receptor (TNFR) families] help drive and control intestinal inflammation and injury/repair responses. Dysregulation of these processes through aberrant ADAM expression or sustained ADAM activity is linked to chronic inflammation, inflammation-associated cancer, and tumorigenesis. PMID:26667078

  13. Neutral serine proteases of neutrophils.

    PubMed

    Kettritz, Ralph

    2016-09-01

    Neutrophil serine proteases (NSPs) exercise tissue-degrading and microbial-killing effects. The spectrum of NSP-mediated functions grows continuously, not least because of methodological progress. Sensitive and specific FRET substrates were developed to study the proteolytic activity of each NSP member. Advanced biochemical methods are beginning to characterize common and specific NSP substrates. The resulting novel information indicates that NSPs contribute not only to genuine inflammatory neutrophil functions but also to autoimmunity, metabolic conditions, and cancer. Tight regulatory mechanisms control the proteolytic potential of NSPs. However, not all NSP functions depend on their enzymatic activity. Proteinase-3 (PR3) is somewhat unique among the NSPs for PR3 functions as an autoantigen. Patients with small-vessel vasculitis develop autoantibodies to PR3 that bind their target antigens on the neutrophil surface and trigger neutrophil activation. These activated cells subsequently contribute to vascular necrosis with life-threatening multiorgan failure. This article discusses various aspects of NSP biology and highlights translational aspects with strong clinical implications. PMID:27558338

  14. Carbohydrate protease conjugates: Stabilized proteases for peptide synthesis

    SciTech Connect

    Wartchow, C.A.; Wang, Peng; Bednarski, M.D.; Callstrom, M.R. |

    1995-12-31

    The synthesis of oligopeptides using stable carbohydrate protease conjugates (CPCs) was examined in acetonitrile solvent systems. CPC[{alpha}-chymotrypsin] was used for the preparation of peptides containing histidine, phenylalanine, tryptophan in the P{sub 1} position in 60-93% yield. The CPC[{alpha}-chymotrypsin]-catalyzed synthesis of octamer Z-Gly-Gly-Phe-Gly-Gly-Phe-Gly-Gly-OEt from Z-Gly-Gly-Phe-Gly-Gly-Phe-OMe was achieved in 71% yield demonstrating that synthesis peptides containing both hydrophylic and hydrophobic amino acids. The P{sub 2} specificity of papain for aromatic residues was utilized for the 2 + 3 coupling of Z-Tyr-Gly-OMe to H{sub 2}N-Gly-Phe-Leu-OH to generate the leucine enkephalin derivative in 79% yield. Although papain is nonspecific for the hydrolysis of N-benzyloxycarbonyl amino acid methyl esters in aqueous solution, the rates of synthesis for these derivitives with nucleophile leucine tert-butyl ester differed by nearly 2 orders of magnitude. CPC[thermolysin] was used to prepare the aspartame precursor Z-Asp-Phe-OMe in 90% yield. The increased stability of CPCs prepared from periodate-modified poly(2-methacryl- amido-2-deoxy-D-glucose), poly(2-methacrylamido-2-deoxy-D-galactose), and poly(5-methacryl-amido-5-deoxy-D-ribose), carbohydrate materials designed to increase the aldehyde concentration in aqueous solution, suggests that the stability of CPCs is directly related to the aldehyde concentration of the carbohydrate material. Periodate oxidation of poly(2-methacrylamido-2-deoxy-D-glucose) followed by covalent attachment to {alpha}-chymotrypsin gave a CPC with catalytic activity in potassium phosphate buffer at 90{degrees}C for 2 h. 1 fig., 1 tab., 40 refs.

  15. Protease inhibitors targeting coronavirus and filovirus entry.

    PubMed

    Zhou, Yanchen; Vedantham, Punitha; Lu, Kai; Agudelo, Juliet; Carrion, Ricardo; Nunneley, Jerritt W; Barnard, Dale; Pöhlmann, Stefan; McKerrow, James H; Renslo, Adam R; Simmons, Graham

    2015-04-01

    In order to gain entry into cells, diverse viruses, including Ebola virus, SARS-coronavirus and the emerging MERS-coronavirus, depend on activation of their envelope glycoproteins by host cell proteases. The respective enzymes are thus excellent targets for antiviral intervention. In cell culture, activation of Ebola virus, as well as SARS- and MERS-coronavirus can be accomplished by the endosomal cysteine proteases, cathepsin L (CTSL) and cathepsin B (CTSB). In addition, SARS- and MERS-coronavirus can use serine proteases localized at the cell surface, for their activation. However, it is currently unclear which protease(s) facilitate viral spread in the infected host. We report here that the cysteine protease inhibitor K11777, ((2S)-N-[(1E,3S)-1-(benzenesulfonyl)-5-phenylpent-1-en-3-yl]-2-{[(E)-4-methylpiperazine-1-carbonyl]amino}-3-phenylpropanamide) and closely-related vinylsulfones act as broad-spectrum antivirals by targeting cathepsin-mediated cell entry. K11777 is already in advanced stages of development for a number of parasitic diseases, such as Chagas disease, and has proven to be safe and effective in a range of animal models. K11777 inhibition of SARS-CoV and Ebola virus entry was observed in the sub-nanomolar range. In order to assess whether cysteine or serine proteases promote viral spread in the host, we compared the antiviral activity of an optimized K11777-derivative with that of camostat, an inhibitor of TMPRSS2 and related serine proteases. Employing a pathogenic animal model of SARS-CoV infection, we demonstrated that viral spread and pathogenesis of SARS-CoV is driven by serine rather than cysteine proteases and can be effectively prevented by camostat. Camostat has been clinically used to treat chronic pancreatitis, and thus represents an exciting potential therapeutic for respiratory coronavirus infections. Our results indicate that camostat, or similar serine protease inhibitors, might be an effective option for treatment of SARS and

  16. Peptide length and prime-side sterics influence potency of peptide phosphonate protease inhibitors

    PubMed Central

    Brown, Christopher M.; Ray, Manisha; Eroy-Reveles, Aura A.; Egea, Pascal; Tajon, Cheryl; Craik, Charles S.

    2010-01-01

    Summary The ability to follow enzyme activity in a cellular context represents a challenging technological frontier that impacts fields ranging from disease pathogenesis to epigenetics. Activity-based probes (ABPs) label the active form of an enzyme via covalent modification of catalytic residues. Here we present an analysis of parameters influencing potency of peptide phosphonate ABPs for trypsin-fold S1A proteases, an abundant and important class of enzymes with similar substrate specificities. We find that peptide length and stability influence potency more than sequence composition and present structural evidence that steric interactions at the prime-side of the substrate-binding cleft affect potency in a protease-dependent manner. We introduce guidelines for the design of peptide phosphonate ABPs and demonstrate their utility in a live-cell labeling application that specifically targets active S1A proteases at the cell surface of cancer cells. PMID:21276938

  17. New soluble ATP-dependent protease, Ti, in Escherichia coli that is distinct from protease La

    SciTech Connect

    Chung, C.H.; Hwang, B.J.; Park, W.J.; Goldberg, A.L.

    1987-05-01

    E. coli must contain other ATP-requiring proteolytic systems in addition to protease La (the lon gene product). A new ATP-dependent protease was purified from lon cells which lack protease La, as shown by immuno-blotting. This enzyme hydrolyzes (TH)casein to acid-soluble products in the presence of ATP (or dATP) and MgS . Nonhydrolyzable ATP analogs, other nucleoside triphosphates and AMP can not replace ATP. Therefore, ATP hydrolysis appears necessary for proteolysis. The enzyme appears to be a serine protease, but also contains essential thiol residues. Unlike protease La, it is not inhibited by vanadate, heparin, or the defective R9 subunit of protease La. On gel filtration, this enzyme has an apparent Mr of 340,000 and is comprised of two components of 190,000D and 130,000D, which can be separated by phosphocellulose chromatography. By themselves, these components do not show ATP-dependent proteolysis, but when mixed, full activity is restored. These finding and similar ones of Maurizi and Gottesman indicate that E. coli contain two soluble ATP-dependent proteases, which function by different mechanisms. This new enzyme may contribute to the rapid breakdown of abnormal polypeptides or of normal proteins during starvation. The authors propose to name it protease Ti.

  18. Escherichia coli contains a soluble ATP-dependent protease (Ti) distinct from protease La

    SciTech Connect

    Hwang, B.J.; Park, W.J.; Chung, C.H.; Goldberg, A.L.

    1987-08-01

    The energy requirement for protein breakdown in Escherichia coli has generally been attributed to the ATP-dependence of protease La, the lon gene product. The authors have partially purified another ATP-dependent protease from lon/sup -/ cells that lack protease La (as shown by immunoblotting). This enzyme hydrolyzes (/sup 3/H)methyl-casein to acid-soluble products in the presence of ATP and Mg/sup 2 +/. ATP hydrolysis appears necessary for proteolytic activity. Since this enzyme is inhibited by diisopropyl fluorophosphate, it appears to be a serine protease, but it also contains essential thiol residues. They propose to name this enzyme protease Ti. It differs from protease La in nucleotide specificity, inhibitor sensitivity, and subunit composition. On gel filtration, protease Ti has an apparent molecular weight of 370,000. It can be fractionated by phosphocellulose chromatography or by DEAE chromatography into two components with apparent molecular weights of 260,000 and 140,000. When separated, they do not show preteolytic activity. One of these components, by itself, has ATPase activity and is labile in the absence of ATP. The other contains the diisopropyl fluorophosphate-sensitive proteolytic site. These results and the similar findings of Katayama-Fujimura et al. indicate that E. coli contains two ATP-hydrolyzing proteases, which differ in many biochemical features and probably in their physiological roles.

  19. A Novel Bioluminescent Protease Assay Using Engineered Firefly Luciferase

    PubMed Central

    Wigdal, Susan S; Anderson, Jessica L; Vidugiris, Gediminas J; Shultz, John; Wood, Keith V; Fan, Frank

    2008-01-01

    Proteases play important roles in a variety of disease processes. Understanding their biological functions underpins the efforts of drug discovery. We have developed a bioluminescent protease assay using a circularly permuted form of firefly luciferase, wherein the native enzyme termini were joined by a peptide containing a protease site of interest. Protease cleavage of these mutant luciferases greatly activates the enzyme, typically over 100 fold. The mutant luciferase substrates are easily generated by molecular cloning and cell-free translation reactions and thus the protease substrates do not need to be chemically synthesized or purchased. The assay has broad applicability using a variety of proteases and their cognate sites and can sensitively detect protease activity. In this report we further demonstrate its utility for the evaluation of protease recognition sequence specificity and subsequent establishment of an optimized assay for the identification and characterization of protease inhibitors using high throughput screening. PMID:20161840

  20. Cleavage of influenza A virus H1 hemagglutinin by swine respiratory bacterial proteases.

    PubMed Central

    Callan, R J; Hartmann, F A; West, S E; Hinshaw, V S

    1997-01-01

    Cleavage of influenza A virus hemagglutinin (HA) is required for expression of fusion activity and virus entry into cells. Extracellular proteases are responsible for the proteolytic cleavage activation of avirulent avian and mammalian influenza viruses and contribute to pathogenicity and tissue tropism. The relative contributions of host and microbial proteases to cleavage activation in natural infection remain to be established. We examined 23 respiratory bacterial pathogens and 150 aerobic bacterial isolates cultured from the nasal cavities of pigs for proteolytic activity. No evidence of secreted proteases was found for the bacterial pathogens, including Haemophilus parasuis, Pasteurella multocida, Actinobacillus pleuropneumoniae, Bordetella bronchiseptica, and Streptococcus suis. Proteolytic bacteria were isolated from 7 of 11 swine nasal samples and included Staphylococcus chromogenes, Staphylococcus hyicus, Aeromonas caviae, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Enterococcus sp. Only P. aeruginosa secreted a protease, elastase, that cleaved influenza virus HA. However, compared to trypsin, the site of cleavage by elastase was shifted one amino acid in the carboxy-terminal direction and resulted in inactivation of the virus. Under the conditions of this study, we identified several bacterial isolates from the respiratory tracts of pigs that secrete proteases in vitro. However, none of these proteolytic isolates demonstrated direct cleavage activation of influenza virus HA. PMID:9311838

  1. Serine Protease Catalysis: A Computational Study of Tetrahedral Intermediates and Inhibitory Adducts.

    PubMed

    Ngo, Phong D; Mansoorabadi, Steven O; Frey, Perry A

    2016-08-01

    Peptide boronic acids and peptidyl trifluoromethyl ketones (TFKs) inhibit serine proteases by forming monoanionic, tetrahedral adducts to serine in the active sites. Investigators regard these adducts as analogs of monoanionic, tetrahedral intermediates. Density functional theory (DFT) calculations and fractional charge analysis show that tetrahedral adducts of model peptidyl TFKs are structurally and electrostatically very similar to corresponding tetrahedral intermediates. In contrast, the DFT calculations show the structures and electrostatic properties of analogous peptide boronate adducts to be significantly different. The peptide boronates display highly electrostatically positive boron, with correspondingly negative ligands in the tetrahedra. In addition, the computed boron-oxygen and boron-carbon bond lengths in peptide boronates (which are identical or very similar to the corresponding bonds in a peptide boronate adduct of α-lytic protease determined by X-ray crystallography at subangstrom resolution) are significantly longer than the corresponding bond lengths in model tetrahedral intermediates. Since protease-peptidyl TFKs incorporate low-barrier hydrogen bonds (LBHBs) between an active site histidine and aspartate, while the protease-peptide boronates do not, these data complement the spectroscopic and chemical evidence for the participation of LBHBs in catalysis by serine proteases. Moreover, while the potency of these classes of inhibitors can be correlated to the structures of the peptide moieties, the present results indicate that the strength of their bonds to serine contribute significantly to their inhibitory properties. PMID:27387593

  2. The Hypervariable Amino-Terminus of P1 Protease Modulates Potyviral Replication and Host Defense Responses

    PubMed Central

    Pasin, Fabio; Simón-Mateo, Carmen; García, Juan Antonio

    2014-01-01

    The replication of many RNA viruses involves the translation of polyproteins, whose processing by endopeptidases is a critical step for the release of functional subunits. P1 is the first protease encoded in plant potyvirus genomes; once activated by an as-yet-unknown host factor, it acts in cis on its own C-terminal end, hydrolyzing the P1-HCPro junction. Earlier research suggests that P1 cooperates with HCPro to inhibit host RNA silencing defenses. Using Plum pox virus as a model, we show that although P1 does not have a major direct role in RNA silencing suppression, it can indeed modulate HCPro function by its self-cleavage activity. To study P1 protease regulation, we used bioinformatic analysis and in vitro activity experiments to map the core C-terminal catalytic domain. We present evidence that the hypervariable region that precedes the protease domain is predicted as intrinsically disordered, and that it behaves as a negative regulator of P1 proteolytic activity in in vitro cleavage assays. In viral infections, removal of the P1 protease antagonistic regulator is associated with greater symptom severity, induction of salicylate-dependent pathogenesis-related proteins, and reduced viral loads. We suggest that fine modulation of a viral protease activity has evolved to keep viral amplification below host-detrimental levels, and thus to maintain higher long-term replicative capacity. PMID:24603811

  3. Phytaspase, a relocalisable cell death promoting plant protease with caspase specificity

    PubMed Central

    Chichkova, Nina V; Shaw, Jane; Galiullina, Raisa A; Drury, Georgina E; Tuzhikov, Alexander I; Kim, Sang Hyon; Kalkum, Markus; Hong, Teresa B; Gorshkova, Elena N; Torrance, Lesley; Vartapetian, Andrey B; Taliansky, Michael

    2010-01-01

    Caspases are cysteine-dependent proteases and are important components of animal apoptosis. They introduce specific breaks after aspartate residues in a number of cellular proteins mediating programmed cell death (PCD). Plants encode only distant homologues of caspases, the metacaspases that are involved in PCD, but do not possess caspase-specific proteolytic activity. Nevertheless, plants do display caspase-like activities indicating that enzymes structurally distinct from classical caspases may operate as caspase-like proteases. Here, we report the identification and characterisation of a novel PCD-related subtilisin-like protease from tobacco and rice named phytaspase (plant aspartate-specific protease) that possesses caspase specificity distinct from that of other known caspase-like proteases. We provide evidence that phytaspase is synthesised as a proenzyme, which is autocatalytically processed to generate the mature enzyme. Overexpression and silencing of the phytaspase gene showed that phytaspase is essential for PCD-related responses to tobacco mosaic virus and abiotic stresses. Phytaspase is constitutively secreted into the apoplast before PCD, but unexpectedly is re-imported into the cell during PCD providing insights into how phytaspase operates. PMID:20111004

  4. Replication of SULT4A1-1 as a pharmacogenetic marker of olanzapine response and evidence of lower weight gain in the high response group

    PubMed Central

    Ramsey, Timothy L; Liu, Qian; Brennan, Mark D

    2014-01-01

    Aim Antipsychotic efficacy biomarkers have the potential to improve outcomes in psychotic patients. This study examined the effect of SULT4A1-1 haplotype status (rs2285162 [A]-rs2285167 [G]) on olanzapine response. Patients & methods We evaluated 87 olanzapine treated subjects from Phases 1, 1B and 2 of the CATIE trial for the impact of SULT4A1-1 status on change in Positive and Negative Syndrome Scale (PANSS) total score using two models of response. We also examined weight change. Results SULT4A1-1-positive status correlated with superior olanzapine response in Phase 1 (p = 0.004 for model 1 and p = 0.001 for model 2) and Phases 1B/2 (p = 0.05 for model 1 and p = 0.007 for model 2). SULT4A1-1-positive subjects gained significantly less weight per month on olanzapine, 0.15 lbs, than did SULT4A1-1-negative subjects, 2.27 lbs (p = 0.04). Conclusion This study provides a second replication of superior olanzapine response in SULT4A1-1-positive subjects compared with SULT4A1-1-negative subjects. SULT4A1-1-positive subjects treated with olanzapine also gained less weight than SULT4A1-1-negative subjects. PMID:24956247

  5. Identification of covalent active site inhibitors of dengue virus protease

    PubMed Central

    Koh-Stenta, Xiaoying; Joy, Joma; Wang, Si Fang; Kwek, Perlyn Zekui; Wee, John Liang Kuan; Wan, Kah Fei; Gayen, Shovanlal; Chen, Angela Shuyi; Kang, CongBao; Lee, May Ann; Poulsen, Anders; Vasudevan, Subhash G; Hill, Jeffrey; Nacro, Kassoum

    2015-01-01

    Dengue virus (DENV) protease is an attractive target for drug development; however, no compounds have reached clinical development to date. In this study, we utilized a potent West Nile virus protease inhibitor of the pyrazole ester derivative class as a chemical starting point for DENV protease drug development. Compound potency and selectivity for DENV protease were improved through structure-guided small molecule optimization, and protease-inhibitor binding interactions were validated biophysically using nuclear magnetic resonance. Our work strongly suggests that this class of compounds inhibits flavivirus protease through targeted covalent modification of active site serine, contrary to an allosteric binding mechanism as previously described. PMID:26677315

  6. 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

  7. Subtilisin-like proteases in nematodes.

    PubMed

    Poole, Catherine B; Jin, Jingmin; McReynolds, Larry A

    2007-09-01

    Cleavage by subtilisin-like proteases (subtilases) is an essential step in post-translational processing of proteins found in organisms ranging from yeast to mammals. Our knowledge of the diversity of this protease family in nematodes is aided by the rapid increase in sequence information, especially from the Brugia malayi genome project. Genetic studies of the subtilases in Caenorhabitis elegans give valuable insight into the biological function of these proteases in other nematode species. In this review, we focus on the subtilases in filarial nematodes as well as other parasitic and free-living nematodes in comparison to what is known in C. elegans. Topics to be addressed include expansion and diversity of the subtilase gene family during evolution, enhanced complexity created by alternative RNA splicing, molecular and biochemical characterization of the different subtilases and the challenges of designing subtilase-specific inhibitors for parasitic nematodes. PMID:17570539

  8. Serine protease inhibitors of parasitic helminths.

    PubMed

    Molehin, Adebayo J; Gobert, Geoffrey N; McManus, Donald P

    2012-05-01

    Serine protease inhibitors (serpins) are a superfamily of structurally conserved proteins that inhibit serine proteases and play key physiological roles in numerous biological systems such as blood coagulation, complement activation and inflammation. A number of serpins have now been identified in parasitic helminths with putative involvement in immune regulation and in parasite survival through interference with the host immune response. This review describes the serpins and smapins (small serine protease inhibitors) that have been identified in Ascaris spp., Brugia malayi, Ancylostoma caninum Onchocerca volvulus, Haemonchus contortus, Trichinella spiralis, Trichostrongylus vitrinus, Anisakis simplex, Trichuris suis, Schistosoma spp., Clonorchis sinensis, Paragonimus westermani and Echinococcus spp. and discusses their possible biological functions, including roles in host-parasite interplay and their evolutionary relationships. PMID:22310379

  9. Dataset of cocoa aspartic protease cleavage sites.

    PubMed

    Janek, Katharina; Niewienda, Agathe; Wöstemeyer, Johannes; Voigt, Jürgen

    2016-09-01

    The data provide information in support of the research article, "The cleavage specificity of the aspartic protease of cocoa beans involved in the generation of the cocoa-specific aroma precursors" (Janek et al., 2016) [1]. Three different protein substrates were partially digested with the aspartic protease isolated from cocoa beans and commercial pepsin, respectively. The obtained peptide fragments were analyzed by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS/MS) and identified using the MASCOT server. The N- and C-terminal ends of the peptide fragments were used to identify the corresponding in-vitro cleavage sites by comparison with the amino acid sequences of the substrate proteins. The same procedure was applied to identify the cleavage sites used by the cocoa aspartic protease during cocoa fermentation starting from the published amino acid sequences of oligopeptides isolated from fermented cocoa beans. PMID:27508221

  10. Coagulation, Protease Activated Receptors and Viral Myocarditis

    PubMed Central

    Antoniak, Silvio; Mackman, Nigel

    2013-01-01

    The coagulation protease cascade plays an essential role in hemostasis. In addition, a clot contributes to host defense by limiting the spread of pathogens. Coagulation proteases induce intracellular signaling by cleavage of cell surface receptors called protease-activated receptors (PARs). These receptors allow cells to sense changes in the extracellular environment, such as infection. Viruses activate the coagulation cascade by inducing tissue factor expression and by disrupting the endothelium. Virus infection of the heart can cause myocarditis, cardiac remodeling and heart failure. Recent studies using a mouse model have shown that tissue factor, thrombin and PAR-1 signaling all positively regulate the innate immune during viral myocarditis. In contrast, PAR-2 signaling was found to inhibit interferon-β expression and the innate immune response. These observations suggest that anticoagulants may impair the innate immune response to viral infection and that inhibition of PAR-2 may be a new target to reduce viral myocarditis.. PMID:24203054

  11. German cockroach proteases and protease-activated receptor-2 regulate chemokine production and dendritic cell recruitment.

    PubMed

    Day, Scottie B; Ledford, John R; Zhou, Ping; Lewkowich, Ian P; Page, Kristen

    2012-01-01

    We recently showed that serine proteases in German cockroach (GC) feces (frass) decreased experimental asthma through the activation of protease-activated receptor (PAR)-2. Since dendritic cells (DCs) play an important role in the initiation of asthma, we queried the role of GC frass proteases in modulating CCL20 (chemokine C-C motif ligand 20) and granulocyte macrophage colony-stimulating factor (GM-CSF) production, factors that regulate pulmonary DCs. A single exposure to GC frass resulted in a rapid, but transient, increase in GM-CSF and a steady increase in CCL20 in the airways of mice. Instillation of protease-depleted GC frass or instillation of GC frass in PAR-2-deficient mice significantly decreased chemokine release. A specific PAR-2-activating peptide was also sufficient to induce CCL20 production. To directly assess the role of the GC frass protease in chemokine release, we enriched the protease from GC frass and confirmed that the protease was sufficient to induce both GM-CSF and CCL20 production in vivo. Primary airway epithelial cells produced both GM-CSF and CCL20 in a protease- and PAR-2-dependent manner. Finally, we show a decreased percentage of myeloid DCs in the lung following allergen exposure in PAR-2-deficient mice compared to wild-type mice. However, there was no difference in GC frass uptake. Our data indicate that, through the activation of PAR-2, allergen-derived proteases are sufficient to induce CCL20 and GM-CSF production in the airways. This leads to increased recruitment and/or differentiation of myeloid DC populations in the lungs and likely plays an important role in the initiation of allergic airway responses. PMID:21876326

  12. Draft Genome Sequence of Cylindrospermopsis raciborskii (Cyanobacteria) Strain ITEP-A1 Isolated from a Brazilian Semiarid Freshwater Body: Evidence of Saxitoxin and Cylindrospermopsin Synthetase Genes

    PubMed Central

    Lorenzi, Adriana Sturion; Silva, Genivaldo Gueiros Z.; Lopes, Fabyano Alvares Cardoso; Chia, Mathias Ahii; Edwards, Robert A.

    2016-01-01

    Cylindrospermopsis raciborskii ITEP-A1 is a saxitoxin-producing cyanobacterium. We report the draft genome sequence of ITEP-A1, which comprised 195 contigs that were assembled with SPAdes and annotated with Rapid Annotation using Subsystem Technology. The identified genome sequence had 3,605,836 bp, 40.1% G+C, and predicted 3,553 coding sequences (including the synthetase genes). PMID:27151783

  13. Deep Sequencing of Protease Inhibitor Resistant HIV Patient Isolates Reveals Patterns of Correlated Mutations in Gag and Protease

    PubMed Central

    Tan, Zhiqiang; Oliveira, Glenn; Yuan, Jinyun; Okulicz, Jason F.; Torbett, Bruce E.; Levy, Ronald M.

    2015-01-01

    While the role of drug resistance mutations in HIV protease has been studied comprehensively, mutations in its substrate, Gag, have not been extensively cataloged. Using deep sequencing, we analyzed a unique collection of longitudinal viral samples from 93 patients who have been treated with therapies containing protease inhibitors (PIs). Due to the high sequence coverage within each sample, the frequencies of mutations at individual positions were calculated with high precision. We used this information to characterize the variability in the Gag polyprotein and its effects on PI-therapy outcomes. To examine covariation of mutations between two different sites using deep sequencing data, we developed an approach to estimate the tight bounds on the two-site bivariate probabilities in each viral sample, and the mutual information between pairs of positions based on all the bounds. Utilizing the new methodology we found that mutations in the matrix and p6 proteins contribute to continued therapy failure and have a major role in the network of strongly correlated mutations in the Gag polyprotein, as well as between Gag and protease. Although covariation is not direct evidence of structural propensities, we found the strongest correlations between residues on capsid and matrix of the same Gag protein were often due to structural proximity. This suggests that some of the strongest inter-protein Gag correlations are the result of structural proximity. Moreover, the strong covariation between residues in matrix and capsid at the N-terminus with p1 and p6 at the C-terminus is consistent with residue-residue contacts between these proteins at some point in the viral life cycle. PMID:25894830

  14. Using specificity to strategically target proteases

    PubMed Central

    Lim, Mark D.; Craik, Charles S.

    2009-01-01

    Proteases are a family of naturally occurring enzymes in the body whose dysregulation has been implicated in numerous diseases and cancers. Their ability to selectively and catalytically turnover substrate adds both signal amplification and functionality as parameters for the detection of disease. This review will focus on the development of activity-based methodologies to characterize proteases, and in particular, the use of positional scanning, synthetic combinatorial libraries (PS-SCL’s), and substrate activity screening (SAS) assays. The use of these approaches to better understand a protease’s natural substrate will be discussed as well as the technologies that emerged. PMID:18434168

  15. 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.

  16. Allosteric Inhibitors of the NS3 Protease from the Hepatitis C Virus

    PubMed Central

    Abian, Olga; Vega, Sonia; Sancho, Javier; Velazquez-Campoy, Adrian

    2013-01-01

    The nonstructural protein 3 (NS3) from the hepatitis C virus processes the non-structural region of the viral precursor polyprotein in infected hepatic cells. The NS3 protease activity has been considered a target for drug development since its identification two decades ago. Although specific inhibitors have been approved for clinical therapy very recently, resistance-associated mutations have already been reported for those drugs, compromising their long-term efficacy. Therefore, there is an urgent need for new anti-HCV agents with low susceptibility to resistance-associated mutations. Regarding NS3 protease, two strategies have been followed: competitive inhibitors blocking the active site and allosteric inhibitors blocking the binding of the accessory viral protein NS4A. In this work we exploit the intrinsic Zn+2-regulated plasticity of the protease to identify a new type of allosteric inhibitors. In the absence of Zn+2, the NS3 protease adopts a partially-folded inactive conformation. We found ligands binding to the Zn+2-free NS3 protease, trap the inactive protein, and block the viral life cycle. The efficacy of these compounds has been confirmed in replicon cell assays. Importantly, direct calorimetric assays reveal a low impact of known resistance-associated mutations, and enzymatic assays provide a direct evidence of their inhibitory activity. They constitute new low molecular-weight scaffolds for further optimization and provide several advantages: 1) new inhibition mechanism simultaneously blocking substrate and cofactor interactions in a non-competitive fashion, appropriate for combination therapy; 2) low impact of known resistance-associated mutations; 3) inhibition of NS4A binding, thus blocking its several effects on NS3 protease. PMID:23936097

  17. An Aspartic Protease of the Scabies Mite Sarcoptes scabiei Is Involved in the Digestion of Host Skin and Blood Macromolecules

    PubMed Central

    Mahmood, Wajahat; Viberg, Linda T.; Fischer, Katja; Walton, Shelley F.; Holt, Deborah C.

    2013-01-01

    Background Scabies is a disease of worldwide significance, causing considerable morbidity in both humans and other animals. The scabies mite Sarcoptes scabiei burrows into the skin of its host, obtaining nutrition from host skin and blood. Aspartic proteases mediate a range of diverse and essential physiological functions such as tissue invasion and migration, digestion, moulting and reproduction in a number of parasitic organisms. We investigated whether aspartic proteases may play role in scabies mite digestive processes. Methodology/Principle Findings We demonstrated the presence of aspartic protease activity in whole scabies mite extract. We then identified a scabies mite aspartic protease gene sequence and produced recombinant active enzyme. The recombinant scabies mite aspartic protease was capable of digesting human haemoglobin, serum albumin, fibrinogen and fibronectin, but not collagen III or laminin. This is consistent with the location of the scabies mites in the upper epidermis of human skin. Conclusions/Significance The development of novel therapeutics for scabies is of increasing importance given the evidence of emerging resistance to current treatments. We have shown that a scabies mite aspartic protease plays a role in the digestion of host skin and serum molecules, raising the possibility that interference with the function of the enzyme may impact on mite survival. PMID:24244770

  18. cDNA cloning and chromosomal mapping of the mouse type VII collagen gene (Col7a1): Evidence for rapid evolutionary divergence of the gene

    SciTech Connect

    Li, Kehua; Christiano, A.M.; Chu, Mon Li; Uitto, J. Thomas Jefferson Univ., Philadelphia, PA ); Copeland, N.G.; Gilbert, D.J. )

    1993-06-01

    Type VII collagen is the major component of anchoring fibrils, critical attachment structures at the dermal-epidermal basement membrane zone. Genetic linkage analyses with recently cloned human type VII collagen cDNAs have indicated that the corresponding gene, COL7A1, is the candidate gene in the dystrophic forms of epidermolysis bullosa. To gain insight into the evolutionary conservation of COL7A1, in this study the authors have isolated mouse type VII collagen cDNAs by screening a mouse epidermal keratinocyte cDNA library with a human COL7A1 cDNA. Two overlapping mouse cDNAs were isolated, and Northern hybridization of mouse epidermal keratinocyte RNA with one of them revealed the presence of a mRNA transcript of [approximately]9.5 kb, the approximate size of the human COL7A1 mRNA. Nucleotide sequencing of the mouse cDNAs revealed a 2760-bp open reading frame that encodes the 5[prime] half of the collagenous domain and a segment of the NC-1, the noncollagenous amino-terminal domain of type VII collagen. Comparison of the mouse amino acid sequences with the corresponding human sequences deduced from cDNAs revealed 82.5% identity. The evolutionary divergence of the gene was relatively rapid in comparison to other collagen genes. Despite the high degree of sequence variation, several sequences, including the size and the position of noncollagenous imperfections and interruptions within the Gly-X-Y repeat sequence, were precisely conserved. Finally, the mouse Col7a1 gene was located by interspecific backcross mapping to mouse Chromosome 9, a region that corresponds to human chromosome 3p21, the position of human COL7Al. This assignment confirms and extends the relationship between the mouse and the human chromosomes in this region of the genome. 33 refs., 5 figs., 1 tab.

  19. Proteases decode the extracellular matrix cryptome.

    PubMed

    Ricard-Blum, Sylvie; Vallet, Sylvain D

    2016-03-01

    The extracellular matrix is comprised of 1100 core-matrisome and matrisome-associated proteins and of glycosaminoglycans. This structural scaffold contributes to the organization and mechanical properties of tissues and modulates cell behavior. The extracellular matrix is dynamic and undergoes constant remodeling, which leads to diseases if uncontrolled. Bioactive fragments, called matricryptins, are released from the extracellular proteins by limited proteolysis and have biological activities on their own. They regulate numerous physiological and pathological processes such as angiogenesis, cancer, diabetes, wound healing, fibrosis and infectious diseases and either improve or worsen the course of diseases depending on the matricryptins and on the molecular and biological contexts. Several protease families release matricryptins from core-matrisome and matrisome-associated proteins both in vitro and in vivo. The major proteases, which decrypt the extracellular matrix, are zinc metalloproteinases of the metzincin superfamily (matrixins, adamalysins and astacins), cysteine proteinases and serine proteases. Some matricryptins act as enzyme inhibitors, further connecting protease and matricryptin fates and providing intricate regulation of major physiopathological processes such as angiogenesis and tumorigenesis. They strengthen the role of the extracellular matrix as a key player in tissue failure and core-matrisome and matrisome-associated proteins as important therapeutic targets. PMID:26382969

  20. Transient ECM protease activity promotes synaptic plasticity.

    PubMed

    Magnowska, Marta; Gorkiewicz, Tomasz; Suska, Anna; Wawrzyniak, Marcin; Rutkowska-Wlodarczyk, Izabela; Kaczmarek, Leszek; Wlodarczyk, Jakub

    2016-01-01

    Activity-dependent proteolysis at a synapse has been recognized as a pivotal factor in controlling dynamic changes in dendritic spine shape and function; however, excessive proteolytic activity is detrimental to the cells. The exact mechanism of control of these seemingly contradictory outcomes of protease activity remains unknown. Here, we reveal that dendritic spine maturation is strictly controlled by the proteolytic activity, and its inhibition by the endogenous inhibitor (Tissue inhibitor of matrix metalloproteinases-1 - TIMP-1). Excessive proteolytic activity impairs long-term potentiation of the synaptic efficacy (LTP), and this impairment could be rescued by inhibition of protease activity. Moreover LTP is altered persistently when the ability of TIMP-1 to inhibit protease activity is abrogated, further demonstrating the role of such inhibition in the promotion of synaptic plasticity under well-defined conditions. We also show that dendritic spine maturation involves an intermediate formation of elongated spines, followed by their conversion into mushroom shape. The formation of mushroom-shaped spines is accompanied by increase in AMPA/NMDA ratio of glutamate receptors. Altogether, our results identify inhibition of protease activity as a critical regulatory mechanism for dendritic spines maturation. PMID:27282248

  1. Transient ECM protease activity promotes synaptic plasticity

    PubMed Central

    Magnowska, Marta; Gorkiewicz, Tomasz; Suska, Anna; Wawrzyniak, Marcin; Rutkowska-Wlodarczyk, Izabela; Kaczmarek, Leszek; Wlodarczyk, Jakub

    2016-01-01

    Activity-dependent proteolysis at a synapse has been recognized as a pivotal factor in controlling dynamic changes in dendritic spine shape and function; however, excessive proteolytic activity is detrimental to the cells. The exact mechanism of control of these seemingly contradictory outcomes of protease activity remains unknown. Here, we reveal that dendritic spine maturation is strictly controlled by the proteolytic activity, and its inhibition by the endogenous inhibitor (Tissue inhibitor of matrix metalloproteinases-1 – TIMP-1). Excessive proteolytic activity impairs long-term potentiation of the synaptic efficacy (LTP), and this impairment could be rescued by inhibition of protease activity. Moreover LTP is altered persistently when the ability of TIMP-1 to inhibit protease activity is abrogated, further demonstrating the role of such inhibition in the promotion of synaptic plasticity under well-defined conditions. We also show that dendritic spine maturation involves an intermediate formation of elongated spines, followed by their conversion into mushroom shape. The formation of mushroom-shaped spines is accompanied by increase in AMPA/NMDA ratio of glutamate receptors. Altogether, our results identify inhibition of protease activity as a critical regulatory mechanism for dendritic spines maturation. PMID:27282248

  2. Protease IV, a quorum sensing-dependent protease of Pseudomonas aeruginosa modulates insect innate immunity.

    PubMed

    Park, Su-Jin; Kim, Soo-Kyoung; So, Yong-In; Park, Ha-Young; Li, Xi-Hui; Yeom, Doo Hwan; Lee, Mi-Nan; Lee, Bok-Luel; Lee, Joon-Hee

    2014-12-01

    In Pseudomonas aeruginosa, quorum sensing (QS) plays an essential role in pathogenesis and the QS response controls many virulence factors. Using a mealworm, Tenebrio molitor as a host model, we found that Protease IV, a QS-regulated exoprotease of P. aeruginosa functions as a key virulence effector causing the melanization and death of T. molitor larvae. Protease IV was able to degrade zymogens of spätzle processing enzyme (SPE) and SPE-activating enzyme (SAE) without the activation of the antimicrobial peptide (AMP) production. Since SPE and SAE function to activate spätzle, a ligand of Toll receptor in the innate immune system of T. molitor, we suggest that Protease IV may interfere with the activation of the Toll signaling. Independently of the Toll pathway, the melanization response, another innate immunity was still generated, since Protease IV directly converted Tenebrio prophenoloxidase into active phenoloxidase. Protease IV also worked as an important factor in the virulence to brine shrimp and nematode. These results suggest that Protease IV provides P. aeruginosa with a sophisticated way to escape the immune attack of host by interfering with the production of AMPs. PMID:25315216

  3. A novel protease activity assay using a protease-responsive chaperone protein

    SciTech Connect

    Sao, Kentaro; Murata, Masaharu; Fujisaki, Yuri; Umezaki, Kaori; Mori, Takeshi; Niidome, Takuro; Katayama, Yoshiki; Hashizume, Makoto

    2009-06-05

    Protease activity assays are important for elucidating protease function and for developing new therapeutic agents. In this study, a novel turbidimetric method for determining the protease activity using a protease-responsive chaperone protein is described. For this purpose, a recombinant small heat-shock protein (sHSP) with an introduced Factor Xa protease recognition site was synthesized in bacteria. This recombinant mutant, FXa-HSP, exhibited chaperone-like activity at high temperatures in cell lysates. However, the chaperone-like activity of FXa-HSP decreased dramatically following treatment with Factor Xa. Protein precipitation was subsequently observed in the cell lysates. The reaction was Factor Xa concentration-dependent and was quantitatively suppressed by a specific inhibitor for Factor Xa. Protein aggregation was detected by a simple method based on turbidimetry. The results clearly demonstrate that this assay is an effective, easy-to-use method for determining protease activities without the requirement of labeling procedures and the use of radioisotopes.

  4. CYP1A1 MspI polymorphism and the risk of oral squamous cell carcinoma: Evidence from a meta-analysis

    PubMed Central

    XIE, SHANG; LUO, CHONGDAI; SHAN, XIAOFENG; ZHAO, SHUSHAN; HE, JING; CAI, ZHIGANG

    2016-01-01

    Numerous case-control studies have investigated whether the CYP1A1 gene polymorphism is involved in the occurrence of oral squamous cell carcinoma (OSCC); however, the conclusions are inconsistent. In order to further explore the correlation and obtain a strong conclusion, a meta-analysis was performed to systematically assess the association between the CYP1A1 MspI polymorphism and risk of OSCC. In the present meta-analysis, the odds ratios (ORs) and the corresponding 95% confidence intervals (CIs) were used to assess the association. The statistical analyses were performed with STATA 11.0 software. The heterogeneity was assessed by Q test and I2test. The final analysis included 10 studies of 1,505 cases and 1,967 controls. The overall results suggested that the CYP1A1 MspI polymorphism was significantly associated with an increased risk of OSCC (CC+TC vs. TT: OR, 1.31; 95% CI, 1.01–1.70; P=0.043; CC vs. TC+TT: OR, 2.38; 95% CI, 1.58–3.58; P<0.001; CC vs. TT: OR, 2.52; 95% CI, 1.60–3.96; P<0.001; and C vs. T: OR, 1.45; 95% CI, 1.15–1.83; P<0.001). In a stratified analysis by ethnicity, a statistically significant correlation existed in the Asian population, but not mixed-race and Caucasian populations. In conclusion, despite several limitations, the present meta-analysis established that the CYP1A1 MspI polymorphism may be a risk factor for OSCC, particularly among the Asian population. PMID:27073686

  5. Coagulation factor XII protease domain crystal structure

    PubMed Central

    Pathak, M; Wilmann, P; Awford, J; Li, C; Hamad, BK; Fischer, PM; Dreveny, I; Dekker, LV; Emsley, J

    2015-01-01

    Background Coagulation factor XII is a serine protease that is important for kinin generation and blood coagulation, cleaving the substrates plasma kallikrein and FXI. Objective To investigate FXII zymogen activation and substrate recognition by determining the crystal structure of the FXII protease domain. Methods and results A series of recombinant FXII protease constructs were characterized by measurement of cleavage of chromogenic peptide and plasma kallikrein protein substrates. This revealed that the FXII protease construct spanning the light chain has unexpectedly weak proteolytic activity compared to β-FXIIa, which has an additional nine amino acid remnant of the heavy chain present. Consistent with these data, the crystal structure of the light chain protease reveals a zymogen conformation for active site residues Gly193 and Ser195, where the oxyanion hole is absent. The Asp194 side chain salt bridge to Arg73 constitutes an atypical conformation of the 70-loop. In one crystal form, the S1 pocket loops are partially flexible, which is typical of a zymogen. In a second crystal form of the deglycosylated light chain, the S1 pocket loops are ordered, and a short α-helix in the 180-loop of the structure results in an enlarged and distorted S1 pocket with a buried conformation of Asp189, which is critical for P1 Arg substrate recognition. The FXII structures define patches of negative charge surrounding the active site cleft that may be critical for interactions with inhibitors and substrates. Conclusions These data provide the first structural basis for understanding FXII substrate recognition and zymogen activation. PMID:25604127

  6. Serine protease autotransporters of enterobacteriaceae (SPATEs): biogenesis and function.

    PubMed

    Dautin, Nathalie

    2010-06-01

    Serine Protease Autotransporters of Enterobacteriaceae (SPATEs) constitute a large family of proteases secreted by Escherichia coli and Shigella. SPATEs exhibit two distinct proteolytic activities. First, a C-terminal catalytic site triggers an intra-molecular cleavage that releases the N-terminal portion of these proteins in the extracellular medium. Second, the secreted N-terminal domains of SPATEs are themselves proteases; each contains a canonical serine-protease catalytic site. Some of these secreted proteases are toxins, eliciting various effects on mammalian cells. Here, we discuss the biogenesis of SPATEs and their function as toxins. PMID:22069633

  7. Detection of protease-resistant cervid prion protein in water from a CWD-endemic area

    PubMed Central

    Nichols, TA; Pulford, Bruce; Wyckoff, A Christy; Meyerett, Crystal; Michel, Brady; Gertig, Kevin; Hoover, Edward A; Jewell, Jean E; Telling, Glenn C

    2009-01-01

    Chronic wasting disease (CWD) is the only known transmissible spongiform encephalopathy affecting free-ranging wildlife. Although the exact mode of natural transmission remains unknown, substantial evidence suggests that prions can persist in the environment, implicating components thereof as potential prion reservoirs and transmission vehicles.1–4 CWD-positive animals may contribute to environmental prion load via decomposing carcasses and biological materials including saliva, blood, urine and feces.5–7 Sensitivity limitations of conventional assays hamper evaluation of environmental prion loads in soil and water. Here we show the ability of serial protein misfolding cyclic amplification (sPMCA) to amplify a 1.3 × 10−7 dilution of CWD-infected brain homogenate spiked into water samples, equivalent to approximately 5 × 107 protease resistant cervid prion protein (PrPCWD) monomers. We also detected PrPCWD in one of two environmental water samples from a CWD endemic area collected at a time of increased water runoff from melting winter snow pack, as well as in water samples obtained concurrently from the flocculation stage of water processing by the municipal water treatment facility. Bioassays indicated that the PrPCWD detected was below infectious levels. These data demonstrate detection of very low levels of PrPCWD in the environment by sPMCA and suggest persistence and accumulation of prions in the environment that may promote CWD transmission. PMID:19823039

  8. Protease activity, localization and inhibition in the human hair follicle

    PubMed Central

    Bhogal, R K; Mouser, P E; Higgins, C A; Turner, G A

    2014-01-01

    Synopsis Objective In humans, the process of hair shedding, referred to as exogen, is believed to occur independently of the other hair cycle phases. Although the actual mechanisms involved in hair shedding are not fully known, it has been hypothesized that the processes leading to the final step of hair shedding may be driven by proteases and/or protease inhibitor activity. In this study, we investigated the presence of proteases and protease activity in naturally shed human hairs and assessed enzyme inhibition activity of test materials. Methods We measured enzyme activity using a fluorescence-based assay and protein localization by indirect immunohistochemistry (IHC). We also developed an ex vivo skin model for measuring the force required to pull hair fibres from skin. Results Our data demonstrate the presence of protease activity in the tissue material surrounding club roots. We also demonstrated the localization of specific serine protease protein expression in human hair follicle by IHC. These data provide evidence demonstrating the presence of proteases around the hair club roots, which may play a role during exogen. We further tested the hypothesis that a novel protease inhibitor system (combination of Trichogen® and climbazole) could inhibit protease activity in hair fibre club root extracts collected from a range of ethnic groups (UK, Brazil, China, first-generation Mexicans in the USA, Thailand and Turkey) in both males and females. Furthermore, we demonstrated that this combination is capable of increasing the force required to remove hair in an ex vivo skin model system. Conclusion These studies indicate the presence of proteolytic activity in the tissue surrounding the human hair club root and show that it is possible to inhibit this activity with a combination of Trichogen® and climbazole. This technology may have potential to reduce excessive hair shedding. Résumé Objectif Chez l'homme, le processus de perte de cheveux, désigné comme exog

  9. Proteases from the Regenerating Gut of the Holothurian Eupentacta fraudatrix

    PubMed Central

    Lamash, Nina E.; Dolmatov, Igor Yu

    2013-01-01

    Four proteases with molecular masses of 132, 58, 53, and 47 kDa were detected in the digestive system of the holothurian Eupentacta fraudatrix. These proteases displayed the gelatinase activity and characteristics of zinc metalloproteinases. The 58 kDa protease had similar protease inhibitor sensitivity to that of mammalian matrix metalloproteinases. Zymographic assay revealed different lytic activities of all four proteases during intestine regeneration in the holothurian. The 132 kDa protease showed the highest activity at the first stage. During morphogenesis (stages 2–4 of regeneration), the highest activity was measured for the 53 and 58 kDa proteases. Inhibition of protease activity exerts a marked effect on regeneration, which was dependent on the time when 1,10-phenanthroline injections commenced. When metalloproteinases were inhibited at the second stage of regeneration, the restoration rates were decreased. However, such an effect proved to be reversible, and when inhibition ceased, the previous rate of regeneration was recovered. When protease activity is inhibited at the first stage, regeneration is completely abolished, and the animals die, suggesting that early activation of the proteases is crucial for triggering the regenerative process in holothurians. The role of the detected proteases in the regeneration processes of holothurians is discussed. PMID:23505505

  10. Comparative study on the protease inhibitors from fish eggs

    NASA Astrophysics Data System (ADS)

    Ustadi; Kim, K. Y.; Kim, S. M.

    2005-07-01

    The protease inhibitor was purified from five different fish eggs. The molecular weights of Pacific herring, chum salmon, pond smelt, glassfish, and Alaska pollock egg protease inhibitors were 120, 89, 84.5, 17, and l6.8kDa, respectively. The specific inhibitory activity of glassfish egg protease inhibitor was the highest followed by those of Pacific herring and Alaska pollock in order. The specific inhibitory activity and purity of glassfish egg protease inhibitor were 19.70 Umg-1 protein and 164.70 folds of purification, respectively. Glassfish egg protease inhibitor was reasonably stable at 50-65°C and pH 8, which was more stable at high temperature and pH than protease inhibitors from the other fish species. Glassfish egg protease inhibitor was noncompetitive with inhibitor constant ( K i) of 4.44 nmolL-1.

  11. New directions for protease inhibitors directed drug discovery.

    PubMed

    Hamada, Yoshio; Kiso, Yoshiaki

    2016-11-01

    Proteases play crucial roles in various biological processes, and their activities are essential for all living organisms-from viruses to humans. Since their functions are closely associated with many pathogenic mechanisms, their inhibitors or activators are important molecular targets for developing treatments for various diseases. Here, we describe drugs/drug candidates that target proteases, such as malarial plasmepsins, β-secretase, virus proteases, and dipeptidyl peptidase-4. Previously, we reported inhibitors of aspartic proteases, such as renin, human immunodeficiency virus type 1 protease, human T-lymphotropic virus type I protease, plasmepsins, and β-secretase, as drug candidates for hypertension, adult T-cell leukaemia, human T-lymphotropic virus type I-associated myelopathy, malaria, and Alzheimer's disease. Our inhibitors are also described in this review article as examples of drugs that target proteases. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 563-579, 2016. PMID:26584340

  12. The association of the CYP1A1 Ile462Val polymorphism with head and neck cancer risk: evidence based on a cumulative meta-analysis

    PubMed Central

    Wang, Yadong; Yang, Haiyan; Duan, Guangcai; Wang, Haiyu

    2016-01-01

    Objective The aim of this study was to address the association between the Ile462Val polymorphism in the gene encoding cytochrome P450 1A1 (CYP1A1) and the risk of head and neck cancer (HNC). Materials and methods The Medline/PubMed, EMBASE, and Web of Science databases were searched. The strength of the association was evaluated by calculating the odds ratio (OR) with a 95% confidence interval (CI). Results Overall, we observed an increased risk of HNC in patients with the Ile/Val+Val/Val genotype compared to those with the Ile/Ile genotype among the 6,367 cases and 6,395 controls evaluated in the 34 eligible studies, with a pooled OR of 1.284 (95% CI: 1.119–1.473). In addition, we observed an increased risk of HNC in patients with the Ile/Val+Val/Val genotype compared to those with the Ile/Ile genotype in the subgroup analyses (OR =1.362, 95% CI: 1.102–1.685 for laryngeal cancer; OR =1.519, 95% CI: 1.253–1.843 for pharyngeal cancer; OR =1.371, 95% CI: 1.111–1.693 for Asians; and OR =1.329, 95% CI: 1.138–1.551 for patients in studies using hospital-based controls). Conclusion This cumulative meta-analysis suggests that the CYP1A1 Ile462Val polymorphism might contribute to the risk of HNC, particularly for pharyngeal cancer and laryngeal cancer. PMID:27274286

  13. NMR Analysis of a Novel Enzymatically Active Unlinked Dengue NS2B-NS3 Protease Complex*

    PubMed Central

    Kim, Young Mee; Gayen, Shovanlal; Kang, CongBao; Joy, Joma; Huang, Qiwei; Chen, Angela Shuyi; Wee, John Liang Kuan; Ang, Melgious Jin Yan; Lim, Huichang Annie; Hung, Alvin W.; Li, Rong; Noble, Christian G.; Lee, Le Tian; Yip, Andy; Wang, Qing-Yin; Chia, Cheng San Brian; Hill, Jeffrey; Shi, Pei-Yong; Keller, Thomas H.

    2013-01-01

    The dengue virus (DENV) is a mosquito-borne pathogen responsible for an estimated 100 million human infections annually. The viral genome encodes a two-component trypsin-like protease that contains the cofactor region from the nonstructural protein NS2B and the protease domain from NS3 (NS3pro). The NS2B-NS3pro complex plays a crucial role in viral maturation and has been identified as a potential drug target. Using a DENV protease construct containing NS2B covalently linked to NS3pro via a Gly4-Ser-Gly4 linker (“linked protease”), previous x-ray crystal structures show that the C-terminal fragment of NS2B is remote from NS3pro and exists in an open state in the absence of an inhibitor; however, in the presence of an inhibitor, NS2B complexes with NS3pro to form a closed state. This linked enzyme produced NMR spectra with severe signal overlap and line broadening. To obtain a protease construct with a resolved NMR spectrum, we expressed and purified an unlinked protease complex containing a 50-residue segment of the NS2B cofactor region and NS3pro without the glycine linker using a coexpression system. This unlinked protease complex was catalytically active at neutral pH in the absence of glycerol and produced dispersed cross-peaks in a 1H-15N heteronuclear single quantum correlation spectrum that enabled us to conduct backbone assignments using conventional techniques. In addition, titration with an active-site peptide aldehyde inhibitor and paramagnetic relaxation enhancement studies demonstrated that the unlinked DENV protease exists predominantly in a closed conformation in solution. This protease complex can serve as a useful tool for drug discovery against DENV. PMID:23511634

  14. Novel Intermediates of Acenaphthylene Degradation by Rhizobium sp. Strain CU-A1: Evidence for Naphthalene-1,8-Dicarboxylic Acid Metabolism

    PubMed Central

    Poonthrigpun, Siriwat; Pattaragulwanit, Kobchai; Paengthai, Sarunya; Kriangkripipat, Thanyanuch; Juntongjin, Kanchana; Thaniyavarn, Suthep; Petsom, Amorn; Pinphanichakarn, Pairoh

    2006-01-01

    The acenaphthylene-degrading bacterium Rhizobium sp. strain CU-A1 was isolated from petroleum-contaminated soil in Thailand. This strain was able to degrade 600 mg/liter acenaphthylene completely within three days. To elucidate the pathway for degradation of acenaphthylene, strain CU-A1 was mutagenized by transposon Tn5 in order to obtain mutant strains deficient in acenaphthylene degradation. Metabolites produced from Tn5-induced mutant strains B1, B5, and A53 were purified by thin-layer chromatography and silica gel column chromatography and characterized by mass spectrometry. The results suggested that this strain cleaved the fused five-membered ring of acenaphthylene to form naphthalene-1,8-dicarboxylic acid via acenaphthenequinone. One carboxyl group of naphthalene-1,8-dicarboxylic acid was removed to form 1-naphthoic acid which was transformed into salicylic acid before metabolization to gentisic acid. This work is the first report of complete acenaphthylene degradation by a bacterial strain. PMID:16957226

  15. The SUMO Protease Verloren Regulates Dendrite and Axon Targeting in Olfactory Projection Neurons

    PubMed Central

    Berdnik, Daniela; Favaloro, Vincenzo; Luo, Liqun

    2012-01-01

    Sumoylation is a post-translational modification regulating numerous biological processes. Small ubiquitin-like modifier (SUMO) proteases are required for the maturation and de-conjugation of SUMO proteins, thereby either promoting or reverting sumoylation to modify protein function. Here, we show a novel role for a predicted SUMO protease, Verloren (Velo), during projection neuron (PN) target selection in the Drosophila olfactory system. PNs target their dendrites to specific glomeruli within the antennal lobe (AL) and their axons stereotypically into higher brain centers. We uncovered mutations in velo that disrupt PN targeting specificity. PN dendrites that normally target to a particular dorsolateral glomerulus instead mistarget to incorrect glomeruli within the AL or to brain regions outside the AL. velo mutant axons also display defects in arborization. These phenotypes are rescued by postmitotic expression of Velo in PNs but not by a catalytic domain mutant of Velo. Two other SUMO proteases, DmUlp1 and CG12717, can partially compensate for the function of Velo in PN dendrite targeting. Additionally, mutations in SUMO and lesswright (which encodes a SUMO conjugating enzyme) similarly disrupt PN targeting, confirming that sumoylation is required for neuronal target selection. Finally, genetic interaction studies suggest that Velo acts in SUMO de-conjugation rather than in maturation. Our study provides the first in vivo evidence for a specific role of a SUMO protease during neuronal target selection that can be dissociated from its functions in neuronal proliferation and survival. PMID:22699913

  16. The SUMO protease Verloren regulates dendrite and axon targeting in olfactory projection neurons.

    PubMed

    Berdnik, Daniela; Favaloro, Vincenzo; Luo, Liqun

    2012-06-13

    Sumoylation is a post-translational modification regulating numerous biological processes. Small ubiquitin-like modifier (SUMO) proteases are required for the maturation and deconjugation of SUMO proteins, thereby either promoting or reverting sumoylation to modify protein function. Here, we show a novel role for a predicted SUMO protease, Verloren (Velo), during projection neuron (PN) target selection in the Drosophila olfactory system. PNs target their dendrites to specific glomeruli within the antennal lobe (AL) and their axons stereotypically into higher brain centers. We uncovered mutations in velo that disrupt PN targeting specificity. PN dendrites that normally target to a particular dorsolateral glomerulus instead mistarget to incorrect glomeruli within the AL or to brain regions outside the AL. velo mutant axons also display defects in arborization. These phenotypes are rescued by postmitotic expression of Velo in PNs but not by a catalytic domain mutant of Velo. Two other SUMO proteases, DmUlp1 and CG12717, can partially compensate for the function of Velo in PN dendrite targeting. Additionally, mutations in SUMO and lesswright (which encodes a SUMO conjugating enzyme) similarly disrupt PN targeting, confirming that sumoylation is required for neuronal target selection. Finally, genetic interaction studies suggest that Velo acts in SUMO deconjugation rather than in maturation. Our study provides the first in vivo evidence for a specific role of a SUMO protease during neuronal target selection that can be dissociated from its functions in neuronal proliferation and survival. PMID:22699913

  17. Drosophila insulin degrading enzyme and rat skeletal muscle insulin protease cleave insulin at similar sites

    SciTech Connect

    Duckworth, W.C.; Garcia, J.V.; Liepnieks, J.J.; Hamel, F.G.; Hermodson, M.A.; Frank, B.H.; Rosner, M.R. )

    1989-03-21

    Insulin degradation is an integral part of the cellular action of insulin. Recent evidence suggests that the enzyme insulin protease is involved in the degradation of insulin in mammalian tissues. Drosophila, which has insulin-like hormones and insulin receptor homologues, also expresses an insulin degrading enzyme with properties that are very similar to those of mammalian insulin protease. In the present study, the insulin cleavage products generated by the Drosophila insulin degrading enzyme were identified and compared with the products generated by the mammalian insulin protease. Both purified enzymes were incubated with porcine insulin specifically labeled with {sup 125}I on either the A19 or B26 position, and the degradation products were analyzed by HPLC before and after sulfitolysis. Isolation and sequencing of the cleavage products indicated that both enzymes cleave the A chain of intact insulin at identical sites between residues A13 and A14 and A14 and A15. These results demonstrate that all the insulin cleavage sites generated by the Drosopohila insulin degrading enzyme are shared in common with the mammalian insulin protease. These data support the hypothesis that there is evolutionary conservation of the insulin degrading enzyme and further suggest that this enzyme plays an important role in cellular function.

  18. Development of a rapid phenotypic test for HCV protease inhibitors with potential use in clinical decisions

    PubMed Central

    Pessoa, Luciana Santos; Vidal, Luãnna Liebscher; da Costa, Emmerson C.B.; Abreu, Celina Monteiro; da Cunha, Rodrigo Delvecchio; Valadão, Ana Luiza Chaves; dos Santos, André Felipe; Tanuri, Amilcar

    2016-01-01

    Abstract Approximately 185 million people worldwide are chronically infected with hepatitis C virus (HCV). The first-wave of approved NS3 protease inhibitors (PIs) were Telaprevir and Boceprevir, which are currently discontinued. Simeprevir is a second-wave PI incorporated into the Brazilian hepatitis C treatment protocol. Drug resistance plays a key role in patients' treatment regimen. Here, we developed a simple phenotypic assay to evaluate the impact of resistance mutations in HCV NS3 protease to PIs, using a protein expression vector containing wild type NS3 protease domain and NS4A co-factor. We analyzed the impact of five resistance mutations (T54A, V36M, V158I, V170I and T54S+V170I) against Telaprevir, Boceprevir and Simeprevir. Protein purifications were performed with low cost methodology, and enzymatic inhibition assays were measured by FRET. We obtained recombinant proteases with detectable activity, and IC50 and fold change values for the evaluated PIs were determined. The variant T54A showed the highest reduction of susceptibility for the PIs, while the other four variants exhibited lower levels of reduced susceptibility. Interestingly, V170I showed 3.2-fold change for Simeprevir, a new evidence about this variant. These results emphasize the importance of enzymatic assays in phenotypic tests to determine which therapeutic regimen should be implemented. PMID:27575432

  19. Development of a rapid phenotypic test for HCV protease inhibitors with potential use in clinical decisions.

    PubMed

    Pessoa, Luciana Santos; Vidal, Luãnna Liebscher; Costa, Emmerson C B da; Abreu, Celina Monteiro; Cunha, Rodrigo Delvecchio da; Valadão, Ana Luiza Chaves; Santos, André Felipe Dos; Tanuri, Amilcar

    2016-01-01

    Approximately 185 million people worldwide are chronically infected with hepatitis C virus (HCV). The first-wave of approved NS3 protease inhibitors (PIs) were Telaprevir and Boceprevir, which are currently discontinued. Simeprevir is a second-wave PI incorporated into the Brazilian hepatitis C treatment protocol. Drug resistance plays a key role in patients' treatment regimen. Here, we developed a simple phenotypic assay to evaluate the impact of resistance mutations in HCV NS3 protease to PIs, using a protein expression vector containing wild type NS3 protease domain and NS4A co-factor. We analyzed the impact of five resistance mutations (T54A, V36M, V158I, V170I and T54S+V170I) against Telaprevir, Boceprevir and Simeprevir. Protein purifications were performed with low cost methodology, and enzymatic inhibition assays were measured by FRET. We obtained recombinant proteases with detectable activity, and IC50 and fold change values for the evaluated PIs were determined. The variant T54A showed the highest reduction of susceptibility for the PIs, while the other four variants exhibited lower levels of reduced susceptibility. Interestingly, V170I showed 3.2-fold change for Simeprevir, a new evidence about this variant. These results emphasize the importance of enzymatic assays in phenotypic tests to determine which therapeutic regimen should be implemented. PMID:27575432

  20. Structural Insights into the Allosteric Operation of the Lon AAA+ Protease.

    PubMed

    Lin, Chien-Chu; Su, Shih-Chieh; Su, Ming-Yuan; Liang, Pi-Hui; Feng, Chia-Cheng; Wu, Shih-Hsiung; Chang, Chung-I

    2016-05-01

    The Lon AAA+ protease (LonA) is an evolutionarily conserved protease that couples the ATPase cycle into motion to drive substrate translocation and degradation. A hallmark feature shared by AAA+ proteases is the stimulation of ATPase activity by substrates. Here we report the structure of LonA bound to three ADPs, revealing the first AAA+ protease assembly where the six protomers are arranged alternately in nucleotide-free and bound states. Nucleotide binding induces large coordinated movements of conserved pore loops from two pairs of three non-adjacent protomers and shuttling of the proteolytic groove between the ATPase site and a previously unknown Arg paddle. Structural and biochemical evidence supports the roles of the substrate-bound proteolytic groove in allosteric stimulation of ATPase activity and the conserved Arg paddle in driving substrate degradation. Altogether, this work provides a molecular framework for understanding how ATP-dependent chemomechanical movements drive allosteric processes for substrate degradation in a major protein-destruction machine. PMID:27041592

  1. A new principle of oligomerization of plant DEG7 protease based on interactions of degenerated protease domains

    PubMed Central

    Schuhmann, Holger; Mogg, Ulrike; Adamska, Iwona

    2011-01-01

    Deg/HtrA proteases are a large group of ATP-independent serine endoproteases found in almost every organism. Their usual domain arrangement comprises a trypsin-type protease domain and one or more PDZ domains. All Deg/HtrA proteases form homo-oligomers with trimers as the basic unit, where the active protease domain mediates the interaction between individual monomers. Among the members of the Deg/HtrA protease family, the plant protease DEG7 is unique since it contains two protease domains (one active and one degenerated) and four PDZ domains. In the present study, we investigated the oligomerization behaviour of this unusual protease using yeast two-hybrid analysis in vivo and with recombinant protein in vitro. We show that DEG7 forms trimeric complexes, but in contrast with other known Deg/HtrA proteases, it shows a new principle of oligomerization, where trimerization is based on the interactions between degenerated protease domains. We propose that, during evolution, a duplicated active protease domain degenerated and specialized in protein–protein interaction and complex formation. PMID:21247409

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

    PubMed Central

    Fyfe, Cameron D.; Grinter, Rhys; Josts, Inokentijs; Mosbahi, Khedidja; Roszak, Aleksander W.; Cogdell, Richard J.; Wall, Daniel M.; Burchmore, Richard J. S.; Byron, Olwyn; Walker, Daniel

    2015-01-01

    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. coli 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. PMID:26143919

  3. Novel Function of Serine Protease HTRA1 in Inhibiting Adipogenic Differentiation of Human Mesenchymal Stem Cells via MAP Kinase-Mediated MMP Upregulation.

    PubMed

    Tiaden, André N; Bahrenberg, Gregor; Mirsaidi, Ali; Glanz, Stephan; Blüher, Matthias; Richards, Peter J

    2016-06-01

    Adipogenesis is the process by which mesenchymal stem cells (MSCs) develop into lipid-laden adipocytes. Being the dominant cell type within adipose tissue, adipocytes play a central role in regulating circulating fatty acid levels, which is considered to be of critical importance in maintaining insulin sensitivity. High temperature requirement protease A1 (HTRA1) is a newly recognized regulator of MSC differentiation, although its role as a mediator of adipogenesis has not yet been defined. The aim of this work was therefore to evaluate HTRA1's influence on human MSC (hMSC) adipogenesis and to establish a potential mode of action. We report that the addition of exogenous HTRA1 to hMSCs undergoing adipogenesis suppressed their ability to develop into lipid laden adipocytes. These effects were demonstrated as being reliant on both its protease and PDZ domain, and were mediated through the actions of c-Jun N-terminal kinase and matrix metalloproteinases (MMPs). The relevance of such findings with regards to HTRA1's potential influence on adipocyte function in vivo is made evident by the fact that HTRA1 and MMP-13 were readily identifiable within crown-like structures present in visceral adipose tissue samples from insulin resistant obese human subjects. These data therefore implicate HTRA1 as a negative regulator of MSC adipogenesis and are suggestive of its potential involvement in adipose tissue remodeling under pathological conditions. Stem Cells 2016;34:1601-1614. PMID:26864869

  4. Neuroserpin, an axonally secreted serine protease inhibitor.

    PubMed Central

    Osterwalder, T; Contartese, J; Stoeckli, E T; Kuhn, T B; Sonderegger, P

    1996-01-01

    We have identified and chromatographically purified an axonally secreted glycoprotein of CNS and PNS neurons. Several peptides derived from it were microsequenced. Based on these sequences, a fragment of the corresponding cDNA was amplified and used as a probe to isolate a full length cDNA from a chicken brain cDNA library. Because the deduced amino acid sequence qualified the protein as a novel member of the serpin family of serine protease inhibitors, we called it neuroserpin. Analysis of the primary structural features further characterized neuroserpin as a heparin-independent, functional inhibitor of a trypsin-like serine protease. In situ hybridization revealed a predominantly neuronal expression during the late stages of neurogenesis and in the adult brain in regions which exhibit synaptic plasticity. Thus, neuroserpin might function as an axonally secreted regulator of the local extracellular proteolysis involved in the reorganization of the synaptic connectivity during development and synapse plasticity in the adult. Images PMID:8670795

  5. Dysregulation of protease and protease inhibitors in a mouse model of human pelvic organ prolapse.

    PubMed

    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 [Serpina1a (α1-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

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

    SciTech Connect

    Sun, Ping; Austin, Brian P.; Tozer, Jozsef; Waugh, David

    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 TVMV 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.

  7. Emergence of Resistance to Protease Inhibitor Amprenavir in Human Immunodeficiency Virus Type 1-Infected Patients: Selection of Four Alternative Viral Protease Genotypes and Influence of Viral Susceptibility to Coadministered Reverse Transcriptase Nucleoside Inhibitors

    PubMed Central

    Maguire, Michael; Shortino, Denise; Klein, Astrid; Harris, Wendy; Manohitharajah, Varsha; Tisdale, Margaret; Elston, Robert; Yeo, Jane; Randall, Sharon; Xu, Fan; Parker, Hayley; May, Jackie; Snowden, Wendy

    2002-01-01

    Previous data have indicated that the development of resistance to amprenavir, an inhibitor of the human immunodeficiency virus type 1 protease, is associated with the substitution of valine for isoleucine at residue 50 (I50V) in the viral protease. We present further findings from retrospective genotypic and phenotypic analyses of plasma samples from protease inhibitor-naïve and nucleoside reverse transcriptase inhibitor (NRTI)-experienced patients who experienced virological failure while participating in a clinical trial where they had been randomized to receive either amprenavir or indinavir in combination with NRTIs. Paired baseline and on-therapy isolates from 31 of 48 (65%) amprenavir-treated patients analyzed demonstrated the selection of protease mutations. These mutations fell into four distinct categories, characterized by the presence of either I50V, I54L/I54M, I84V, or V32I+I47V and often included accessory mutations, commonly M46I/L. The I50V and I84V genotypes displayed the greatest reductions in susceptibility to amprenavir, although each of the amprenavir-selected genotypes conferred little or no cross-resistance to other protease inhibitors. There was a significant association, for both amprenavir and indinavir, between preexisting baseline resistance to NRTIs subsequently received during the study and development of protease mutations (P = 0.014 and P = 0.031, respectively). Our data provide a comprehensive analysis of the mechanisms by which amprenavir resistance develops during clinical use and present evidence that resistance to concomitant agents in the treatment regimen predisposes to the development of mutations associated with protease inhibitor resistance and treatment failure. PMID:11850255

  8. Stable knock-down of efflux transporters leads to reduced glucuronidation in UGT1A1-overexpressing HeLa cells: the evidence for glucuronidation-transport interplay.

    PubMed

    Zhang, Xingwang; Dong, Dong; Wang, Huailing; Ma, Zhiguo; Wang, Yifei; Wu, Baojian

    2015-04-01

    Efflux of glucuronide is facilitated by the membrane transporters including BCRP and MRPs. In this study, we aimed to determine the effects of transporter expression on glucuronide efflux and cellular glucuronidation. Single efflux transporter (i.e., BCRP, MRP1, MRP3, or MRP4) was stably knocked-down in UGT1A1-overexpressing HeLa cells. Knock-down of transporters was performed by stable transfection of short-hairpin RNA (shRNA) using lentiviral vectors. Glucuronidation and glucuronide transport in the cells were characterized using three different aglycones (i.e., genistein, apigenin, and emodin) with distinct metabolic activities. BCRP knock-down resulted in significant reductions in excretion of glucuronides (42.9% for genistein glucuronide (GG), 21.1% for apigenin glucuronide (AG) , and 33.7% for emodin glucuronide (EG); p < 0.01) and in cellular glucuronidation (38.3% for genistein, 38.6% for apigenin, and 34.7% for emodin; p < 0.01). Knock-down of a MRP transporter led to substantial decreases in excretion of GG (32.3% for MRP1, 36.7% for MRP3, and 36.6% for MRP4; p < 0.01) and AG (59.3% for MRP1, 24.7% for MRP3, and 34.1% for MRP4; p < 0.01). Also, cellular glucuronidation of genistein (38.3% for MRP1, 32.3% for MRP3, and 31.1% for MRP4; p < 0.01) and apigenin (40.6% for MRP1, 32.4% for MRP3, and 34.6% for MRP4; p < 0.001) was markedly suppressed. By contrast, silencing of MRPs did not cause any changes in either excretion of EG or cellular glucuronidation of emodin. In conclusion, cellular glucuronidation was significantly altered by decreasing expression of efflux transporters, revealing a strong interplay of glucuronidation with efflux transport. PMID:25741749

  9. Association between the CYP1A1 A2455G polymorphism and risk of cancer: evidence from 272 case-control studies.

    PubMed

    Qin, Jun; Zhang, Jin-Xia; Li, Xiao-Ping; Wu, Bu-Qiang; Chen, Guang-Bin; He, Xiao-Feng

    2014-04-01

    A2455G is a common polymorphism in CYP1A1, showing differences in its biological functions. Case-control studies have been performed to elucidate the role of A2455G in cancer; however, the results are conflicting and heterogeneous. Hence, we performed a meta-analysis to investigate the association between cancer susceptibility and A2455G (64,593 cases and 91,056 controls from 272 studies) polymorphism in different inheritance models. We used odds ratios with 95% confidence intervals to assess the strength of the association. Overall, significantly increased cancer risk was observed in any genetic model (dominant model, odds ration [OR] = 1.19, 95% confidence interval [CI] = 1.13-1.25; recessive model: OR = 1.41, 95% CI = 1.29-1.54; additive model: OR = 1.49, 95% CI = 1.35-1.65) when all eligible studies were pooled into the meta-analysis. In further stratified and sensitivity analyses, the elevated risk remained for subgroups of breast cancer, colorectal cancer, esophageal cancer, hepatocellular cancer, head and neck cancer, leukemia, lung cancer, and prostate cancer, but these associations vary in different ethnic populations. In summary, this meta-analysis suggests the participation of A2455G in the susceptibility for some cancers, such as breast cancer, colorectal cancer, lung cancer, and so on. Moreover, ethnicity, histological type of cancer, and smokers seem to contribute to varying expressions of the A2455G on some cancers risk. In addition, our work also points out the importance of new studies for A2455G polymorphism in some cancer types, such as gallbladder cancer, Indians of breast cancer, and Caucasians of ovarians, because these cancer types had high heterogeneity in this meta-analysis (I(2) > 75%). PMID:24307623

  10. Mitochondrial Proteases as Emerging Pharmacological Targets.

    PubMed

    Gibellini, Lara; De Biasi, Sara; Nasi, Milena; Iannone, Anna; Cossarizza, Andrea; Pinti, Marcello

    2016-01-01

    The preservation of mitochondrial function and integrity is critical for cell viability. Under stress conditions, unfolded, misfolded or damaged proteins accumulate in a certain compartment of the organelle, interfering with oxidative phosphorylation and normal mitochondrial functions. In stress conditions, several mechanisms, including mitochondrial unfolded protease response (UPRmt), fusion and fission, and mitophagy are engaged to restore normal proteostasis of the organelle. Mitochondrial proteases are a family of more than 20 enzymes that not only are involved in the UPRmt, but actively participate at multiple levels in the stress-response system. Alterations in their expression levels, or mutations that determine loss or gain of function of these proteases deeply impair mitochondrial functionality and can be associated with the onset of inherited diseases, with the development of neurodegenerative disorders and with the process of carcinogenesis. In this review, we focus our attention on six of them, namely CLPP, HTRA2 and LONP1, by analysing the current knowledge about their functions, their involvement in the pathogenesis of human diseases, and the compounds currently available for inhibiting their functions. PMID:26831646

  11. Proteases of human rhinovirus: role in infection.

    PubMed

    Jensen, Lora M; Walker, Erin J; Jans, David A; Ghildyal, Reena

    2015-01-01

    Human rhinoviruses (HRV) are the major etiological agents of the common cold and asthma exacerbations, with significant worldwide health and economic impact. Although large-scale population vaccination has proved successful in limiting or even eradicating many viruses, the more than 100 distinct serotypes mean that conventional vaccination is not a feasible strategy to combat HRV. An alternative strategy is to target conserved viral proteins such as the HRV proteases, 2A(pro) and 3C(pro), the focus of this review. Necessary for host cell shutoff, virus replication, and pathogenesis, 2A(pro) and 3C(pro) are clearly viable drug targets, and indeed, 3C(pro) has been successfully targeted for treating the common cold in experimental infection. 2A(pro) and 3C(pro) are crucial for virus replication due to their role in polyprotein processing as well as cleavage of key cellular proteins to inhibit cellular transcription and translation. Intriguingly, the action of the HRV proteases also disrupts nucleocytoplasmic trafficking, contributing to HRV cytopathic effects. Improved understanding of the protease-cell interactions should enable new therapeutic approaches to be identified for drug development. PMID:25261311

  12. Corruption of Innate Immunity by Bacterial Proteases

    PubMed Central

    Potempa, Jan; Pike, Robert N.

    2009-01-01

    The innate immune system of the human body has developed numerous mechanisms to control endogenous and exogenous bacteria and thus prevent infections by these microorganisms. These mechanisms range from physical barriers such as the skin or mucosal epithelium to a sophisticated array of molecules and cells that function to suppress or prevent bacterial infection. Many bacteria express a variety of proteases, ranging from non-specific and powerful enzymes that degrade many proteins involved in innate immunity to proteases that are extremely precise and specific in their mode of action. Here we have assembled a comprehensive picture of how bacterial proteases affect the host’s innate immune system to gain advantage and cause infection. This picture is far from being complete since the numbers of mechanisms utilized are as astonishing as they are diverse, ranging from degradation of molecules vital to innate immune mechanisms to subversion of the mechanisms to allow the bacterium to hide from the system or take advantage of it. It is vital that such mechanisms are elucidated to allow strategies to be developed to aid the innate immune system in controlling bacterial infections. PMID:19756242

  13. Global Substrate Profiling of Proteases in Human Neutrophil Extracellular Traps Reveals Consensus Motif Predominantly Contributed by Elastase

    PubMed Central

    Knudsen, Giselle M.; Perera, Natascha C.; Jenne, Dieter E.; Murphy, John E.; Craik, Charles S.; Hermiston, Terry W.

    2013-01-01

    Neutrophil extracellular traps (NETs) consist of antimicrobial molecules embedded in a web of extracellular DNA. Formation of NETs is considered to be a defense mechanism utilized by neutrophils to ensnare and kill invading pathogens, and has been recently termed NETosis. Neutrophils can be stimulated to undergo NETosis ex vivo, and are predicted to contain high levels of serine proteases, such as neutrophil elastase (NE), cathepsin G (CG) and proteinase 3 (PR3). Serine proteases are important effectors of neutrophil-mediated immunity, which function directly by degrading pathogenic virulent factors and indirectly via proteolytic activation or deactivation of cytokines, chemokines and receptors. In this study, we utilized a diverse and unbiased peptide library to detect and profile protease activity associated with NETs induced by phorbol-12-myristate-13-acetate (PMA). We obtained a “proteolytic signature” from NETs derived from healthy donor neutrophils and used proteomics to assist in the identification of the source of this proteolytic activity. In addition, we profiled each neutrophil serine protease and included the newly identified enzyme, neutrophil serine protease 4 (NSP4). Each enzyme had overlapping yet distinct endopeptidase activities and often cleaved at unique sites within the same peptide substrate. The dominant proteolytic activity in NETs was attributed to NE; however, cleavage sites corresponding to CG and PR3 activity were evident. When NE was immunodepleted, the remaining activity was attributed to CG and to a lesser extent PR3 and NSP4. Our results suggest that blocking NE activity would abrogate the major protease activity associated with NETs. In addition, the newly identified substrate specificity signatures will guide the design of more specific probes and inhibitors that target NET-associated proteases. PMID:24073241

  14. Cysteine Protease Profiles of the Medicinal Plant Calotropis procera R. Br. revealed by de novo transcriptome analysis.

    PubMed

    Kwon, Chang Woo; Park, Kyung-Min; Kang, Byoung-Cheorl; Kweon, Dae-Hyuk; Kim, Myoung-Dong; Shin, Sang Woon; Je, Yeon Ho; Chang, Pahn-Shick

    2015-01-01

    Calotropis procera R. Br., a traditional medicinal plant in India, is a promising source of commercial proteases, because the cysteine proteases from the plant exhibit high thermo-stability, broad pH optima, and plasma-clotting activity. Though several proteases such as Procerain, Procerain B, CpCp-1, CpCp-2, and CpCp-3 have been isolated and characterized, the information of their transcripts is limited to cDNAs encoding their mature peptides. Due to this limitation, in this study, to determine the cDNA sequences encoding full open reading frame of these cysteine proteases, transcripts were sequenced with an Illumina Hiseq2000 sequencer. A total of 171,253,393 clean reads were assembled into 106,093 contigs with an average length of 1,614 bp and an N50 of 2,703 bp, and 70,797 contigs with an average length of 1,565 bp and N50 of 2,082 bp using Trinity and Velvet-Oases software, respectively. Among these contigs, we found 20 unigenes related to papain-like cysteine proteases by BLASTX analysis against a non-redundant NCBI protein database. Our expression analysis revealed that the cysteine protease contains an N-terminal pro-peptide domain (inhibitor region), which is necessary for correct folding and proteolytic activity. It was evident that expression yields using an inducible T7 expression system in Escherichia coli were considerably higher with the pro-peptide domain than without the domain, which could contribute to molecular cloning of the Calotropis procera protease as an active form with correct folding. PMID:25786229

  15. Initiating protease with modular domains interacts with β-glucan recognition protein to trigger innate immune response in insects

    PubMed Central

    Takahashi, Daisuke; Garcia, Brandon L.; Kanost, Michael R.

    2015-01-01

    The autoactivation of an initiating serine protease upon binding of pattern recognition proteins to pathogen surfaces is a crucial step in eliciting insect immune responses such as the activation of Toll and prophenoloxidase pathways. However, the molecular mechanisms responsible for autoactivation of the initiating protease remains poorly understood. Here, we investigated the molecular basis for the autoactivation of hemolymph protease 14 (HP14), an initiating protease in hemolymph of Manduca sexta, upon the binding of β-1,3-glucan by its recognition protein, βGRP2. Biochemical analysis using HP14 zymogen (proHP14), βGRP2, and the recombinant proteins as truncated forms showed that the amino-terminal modular low-density lipoprotein receptor class A (LA) domains within HP14 are required for proHP14 autoactivation that is stimulated by its interaction with βGRP2. Consistent with this result, recombinant LA domains inhibit the activation of proHP14 and prophenoloxidase, likely by competing with the interaction between βGRP2 and LA domains within proHP14. Using surface plasmon resonance, we demonstrated that immobilized LA domains directly interact with βGRP2 in a calcium-dependent manner and that high-affinity interaction requires the C-terminal glucanase-like domain of βGRP2. Importantly, the affinity of LA domains for βGRP2 increases nearly 100-fold in the presence of β-1,3-glucan. Taken together, these results present the first experimental evidence to our knowledge that LA domains of an insect modular protease and glucanase-like domains of a βGRP mediate their interaction, and that this binding is essential for the protease autoactivation. Thus, our study provides important insight into the molecular basis underlying the initiation of protease cascade in insect immune responses. PMID:26504233

  16. Cysteine Protease Profiles of the Medicinal Plant Calotropis procera R. Br. Revealed by De Novo Transcriptome Analysis

    PubMed Central

    Kwon, Chang Woo; Park, Kyung-Min; Kang, Byoung-Cheorl; Kweon, Dae-Hyuk; Kim, Myoung-Dong; Shin, Sang Woon; Je, Yeon Ho; Chang, Pahn-Shick

    2015-01-01

    Calotropis procera R. Br., a traditional medicinal plant in India, is a promising source of commercial proteases, because the cysteine proteases from the plant exhibit high thermo-stability, broad pH optima, and plasma-clotting activity. Though several proteases such as Procerain, Procerain B, CpCp-1, CpCp-2, and CpCp-3 have been isolated and characterized, the information of their transcripts is limited to cDNAs encoding their mature peptides. Due to this limitation, in this study, to determine the cDNA sequences encoding full open reading frame of these cysteine proteases, transcripts were sequenced with an Illumina Hiseq2000 sequencer. A total of 171,253,393 clean reads were assembled into 106,093 contigs with an average length of 1,614 bp and an N50 of 2,703 bp, and 70,797 contigs with an average length of 1,565 bp and N50 of 2,082 bp using Trinity and Velvet-Oases software, respectively. Among these contigs, we found 20 unigenes related to papain-like cysteine proteases by BLASTX analysis against a non-redundant NCBI protein database. Our expression analysis revealed that the cysteine protease contains an N-terminal pro-peptide domain (inhibitor region), which is necessary for correct folding and proteolytic activity. It was evident that expression yields using an inducible T7 expression system in Escherichia coli were considerably higher with the pro-peptide domain than without the domain, which could contribute to molecular cloning of the Calotropis procera protease as an active form with correct folding. PMID:25786229

  17. The value of using multiple proteases for large-scale mass spectrometry-based proteomics

    PubMed Central

    Swaney, Danielle L.; Wenger, Craig D.; Coon, Joshua J.

    2009-01-01

    Large-scale protein sequencing methods rely on enzymatic digestion of complex protein mixtures to generate a collection of peptides for mass spectrometric analysis. Here we examine the use of multiple proteases (trypsin, LysC, ArgC, AspN, and GluC) to improve both protein identification and characterization in the model organism Saccharomyces cerevisiae. Using a data-dependent, decision tree-based algorithm to tailor MS2 fragmentation method to peptide precursor, we identified 92,095 unique peptides (609,665 total) mapping to 3,908 proteins at a 1% false discovery rate (FDR). These results were a significant improvement upon data from a single protease digest (trypsin) – 27,822 unique peptides corresponding to 3,313 proteins. The additional 595 protein identifications were mainly from those at low abundances (i.e., < 1,000 copies/cell); sequence coverage for these proteins was likewise improved nearly 3-fold. We demonstrate that large portions of the proteome are simply inaccessible following digestion with a single protease and that multiple proteases, rather than technical replicates, provide a direct route to increase both protein identifications and proteome sequence coverage. PMID:20113005

  18. Value of using multiple proteases for large-scale mass spectrometry-based proteomics.

    PubMed

    Swaney, Danielle L; Wenger, Craig D; Coon, Joshua J

    2010-03-01

    Large-scale protein sequencing methods rely on enzymatic digestion of complex protein mixtures to generate a collection of peptides for mass spectrometric analysis. Here we examine the use of multiple proteases (trypsin, LysC, ArgC, AspN, and GluC) to improve both protein identification and characterization in the model organism Saccharomyces cerevisiae. Using a data-dependent, decision tree-based algorithm to tailor MS(2) fragmentation method to peptide precursor, we identified 92 095 unique peptides (609 665 total) mapping to 3908 proteins at a 1% false discovery rate (FDR). These results were a significant improvement upon data from a single protease digest (trypsin) - 27 822 unique peptides corresponding to 3313 proteins. The additional 595 protein identifications were mainly from those at low abundances (i.e., < 1000 copies/cell); sequence coverage for these proteins was likewise improved nearly 3-fold. We demonstrate that large portions of the proteome are simply inaccessible following digestion with a single protease and that multiple proteases, rather than technical replicates, provide a direct route to increase both protein identifications and proteome sequence coverage. PMID:20113005

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

    PubMed Central

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

    2016-01-01

    Background 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. Results 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. Conclusions 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. PMID:26982200

  20. StAR enhances transcription of genes encoding the mitochondrial proteases involved in its own degradation.

    PubMed

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

    2014-02-01

    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

  1. Four Amino Acid Changes in HIV-2 Protease Confer Class-Wide Sensitivity to Protease Inhibitors

    PubMed Central

    Smith, Robert A.; Gottlieb, Geoffrey S.

    2015-01-01

    ABSTRACT Protease is essential for retroviral replication, and protease inhibitors (PI) are important for treating HIV infection. HIV-2 exhibits intrinsic resistance to most FDA-approved HIV-1 PI, retaining clinically useful susceptibility only to lopinavir, darunavir, and saquinavir. The mechanisms for this resistance are unclear; although HIV-1 and HIV-2 proteases share just 38 to 49% sequence identity, all critical structural features of proteases are conserved. Structural studies have implicated four amino acids in the ligand-binding pocket (positions 32, 47, 76, and 82). We constructed HIV-2ROD9 molecular clones encoding the corresponding wild-type HIV-1 amino acids (I32V, V47I, M76L, and I82V) either individually or together (clone PRΔ4) and compared the phenotypic sensitivities (50% effective concentration [EC50]) of mutant and wild-type viruses to nine FDA-approved PI. Single amino acid replacements I32V, V47I, and M76L increased the susceptibility of HIV-2 to multiple PI, but no single change conferred class-wide sensitivity. In contrast, clone PRΔ4 showed PI susceptibility equivalent to or greater than that of HIV-1 for all PI. We also compared crystallographic structures of wild-type HIV-1 and HIV-2 proteases complexed with amprenavir and darunavir to models of the PRΔ4 enzyme. These models suggest that the amprenavir sensitivity of PRΔ4 is attributable to stabilizing enzyme-inhibitor interactions in the P2 and P2′ pockets of the protease dimer. Together, our results show that the combination of four amino acid changes in HIV-2 protease confer a pattern of PI susceptibility comparable to that of HIV-1, providing a structural rationale for intrinsic HIV-2 PI resistance and resolving long-standing questions regarding the determinants of differential PI susceptibility in HIV-1 and HIV-2. IMPORTANCE Proteases are essential for retroviral replication, and HIV-1 and HIV-2 proteases share a great deal of structural similarity. However, only three of nine

  2. High Throughput Substrate Phage Display for Protease Profiling

    PubMed Central

    Ratnikov, Boris; Cieplak, Piotr; Smith, Jeffrey W.

    2012-01-01

    Summary The interplay between a protease and its substrates is controlled at many different levels, including coexpression, colocalization, binding driven by ancillary contacts, and the presence of natural inhibitors. Here we focus on the most basic parameter that guides substrate recognition by a protease, the recognition specificity at the catalytic cleft. An understanding of this substrate specificity can be used to predict the putative substrates of a protease, to design protease activated imaging agents, and to initiate the design of active site inhibitors. Our group has characterized protease specificities of several matrix metalloproteinases using substrate phage display. Recently, we have adapted this method to a semiautomated platform that includes several high-throughput steps. The semiautomated platform allows one to obtain an order of magnitude more data, thus permitting precise comparisons among related proteases to define their functional distinctions. PMID:19377968

  3. High throughput substrate phage display for protease profiling.

    PubMed

    Ratnikov, Boris; Cieplak, Piotr; Smith, Jeffrey W

    2009-01-01

    The interplay between a protease and its substrates is controlled at many different levels, including coexpression, colocalization, binding driven by ancillary contacts, and the presence of natural inhibitors. Here we focus on the most basic parameter that guides substrate recognition by a protease, the recognition specificity at the catalytic cleft. An understanding of this substrate specificity can be used to predict the putative substrates of a protease, to design protease activated imaging agents, and to initiate the design of active site inhibitors. Our group has characterized protease specificities of several matrix metalloproteinases using substrate phage display. Recently, we have adapted this method to a semiautomated platform that includes several high-throughput steps. The semiautomated platform allows one to obtain an order of magnitude more data, thus permitting precise comparisons among related proteases to define their functional distinctions. PMID:19377968

  4. Protease susceptibility and toxicity of heat-labile enterotoxins with a mutation in the active site or in the protease-sensitive loop.

    PubMed Central

    Giannelli, V; Fontana, M R; Giuliani, M M; Guangcai, D; Rappuoli, R; Pizza, M

    1997-01-01

    To generate nontoxic derivatives of Escherichia coli heat-labile enterotoxin (LT), site-directed mutagenesis has been used to change either the amino acid residues located in the catalytic site (M. Pizza, M. Domenighini, W. Hol, V. Giannelli, M. R. Fontana, M. M. Giuliani, C. Magagnoli, S. Peppoloni, R. Manetti, and R. Rappuoli, Mol. Microbiol. 14:51-60, 1994) or those located in the proteolytically sensitive loop that joins the A1 and A2 moieties of the A subunit (C. C. R. Grant, R. J. Messer, and W. J. Cieplack, Infect. Immun. 62:4270-4278, 1994; B. L. Dickinson and J. D. Clements, Infect. Immun. 63:1617-1623, 1995). In this work, we compared the in vitro and in vivo toxic properties and the resistance to protease digestion of the prototype molecules obtained by both approaches (LT-K63 and LT-R192G, respectively). As expected, LT-K63 was normally processed by proteases, while LT-R192G showed increased resistance to trypsin in vitro and was digested by trypsin only under denaturing conditions (3.5 M urea) or by intestinal proteases. No toxicity was detected with the LT-K63 mutant, even when 40 micrograms and 1 mg were used in the in vitro and in vivo assays, respectively. In marked contrast, LT-R192G showed only a modest (10-fold) reduction in toxicity in Y1 cells with a delay in the appearance of the toxic activity and had toxicity comparable to that of wild-type LT in the rabbit ileal loop assay. We conclude that mutagenesis of the active site generates molecules that are fully devoid of toxicity, while mutagenesis of the A1-A2 loop generates molecules that are resistant to trypsin in vitro but still susceptible to proteolytic activation by proteases other than trypsin, and therefore they may still be toxic in tissue culture and in vivo. PMID:8975934

  5. Serum albumin as a probe for testing the selectivity of irreversible cysteine protease inhibitors: The case of vinyl sulfones.

    PubMed

    Regazzoni, Luca; Colombo, Simone; Mazzolari, Angelica; Vistoli, Giulio; Carini, Marina

    2016-05-30

    Vinyl sulfones are used for drug design of irreversible inhibitors of cysteine proteases since they are able to alkylate cysteine thiols inside the catalytic pocket of this class of enzymes. Some authors have reported the lack of reactivity towards glutathione as sufficient evidence of the selectivity of such a mechanism. Herein, we demonstrate that some simple molecules containing a vinyl sulfone moiety are not thiol-specific alkylants since they react with some albumin nucleophiles including side chains of Cys34 and His146. Such side-reactions are not desirable for any drug candidate since they limit serum stability, bioavailability and they possibly trigger toxicity mechanisms. In silico predictions, indicate that the compounds tested share similar structural features with reported inhibitors of cysteine proteases, as well as similar poses around the main albumin nucleophiles. Altogether, the data suggest that albumin is better than glutathione for the setup of early in vitro tests probing the selectivity of cysteine protease inhibitors. PMID:26970985

  6. Protein protease inhibitors in insects and comparison with mammalian inhibitors.

    PubMed

    Eguchi, M

    1993-01-01

    1. Studies on insect protein protease inhibitors are summarized. Biochemical, genetic and physiological investigations of the silkworm are performed. 2. In addition, the properties and characteristics of fungal protease inhibitors from the silkworm (Bombyx mori) are described and their importance as defensive functions is emphasized. 3. This review also concerns comparative and evolutionary studies of protease inhibitors from various sources. 4. The biological significance of inhibitors is discussed in view of the extensive experimental results. PMID:8365101

  7. Economic Methods of Ginger Protease'sextraction and Purification

    NASA Astrophysics Data System (ADS)

    Qiao, Yuanyuan; Tong, Junfeng; Wei, Siqing; Du, Xinyong; Tang, Xiaozhen

    This article reports the ginger protease extraction and purification methods from fresh ginger rhizome. As to ginger protease extraction, we adapt the steps of organic solvent dissolving, ammonium sulfate depositing and freeze-drying, and this method can attain crude enzyme powder 0.6% weight of fresh ginger rhizome. The purification part in this study includes two steps: cellulose ion exchange (DEAE-52) and SP-Sephadex 50 chromatography, which can purify crude ginger protease through ion and molecular weight differences respectively.

  8. HvPap-1 C1A protease actively participates in barley proteolysis mediated by abiotic stresses.

    PubMed

    Velasco-Arroyo, Blanca; Diaz-Mendoza, Mercedes; Gandullo, Jacinto; Gonzalez-Melendi, Pablo; Santamaria, M Estrella; Dominguez-Figueroa, Jose D; Hensel, Goetz; Martinez, Manuel; Kumlehn, Jochen; Diaz, Isabel

    2016-07-01

    Protein breakdown and mobilization from old or stressed tissues to growing and sink organs are some of the metabolic features associated with abiotic/biotic stresses, essential for nutrient recycling. The massive degradation of proteins implies numerous proteolytic events in which cysteine-proteases are the most abundant key players. Analysing the role of barley C1A proteases in response to abiotic stresses is crucial due to their impact on plant growth and grain yield and quality. In this study, dark and nitrogen starvation treatments were selected to induce stress in barley. Results show that C1A proteases participate in the proteolytic processes triggered in leaves by both abiotic treatments, which strongly induce the expression of the HvPap-1 gene encoding a cathepsin F-like protease. Differences in biochemical parameters and C1A gene expression were found when comparing transgenic barley plants overexpressing or silencing the HvPap-1 gene and wild-type dark-treated leaves. These findings associated with morphological changes evidence a lifespan-delayed phenotype of HvPap-1 silenced lines. All these data elucidate on the role of this protease family in response to abiotic stresses and the potential of their biotechnological manipulation to control the timing of plant growth. PMID:27217548

  9. Caspase-dependant activation of chymotrypsin-like proteases mediates nuclear events during Jurkat T cell apoptosis

    SciTech Connect

    O'Connell, A.R.; Lee, B.W.; Stenson-Cox, C. . E-mail: catherine.stenson@nuigalway.ie

    2006-06-30

    Apoptosis involves a cascade of biochemical and morphological changes resulting in the systematic disintegration of the cell. Caspases are central mediators of this process. Supporting and primary roles for serine proteases as pro-apoptotic mediators have also been highlighted. Evidence for such roles comes largely from the use of pharmacological inhibitors; as a consequence information regarding their apoptotic function and biochemical properties has been limited. Here, we circumvented limitations associated with traditional serine protease inhibitors through use of a fluorescently labelled inhibitor of serine proteases (FLISP) that allowed for analysis of the specificity, regulation and positioning of apoptotic serine proteases within a classical apoptotic cascade. We demonstrate that staurosporine triggers a caspase-dependant induction of chymotrypsin-like activity in the nucleus of apoptotic Jurkat T cells. We show that serine protease activity is required for the generation of late stage nuclear events including condensation, fragmentation and DNA degradation. Furthermore, we reveal caspase-dependant activation of two chymotrypsin-like protein species that we hypothesize mediate cell death-associated nuclear events.

  10. Cloning, expression, and sequencing of a protease gene (tpr) from Porphyromonas gingivalis W83 in Escherichia coli.

    PubMed Central

    Bourgeau, G; Lapointe, H; Péloquin, P; Mayrand, D

    1992-01-01

    Porphyromonas gingivalis is a highly proteolytic organism which metabolizes small peptides and amino acids. Indirect evidence suggests that the proteases produced by this microorganism constitute an important virulence factor. In this study, a gene bank of P. gingivalis W83 DNA was constructed by cloning 0.5- to 20-kb HindIII-cut DNA fragments into Escherichia coli DH5 alpha by using the plasmid vector pUC19. A clone expressing a protease from P. gingivalis was isolated on LB agar containing 1% skim milk. The clone contained a 3.0-kb insert that coded for a protease with an apparent molecular mass of 64 kDa. Sequencing part of the 3.0-kb DNA fragment revealed an open reading frame encoding a protein of 482 amino acids with a molecular mass of 62.5 kDa. Putative promoter and termination elements flanking the open reading frame were identified. The activity expressed in E. coli was extensively characterized by using various substrates and protease inhibitors, and the results suggest that it is possibly a thiol protease. Images PMID:1322368

  11. Uptake and Degradation of Protease-Sensitive and -Resistant Forms of Abnormal Human Prion Protein Aggregates by Human Astrocytes

    PubMed Central

    Choi, Young Pyo; Head, Mark W.; Ironside, James W.; Priola, Suzette A.

    2015-01-01

    Sporadic Creutzfeldt-Jakob disease is the most common of the human prion diseases, a group of rare, transmissible, and fatal neurologic diseases associated with the accumulation of an abnormal form (PrPSc) of the host prion protein. In sporadic Creutzfeldt-Jakob disease, disease-associated PrPSc is present not only as an aggregated, protease-resistant form but also as an aggregated protease-sensitive form (sPrPSc). Although evidence suggests that sPrPSc may play a role in prion pathogenesis, little is known about how it interacts with cells during prion infection. Here, we show that protease-sensitive abnormal PrP aggregates derived from patients with sporadic Creutzfeldt-Jakob disease are taken up and degraded by immortalized human astrocytes similarly to abnormal PrP aggregates that are resistant to proteases. Our data suggest that relative proteinase K resistance does not significantly influence the astrocyte's ability to degrade PrPSc. Furthermore, the cell does not appear to distinguish between sPrPSc and protease-resistant PrPSc, suggesting that sPrPSc could contribute to prion infection. PMID:25280631

  12. Production and characterization of thermostable alkaline protease of Bacillus subtilis (ATCC 6633) from optimized solid-state fermentation.

    PubMed

    Chatterjee, Joyee; Giri, Sudipta; Maity, Sujan; Sinha, Ankan; Ranjan, Ashish; Rajshekhar; Gupta, Suvroma

    2015-01-01

    Proteases are the most important group of enzymes utilized commercially in various arenas of industries, such as food, detergent, leather, dairy, pharmaceutical, diagnostics, and waste management, accounting for nearly 20% of the world enzyme market. Microorganisms of specially Bacillus genera serve as a vast repository of diverse set of industrially important enzymes and utilized for the large-scale enzyme production using a fermentation technology. Approximately 30%-40% of the cost of industrial enzymes originates from the cost of the growth medium. This study is attempted to produce protease from Bacillus subtilis (ATCC 6633) after optimization of various process parameters with the aid of solid-state fermentation using a cheap nutrient source such as wheat bran. B. subtilis (ATCC 6633) produces proteases of molecular weight 36 and 20 kDa, respectively, in the fermented medium as evident from SDS zymogram. Alkaline protease activity has been detected with optimum temperature at 50 °C and is insensitive to ethylenediaminetetraacetic acid. This thermostable alkaline protease exhibits dual pH optimum at 7 and 10 with moderate pH stability at alkaline pH range. It preserves its activity in the presence of detergent such as SDS, Tween 20, and Triton X-100 and may be considered as an effective additive to detergent formulation with some industrial importance. PMID:25323045

  13. Role of Protease-Inhibitors in Ocular Diseases.

    PubMed

    Pescosolido, Nicola; Barbato, Andrea; Pascarella, Antonia; Giannotti, Rossella; Genzano, Martina; Nebbioso, Marcella

    2014-01-01

    It has been demonstrated that the balance between proteases and protease-inhibitors system plays a key role in maintaining cellular and tissue homeostasis. Indeed, its alteration has been involved in many ocular and systemic diseases. In particular, research has focused on keratoconus, corneal wounds and ulcers, keratitis, endophthalmitis, age-related macular degeneration, Sorsby fundus dystrophy, loss of nerve cells and photoreceptors during optic neuritis both in vivo and in vitro models. Protease-inhibitors have been extensively studied, rather than proteases, because they may represent a therapeutic approach for some ocular diseases. The protease-inhibitors mainly involved in the onset of the above-mentioned ocular pathologies are: α2-macroglobulin, α1-proteinase inhibitor (α1-PI), metalloproteinase inhibitor (TIMP), maspin, SERPINA3K, SERPINB13, secretory leukocyte protease inhibitor (SLPI), and calpeptin. This review is focused on the several characteristics of dysregulation of this system and, particularly, on a possible role of proteases and protease-inhibitors in molecular remodeling that may lead to some ocular diseases. Recently, researchers have even hypothesized a possible therapeutic effect of the protease-inhibitors in the treatment of injured eye in animal models. PMID:25493637

  14. 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. PMID:17630120

  15. 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. PMID:25182493

  16. 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

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

    SciTech Connect

    Fyfe, Cameron D.; Grinter, Rhys; Josts, Inokentijs; Mosbahi, Khedidja; Roszak, Aleksander W.; Cogdell, Richard J.; Wall, Daniel M.; Burchmore, Richard J. S.; Byron, Olwyn; Walker, Daniel

    2015-06-30

    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. coli 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.

  18. Structural and functional analysis of human HtrA3 protease and its subdomains

    SciTech Connect

    Glaza, Przemyslaw; Osipiuk, Jerzy; Wenta, Tomasz; Zurawa-Janicka, Dorota; Jarzab, Miroslaw; Lesner, Adam; Banecki, Bogdan; Skorko-Glonek, Joanna; Joachimiak, Andrzej; Lipinska, Barbara; van Raaij, Mark J.

    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 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.

  19. Structural and functional analysis of human HtrA3 protease and its subdomains

    DOE PAGESBeta

    Glaza, Przemyslaw; Osipiuk, Jerzy; Wenta, Tomasz; Zurawa-Janicka, Dorota; Jarzab, Miroslaw; Lesner, Adam; Banecki, Bogdan; Skorko-Glonek, Joanna; Joachimiak, Andrzej; Lipinska, Barbara; et al

    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

  20. Origin and Diversification of Meprin Proteases.

    PubMed

    Marín, Ignacio

    2015-01-01

    Meprins are astacin metalloproteases with a characteristic, easily recognizable structure, given that they are the only proteases with both MAM and MATH domains plus a transmembrane region. So far assumed to be vertebrate-specific, it is shown here, using a combination of evolutionary and genomic analyses, that meprins originated before the urochordates/vertebrates split. In particular, three genes encoding structurally typical meprin proteins are arranged in tandem in the genome of the urochordate Ciona intestinalis. Phylogenetic analyses showed that the protease and MATH domains present in the meprin-like proteins encoded by the Ciona genes are very similar in sequence to the domains found in vertebrate meprins, which supports them having a common origin. While many vertebrates have the two canonical meprin-encoding genes orthologous to human MEP1A and MEP1B (which respectively encode for the proteins known as meprin α and meprin β), a single gene has been found so far in the genome of the chondrichthyan fish Callorhinchus milii, and additional meprin-encoding genes are present in some species. Particularly, a group of bony fish species have genes encoding highly divergent meprins, here named meprin-F. Genes encoding meprin-F proteins, derived from MEP1B genes, are abundant in some species, as the Amazon molly, Poecilia formosa, which has 7 of them. Finally, it is confirmed that the MATH domains of meprins are very similar to the ones in TRAF ubiquitin ligases, which suggests that meprins originated when protease and TRAF E3-encoding sequences were combined. PMID:26288188

  1. Reversible Unfolding of Rhomboid Intramembrane Proteases.

    PubMed

    Panigrahi, Rashmi; Arutyunova, Elena; Panwar, Pankaj; Gimpl, Katharina; Keller, Sandro; Lemieux, M Joanne

    2016-03-29

    Denaturant-induced unfolding of helical membrane proteins provides insights into their mechanism of folding and domain organization, which take place in the chemically heterogeneous, anisotropic environment of a lipid membrane. Rhomboid proteases are intramembrane proteases that play key roles in various diseases. Crystal structures have revealed a compact helical bundle with a buried active site, which requires conformational changes for the cleavage of transmembrane substrates. A dimeric form of the rhomboid protease has been shown to be important for activity. In this study, we examine the mechanism of refolding for two distinct rhomboids to gain insight into their secondary structure-activity relationships. Although helicity is largely abolished in the unfolded states of both proteins, unfolding is completely reversible for HiGlpG but only partially reversible for PsAarA. Refolding of both proteins results in reassociation of the dimer, with a 90% regain of catalytic activity for HiGlpG but only a 70% regain for PsAarA. For both proteins, a broad, gradual transition from the native, folded state to the denatured, partly unfolded state was revealed with the aid of circular dichroism spectroscopy as a function of denaturant concentration, thus arguing against a classical two-state model as found for many globular soluble proteins. Thermal denaturation has irreversible destabilizing effects on both proteins, yet reveals important functional details regarding substrate accessibility to the buried active site. This concerted biophysical and functional analysis demonstrates that HiGlpG, with a simple six-transmembrane-segment organization, is more robust than PsAarA, which has seven predicted transmembrane segments, thus rendering HiGlpG amenable to in vitro studies of membrane-protein folding. PMID:27028647

  2. Origin and Diversification of Meprin Proteases

    PubMed Central

    Marín, Ignacio

    2015-01-01

    Meprins are astacin metalloproteases with a characteristic, easily recognizable structure, given that they are the only proteases with both MAM and MATH domains plus a transmembrane region. So far assumed to be vertebrate-specific, it is shown here, using a combination of evolutionary and genomic analyses, that meprins originated before the urochordates/vertebrates split. In particular, three genes encoding structurally typical meprin proteins are arranged in tandem in the genome of the urochordate Ciona intestinalis. Phylogenetic analyses showed that the protease and MATH domains present in the meprin-like proteins encoded by the Ciona genes are very similar in sequence to the domains found in vertebrate meprins, which supports them having a common origin. While many vertebrates have the two canonical meprin-encoding genes orthologous to human MEP1A and MEP1B (which respectively encode for the proteins known as meprin α and meprin β), a single gene has been found so far in the genome of the chondrichthyan fish Callorhinchus milii, and additional meprin-encoding genes are present in some species. Particularly, a group of bony fish species have genes encoding highly divergent meprins, here named meprin-F. Genes encoding meprin-F proteins, derived from MEP1B genes, are abundant in some species, as the Amazon molly, Poecilia formosa, which has 7 of them. Finally, it is confirmed that the MATH domains of meprins are very similar to the ones in TRAF ubiquitin ligases, which suggests that meprins originated when protease and TRAF E3-encoding sequences were combined. PMID:26288188

  3. Burden of Diabetes and First Evidence for the Utility of HbA1c for Diagnosis and Detection of Diabetes in Urban Black South Africans: The Durban Diabetes Study

    PubMed Central

    Hird, Thomas R.; Pirie, Fraser J.; Esterhuizen, Tonya M.; O’Leary, Brian; McCarthy, Mark I.; Young, Elizabeth H.; Sandhu, Manjinder S.; Motala, Ayesha A.

    2016-01-01

    Objective Glycated haemoglobin (HbA1c) is recommended as an additional tool to glucose-based measures (fasting plasma glucose [FPG] and 2-hour plasma glucose [2PG] during oral glucose tolerance test [OGTT]) for the diagnosis of diabetes; however, its use in sub-Saharan African populations is not established. We assessed prevalence estimates and the diagnosis and detection of diabetes based on OGTT, FPG, and HbA1c in an urban black South African population. Research Design and Methods We conducted a population-based cross-sectional survey using multistage cluster sampling of adults aged ≥18 years in Durban (eThekwini municipality), KwaZulu-Natal. All participants had a 75-g OGTT and HbA1c measurements. Receiver operating characteristic (ROC) analysis was used to assess the overall diagnostic accuracy of HbA1c, using OGTT as the reference, and to determine optimal HbA1c cut-offs. Results Among 1190 participants (851 women, 92.6% response rate), the age-standardised prevalence of diabetes was 12.9% based on OGTT, 11.9% based on FPG, and 13.1% based on HbA1c. In participants without a previous history of diabetes (n = 1077), using OGTT as the reference, an HbA1c ≥48 mmol/mol (6.5%) detected diabetes with 70.3% sensitivity (95%CI 52.7–87.8) and 98.7% specificity (95%CI 97.9–99.4) (AUC 0.94 [95%CI 0.89–1.00]). Additional analyses suggested the optimal HbA1c cut-off for detection of diabetes in this population was 42 mmol/mol (6.0%) (sensitivity 89.2% [95%CI 78.6–99.8], specificity 92.0% [95%CI: 90.3–93.7]). Conclusions In an urban black South African population, we found a high prevalence of diabetes and provide the first evidence for the utility of HbA1c for the diagnosis and detection of diabetes in black Africans in sub-Saharan Africa. PMID:27560687

  4. Effects of exogenous proteases without or with carbohydrases on nutrient digestibility and disappearance of non-starch polysaccharides in broiler chickens.

    PubMed

    Olukosi, O A; Beeson, L A; Englyst, K; Romero, L F

    2015-11-01

    The objective of the current study was to evaluate the effect of a subtilisin protease, without or with inclusion of carbohydrases, on digestibility and retention of energy and protein, as well as the solubilization and disappearance of non-starch polysaccharides (NSP) from corn-soybean meal based diets fed to broiler chickens. Two hundred eighty-eight Ross 308 male broiler chickens were used for the experiment. On d 14, the birds were weighed and allocated to 6 treatments and 8 replicates per treatment with 6 birds per replicate. Treatments were: 1) corn-soybean meal based control diet; 2) control diet plus supplemental protease at 5,000 (P5000) protease units (PU)/kg); 3) control plus 10,000 PU/kg protease (P10000); or control plus an enzyme combination containing xylanase, amylase, and protease (XAP) added to achieve protease activity of: 4) 2,500 PU/kg (XAP2500); 5) 5,000 PU/kg (XAP5000); or 6) 10,000 PU/kg (XAP10000). The enzymes in XAP were combined at fixed ratios of 10:1:25 of xylanase:amylase:protease. Data were analyzed by ANOVA and specific orthogonal contrasts between treatments were performed. Addition of xylanase and amylase increased (P < 0.05) the ileal digestibility of protein by 4.2% and 2.1% at XAP5000 and XAP10000, respectively (relative to P5000 and P10000, respectively), exhibiting a plateau after the XAP5000 dose. Increment (P < 0.05) in AME due to protease was evident, particularly in P10000. At the ileal level, XAP reduced (P < 0.05) the flow of insoluble xylose and arabinose, which indicates an increase in the solubilization of arabinoxylan polymers in the small intestine. Protease on its own reduced (P < 0.05) the flow of insoluble arabinose but did not affect the flow of insoluble xylose. XAP reduced (P < 0.05) the pre-cecal flow of insoluble and total glucose and galactose. It was concluded that whereas protease by itself improved nutrient utilization and increased solubilization of NSP components, at the lower dose, a

  5. Effects of exogenous proteases without or with carbohydrases on nutrient digestibility and disappearance of non-starch polysaccharides in broiler chickens

    PubMed Central

    Olukosi, O. A.; Beeson, L. A.; Englyst, K.; Romero, L. F.

    2015-01-01

    The objective of the current study was to evaluate the effect of a subtilisin protease, without or with inclusion of carbohydrases, on digestibility and retention of energy and protein, as well as the solubilization and disappearance of non-starch polysaccharides (NSP) from corn-soybean meal based diets fed to broiler chickens. Two hundred eighty-eight Ross 308 male broiler chickens were used for the experiment. On d 14, the birds were weighed and allocated to 6 treatments and 8 replicates per treatment with 6 birds per replicate. Treatments were: 1) corn-soybean meal based control diet; 2) control diet plus supplemental protease at 5,000 (P5000) protease units (PU)/kg); 3) control plus 10,000 PU/kg protease (P10000); or control plus an enzyme combination containing xylanase, amylase, and protease (XAP) added to achieve protease activity of: 4) 2,500 PU/kg (XAP2500); 5) 5,000 PU/kg (XAP5000); or 6) 10,000 PU/kg (XAP10000). The enzymes in XAP were combined at fixed ratios of 10:1:25 of xylanase:amylase:protease. Data were analyzed by ANOVA and specific orthogonal contrasts between treatments were performed. Addition of xylanase and amylase increased (P < 0.05) the ileal digestibility of protein by 4.2% and 2.1% at XAP5000 and XAP10000, respectively (relative to P5000 and P10000, respectively), exhibiting a plateau after the XAP5000 dose. Increment (P < 0.05) in AME due to protease was evident, particularly in P10000. At the ileal level, XAP reduced (P < 0.05) the flow of insoluble xylose and arabinose, which indicates an increase in the solubilization of arabinoxylan polymers in the small intestine. Protease on its own reduced (P < 0.05) the flow of insoluble arabinose but did not affect the flow of insoluble xylose. XAP reduced (P < 0.05) the pre-cecal flow of insoluble and total glucose and galactose. It was concluded that whereas protease by itself improved nutrient utilization and increased solubilization of NSP components, at the lower dose, a

  6. Sequence conservation, phylogenetic relationships, and expression profiles of nondigestive serine proteases and serine protease homologs in Manduca sexta.

    PubMed

    Cao, Xiaolong; He, Yan; Hu, Yingxia; Zhang, Xiufeng; Wang, Yang; Zou, Zhen; Chen, Yunru; Blissard, Gary W; Kanost, Michael R; Jiang, Haobo

    2015-07-01

    Serine protease (SP) and serine protease homolog (SPH) genes in insects encode a large family of proteins involved in digestion, development, immunity, and other processes. While 68 digestive SPs and their close homologs are reported in a companion paper (Kuwar et al., in preparation), we have identified 125 other SPs/SPHs in Manduca sexta and studied their structure, evolution, and expression. Fifty-two of them contain cystine-stabilized structures for molecular recognition, including clip, LDLa, Sushi, Wonton, TSP, CUB, Frizzle, and SR domains. There are nineteen groups of genes evolved from relatively recent gene duplication and sequence divergence. Thirty-five SPs and seven SPHs contain 1, 2 or 5 clip domains. Multiple sequence alignment and molecular modeling of the 54 clip domains have revealed structural diversity of these regulatory modules. Sequence comparison with their homologs in Drosophila melanogaster, Anopheles gambiae and Tribolium castaneum allows us to classify them into five subfamilies: A are SPHs with 1 or 5 group-3 clip domains, B are SPs with 1 or 2 group-2 clip domains, C, D1 and D2 are SPs with a single clip domain in group-1a, 1b and 1c, respectively. We have classified into six categories the 125 expression profiles of SP-related proteins in fat body, brain, midgut, Malpighian tubule, testis, and ovary at different stages, suggesting that they participate in various physiological processes. Through RNA-Seq-based gene annotation and expression profiling, as well as intragenomic sequence comparisons, we have established a framework of information for future biochemical research of nondigestive SPs and SPHs in this model species. PMID:25530503

  7. A NOVEL APPROACH TO REGULATE NITROGEN MINERALIZATION USING PROTEASE INHIBITORS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mineralization of organic N sources by extracellular proteases affects both the availability of inorganic N to plants and losses of N to the environment. We hypothesized that (i) application of purified protease inhibitors would slow down soil N mineralization, and (ii) elevated concentrations of pr...

  8. Effect of proteases on the. beta. -thromboglobulin radioimmunoassay

    SciTech Connect

    Donlon, J.A.; Helgeson, E.A.; Donlon, M.A.

    1985-02-11

    Rat peritoneal mast cells and mast cell granules were evaluated by radioimmunoassay for the presence of ..beta..-thromboglobulin and platelet factor 4. The initial assays indicated that a ..beta..-thromboglobulin cross reacting material was released from mast cells by compound 48/80 in a similar dose-dependent manner as histamine release. The material was also found to be associated with purified granules. However, the use of protease inhibitors in the buffers completely abolished the positive assays. Further evaluation of the effects of various proteases on the ..beta..-thromboglobulin assay indicated that elastase would also generate a false positive assay which could then be neutralized by the use of ..cap alpha../sub 1/-antitrypsin as a protease inhibitor. There was no protease effect on the platelet factor 4 radioimmunoassay which always showed no detectable amounts with mast cells, granules or proteases. These results clearly indicate the artifactual positive assays which can arise when using certain radioimmunoassay tests in the presence of cell proteases. The use of protease inhibitors is a necessary control when applying a radioimmunoassay to a system with potentially active proteases. 24 references, 2 figures, 4 tables.

  9. Broad-Spectrum Allosteric Inhibition of Herpesvirus Proteases

    PubMed Central

    2015-01-01

    Herpesviruses rely on a homodimeric protease for viral capsid maturation. A small molecule, DD2, previously shown to disrupt dimerization of Kaposi’s sarcoma-associated herpesvirus protease (KSHV Pr) by trapping an inactive monomeric conformation and two analogues generated through carboxylate bioisosteric replacement (compounds 2 and 3) were shown to inhibit the associated proteases of all three human herpesvirus (HHV) subfamilies (α, β, and γ). Inhibition data reveal that compound 2 has potency comparable to or better than that of DD2 against the tested proteases. Nuclear magnetic resonance spectroscopy and a new application of the kinetic analysis developed by Zhang and Poorman [Zhang, Z. Y., Poorman, R. A., et al. (1991) J. Biol. Chem. 266, 15591–15594] show DD2, compound 2, and compound 3 inhibit HHV proteases by dimer disruption. All three compounds bind the dimer interface of other HHV proteases in a manner analogous to binding of DD2 to KSHV protease. The determination and analysis of cocrystal structures of both analogues with the KSHV Pr monomer verify and elaborate on the mode of binding for this chemical scaffold, explaining a newly observed critical structure–activity relationship. These results reveal a prototypical chemical scaffold for broad-spectrum allosteric inhibition of human herpesvirus proteases and an approach for the identification of small molecules that allosterically regulate protein activity by targeting protein–protein interactions. PMID:24977643

  10. Expression and characterization of Coprothermobacter proteolyticus alkaline serine protease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  11. Cathepsin Protease Inhibition Reduces Endometriosis Lesion Establishment.

    PubMed

    Porter, Kristi M; Wieser, Friedrich A; Wilder, Catera L; Sidell, Neil; Platt, Manu O

    2016-05-01

    Endometriosis is a gynecologic disease characterized by the ectopic presence of endometrial tissue on organs within the peritoneal cavity, causing debilitating abdominal pain and infertility. Current treatments alleviate moderate pain symptoms associated with the disorder but exhibit limited ability to prevent new or recurring lesion establishment and growth. Retrograde menstruation has been implicated for introducing endometrial tissue into the peritoneal cavity, but molecular mechanisms underlying attachment and invasion are not fully understood. We hypothesize that cysteine cathepsins, a group of powerful extracellular matrix proteases, facilitate endometrial tissue invasion and endometriosis lesion establishment in the peritoneal wall and inhibiting this activity would decrease endometriosis lesion implantation. To test this, we used an immunocompetent endometriosis mouse model and found that endometriotic lesions exhibited a greater than 5-fold increase in active cathepsins compared to tissue from peritoneal wall or eutopic endometrium, with cathepsins L and K specifically implicated. Human endometriosis lesions also exhibited greater cathepsin activity than adjacent peritoneum tissue, supporting the mouse results. Finally, we tested the hypothesis that inhibiting cathepsin activity could block endometriosis lesion attachment and implantation in vivo. Intraperitoneal injection of the broad cysteine cathepsin inhibitor, E-64, significantly reduced the number of attached endometriosis lesions in our murine model compared to vehicle-treated controls demonstrating that cathepsin proteases contribute to endometriosis lesion establishment, and their inhibition may provide a novel, nonhormonal therapy for endometriosis. PMID:26482207

  12. Allosteric antibody inhibition of human hepsin protease.

    PubMed

    Koschubs, Tobias; Dengl, Stefan; Dürr, Harald; Kaluza, Klaus; Georges, Guy; Hartl, Christiane; Jennewein, Stefan; Lanzendörfer, Martin; Auer, Johannes; Stern, Alvin; Huang, Kuo-Sen; Packman, Kathryn; Gubler, Ueli; Kostrewa, Dirk; Ries, Stefan; Hansen, Silke; Kohnert, Ulrich; Cramer, Patrick; Mundigl, Olaf

    2012-03-15

    Hepsin is a type II transmembrane serine protease that is expressed in several human tissues. Overexpression of hepsin has been found to correlate with tumour progression and metastasis, which is so far best studied for prostate cancer, where more than 90% of such tumours show this characteristic. To enable improved future patient treatment, we have developed a monoclonal humanized antibody that selectively inhibits human hepsin and does not inhibit other related proteases. We found that our antibody, hH35, potently inhibits hepsin enzymatic activity at nanomolar concentrations. Kinetic characterization revealed non-linear, slow, tight-binding inhibition. This correlates with the crystal structure we obtained for the human hepsin-hH35 antibody Fab fragment complex, which showed that the antibody binds hepsin around α3-helix, located far from the active centre. The unique allosteric mode of inhibition of hH35 is distinct from the recently described HGFA (hepatocyte growth factor activator) allosteric antibody inhibition. We further explain how a small change in the antibody design induces dramatic structural rearrangements in the hepsin antigen upon binding, leading to complete enzyme inactivation. PMID:22132769

  13. Viral proteases as targets for drug design.

    PubMed

    Skoreński, Marcin; Sieńczyk, Marcin

    2013-01-01

    In order to productively infect a host, viruses must enter the cell and force host cell replication mechanisms to produce new infectious virus particles. The success of this process unfortunately results in disease progression and, in the case of infection with many viral species, may cause mortality. The discoveries of Louis Pasteur and Edward Jenner led to one of the greatest advances in modern medicine - the development of vaccines that generate long-lasting memory immune responses to combat viral infection. Widespread use of vaccines has reduced mortality and morbidity associated with viral infection and, in some cases, has completely eradicated virus from the human population. Unfortunately, several viral species maintain a significant ability to mutate and "escape" vaccine-induced immune responses. Thus, novel anti-viral agents are required for treatment and prevention of viral disease. Targeting proteases that are crucial in the viral life cycle has proven to be an effective method to control viral infection, and this avenue of investigation continues to generate anti-viral treatments. Herein, we provide the reader with a brief history as well as a comprehensive review of the most recent advances in the design and synthesis of viral protease inhibitors. PMID:23016690

  14. Purification and characterization of an alkaline protease from Acetes chinensis

    NASA Astrophysics Data System (ADS)

    Xu, Jiachao; Liu, Xin; Li, Zhaojie; Xu, Jie; Xue, Changhu; Gao, Xin

    2005-07-01

    An alkaline protease from Acetes chinensis was purified and characterized in this study. The steps of purification include ammonium sulfate precipitation, ion-exchange chromatography with Q-sepharose Fast Flow, gel filtration chromatography with S300 and the second ion-exchange chromatography with Q-sepharose Fast Flow. The protease was isolated and purified, which was present and active on protein substrates (azocasein and casein). The specific protease activity was 17.15 folds and the recovery was 4.67. The molecular weight of the protease was estimated at 23.2 kD by SDS-PAGE. With azocasein as the susbstrate, the optimal temperature was 55°C and the optimal pH value was 5.5. Ion Ca2+ could enhance the proteolytic activity of the protease, while Cu2+, EDTA and PMSF could inhibit its activity.

  15. Phosphoramidates as novel activity-based probes for serine proteases.

    PubMed

    Haedke, Ute R; Frommel, Sandra C; Hansen, Fabian; Hahne, Hannes; Kuster, Bernhard; Bogyo, Matthew; Verhelst, Steven H L

    2014-05-26

    Activity-based probes (ABPs) are small molecules that exclusively form covalent bonds with catalytically active enzymes. In the last decade, they have especially been used in functional proteomics studies of proteases. Here, we present phosphoramidate peptides as a novel type of ABP for serine proteases. These molecules can be made in a straightforward manner by standard Fmoc-based solid-phase peptide synthesis, allowing rapid diversification. The resulting ABPs covalently bind different serine proteases, depending on the amino acid recognition element adjacent to the reactive group. A reporter tag enables downstream gel-based analysis or LC-MS/MS-mediated identification of the targeted proteases. Overall, we believe that these readily accessible probes will provide new avenues for the functional study of serine proteases in complex proteomes. PMID:24817682

  16. Fluorous-based Peptide Microarrays for Protease Screening

    PubMed Central

    Collet, Beatrice Y. M.; Nagashima, Tadamichi; Yu, Marvin S.; Pohl, Nicola L. B.

    2009-01-01

    As ever more protease sequences are uncovered through genome sequencing projects, efficient parallel methods to discover the potential substrates of these proteases becomes crucial. Herein we describe the first use of fluorous-based microarrays to probe peptide sequences and begin to define the scope and limitations of fluorous microarray technologies for the screening of proteases. Comparison of a series of serine proteases showed that their ability to cleave peptide substrates in solution was maintained upon immobilization of these substrates onto fluorous-coated glass slides. The fluorous surface did not serve to significantly inactivate the enzymes. However, addition of hydrophilic components to the peptide sequences could induce lower rates of substrate cleavage with enzymes such as chymotrypsin with affinities to hydrophobic moieties. This work represents the first step to creating robust protease screening platforms using noncovalent microarray interface that can easily incorporate a range of compounds on the same slide. PMID:20161483

  17. Alkaline protease production by a strain of marine yeasts

    NASA Astrophysics Data System (ADS)

    Ping, Wang; Zhenming, Chi; Chunling, Ma

    2006-07-01

    Yeast strain 10 with high yield of protease was isolated from sediments of saltern near Qingdao, China. The protease had the highest activity at pH 9.0 and 45°C. The optimal medium for the maximum alkaline protease production of strain 10 was 2.5g soluble starch and 2.0g NaNO3 in 100mL seawater with initial pH 6.0. The optimal cultivation conditions for the maximum protease production were temperature 24.5°C, aeration rate 8.0L min-1 and agitation speed 150r min-1 Under the optimal conditions, 623.1 U mg-1 protein of alkaline protease was reached in the culture within 30h of fermentation.

  18. Positive selection of digestive Cys proteases in herbivorous Coleoptera.

    PubMed

    Vorster, Juan; Rasoolizadeh, Asieh; Goulet, Marie-Claire; Cloutier, Conrad; Sainsbury, Frank; Michaud, Dominique

    2015-10-01

    Positive selection is thought to contribute to the functional diversification of insect-inducible protease inhibitors in plants in response to selective pressures exerted by the digestive proteases of their herbivorous enemies. Here we assessed whether a reciprocal evolutionary process takes place on the insect side, and whether ingestion of a positively selected plant inhibitor may translate into a measurable rebalancing of midgut proteases in vivo. Midgut Cys proteases of herbivorous Coleoptera, including the major pest Colorado potato beetle (Leptinotarsa decemlineata), were first compared using a codon-based evolutionary model to look for the occurrence of hypervariable, positively selected amino acid sites among the tested sequences. Hypervariable sites were found, distributed within -or close to- amino acid regions interacting with Cys-type inhibitors of the plant cystatin protein family. A close examination of L. decemlineata sequences indicated a link between their assignment to protease functional families and amino acid identity at positively selected sites. A function-diversifying role for positive selection was further suggested empirically by in vitro protease assays and a shotgun proteomic analysis of L. decemlineata Cys proteases showing a differential rebalancing of protease functional family complements in larvae fed single variants of a model cystatin mutated at positively selected amino acid sites. These data confirm overall the occurrence of hypervariable, positively selected amino acid sites in herbivorous Coleoptera digestive Cys proteases. They also support the idea of an adaptive role for positive selection, useful to generate functionally diverse proteases in insect herbivores ingesting functionally diverse, rapidly evolving dietary cystatins. PMID:26264818

  19. A symmetric inhibitor binds HIV-1 protease asymmetrically.

    PubMed

    Dreyer, G B; Boehm, J C; Chenera, B; DesJarlais, R L; Hassell, A M; Meek, T D; Tomaszek, T A; Lewis, M

    1993-01-26

    Potential advantages of C2-symmetric inhibitors designed for the symmetric HIV-1 protease include high selectivity, potency, stability, and bioavailability. Pseudo-C2-symmetric monools and C2-symmetric diols, containing central hydroxymethylene and (R,R)-dihydroxyethylene moieties flanked by a variety of hydrophobic P1/P1' side chains, were studied as HIV-1 protease inhibitors. The monools and diols were synthesized in 8-10 steps from D-(+)-arabitol and D-(+)-mannitol, respectively. Monools with ethyl or isobutyl P1/P1' side chains were weak inhibitors of recombinant HIV-1 protease (Ki > 10 microM), while benzyl P1/P1' side chains afforded a moderately potent inhibitor (apparent Ki = 230 nM). Diols were 100-10,000x more potent than analogous monools, and a wider range of P1/P1' side chains led to potent inhibition. Both classes of compounds exhibited lower apparent Ki values under high-salt conditions. Surprisingly, monool and diol HIV-1 protease inhibitors were potent inhibitors of porcine pepsin, a prototypical asymmetric monomeric aspartic protease. These results were evaluated in the context of the pseudosymmetric structure of monomeric aspartic proteases and their evolutionary kinship with the retroviral proteases. The X-ray crystal structure of HIV-1 protease complexed with a symmetric diol was determined at 2.6 A. Contrary to expectations, the diol binds the protease asymmetrically and exhibits 2-fold disorder in the electron density map. Molecular dynamics simulations were conducted beginning with asymmetric and symmetric HIV-1 protease/inhibitor model complexes. A more stable trajectory resulted from the asymmetric complex, in agreement with the observed asymmetric binding mode. A simple four-point model was used to argue more generally that van der Waals and electrostatic force fields can commonly lead to an asymmetric association between symmetric molecules. PMID:8422397

  20. Mechanism of 17α,20-Lyase and New Hydroxylation Reactions of Human Cytochrome P450 17A1: 18O LABELING AND OXYGEN SURROGATE EVIDENCE FOR A ROLE OF A PERFERRYL OXYGEN.

    PubMed

    Yoshimoto, Francis K; Gonzalez, Eric; Auchus, Richard J; Guengerich, F Peter

    2016-08-12

    Cytochrome P450 (P450) reactions can involve C-C bond cleavage, and several of these are critical in steroid and sterol biosynthesis. The mechanisms of P450s 11A1, 17A1, 19A1, and 51A1 have been controversial, in the context of the role of ferric peroxide (FeO2 (-)) versus perferryl (FeO(3+), compound I) chemistry. We reinvestigated the 17α-hydroxyprogesterone and 17α-hydroxypregnenolone 17α,20-lyase reactions of human P450 17A1 and found incorporation of one (18)O atom (from (18)O2) into acetic acid, consonant with proposals for a ferric peroxide mechanism (Akhtar, M., Lee-Robichaud, P., Akhtar, M. E., and Wright, J. N. (1997) J. Steroid Biochem. Mol. Biol. 61, 127-132; Akhtar, M., Wright, J. N., and Lee-Robichaud, P. (2011) J. Steroid Biochem. Mol. Biol. 125, 2-12). However, the reactions were supported by iodosylbenzene (a precursor of the FeO(3+) species) but not by H2O2 We propose three mechanisms that can involve the FeO(3+) entity and that explain the (18)O label in the acetic acid, two involving the intermediacy of an acetyl radical and one a steroid 17,20-dioxetane. P450 17A1 was found to perform 16-hydroxylation reactions on its 17α-hydroxylated products to yield 16,17α-dihydroxypregnenolone and progesterone, suggesting the presence of an active perferryloxo active species of P450 17A1 when its lyase substrate is bound. The 6β-hydroxylation of 16α,17α-dihydroxyprogesterone and the oxidation of both 16α,17α-dihydroxyprogesterone and 16α,17α-dihydroxypregnenolone to 16-hydroxy lyase products were also observed. We provide evidence for the contribution of a compound I mechanism, although contribution of a ferric peroxide pathway in the 17α,20-lyase reaction cannot be excluded. PMID:27339894

  1. Force generation and protease gene expression in organotypic co-cultures of fibroblasts and keratinocytes.

    PubMed

    Wall, Ivan B; Bhadal, Navneet; Broad, Simon; Whawell, Simon A; Mudera, Vivek; Lewis, Mark P

    2009-12-01

    Fibroblast-epithelium interactions are crucial for successful tissue engineering of skin and oral mucosal equivalents. In this study, we assessed early force generation in organotypic fibroblast-epithelium co-cultures, using normal human keratinocytes (NHK) and HPV16-transformed (UP) cells. During the initial 2 h period, organotypic co-cultures containing both epithelial cell types produced significantly more force than fibroblasts alone (p < 0.05). After 2 h, the epithelial contribution became diminished and did not significantly contribute to intrinsic force generation by fibroblasts, and no differences were observed when using UP vs. NHK. We then measured protease gene expression at the end of the experimental period. Distinct differences were evident in protease expression both between NHK-human skin fibroblast (HSF) vs. UP-HSF co-cultures and compared to fibroblasts alone. We conclude that whilst the very early contractile response of fibroblasts is enhanced by the overlying epithelium, this becomes diminished as the fibroblast response becomes predominant and it does contribute to tissue remodelling via regulation of protease expression. PMID:19701934

  2. Post-Translational Regulation via Clp Protease Is Critical for Survival of Mycobacterium tuberculosis

    PubMed Central

    Raju, Ravikiran M.; Jedrychowski, Mark P.; Wei, Jun-Rong; Pinkham, Jessica T.; Park, Annie S.; O'Brien, Kathryn; Rehren, German; Schnappinger, Dirk; Gygi, Steven P.; Rubin, Eric J.

    2014-01-01

    Unlike most bacterial species, Mycobacterium tuberculosis depends on the Clp proteolysis system for survival even in in vitro conditions. We hypothesized that Clp is required for the physiologic turnover of mycobacterial proteins whose accumulation is deleterious to bacterial growth and survival. To identify cellular substrates, we employed quantitative proteomics and transcriptomics to identify the set of proteins that accumulated upon the loss of functional Clp protease. Among the set of potential Clp substrates uncovered, we were able to unambiguously identify WhiB1, an essential transcriptional repressor capable of auto-repression, as a substrate of the mycobacterial Clp protease. Dysregulation of WhiB1 turnover had a toxic effect that was not rescued by repression of whiB1 transcription. Thus, under normal growth conditions, Clp protease is the predominant regulatory check on the levels of potentially toxic cellular proteins. Our findings add to the growing evidence of how post-translational regulation plays a critical role in the regulation of bacterial physiology. PMID:24603869

  3. α1-Antichymotrypsin inactivates staphylococcal cysteine protease in cross-class inhibition.

    PubMed

    Wladyka, Benedykt; Kozik, Agata J; Bukowski, Michal; Rojowska, Anna; Kantyka, Tomasz; Dubin, Grzegorz; Dubin, Adam

    2011-05-01

    Staphylococcal cysteine proteases are implicated as virulence factors in human and avian infections. Human strains of Staphylococcus aureus secrete two cysteine proteases (staphopains A and B), whereas avian strains express staphopain C (ScpA2), which is distinct from both human homologues. Here, we describe probable reasons why the horizontal transfer of a plasmid encoding staphopain C between avian and human strains has never been observed. The human plasma serine protease inhibitor α(1)-antichymotrypsin (ACHT) inhibits ScpA2. Together with the lack of ScpA2 inhibition by chicken plasma, these data may explain the exclusively avian occurrence of ScpA2. We also clarify the mechanistic details of this unusual cross-class inhibition. Analysis of mutated ACHT variants revealed that the cleavage of the Leu383-Ser384 peptide bond results in ScpA2 inhibition, whereas hydrolysis of the preceding peptide bond leads to ACHT inactivation. This evidence is consistent with the suicide-substrate-like mechanism of inhibition. PMID:21296644

  4. Using the SUBcellular database for Arabidopsis proteins to localize the Deg protease family.

    PubMed

    Tanz, Sandra K; Castleden, Ian; Hooper, Cornelia M; Small, Ian; Millar, A Harvey

    2014-01-01

    Sub-functionalization during the expansion of gene families in eukaryotes has occurred in part through specific subcellular localization of different family members. To better understand this process in plants, compiled records of large-scale proteomic and fluorescent protein localization datasets can be explored and bioinformatic predictions for protein localization can be used to predict the gaps in experimental data. This process can be followed by targeted experiments to test predictions. The SUBA3 database is a free web-service at http://suba.plantenergy.uwa.edu.au that helps users to explore reported experimental data and predictions concerning proteins encoded by gene families and to define the experiments required to locate these homologous sets of proteins. Here we show how SUBA3 can be used to explore the subcellular location of the Deg protease family of ATP-independent serine endopeptidases (Deg1-Deg16). Combined data integration and new experiments refined location information for Deg1 and Deg9, confirmed Deg2, Deg5, and Deg8 in plastids and Deg 15 in peroxisomes and provide substantial experimental evidence for mitochondrial localized Deg proteases. Two of these, Deg3 and Deg10, additionally localized to the plastid, revealing novel dual-targeted Deg proteases in the plastid and the mitochondrion. SUBA3 is continually updated to ensure that researchers can use the latest published data when planning the experimental steps remaining to localize gene family functions. PMID:25161662

  5. Using the SUBcellular database for Arabidopsis proteins to localize the Deg protease family

    PubMed Central

    Tanz, Sandra K.; Castleden, Ian; Hooper, Cornelia M.; Small, Ian; Millar, A. Harvey

    2014-01-01

    Sub-functionalization during the expansion of gene families in eukaryotes has occurred in part through specific subcellular localization of different family members. To better understand this process in plants, compiled records of large-scale proteomic and fluorescent protein localization datasets can be explored and bioinformatic predictions for protein localization can be used to predict the gaps in experimental data. This process can be followed by targeted experiments to test predictions. The SUBA3 database is a free web-service at http://suba.plantenergy.uwa.edu.au that helps users to explore reported experimental data and predictions concerning proteins encoded by gene families and to define the experiments required to locate these homologous sets of proteins. Here we show how SUBA3 can be used to explore the subcellular location of the Deg protease family of ATP-independent serine endopeptidases (Deg1–Deg16). Combined data integration and new experiments refined location information for Deg1 and Deg9, confirmed Deg2, Deg5, and Deg8 in plastids and Deg 15 in peroxisomes and provide substantial experimental evidence for mitochondrial localized Deg proteases. Two of these, Deg3 and Deg10, additionally localized to the plastid, revealing novel dual-targeted Deg proteases in the plastid and the mitochondrion. SUBA3 is continually updated to ensure that researchers can use the latest published data when planning the experimental steps remaining to localize gene family functions. PMID:25161662

  6. Distinct protease pathways control cell shape and apoptosis in v-src-transformed quail neuroretina cells

    SciTech Connect

    Neel, Benjamin D.; Gillet, Germain . E-mail: g.gillet@ibcp.fr

    2005-11-15

    Intracellular proteases play key roles in cell differentiation, proliferation and apoptosis. In nerve cells, little is known about their relative contribution to the pathways which control cell physiology, including cell death. Neoplastic transformation of avian neuroretina cells by p60 {sup v-src} tyrosine kinase results in dramatic morphological changes and deregulation of apoptosis. To identify the proteases involved in the cellular response to p60 {sup v-src}, we evaluated the effect of specific inhibitors of caspases, calpains and the proteasome on cell shape changes and apoptosis induced by p60 {sup v-src} inactivation in quail neuroretina cells transformed by tsNY68, a thermosensitive strain of Rous sarcoma virus. We found that the ubiquitin-proteasome pathway is recruited early after p60 {sup v-src} inactivation and is critical for morphological changes, whereas caspases are essential for cell death. This study provides evidence that distinct intracellular proteases are involved in the control of the morphology and fate of v-src-transformed cells.

  7. Enhanced Secretory Leukocyte Protease Inhibitor in Human Immunodeficiency Virus Type 1-Infected Patients

    PubMed Central

    Baqui, A. A. M. A.; Meiller, Timothy F.; Falkler, William A.

    1999-01-01

    Secretory leukocyte protease inhibitor (SLPI) has been found to possess activity against the human immunodeficiency virus type 1 (HIV-1) in vitro at physiological concentrations. A study was undertaken to evaluate SLPI levels in human saliva and plasma among HIV-positive (HIV+) patients with various HIV-1 viral loads in comparison to uninfected controls. Whole blood in EDTA and unstimulated saliva samples were collected from 37 HIV+ patients, of whom 20 had a history of intravenous drug abuse (IVDA). Control samples were collected from 20 appropriate age- and sex-matched HIV-1-negative individuals. SLPI was estimated from both saliva and serum samples by an enzyme-linked immunosorbent assay. HIV viral load was determined using a quantitative reverse transcription-PCR. SLPI levels were increased 16.7% in plasma and 10.3% in saliva among HIV+ patients in comparison to uninfected controls. SLPI levels were increased 5.9% in saliva and 3.9% in plasma among HIV+ patients with a high viral load (>10,000 copies/ml) as compared to patients with a low viral load (<400 copies/ml). Only 23% of patients with a high viral load used combination therapy with protease inhibitor drugs, whereas 92.9% of HIV+ patients with a low viral load used protease inhibitors. SLPI levels did not differ significantly among the IVDA patients, patients with different viral loads, or patients using protease inhibitor drugs. There was a statistically significant increase in SLPI levels in saliva among HIV patients in comparison to non-HIV-infected controls. An increase in SLPI levels among HIV+ patients may be a natural consequence of HIV pathogenesis and an important factor in preventing oral transmission of HIV, but this increase may not be evident during plasma viremia in patients with a high viral load. PMID:10548568

  8. Synergistic Defensive Function of Raphides and Protease through the Needle Effect

    PubMed Central

    Konno, Kotaro; Inoue, Takashi A.; Nakamura, Masatoshi

    2014-01-01

    Raphides, needle-shaped calcium oxalate crystals in tissues of many plants, have been thought to play defensive roles against herbivores without detailed bioassays for their defensive roles and modes of function using purified raphides. In order to examine the defensive roles and modes of function of raphides in a clear experimental system, we performed bioassays giving the larvae of the Eri silkmoth, Samia ricini (Saturniidae), leaves of their host plant, the castor oil plant, Ricinus communis (Euphorbiaceae), painted with the raphides purified from kiwifruits, Actinidia deliciosa (Actinidiaceae), in presence or absence of cysteine protease, which often coincide with raphides in plant tissues. Raphides alone or cysteine protease alone showed only weak defensive activities around experimental concentrations. However, when raphides and cysteine protease coexisted, they synergistically showed very strong growth-reducing activities, and the mortality of caterpillars was very high. In contrast, amorphous calcium oxalate did not show synergism with cysteine protease on defensive activities, indicating that the needle-shape of raphides is essential for the synergism. The present study provides the first clear experimental evidence for the synergism between raphides and other defensive factors. Further, the study suggests that “the needle effect”, which intensify the bioactivities of other bioactive factors by making holes to the barriers (cell membrane, cuticle, epithelium, the nuclear membrane, etc.) and facilitate the bioactive factors to go through them and reach the targets, is important in the defensive activities of raphides, and possibly in the allergy caused by raphides, and in the carcinogenic activities of other needle-shaped components including asbestos and plant derived silica needles. PMID:24621613

  9. Determinants of Affinity and Proteolytic Stability in Interactions of Kunitz Family Protease Inhibitors with Mesotrypsin

    SciTech Connect

    M Salameh; A Soares; D Navaneetham; D Sinha; P Walsh; E Radisky

    2011-12-31

    An important functional property of protein protease inhibitors is their stability to proteolysis. Mesotrypsin is a human trypsin that has been implicated in the proteolytic inactivation of several protein protease inhibitors. We have found that bovine pancreatic trypsin inhibitor (BPTI), a Kunitz protease inhibitor, inhibits mesotrypsin very weakly and is slowly proteolyzed, whereas, despite close sequence and structural homology, the Kunitz protease inhibitor domain of the amyloid precursor protein (APPI) binds to mesotrypsin 100 times more tightly and is cleaved 300 times more rapidly. To define features responsible for these differences, we have assessed the binding and cleavage by mesotrypsin of APPI and BPTI reciprocally mutated at two nonidentical residues that make direct contact with the enzyme. We find that Arg at P{sub 1} (versus Lys) favors both tighter binding and more rapid cleavage, whereas Met (versus Arg) at P'{sub 2} favors tighter binding but has minimal effect on cleavage. Surprisingly, we find that the APPI scaffold greatly enhances proteolytic cleavage rates, independently of the binding loop. We draw thermodynamic additivity cycles analyzing the interdependence of P{sub 1} and P'{sub 2} substitutions and scaffold differences, finding multiple instances in which the contributions of these features are nonadditive. We also report the crystal structure of the mesotrypsin-APPI complex, in which we find that the binding loop of APPI displays evidence of increased mobility compared with BPTI. Our data suggest that the enhanced vulnerability of APPI to mesotrypsin cleavage may derive from sequence differences in the scaffold that propagate increased flexibility and mobility to the binding loop.

  10. Determinants of Affinity and Proteolytic Stability in Interactions of Kunitz Family Protease Inhibitors with Mesotrypsin

    SciTech Connect

    Salameh, M.A.; Soares, A.; Navaneetham, D.; Sinha, D.; Walsh, P. N.; Radisky, E. S.

    2010-11-19

    An important functional property of protein protease inhibitors is their stability to proteolysis. Mesotrypsin is a human trypsin that has been implicated in the proteolytic inactivation of several protein protease inhibitors. We have found that bovine pancreatic trypsin inhibitor (BPTI), a Kunitz protease inhibitor, inhibits mesotrypsin very weakly and is slowly proteolyzed, whereas, despite close sequence and structural homology, the Kunitz protease inhibitor domain of the amyloid precursor protein (APPI) binds to mesotrypsin 100 times more tightly and is cleaved 300 times more rapidly. To define features responsible for these differences, we have assessed the binding and cleavage by mesotrypsin of APPI and BPTI reciprocally mutated at two nonidentical residues that make direct contact with the enzyme. We find that Arg at P{sub 1} (versus Lys) favors both tighter binding and more rapid cleavage, whereas Met (versus Arg) at P'{sub 2} favors tighter binding but has minimal effect on cleavage. Surprisingly, we find that the APPI scaffold greatly enhances proteolytic cleavage rates, independently of the binding loop. We draw thermodynamic additivity cycles analyzing the interdependence of P1 and P'{sub 2} substitutions and scaffold differences, finding multiple instances in which the contributions of these features are nonadditive. We also report the crystal structure of the mesotrypsin {center_dot} APPI complex, in which we find that the binding loop of APPI displays evidence of increased mobility compared with BPTI. Our data suggest that the enhanced vulnerability of APPI to mesotrypsin cleavage may derive from sequence differences in the scaffold that propagate increased flexibility and mobility to the binding loop.

  11. Sweet potato cysteine proteases SPAE and SPCP2 participate in sporamin degradation during storage root sprouting.

    PubMed

    Chen, Hsien-Jung; Liang, Shu-Hao; Huang, Guan-Jhong; Lin, Yaw-Huei

    2015-08-15

    Sweet potato sporamins are trypsin inhibitors and exhibit strong resistance to digestion by pepsin, trypsin and chymotrypsin. In addition, they constitute the major storage proteins in the sweet potato and, after degradation, provide nitrogen as a nutrient for seedling regrowth in sprouting storage roots. In this report, four cysteine proteases-one asparaginyl endopeptidase (SPAE), two papain-like cysteine proteases (SPCP1 and SPCP2), and one granulin-containing cysteine protease (SPCP3)-were studied to determine their association with sporamin degradation in sprouting storage roots. Sporamin degradation became significant in the flesh of storage roots starting from week 4 after sprouting and this correlated with expression levels of SPAE and SPCP2, but not of SPCP1 and SPCP3. In the outer flesh near the skin, sporamin degradation was more evident and occurred earlier than in the inner flesh of storage roots. Degradation of sporamins in the outer flesh was inversely correlated with the distance of the storage root from the sprout. Exogenous application of SPAE and SPCP2, but not SPCP3, fusion proteins to crude extracts of the outer flesh (i.e., extracted from a depth of 0.3cm and within 2cm of one-week-old sprouts) promoted in vitro sporamin degradation in a dose-dependent manner. Pre-treatment of SPAE and SPCP2 fusion proteins at 95°C for 5min prior to their application to the crude extracts reduced sporamin degradation. These data show that sweet potato asparaginyl endopeptidase SPAE and papain-like cysteine protease SPCP2 participate in sporamin degradation during storage root sprouting. PMID:26363719

  12. PEGylated substrates of NSP4 protease: A tool to study protease specificity

    NASA Astrophysics Data System (ADS)

    Wysocka, Magdalena; Gruba, Natalia; Grzywa, Renata; Giełdoń, Artur; Bąchor, Remigiusz; Brzozowski, Krzysztof; Sieńczyk, Marcin; Dieter, Jenne; Szewczuk, Zbigniew; Rolka, Krzysztof; Lesner, Adam

    2016-03-01

    Herein we present the synthesis of a novel type of peptidomimetics composed of repeating diaminopropionic acid residues modified with structurally diverse heterobifunctional polyethylene glycol chains (abbreviated as DAPEG). Based on the developed compounds, a library of fluorogenic substrates was synthesized. Further library deconvolution towards human neutrophil serine protease 4 (NSP4) yielded highly sensitive and selective internally quenched peptidomimetic substrates. In silico analysis of the obtained peptidomimetics revealed the presence of an interaction network with distant subsites located on the enzyme surface.

  13. Cleavage and activation of human factor IX by serine proteases

    SciTech Connect

    Enfield, D.L.; Thompson, A.R.

    1984-10-01

    Human factor IX circulates as a single-chain glycoprotein. Upon activation in vitro, it is cleaved into disulfide-linked light and heavy chains and an activation peptide. After reduction of activated /sup 125/I-factor IX, the heavy and light chains are readily identified by gel electrophoresis. A direct, immunoradiometric assay for factor IXa was developed to assess activation of factor IX for proteases that cleaved it. The assay utilized radiolabeled antithrombin III with heparin to identify the active site and antibodies to distinguish factor IX. After cleavage of factor IX by factor XIa, factor VIIa-tissue thromboplastin complex, or the factor X-activating enzyme from Russell's viper venom, antithrombin III bound readily to factor IXa. Cleavage of /sup 125/I-factor IX by trypsin, chymotrypsin, and granulocyte elastase in the presence of calcium yielded major polypeptide fragments of the sizes of the factor XIa-generated light and heavy chains. When the immunoradiometric assay was used to assess trypsin-cleaved factor IX, the product bound antithrombin III, but not maximally. After digesting with insolubilized trypsin, clotting activity confirmed activation. In evaluating activation of factor IX, physical evidence of activation cleavages does not necessarily correlate with generation of an active site.

  14. The yeast autophagy protease Atg4 is regulated by thioredoxin

    PubMed Central

    Pérez-Pérez, María Esther; Zaffagnini, Mirko; Marchand, Christophe H; Crespo, José L; Lemaire, Stéphane D

    2014-01-01

    Autophagy is a membrane-trafficking process whereby double-membrane vesicles called autophagosomes engulf and deliver intracellular material to the vacuole for degradation. Atg4 is a cysteine protease with an essential function in autophagosome formation. Mounting evidence suggests that reactive oxygen species may play a role in the control of autophagy and could regulate Atg4 activity but the precise mechanisms remain unclear. In this study, we showed that reactive oxygen species activate autophagy in the model yeast Saccharomyces cerevisiae and unraveled the molecular mechanism by which redox balance controls Atg4 activity. A combination of biochemical assays, redox titrations, and site-directed mutagenesis revealed that Atg4 is regulated by oxidoreduction of a single disulfide bond between Cys338 and Cys394. This disulfide has a low redox potential and is very efficiently reduced by thioredoxin, suggesting that this oxidoreductase plays an important role in Atg4 regulation. Accordingly, we found that autophagy activation by rapamycin was more pronounced in a thioredoxin mutant compared with wild-type cells. Moreover, in vivo studies indicated that Cys338 and Cys394 are required for the proper regulation of autophagosome biogenesis, since mutation of these cysteines resulted in increased recruitment of Atg8 to the phagophore assembly site. Thus, we propose that the fine-tuning of Atg4 activity depending on the intracellular redox state may regulate autophagosome formation. PMID:25483965

  15. The yeast autophagy protease Atg4 is regulated by thioredoxin.

    PubMed

    Pérez-Pérez, María Esther; Zaffagnini, Mirko; Marchand, Christophe H; Crespo, José L; Lemaire, Stéphane D

    2014-01-01

    Autophagy is a membrane-trafficking process whereby double-membrane vesicles called autophagosomes engulf and deliver intracellular material to the vacuole for degradation. Atg4 is a cysteine protease with an essential function in autophagosome formation. Mounting evidence suggests that reactive oxygen species may play a role in the control of autophagy and could regulate Atg4 activity but the precise mechanisms remain unclear. In this study, we showed that reactive oxygen species activate autophagy in the model yeast Saccharomyces cerevisiae and unraveled the molecular mechanism by which redox balance controls Atg4 activity. A combination of biochemical assays, redox titrations, and site-directed mutagenesis revealed that Atg4 is regulated by oxidoreduction of a single disulfide bond between Cys338 and Cys394. This disulfide has a low redox potential and is very efficiently reduced by thioredoxin, suggesting that this oxidoreductase plays an important role in Atg4 regulation. Accordingly, we found that autophagy activation by rapamycin was more pronounced in a thioredoxin mutant compared with wild-type cells. Moreover, in vivo studies indicated that Cys338 and Cys394 are required for the proper regulation of autophagosome biogenesis, since mutation of these cysteines resulted in increased recruitment of Atg8 to the phagophore assembly site. Thus, we propose that the fine-tuning of Atg4 activity depending on the intracellular redox state may regulate autophagosome formation. PMID:25483965

  16. Highly potent fibrinolytic serine protease from Streptomyces.

    PubMed

    Uesugi, Yoshiko; Usuki, Hirokazu; Iwabuchi, Masaki; Hatanaka, Tadashi

    2011-01-01

    We introduce a highly potent fibrinolytic serine protease from Streptomyces omiyaensis (SOT), which belongs to the trypsin family. The fibrinolytic activity of SOT was examined using in vitro assays and was compared with those of known fibrinolytic enzymes such as plasmin, tissue-type plasminogen activator (t-PA), urokinase, and nattokinase. Compared to other enzymes, SOT showed remarkably higher hydrolytic activity toward mimic peptides of fibrin and plasminogen. The fibrinolytic activity of SOT is about 18-fold higher than that of plasmin, and is comparable to that of t-PA by fibrin plate assays. Furthermore, SOT had some plasminogen activator-like activity. Results show that SOT and nattokinase have very different fibrinolytic and fibrinogenolytic modes, engendering significant synergetic effects of SOT and nattokinase on fibrinolysis. These results suggest that SOT presents important possibilities for application in the therapy of thrombosis. PMID:22112764

  17. Laundry detergent compatibility of the alkaline protease from Bacillus cereus.

    PubMed

    Banik, Rathindra Mohan; Prakash, Monika

    2004-01-01

    The endogenous protease activity in various commercially available laundry detergents of international companies was studied. The maximum protease activity was found at 50 degrees C in pH range 10.5-11.0 in all the tested laundry detergents. The endogenous protease activity in the tested detergents retained up to 70% on incubation at 40 degrees C for 1 h, whereas less than 30% activity was only found on incubation at 50 degrees C for 1 h. The alkaline protease from an alkalophilic strain of Bacillus cereus was studied for its compatibility in commercial detergents. The cell free fermented broth from shake flask culture of the organism showed maximum activity at pH 10.5 and 50 degrees C. The protease from B. cereus showed much higher residual activity (more than 80%) on incubation with laundry detergents at 50 degrees C for 1 h or longer. The protease enzyme from B. cereus was found to be superior over the endogenous proteases present in the tested commercial laundry detergents in comparison to the enzyme stability during the washing at higher temperature, e.g., 40-50 degrees C. PMID:15293947

  18. Protease production by Streptococcus sanguis associated with subacute bacterial endocarditis.

    PubMed Central

    Straus, D C

    1982-01-01

    A viridans streptococcus (Streptococcus sanguis biotype II) isolated from the blood of a patient with subacute bacterial endocarditis was examined for protease production. In broth culture, extracellular proteolytic enzymes were not produced by this organism until after the early exponential phase of growth, with maximal protease production occurring during the stationary phase. Four distinct proteases were isolated and purified from the supernatant fluids of stationary-phase cultures, employing a combination of ion-exchange column chromatography, gel filtration column chromatography, and polyacrylamide gel electrophoresis. All four proteases could be eluted from a diethylaminoethyl cellulose column at a sodium chloride gradient concentration of 0.25 M but were separable by gel filtration chromatography on a Sephadex G-100 column. They varied in molecular weights as determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis from approximately 13,000 to 230,000. All four proteases had pH optima of between 8.0 and 9.0, and two of the proteases were active against casein, human serum albumin, and gelatin but were not active against elastin and collagen. The remaining two proteases were able to degrade only casein and gelatin. These results show that S. sanguis is able to excrete maximal levels of potentially destructive enzymes when the organisms are not actively multiplying. This finding may explain some of the damage caused in heart tissue by these organisms during subacute bacterial endocarditis. Images PMID:6759404

  19. Screening and characterization of protease producing actinomycetes from marine saltern.

    PubMed

    Suthindhiran, Krish; Jayasri, Mangalam Achuthananda; Dipali, Dipa; Prasar, Apurva

    2014-10-01

    In the course of systematic screening program for bioactive actinomycetes, an alkaline protease producing halophilic strain Actinopolyspora sp. VITSDK2 was isolated from marine saltern, Southern India. The strain was identified as Actinopolyspora based on its phenotypic and phylogenetic characters. The protease was partially purified using ammonium sulfate precipitation and subsequently by DEAE cellulose column chromatography. The enzyme was further purified using HPLC and the molecular weight was found to be 22 kDa as determined by SDS-PAGE analysis. The purified protease exhibited pH stability in a wide range of 4-12 with optimum at 10.0. The enzyme was found to be stable between 25 and 80 °C and displayed a maximum activity at 60 °C. The enzyme activity was increased marginally in presence of Mn(2+) , Mg(2+) , and Ca(2+) and decreased in presence of Cu(2+) . PMSF and DFP completely inhibited the activity suggesting it belongs to serine protease. Further, the proteolytic activity was abolished in presence of N-tosyl-L-lysine chloromethyl ketone suggesting this might be chymotrypsin-like serine protease. The protease was 96% active when kept for 10 days at room temperature. The results indicate that the enzyme belong to chymotrypsin-like serine protease exhibiting both pH and thermostability, which can be used for various applications in industries. PMID:24136565

  20. 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.

  1. Exploring a new serine protease from Cucumis sativus L.

    PubMed

    Nafeesa, Zohara; Shivalingu, B R; Vivek, H K; Priya, B S; Swamy, S Nanjunda

    2015-03-01

    Coagulation is an important physiological process in hemostasis which is activated by sequential action of proteases. This study aims to understand the involvement of aqueous fruit extract of Cucumis sativus L. (AqFEC) European burp less variety in blood coagulation cascade. AqFEC hydrolyzed casein in a dose-dependent manner. The presence of protease activity was further confirmed by casein zymography which revealed the possible presence of two high molecular weight protease(s). The proteolytic activity was inhibited only by phenyl methyl sulphonyl fluoride suggesting the presence of serine protease(s). In a dose-dependent manner, AqFEC also hydrolysed Aα and Bβ subunits of fibrinogen, whereas it failed to degrade the γ subunit of fibrinogen even at a concentration as high as 100 μg and incubation time up to 4 h. AqFEC reduced the clotting time of citrated plasma by 87.65%. The protease and fibrinogenolytic activity of AqFEC suggests its possible role in stopping the bleeding and ensuing wound healing process. PMID:25577345

  2. Inferring selection in the Anopheles gambiae species complex: an example from immune-related serine protease inhibitors

    PubMed Central

    Obbard, Darren J; Welch, John J; Little, Tom J

    2009-01-01

    Background Mosquitoes of the Anopheles gambiae species complex are the primary vectors of human malaria in sub-Saharan Africa. Many host genes have been shown to affect Plasmodium development in the mosquito, and so are expected to engage in an evolutionary arms race with the pathogen. However, there is little conclusive evidence that any of these mosquito genes evolve rapidly, or show other signatures of adaptive evolution. Methods Three serine protease inhibitors have previously been identified as candidate immune system genes mediating mosquito-Plasmodium interaction, and serine protease inhibitors have been identified as hot-spots of adaptive evolution in other taxa. Population-genetic tests for selection, including a recent multi-gene extension of the McDonald-Kreitman test, were applied to 16 serine protease inhibitors and 16 other genes sampled from the An. gambiae species complex in both East and West Africa. Results Serine protease inhibitors were found to show a marginally significant trend towards higher levels of amino acid diversity than other genes, and display extensive genetic structuring associated with the 2La chromosomal inversion. However, although serpins are candidate targets for strong parasite-mediated selection, no evidence was found for rapid adaptive evolution in these genes. Conclusion It is well known that phylogenetic and population history in the An. gambiae complex can present special problems for the application of standard population-genetic tests for selection, and this may explain the failure of this study to detect selection acting on serine protease inhibitors. The pitfalls of uncritically applying these tests in this species complex are highlighted, and the future prospects for detecting selection acting on the An. gambiae genome are discussed. PMID:19497100

  3. Characterization of protease IV expression in Pseudomonas aeruginosa clinical isolates.

    PubMed

    Conibear, Tim C R; Willcox, Mark D P; Flanagan, Judith L; Zhu, Hua

    2012-02-01

    Expression of protease IV by Pseudomonas aeruginosa during ocular infections contributes significantly to tissue damage. However, several P. aeruginosa strains isolated from ocular infections or inflammatory events produce very low levels of protease IV. The aim of the present study was to characterize, genetically and phenotypically, the presence and expression of the protease IV gene in a group of clinical isolates that cause adverse ocular events of varying degrees, and to elucidate the possible control mechanisms of expression associated with this virulence factor. Protease IV gene sequences from seven clinical isolates of P. aeruginosa were determined and compared to P. aeruginosa strains PAO1 and PA103-29. Production and enzyme activity of protease IV were measured in test strains and compared to that of quorum-sensing gene (lasRI) mutants and the expression of other virulence factors. Protease IV gene sequence similarities between the isolates were 97.5-99.5 %. The strains were classified into two distinct phylogenetic groups that correlated with the presence of exo-enzymes from type three secretion systems (TTSS). Protease IV concentrations produced by PAOΔlasRI mutants and the two clinical isolates with a lasRI gene deficiency were restored to levels comparable to strain PAO1 following complementation of the quorum-sensing gene deficiencies. The protease IV gene is highly conserved in P. aeruginosa clinical isolates that cause a range of adverse ocular events. Observed variations within the gene sequence appear to correlate with presence of specific TTSS genes. Protease IV expression was shown to be regulated by the Las quorum-sensing system. PMID:21921113

  4. A Bacillus anthracis strain deleted for six proteases serves as an effective host for production of recombinant proteins.

    PubMed

    Pomerantsev, Andrei P; Pomerantseva, Olga M; Moayeri, Mahtab; Fattah, Rasem; Tallant, Cynthia; Leppla, Stephen H

    2011-11-01

    Bacillus anthracis produces a number of extracellular proteases that impact the integrity and yield of other proteins in the B. anthracis secretome. In this study we show that anthrolysin O (ALO) and the three anthrax toxin proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF), produced from the B. anthracis Ames 35 strain (pXO1⁺, pXO2⁻), are completely degraded at the onset of stationary phase due to the action of proteases. An improved Cre-loxP gene knockout system was used to sequentially delete the genes encoding six proteases (InhA1, InhA2, camelysin, TasA, NprB, and MmpZ). The role of each protease in degradation of the B. anthracis toxin components and ALO was demonstrated. Levels of the anthrax toxin components and ALO in the supernatant of the sporulation defective, pXO1⁺ A35HMS mutant strain deleted for the six proteases were significantly increased and remained stable over 24 h. A pXO1-free variant of this six-protease mutant strain, designated BH460, provides an improved host strain for the preparation of recombinant proteins. As an example, BH460 was used to produce recombinant EF, which previously has been difficult to obtain from B. anthracis. The EF protein produced from BH460 had the highest in vivo potency of any EF previously purified from B. anthracis or Escherichia coli hosts. BH460 is recommended as an effective host strain for recombinant protein production, typically yielding greater than 10mg pure protein per liter of culture. PMID:21827967

  5. Protease-associated cellular networks in malaria parasite Plasmodium falciparum

    PubMed Central

    2011-01-01

    Background Malaria continues to be one of the most severe global infectious diseases, responsible for 1-2 million deaths yearly. The rapid evolution and spread of drug resistance in parasites has led to an urgent need for the development of novel antimalarial targets. Proteases are a group of enzymes that play essential roles in parasite growth and invasion. The possibility of designing specific inhibitors for proteases makes them promising drug targets. Previously, combining a comparative genomics approach and a machine learning approach, we identified the complement of proteases (degradome) in the malaria parasite Plasmodium falciparum and its sibling species [1-3], providing a catalog of targets for functional characterization and rational inhibitor design. Network analysis represents another route to revealing the role of proteins in the biology of parasites and we use this approach here to expand our understanding of the systems involving the proteases of P. falciparum. Results We investigated the roles of proteases in the parasite life cycle by constructing a network using protein-protein association data from the STRING database [4], and analyzing these data, in conjunction with the data from protein-protein interaction assays using the yeast 2-hybrid (Y2H) system [5], blood stage microarray experiments [6-8], proteomics [9-12], literature text mining, and sequence homology analysis. Seventy-seven (77) out of 124 predicted proteases were associated with at least one other protein, constituting 2,431 protein-protein interactions (PPIs). These proteases appear to play diverse roles in metabolism, cell cycle regulation, invasion and infection. Their degrees of connectivity (i.e., connections to other proteins), range from one to 143. The largest protease-associated sub-network is the ubiquitin-proteasome system which is crucial for protein recycling and stress response. Proteases are also implicated in heat shock response, signal peptide processing, cell cycle

  6. In vitro digestion with proteases producing MHC class II ligands.

    PubMed

    Tohmé, Mira; Maschalidi, Sophia; Manoury, Bénédicte

    2013-01-01

    Proteases generate peptides that bind to MHC class II molecules to interact with a wide diversity of CD4(+) T cells. They are expressed in dedicated organelles: endosomes and lysosomes of professional antigen presenting cells (pAPCs) such as B cells, macrophages, and dendritic cells. The identification of endosomal proteases which produce antigenic peptides is important, for example, for better vaccination and to prevent autoimmune diseases. Here, we describe a panel of technics (in vitro digestion assays of protein with recombinant proteases or purified endosomes/lysosomes, T cell stimulation) to monitor the production of MHC class II ligands. PMID:23329510

  7. Detection of Legume Protease Inhibitors by the Gel-X-ray Film Contact Print Technique

    ERIC Educational Resources Information Center

    Mulimani, Veerappa H.; Sudheendra, Kulkarni; Giri, Ashok P.

    2002-01-01

    Redgram (Cajanus cajan L.) extracts have been analyzed for the protease inhibitors using a new, sensitive, simple, and rapid method for detection of electrophoretically separated protease inhibitors. The detection involves equilibrating the gel successively in the protease assay buffer and protease solution, rinsing the gel in assay buffer, and…

  8. Protease inhibitor expression in soybean roots exhibiting susceptible and resistance reactions to soybean cyst nematode

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protease inhibitors play a role in regulating proteases during cellular development and in plant defense against insects and nematodes. We identified, cloned and sequenced cDNAs encoding six protease inhibitors expressed in soybean roots infected with soybean cyst nematode. Four of these protease in...

  9. 21 CFR 184.1027 - Mixed carbohydrase and protease enzyme product.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Mixed carbohydrase and protease enzyme product... Substances Affirmed as GRAS § 184.1027 Mixed carbohydrase and protease enzyme product. (a) Mixed carbohydrase and protease enzyme product is an enzyme preparation that includes carbohydrase and protease...

  10. Structural Basis for the Magnesium-Dependent Activation and Hexamerization of the Lon AAA+ Protease.

    PubMed

    Su, Shih-Chieh; Lin, Chien-Chu; Tai, Hui-Chung; Chang, Mu-Yueh; Ho, Meng-Ru; Babu, C Satheesan; Liao, Jiahn-Haur; Wu, Shih-Hsiung; Chang, Yuan-Chih; Lim, Carmay; Chang, Chung-I

    2016-05-01

    The Lon AAA+ protease (LonA) plays important roles in protein homeostasis and regulation of diverse biological processes. LonA behaves as a homomeric hexamer in the presence of magnesium (Mg(2+)) and performs ATP-dependent proteolysis. However, it is also found that LonA can carry out Mg(2+)-dependent degradation of unfolded protein substrate in an ATP-independent manner. Here we show that in the presence of Mg(2+) LonA forms a non-secluded hexameric barrel with prominent openings, which explains why Mg(2+)-activated LonA can operate as a diffusion-based chambered protease to degrade unstructured protein and peptide substrates efficiently in the absence of ATP. A 1.85 Å crystal structure of Mg(2+)-activated protease domain reveals Mg(2+)-dependent remodeling of a substrate-binding loop and a potential metal-binding site near the Ser-Lys catalytic dyad, supported by biophysical binding assays and molecular dynamics simulations. Together, these findings reveal the specific roles of Mg(2+) in the molecular assembly and activation of LonA. PMID:27041593

  11. Evaluation of trypanocidal activity of combinations of anti-sleeping sickness drugs with cysteine protease inhibitors.

    PubMed

    Steverding, Dietmar

    2015-01-01

    Chemotherapy of human African trypanosomiasis (HAT) is unsatisfactory because only a few drugs, with serious side effects and poor efficacy, are available. As drug combination regimes often achieve greater therapeutic efficacy than monotherapies, here the trypanocidal activity of the cysteine protease inhibitor K11777 in combination with current anti-HAT drugs using bloodstream forms of Trypanosoma brucei was investigated. Isobolographic analysis was used to determine the interaction between cysteine protease inhibitors (K11777, CA-074Me and CAA0225) and anti-HAT drugs (suramin, pentamidine, melarsoprol and eflornithine). Bloodstream forms of T. brucei were incubated in culture medium containing cysteine protease inhibitors or anti-HAT drugs alone or in combination at a 1:1 fixed-dose ratio. After 48 h incubation, live cells were counted, the 50% growth inhibition values determined and combination indices calculated. The general cytotoxicity of drug combinations was evaluated with human leukaemia HL-60 cells. Combinations of K11777 with suramin, pentamidine and melarsoprol showed antagonistic effects while with eflornithine a synergistic effect was observed. Whereas eflornithine antagonises with CA-074Me, an inhibitor inactivating the targeted TbCATL only under reducing conditions, it synergises with CAA0255, an inhibitor structurally related to CA-074Me which inactivates TbCATL independently of thiols. These findings indicate an essential role of thiols for the synergistic interaction between K11777 and eflornithine. Encouragingly, the K11777/eflornithine combination displayed higher trypanocidal than cytotoxic activity. The results of this study suggest that the combination of the cysteine protease inhibitor K11777 and eflornithine display promising synergistic trypanocidal activity that warrants further investigation of the drug combination as possible alternative treatment of HAT. PMID:25662707

  12. Effects of Mutational Loss of Specific Intracellular Proteases on the Sporulation of Bacillus subtilis

    PubMed Central

    Hageman, James H.; Carlton, Bruce C.

    1973-01-01

    Two protease-deficient mutants of Bacillus subtilis 168 (Trp−) were isolated and compared with the parental strain with respect to production of intracellular proteases and sporulation. A mutant lacking the metal-requiring “neutral” protease intracellularly sporulated as well as the parental strain. A second mutant, deficient in an as yet uncharacterized intracellular protease, failed to sporulate normally. It is proposed that this new protease is also involved in intracellular protein turnover. Images PMID:4196247

  13. Mini review: ATP-dependent proteases in bacteria.

    PubMed

    Bittner, Lisa-Marie; Arends, Jan; Narberhaus, Franz

    2016-08-01

    AAA(+) proteases are universal barrel-like and ATP-fueled machines preventing the accumulation of aberrant proteins and regulating the proteome according to the cellular demand. They are characterized by two separate operating units, the ATPase and peptidase domains. ATP-dependent unfolding and translocation of a substrate into the proteolytic chamber is followed by ATP-independent degradation. This review addresses the structure and function of bacterial AAA(+) proteases with a focus on the ATP-driven mechanisms and the coordinated movements in the complex mainly based on the knowledge of ClpXP. We conclude by discussing strategies how novel protease substrates can be trapped by mutated AAA(+) protease variants. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 505-517, 2016. PMID:26971705

  14. A new cuticle scale hydrolysing protease from Beauveria brongniartii.

    PubMed

    Erlacher, A; Sousa, F; Schroeder, M; Jus, S; Kokol, V; Cavaco-Paulo, A; Guebitz, G M

    2006-05-01

    From a screening for the production of new proteases specific for cuticle scales, Beauveria brongniartii was selected producing an alkaline Ca(++) dependent protease. The purified had a molecular weight of 27 kDa and a pI value of 8.0. Substrate specificities of model substrates (wool with partially removed cuticles treated with SDS) were analyzed by protein release, dissolved organic carbon (DOC) and nitrogen analysis. The C/N ratio of released material turned out to be a good parameter to determine the site of action of proteases on fibres. Compared to other enzymes, the fungal protease preferentially hydrolyzed cuticle scales and has thus a potential for anti-shrinking pre-treatment of wool fabrics. PMID:16791724

  15. Improving Viral Protease Inhibitors to Counter Drug Resistance.

    PubMed

    Kurt Yilmaz, Nese; Swanstrom, Ronald; Schiffer, Celia A

    2016-07-01

    Drug resistance is a major problem in health care, undermining therapy outcomes and necessitating novel approaches to drug design. Extensive studies on resistance to viral protease inhibitors, particularly those of HIV-1 and hepatitis C virus (HCV) protease, revealed a plethora of information on the structural and molecular mechanisms underlying resistance. These insights led to several strategies to improve viral protease inhibitors to counter resistance, such as exploiting the essential biological function and leveraging evolutionary constraints. Incorporation of these strategies into structure-based drug design can minimize vulnerability to resistance, not only for viral proteases but for other quickly evolving drug targets as well, toward designing inhibitors one step ahead of evolution to counter resistance with more intelligent and rational design. PMID:27090931

  16. Proteases in cardiometabolic diseases: Pathophysiology, molecular mechanisms and clinical applications.

    PubMed

    Hua, Yinan; Nair, Sreejayan

    2015-02-01

    Cardiovascular disease is the leading cause of death in the U.S. and other developed countries. Metabolic syndrome, including obesity, diabetes/insulin resistance, hypertension and dyslipidemia is a major threat for public health in the modern society. It is well established that metabolic syndrome contributes to the development of cardiovascular disease collective called as cardiometabolic disease. Despite documented studies in the research field of cardiometabolic disease, the underlying mechanisms are far from clear. Proteases are enzymes that break down proteins, many of which have been implicated in various diseases including cardiac disease. Matrix metalloproteinase (MMP), calpain, cathepsin and caspase are among the major proteases involved in cardiac remodeling. Recent studies have also implicated proteases in the pathogenesis of cardiometabolic disease. Elevated expression and activities of proteases in atherosclerosis, coronary heart disease, obesity/insulin-associated heart disease as well as hypertensive heart disease have been documented. Furthermore, transgenic animals that are deficient in or over-express proteases allow scientists to understand the causal relationship between proteases and cardiometabolic disease. Mechanistically, MMPs and cathepsins exert their effect on cardiometabolic diseases mainly through modifying the extracellular matrix. However, MMP and cathepsin are also reported to affect intracellular proteins, by which they contribute to the development of cardiometabolic diseases. On the other hand, activation of calpain and caspases has been shown to influence intracellular signaling cascade including the NF-κB and apoptosis pathways. Clinically, proteases are reported to function as biomarkers of cardiometabolic diseases. More importantly, the inhibitors of proteases are credited with beneficial cardiometabolic profile, although the exact molecular mechanisms underlying these salutary effects are still under investigation. A better

  17. Current strategies for probing substrate specificity of proteases.

    PubMed

    Poreba, M; Drag, M

    2010-01-01

    In this review we describe in detail the available technologies used for investigating the substrate specificity of proteases. Critical comparison of the available detection methods and their choice for certain type of screening is discussed. We present successful strategies along with appropriate examples for the design and synthesis of combinatorial libraries of substrates using both chemical and biological approaches. Proteomic tools for the identification of natural substrates of proteases are also discussed. PMID:20939826

  18. Proteases in cardiometabolic diseases: Pathophysiology, molecular mechanisms and clinical applications

    PubMed Central

    Hua, Yinan; Nair, Sreejayan

    2014-01-01

    Cardiovascular disease is the leading cause of death in the U.S. and other developed country. Metabolic syndrome, including obesity, diabetes/insulin resistance, hypertension and dyslipidemia is major threat for public health in the modern society. It is well established that metabolic syndrome contributes to the development of cardiovascular disease collective called as cardiometabolic disease. Despite documented studies in the research field of cardiometabolic disease, the underlying mechanisms are far from clear. Proteases are enzymes that break down proteins, many of which have been implicated in various diseases including cardiac disease. Matrix metalloproteinase (MMP), calpain, cathepsin and caspase are among the major proteases involved in cardiac remodeling. Recent studies have also implicated proteases in the pathogenesis of cardiometabolic disease. Elevated expression and activities of proteases in atherosclerosis, coronary heart disease, obesity/insulin-associated heart disease as well as hypertensive heart disease have been documented. Furthermore, transgenic animals that are deficient in or overexpress proteases allow scientists to understand the causal relationship between proteases and cardiometabolic disease. Mechanistically, MMPs and cathepsins exert their effect on cardiometabolic diseases mainly through modifying the extracellular matrix. However, MMP and cathepsin are also reported to affect intracellular proteins, by which they contribute to the development of cardiometabolic diseases. On the other hand, activation of calpain and caspases has been shown to influence intracellular signaling cascade including the NF-κB and apoptosis pathways. Clinically, proteases are reported to function as biomarkers of cardiometabolic diseases. More importantly, the inhibitors of proteases are credited with beneficial cardiometabolic profile, although the exact molecular mechanisms underlying these salutary effects are still under investigation. A better

  19. Autocatalytic processing of m-AAA protease subunits in mitochondria.

    PubMed

    Koppen, Mirko; Bonn, Florian; Ehses, Sarah; Langer, Thomas

    2009-10-01

    m-AAA proteases are ATP-dependent proteolytic machines in the inner membrane of mitochondria which are crucial for the maintenance of mitochondrial activities. Conserved nuclear-encoded subunits, termed paraplegin, Afg3l1, and Afg3l2, form various isoenzymes differing in their subunit composition in mammalian mitochondria. Mutations in different m-AAA protease subunits are associated with distinct neuronal disorders in human. However, the biogenesis of m-AAA protease complexes or of individual subunits is only poorly understood. Here, we have examined the processing of nuclear-encoded m-AAA protease subunits upon import into mitochondria and demonstrate autocatalytic processing of Afg3l1 and Afg3l2. The mitochondrial processing peptidase MPP generates an intermediate form of Afg3l2 that is matured autocatalytically. Afg3l1 or Afg3l2 are also required for maturation of newly imported paraplegin subunits after their cleavage by MPP. Our results establish that mammalian m-AAA proteases can act as processing enzymes in vivo and reveal overlapping activities of Afg3l1 and Afg3l2. These findings might be of relevance for the pathogenesis of neurodegenerative disorders associated with mutations in different m-AAA protease subunits. PMID:19656850

  20. The Crystal Structure of GXGD Membrane Protease FlaK

    SciTech Connect

    J Hu; Y Xue; S Lee; Y Ha

    2011-12-31

    The GXGD proteases are polytopic membrane proteins with catalytic activities against membrane-spanning substrates that require a pair of aspartyl residues. Representative members of the family include preflagellin peptidase, type 4 prepilin peptidase, presenilin and signal peptide peptidase. Many GXGD proteases are important in medicine. For example, type 4 prepilin peptidase may contribute to bacterial pathogenesis, and mutations in presenilin are associated with Alzheimer's disease. As yet, there is no atomic-resolution structure in this protease family. Here we report the crystal structure of FlaK, a preflagellin peptidase from Methanococcus maripaludis, solved at 3.6 {angstrom} resolution. The structure contains six transmembrane helices. The GXGD motif and a short transmembrane helix, helix 4, are positioned at the centre, surrounded by other transmembrane helices. The crystal structure indicates that the protease must undergo conformational changes to bring the GXGD motif and a second essential aspartyl residue from transmembrane helix 1 into close proximity for catalysis. A comparison of the crystal structure with models of presenilin derived from biochemical analysis reveals three common transmembrane segments that are similarly arranged around the active site. This observation reinforces the idea that the prokaryotic and human proteases are evolutionarily related. The crystal structure presented here provides a framework for understanding the mechanism of the GXGD proteases, and may facilitate the rational design of inhibitors that target specific members of the family.

  1. The crystal structure of GXGD membrane protease FlaK

    SciTech Connect

    Hu, Jian; Xue, Yi; Lee, Sangwon; Ha, Ya

    2011-09-20

    The GXGD proteases are polytopic membrane proteins with catalytic activities against membrane-spanning substrates that require a pair of aspartyl residues. Representative members of the family include preflagellin peptidase, type 4 prepilin peptidase, presenilin and signal peptide peptidase. Many GXGD proteases are important in medicine. For example, type 4 prepilin peptidase may contribute to bacterial pathogenesis, and mutations in presenilin are associated with Alzheimer's disease. As yet, there is no atomic-resolution structure in this protease family. Here we report the crystal structure of FlaK, a preflagellin peptidase from Methanococcus maripaludis, solved at 3.6 {angstrom} resolution. The structure contains six transmembrane helices. The GXGD motif and a short transmembrane helix, helix 4, are positioned at the centre, surrounded by other transmembrane helices. The crystal structure indicates that the protease must undergo conformational changes to bring the GXGD motif and a second essential aspartyl residue from transmembrane helix 1 into close proximity for catalysis. A comparison of the crystal structure with models of presenilin derived from biochemical analysis reveals three common transmembrane segments that are similarly arranged around the active site. This observation reinforces the idea that the prokaryotic and human proteases are evolutionarily related. The crystal structure presented here provides a framework for understanding the mechanism of the GXGD proteases, and may facilitate the rational design of inhibitors that target specific members of the family.

  2. Insights into the Cyanobacterial Deg/HtrA Proteases

    PubMed Central

    Cheregi, Otilia; Wagner, Raik; Funk, Christiane

    2016-01-01

    Proteins are the main machinery for all living processes in a cell; they provide structural elements, regulate biochemical reactions as enzymes, and are the interface to the outside as receptors and transporters. Like any other machinery proteins have to be assembled correctly and need maintenance after damage, e.g., caused by changes in environmental conditions, genetic mutations, and limitations in the availability of cofactors. Proteases and chaperones help in repair, assembly, and folding of damaged and misfolded protein complexes cost-effective, with low energy investment compared with neo-synthesis. Despite their importance for viability, the specific biological role of most proteases in vivo is largely unknown. Deg/HtrA proteases, a family of serine-type ATP-independent proteases, have been shown in higher plants to be involved in the degradation of the Photosystem II reaction center protein D1. The objective of this review is to highlight the structure and function of their cyanobacterial orthologs. Homology modeling was used to find specific features of the SynDeg/HtrA proteases of Synechocystis sp. PCC 6803. Based on the available data concerning their location and their physiological substrates we conclude that these Deg proteases not only have important housekeeping and chaperone functions within the cell, but also are needed for remodeling the cell exterior. PMID:27252714

  3. Isolation and molecular characterisation of alkaline protease producing Bacillus thuringiensis.

    PubMed

    Agasthya, Annapurna S; Sharma, Naresh; Mohan, Anand; Mahal, Prabhpreet

    2013-05-01

    Proteases are of particular interest because of their action on insoluble keratin substrates and generally on a broad range of protein substrates. Proteases are one of the most important groups of industrial enzymes used in detergent, protein, brewing, meat, photographic, leather, dairy, pharmaceutical and food industry. In the present study, the organism isolated from the protein rich soil sample was identified by biochemical and molecular characterisation as Bacillus thuringiensis and further optimum conditions for alkaline protease synthesis were determined. The growth conditions for B. thuringiensis was optimised by inoculating into yeast extract casein medium at different pH and incubating at different temperatures. The maximum protease production occurred at pH 8 and at 37 °C. B. thuringiensis showed proteolytic activity at various culture conditions. Optimum conditions for the protease activity were found to be 47 °C and pH 8. In the later stage, the blood removing action of crude and partially purified protease was found to be effective within 25 min in the presence of commercial detergents indicating the possible use of this enzyme in detergent industry. Enzyme also showed good activity against hair substrate keratin and can be used for dehairing. PMID:22826099

  4. Signaling pathways activated by a protease allergen in basophils

    PubMed Central

    Rosenstein, Rachel K.; Bezbradica, Jelena S.; Yu, Shuang; Medzhitov, Ruslan

    2014-01-01

    Allergic diseases represent a significant burden in industrialized countries, but why and how the immune system responds to allergens remain largely unknown. Because many clinically significant allergens have proteolytic activity, and many helminths express proteases that are necessary for their life cycles, host mechanisms likely have evolved to detect the proteolytic activity of helminth proteases, which may be incidentally activated by protease allergens. A cysteine protease, papain, is a prototypic protease allergen that can directly activate basophils and mast cells, leading to the production of cytokines, including IL-4, characteristic of the type 2 immune response. The mechanism of papain’s immunogenic activity remains unknown. Here we have characterized the cellular response activated by papain in basophils. We find that papain-induced IL-4 production requires calcium flux and activation of PI3K and nuclear factor of activated T cells. Interestingly, papain-induced IL-4 production was dependent on the immunoreceptor tyrosine-based activation motif (ITAM) adaptor protein Fc receptor γ-chain, even though the canonical ITAM signaling was not activated by papain. Collectively, these data characterize the downstream signaling pathway activated by a protease allergen in basophils. PMID:25369937

  5. Delay of Iris flower senescence by protease inhibitors.

    PubMed

    Pak, Caroline; van Doorn, Wouter G

    2005-02-01

    Visible senescence of the flag tepals in Iris x hollandica (cv. Blue Magic) was preceded by a large increase in endoprotease activity. Just before visible senescence about half of total endoprotease activity was apparently due to cysteine proteases, somewhat less than half to serine proteases, with a minor role of metalloproteases. Treatment of isolated tepals with the purported serine protease inhibitors AEBSF [4-(2-aminoethyl)-benzenesulfonyl fluoride] or DFP (diisopropyl-fluorophosphate) prevented the increase in endoprotease activity and considerably delayed or prevented the normal senescence symptoms. The specific cysteine protease-specific E-64d reduced maximum endoprotease activity by 30%, but had no effect on the time to visible senescence. Zinc chloride and aprotinin reduced maximum endoprotease activity by c. 50 and 40%, respectively, and slightly delayed visible senescence. A proteasome inhibitor (Z-leu-leu-Nva-H) slightly delayed tepal senescence, which indicates that protein degradation in the proteasome may play a role in induction of the visible senescence symptoms. It is concluded that visible senescence is preceded by large-scale protein degradation, which is apparently mainly due to cysteine- and serine protease activity, and that two (unspecific) inhibitors of serine proteases considerably delay the senescence symptoms. PMID:15720658

  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. Effect of protease inhibitors on the sense of taste.

    PubMed

    Schiffman, S S; Zervakis, J; Heffron, S; Heald, A E

    1999-10-01

    The purpose of this study was to investigate the taste properties of protease inhibitors which are essential components of drug regimes used to treat human immunodeficiency virus (HIV) infection. In this study, the taste properties of four protease inhibitors (indinavir, ritonavir, saquinavir, and nelfinavir) were investigated in unmedicated HIV-infected patients and healthy controls. Three of the four protease inhibitors (indinavir, ritonavir, and saquinavir) were found to be predominantly bitter (with additional qualities of medicinal, metallic, astringent, sour, and burning). Nelfinavir was found to be relatively tasteless. HIV-infected and uninfected control subjects detected protease inhibitors at similar concentrations, but HIV-infected subjects perceived suprathreshold concentrations as more bitter than controls. Detection thresholds ranged from 0.0061 mM for saquinavir in HIV-infected patients to 0.0702 mM for ritonavir in uninfected control subjects. Suprathreshold studies indicated that protease inhibitors modified the taste perception of a variety of other taste compounds. These results are consistent with clinical findings that protease inhibitors produce taste complaints that can impact patient compliance. PMID:10501290

  8. Protease inhibitors decrease the resistance of Vitaceae to Plasmopara viticola.

    PubMed

    Gindro, Katia; Berger, Valentine; Godard, Sophie; Voinesco, Francine; Schnee, Sylvain; Viret, Olivier; Alonso-Villaverde, Virginia

    2012-11-01

    Plasmopara viticola must successfully infect susceptible grapevine cultivars to complete its biological cycle. In resistant grapevine varieties, P. viticola is blocked by the activation of defense mechanisms; these defense mechanisms produce hypersensitive reactions, which are related to programmed cell death. In animals, programmed cell death is dependent on caspase activities. In plants, different caspase-like proteases assume the same functions. To examine the roles of caspase-like proteases in P. viticola-grapevine interactions, three varieties of grapevine with different levels of P. viticola resistance were chosen. These grapevine varieties were treated with either PMSF, a serine protease inhibitor, or E-64, a cysteine protease inhibitor. The development of the pathogen was followed microscopically, and the plant defense reactions were estimated through stilbene quantification. Both protease inhibitor treatments increased the infection rate in the resistant and immune varieties, diminished the production of toxic stilbenes and changed the level of the plants' susceptibility to the pathogen. In particular, after either protease treatment, the cultivar that was originally immune became resistant (hyphae and haustoria were observed), the resistant cultivar reached the level of a susceptible cultivar (sporulation was observed) and the susceptible cultivar became more sensitive (P. viticola colonized the entirety of the leaf mesophyll). PMID:22906813

  9. PEGylated substrates of NSP4 protease: A tool to study protease specificity.

    PubMed

    Wysocka, Magdalena; Gruba, Natalia; Grzywa, Renata; Giełdoń, Artur; Bąchor, Remigiusz; Brzozowski, Krzysztof; Sieńczyk, Marcin; Dieter, Jenne; Szewczuk, Zbigniew; Rolka, Krzysztof; Lesner, Adam

    2016-01-01

    Herein we present the synthesis of a novel type of peptidomimetics composed of repeating diaminopropionic acid residues modified with structurally diverse heterobifunctional polyethylene glycol chains (abbreviated as DAPEG). Based on the developed compounds, a library of fluorogenic substrates was synthesized. Further library deconvolution towards human neutrophil serine protease 4 (NSP4) yielded highly sensitive and selective internally quenched peptidomimetic substrates. In silico analysis of the obtained peptidomimetics revealed the presence of an interaction network with distant subsites located on the enzyme surface. PMID:26955973

  10. PEGylated substrates of NSP4 protease: A tool to study protease specificity

    PubMed Central

    Wysocka, Magdalena; Gruba, Natalia; Grzywa, Renata; Giełdoń, Artur; Bąchor, Remigiusz; Brzozowski, Krzysztof; Sieńczyk, Marcin; Dieter, Jenne; Szewczuk, Zbigniew; Rolka, Krzysztof; Lesner, Adam

    2016-01-01

    Herein we present the synthesis of a novel type of peptidomimetics composed of repeating diaminopropionic acid residues modified with structurally diverse heterobifunctional polyethylene glycol chains (abbreviated as DAPEG). Based on the developed compounds, a library of fluorogenic substrates was synthesized. Further library deconvolution towards human neutrophil serine protease 4 (NSP4) yielded highly sensitive and selective internally quenched peptidomimetic substrates. In silico analysis of the obtained peptidomimetics revealed the presence of an interaction network with distant subsites located on the enzyme surface. PMID:26955973

  11. Retrovirus Entry by Endocytosis and Cathepsin Proteases

    PubMed Central

    Kubo, Yoshinao; Hayashi, Hideki; Matsuyama, Toshifumi; Sato, Hironori; Yamamoto, Naoki

    2012-01-01

    Retroviruses include infectious agents inducing severe diseases in humans and animals. In addition, retroviruses are widely used as tools to transfer genes of interest to target cells. Understanding the entry mechanism of retroviruses contributes to developments of novel therapeutic approaches against retrovirus-induced diseases and efficient exploitation of retroviral vectors. Entry of enveloped viruses into host cell cytoplasm is achieved by fusion between the viral envelope and host cell membranes at either the cell surface or intracellular vesicles. Many animal retroviruses enter host cells through endosomes and require endosome acidification. Ecotropic murine leukemia virus entry requires cathepsin proteases activated by the endosome acidification. CD4-dependent human immunodeficiency virus (HIV) infection is thought to occur via endosomes, but endosome acidification is not necessary for the entry whereas entry of CD4-independent HIVs, which are thought to be prototypes of CD4-dependent viruses, is low pH dependent. There are several controversial results on the retroviral entry pathways. Because endocytosis and endosome acidification are complicatedly controlled by cellular mechanisms, the retrovirus entry pathways may be different in different cell lines. PMID:23304142

  12. Protease Production by Different Thermophilic Fungi

    NASA Astrophysics Data System (ADS)

    Macchione, Mariana M.; Merheb, Carolina W.; Gomes, Eleni; da Silva, Roberto

    A comparative study was carried out to evaluate protease production in solid-state fermentation (SSF) and submerged fermentation (SmF) by nine different thermophilic fungi — Thermoascus aurantiacus Miehe, Thermomyces lanuginosus, T. lanuginosus TO.03, Aspergillus flavus 1.2, Aspergillus sp. 13.33, Aspergillus sp. 13.34, Aspergillus sp. 13.35, Rhizomucor pusillus 13.36 and Rhizomucor sp. 13.37 — using substrates containing proteins to induce enzyme secretion. Soybean extract (soybean milk), soybean flour, milk powder, rice, and wheat bran were tested. The most satisfactory results were obtained when using wheat bran in SSF. The fungi that stood out in SSF were T. lanuginosus, T. lanuginosus TO.03, Aspergillus sp. 13.34, Aspergillus sp. 13.35, and Rhizomucor sp. 13.37, and those in SmF were T. aurantiacus, T. lanuginosus TO.03, and 13.37. In both fermentation systems, A. flavus 1.2 and R. pusillus 13.36 presented the lowest levels of proteolytic activity.

  13. Gene identification and molecular characterization of solvent stable protease from a moderately haloalkaliphilic bacterium, Geomicrobium sp. EMB2.

    PubMed

    Karan, Ram; Singh, Raj Kumar Mohan; Kapoor, Sanjay; Khare, S K

    2011-02-01

    Cloning and characterization of the gene encoding a solvent-tolerant protease from the haloalkaliphilic bacterium Geomicrobium sp. EMB2 are described. Primers designed based on the N-terminal amino acid sequence of the purified EMB2 protease helped in the amplification of a 1,505-bp open reading frame that had a coding potential of a 42.7-kDa polypeptide. The deduced EMB2 protein contained a 35.4-kDa mature protein of 311 residues, with a high proportion of acidic amino acid residues. Phylogenetic analysis placed the EMB2 gene close to a known serine protease from Bacillus clausii KSM-K16. Primary sequence analysis indicated a hydrophobic inclination of the protein; and the 3D structure modeling elucidated a relatively higher percentage of small (glycine, alanine, and valine) and borderline (serine and threonine) hydrophobic residues on its surface. The structure analysis also highlighted enrichment of acidic residues at the cost of basic residues. The study indicated that solvent and salt stabilities in Geomicrobium sp. protease may be accorded to different structural features; that is, the presence of a number of small hydrophobic amino acid residues on the surface and a higher content of acidic amino acid residues, respectively. PMID:21364294

  14. The Majority of 9,729 Group A Streptococcus Strains Causing Disease Secrete SpeB Cysteine Protease: Pathogenesis Implications

    PubMed Central

    Raghuram, Anjali; Cantu, Concepcion; Hartman, Meredith H.; Jimenez, Francisco E.; Lee, Susan; Ngo, Ashley; Rice, Kelsey A.; Saddington, Deborah; Spillman, Hannaka; Valson, Chandni; Flores, Anthony R.; Beres, Stephen B.; Long, S. Wesley; Nasser, Waleed; Musser, James M.

    2015-01-01

    Group A streptococcus (GAS), the causative agent of pharyngitis and necrotizing fasciitis, secretes the potent cysteine protease SpeB. Several lines of evidence suggest that SpeB is an important virulence factor. SpeB is expressed in human infections, protects mice from lethal challenge when used as a vaccine, and contributes significantly to tissue destruction and dissemination in animal models. However, recent descriptions of mutations in genes implicated in SpeB production have led to the idea that GAS may be under selective pressure to decrease secreted SpeB protease activity during infection. Thus, two divergent hypotheses have been proposed. One postulates that SpeB is a key contributor to pathogenesis; the other, that GAS is under selection to decrease SpeB during infection. In order to distinguish between these alternative hypotheses, we performed casein hydrolysis assays to measure the SpeB protease activity secreted by 6,775 GAS strains recovered from infected humans. The results demonstrated that 84.3% of the strains have a wild-type SpeB protease phenotype. The availability of whole-genome sequence data allowed us to determine the relative frequencies of mutations in genes implicated in SpeB production. The most abundantly mutated genes were direct transcription regulators. We also sequenced the genomes of 2,954 GAS isolates recovered from nonhuman primates with experimental necrotizing fasciitis. No mutations that would result in a SpeB-deficient phenotype were identified. Taken together, these data unambiguously demonstrate that the great majority of GAS strains recovered from infected humans secrete wild-type levels of SpeB protease activity. Our data confirm the important role of SpeB in GAS pathogenesis and help end a long-standing controversy. PMID:26416912

  15. Stress Conditions Increase Vimentin Cleavage by Omi/HtrA2 Protease in Human Primary Neurons and Differentiated Neuroblastoma Cells.

    PubMed

    Lucotte, Bérangère; Tajhizi, Mehdi; Alkhatib, Dareen; Samuelsson, Eva-Britt; Wiehager, Birgitta; Schedin-Weiss, Sophia; Sundström, Erik; Winblad, Bengt; Tjernberg, Lars O; Behbahani, Homira

    2015-12-01

    Dysfunctional Omi/HtrA2, a mitochondrial serine protease, has been implicated in various neurodegenerative disorders. Despite the wealth of evidence on the roles of Omi/HtrA2 in apoptosis, little is known about its cytosolic targets, the cleavage of which could account for the observed morphological changes such as cytoskeletal reorganizations in axons. By proteomic analysis, vimentin was identified as a substrate for Omi/HtrA2 and we have reported increased Omi/HtrA2 protease activity in Alzheimer disease (AD) brain. Here, we investigated a possible link between Omi/HtrA2 and vimentin cleavage, and consequence of this cleavage on mitochondrial distribution in neurons. In vitro protease assays showed vimentin to be cleaved by Omi/HtrA2 protease, and proximity ligation assay demonstrated an increased interaction between Omi/HtrA2 and vimentin in human primary neurons upon stress stimuli. Using differentiated neuroblastoma SH-SY5Y cells, we showed that Omi/HtrA2 under several different stress conditions induces cleavage of vimentin in wild-type as well as SH-SY5Y cells transfected with amyloid precursor protein with the Alzheimer disease-associated Swedish mutation. After stress treatment, inhibition of Omi/HtrA2 protease activity by the Omi/HtrA2 specific inhibitor, Ucf-101, reduced the cleavage of vimentin in wild-type cells. Following altered vimentin filaments integrity by stress stimuli, mitochondria was redistributed in differentiated SH-SY5Y cells and human primary neurons. In summary, the findings outlined in this paper suggest a role of Omi/HtrA2 in modulation of vimentin filamentous structure in neurons. Our results provide important findings for understanding the biological role of Omi/HtrA2 activity during stress conditions, and give knowledge of interplay between Omi/HtrA2 and vimentin which might affect mitochondrial distribution in neurons. PMID:25288153

  16. The majority of 9,729 group A streptococcus strains causing disease secrete SpeB cysteine protease: pathogenesis implications.

    PubMed

    Olsen, Randall J; Raghuram, Anjali; Cantu, Concepcion; Hartman, Meredith H; Jimenez, Francisco E; Lee, Susan; Ngo, Ashley; Rice, Kelsey A; Saddington, Deborah; Spillman, Hannaka; Valson, Chandni; Flores, Anthony R; Beres, Stephen B; Long, S Wesley; Nasser, Waleed; Musser, James M

    2015-12-01

    Group A streptococcus (GAS), the causative agent of pharyngitis and necrotizing fasciitis, secretes the potent cysteine protease SpeB. Several lines of evidence suggest that SpeB is an important virulence factor. SpeB is expressed in human infections, protects mice from lethal challenge when used as a vaccine, and contributes significantly to tissue destruction and dissemination in animal models. However, recent descriptions of mutations in genes implicated in SpeB production have led to the idea that GAS may be under selective pressure to decrease secreted SpeB protease activity during infection. Thus, two divergent hypotheses have been proposed. One postulates that SpeB is a key contributor to pathogenesis; the other, that GAS is under selection to decrease SpeB during infection. In order to distinguish between these alternative hypotheses, we performed casein hydrolysis assays to measure the SpeB protease activity secreted by 6,775 GAS strains recovered from infected humans. The results demonstrated that 84.3% of the strains have a wild-type SpeB protease phenotype. The availability of whole-genome sequence data allowed us to determine the relative frequencies of mutations in genes implicated in SpeB production. The most abundantly mutated genes were direct transcription regulators. We also sequenced the genomes of 2,954 GAS isolates recovered from nonhuman primates with experimental necrotizing fasciitis. No mutations that would result in a SpeB-deficient phenotype were identified. Taken together, these data unambiguously demonstrate that the great majority of GAS strains recovered from infected humans secrete wild-type levels of SpeB protease activity. Our data confirm the important role of SpeB in GAS pathogenesis and help end a long-standing controversy. PMID:26416912

  17. Expression, purification and molecular modeling of the NIa protease of Cardamom mosaic virus.

    PubMed

    Jebasingh, T; Pandaranayaka, Eswari P J; Mahalakshmi, A; Kasin Yadunandam, A; Krishnaswamy, S; Usha, R

    2013-01-01

    The NIa protease of Potyviridae is the major viral protease that processes potyviral polyproteins. The NIa protease coding region of Cardamom mosaic virus (CdMV) is amplified from the viral cDNA, cloned and expressed in Escherichia coli. NIa protease forms inclusion bodies in E.coli. The inclusion bodies are solubilized with 8 M urea, refolded and purified by Nickel-Nitrilotriacetic acid affinity chromatography. Three-dimensional modeling of the CdMV NIa protease is achieved by threading approach using the homologous X-ray crystallographic structure of Tobacco etch mosaic virus NIa protease. The model gave an insight in to the substrate specificities of the NIa proteases and predicted the complementation of nearby residues in the catalytic triad (H42, D74 and C141) mutants in the cis protease activity of CdMV NIa protease. PMID:22888800

  18. The structure of a universally employed enzyme: V8 protease from Staphylococcus aureus

    SciTech Connect

    Prasad, Lata; Leduc, Yvonne; Hayakawa, Koto; Delbaere, Louis T.J.

    2008-06-27

    V8 protease, an extracellular protease of Staphylococcus aureus, is related to the pancreatic serine proteases. The enzyme cleaves peptide bonds exclusively on the carbonyl side of aspartate and glutamate residues. Unlike the pancreatic serine proteases, V8 protease possesses no disulfide bridges. This is a major evolutionary difference, as all pancreatic proteases have at least two disulfide bridges. The structure of V8 protease shows structural similarity with several other serine proteases, specifically the epidermolytic toxins A and B from S. aureus and trypsin, in which the conformation of the active site is almost identical. V8 protease is also unique in that the positively charged N-terminus is involved in determining the substrate-specificity of the enzyme.

  19. Diversity, Structures, and Collagen-Degrading Mechanisms of Bacterial Collagenolytic Proteases

    PubMed Central

    Zhang, Yu-Zhong; Ran, Li-Yuan; Li, Chun-Yang

    2015-01-01

    Bacterial collagenolytic proteases are important because of their essential role in global collagen degradation and because of their virulence in some human bacterial infections. Bacterial collagenolytic proteases include some metalloproteases of the M9 family from Clostridium or Vibrio strains, some serine proteases distributed in the S1, S8, and S53 families, and members of the U32 family. In recent years, there has been remarkable progress in discovering new bacterial collagenolytic proteases and in investigating the collagen-degrading mechanisms of bacterial collagenolytic proteases. This review provides comprehensive insight into bacterial collagenolytic proteases, especially focusing on the structures and collagen-degrading mechanisms of representative bacterial collagenolytic proteases in each family. The roles of bacterial collagenolytic proteases in human diseases and global nitrogen cycling, together with the biotechnological and medical applications for these proteases, are also briefly discussed. PMID:26150451

  20. Protease inhibitor from Moringa oleifera with potential for use as therapeutic drug and as seafood preservative

    PubMed Central

    Bijina, B.; Chellappan, Sreeja; Krishna, Jissa G.; Basheer, Soorej M.; Elyas, K.K.; Bahkali, Ali H.; Chandrasekaran, M.

    2011-01-01

    Protease inhibitors are well known to have several applications in medicine and biotechnology. Several plant sources are known to return potential protease inhibitors. In this study plants belonging to different families of Leguminosae, Malvaceae, Rutaceae, Graminae and Moringaceae were screened for the protease inhibitor. Among them Moringa oleifera, belonging to the family Moringaceae, recorded high level of protease inhibitor activity after ammonium sulfate fractionation. M. oleifera, which grows throughout most of the tropics and having several industrial and medicinal uses, was selected as a source of protease inhibitor since so far no reports were made on isolation of the protease inhibitor. Among the different parts of M. oleifera tested, the crude extract isolated from the mature leaves and seeds showed the highest level of inhibition against trypsin. Among the various extraction media evaluated, the crude extract prepared in phosphate buffer showed maximum recovery of the protease inhibitor. The protease inhibitor recorded high inhibitory activity toward the serine proteases thrombin, elastase, chymotrypsin and the cysteine proteases cathepsin B and papain which have more importance in pharmaceutical industry. The protease inhibitor also showed complete inhibition of activities of the commercially available proteases of Bacillus licheniformis and Aspergillus oryzae. However, inhibitory activities toward subtilisin, esperase, pronase E and proteinase K were negligible. Further, it was found that the protease inhibitor could prevent proteolysis in a commercially valuable shrimp Penaeus monodon during storage indicating the scope for its application as a seafood preservative. This is the first report on isolation of a protease inhibitor from M. oleifera. PMID:23961135

  1. Expression and characterization of group A Streptococcus extracellular cysteine protease recombinant mutant proteins and documentation of seroconversion during human invasive disease episodes.

    PubMed

    Gubba, S; Low, D E; Musser, J M

    1998-02-01

    A recent study with isogenic strains constructed by recombinant DNA strategies unambiguously documented that a highly conserved extracellular cysteine protease expressed by Streptococcus pyogenes (group A Streptococcus [GAS]) is a critical virulence factor in a mouse model of invasive disease (S. Lukomski, S. Sreevatsan, C. Amberg, W. Reichardt, M. Woischnik, A. Podbielski, and J. M. Musser, J. Clin. Invest. 99:2574-2580, 1997). To facilitate further investigations of the streptococcal cysteine protease, recombinant proteins composed of a 40-kDa zymogen containing a C192S amino acid substitution that ablates enzymatic activity, a 28-kDa mature protein with the C192S replacement, and a 12-kDa propeptide were purified from Escherichia coli containing His tag expression vectors. The recombinant C192S zymogen retained apparently normal structural integrity, as assessed by the ability of purified wild-type streptococcal cysteine protease to process the 40-kDa molecule to the 28-kDa mature form. All three recombinant purified proteins retained immunologic reactivity with polyclonal and monoclonal antibodies. Humans with a diverse range of invasive disease episodes (erysipelas, cellulitis, pneumonia, bacteremia, septic arthritis, streptococcal toxic shock syndrome, and necrotizing fasciitis) caused by six distinct M types of GAS seroconverted to the streptococcal cysteine protease. These results demonstrate that this GAS protein is expressed in vivo during the course of human infections and thereby provide additional evidence that the cysteine protease participates in host-pathogen interactions in some patients. PMID:9453639

  2. Expression and Characterization of Group A Streptococcus Extracellular Cysteine Protease Recombinant Mutant Proteins and Documentation of Seroconversion during Human Invasive Disease Episodes

    PubMed Central

    Gubba, Siddeswar; Low, Donald E.; Musser, James M.

    1998-01-01

    A recent study with isogenic strains constructed by recombinant DNA strategies unambiguously documented that a highly conserved extracellular cysteine protease expressed by Streptococcus pyogenes (group A Streptococcus [GAS]) is a critical virulence factor in a mouse model of invasive disease (S. Lukomski, S. Sreevatsan, C. Amberg, W. Reichardt, M. Woischnik, A. Podbielski, and J. M. Musser, J. Clin. Invest. 99:2574–2580, 1997). To facilitate further investigations of the streptococcal cysteine protease, recombinant proteins composed of a 40-kDa zymogen containing a C192S amino acid substitution that ablates enzymatic activity, a 28-kDa mature protein with the C192S replacement, and a 12-kDa propeptide were purified from Escherichia coli containing His tag expression vectors. The recombinant C192S zymogen retained apparently normal structural integrity, as assessed by the ability of purified wild-type streptococcal cysteine protease to process the 40-kDa molecule to the 28-kDa mature form. All three recombinant purified proteins retained immunologic reactivity with polyclonal and monoclonal antibodies. Humans with a diverse range of invasive disease episodes (erysipelas, cellulitis, pneumonia, bacteremia, septic arthritis, streptococcal toxic shock syndrome, and necrotizing fasciitis) caused by six distinct M types of GAS seroconverted to the streptococcal cysteine protease. These results demonstrate that this GAS protein is expressed in vivo during the course of human infections and thereby provide additional evidence that the cysteine protease participates in host-pathogen interactions in some patients. PMID:9453639

  3. Production of protease and lipase by solvent tolerant Pseudomonas aeruginosa PseA in solid-state fermentation using Jatropha curcas seed cake as substrate.

    PubMed

    Mahanta, Nilkamal; Gupta, Anshu; Khare, S K

    2008-04-01

    Deoiled Jatropha seed cake was assessed for its suitability as substrate for enzyme production by solid-state fermentation (SSF). Solvent tolerant Pseudomonas aeruginosa PseA strain previously reported by us was used for fermentation. The seed cake supported good bacterial growth and enzyme production (protease, 1818 U/g of substrate and lipase, 625 U/g of substrate) as evident by its chemical composition. Maximum protease and lipase production was observed at 50% substrate moisture, a growth period of 72 and 120 h, and a substrate pH of 6.0 and 7.0, respectively. Enrichment with maltose as carbon source increased protease and lipase production by 6.3- and 1.6-fold, respectively. Nitrogen supplementation with peptone for protease and NaNO(3) for lipase production also enhanced the enzyme yield reaching 11,376 U protease activity and 1084 U lipase activity per gram of Jatropha seed cake. These results demonstrated viable approach for utilization of this huge biomass by solid-state fermentation for the production of industrial enzymes. This offers significant benefit due to low cost and abundant availability of cake during biodiesel production. PMID:17509877

  4. Kinetics of alkaline protease production by Streptomyces griseoflavus PTCC1130

    PubMed Central

    Hosseini, Seyed Vesal; Saffari, Zahra; Farhanghi, Ali; Atyabi, Seyed Mohammad; Norouzian, Dariush

    2016-01-01

    Background and Objectives: Proteases are a group of enzymes that catalyze the degradation of proteins resulting in the production of their amino acid constituents. They are the most important group of industrial enzymes which account for about 60% of total enzymes in the market and produced mainly by microorganisms. The attempts were made to study the kinetic parameters of protease produced by Streptomyces griseoflavus PTCC1130. Materials and Methods: Streptomyces griseoflavus PTCC1130 was grown on casein agar. Different media such as BM1, BM2, BM3 and BM4 were prepared. Data obtained from growth and protease production were subjected to kinetics evaluation. Casein was used as substrate for protease activity and the released soluble peptide bearing aromatic amino acid were quantified by Folin Cioclateaue reagent. Protein content of the enzyme and the sugar utilized by the organism were estimated by Bradford and Miller’s methods respectively. Results: Basal Medium named as BM1, BM2, BM3 and BM4(50 mL in 250 mL Erlen Meyer flasks) were screened out to evaluate protease production by Streptomyces griseoflavus PTCC1130. They were inoculated with known amount of seed culture and kept on rotary shaker. To obtain the specific growth rate, wet weight of biomass was plotted against the time. The clarified supernatant was used for the analysis of protease by measuring the soluble peptide containing aromatic amino acid residues employing Folin Cioclateaue reagent. Our results showed that maximum level of enzyme production (14035 U/L) was occurred at late exponential phase using Basal Medium supplemented with zinc sulfate (0.5g/L), casein (10g/L) at pH 6.5. Conclusions: A kinetic study of protease production by Streptomyces griseoflavus PTCC1130 provided highly quantitative information regarding the behavior of a system, which is essential to study the fermentation process. Exploitation of such kinetics analysis would be useful in commercialization of microbial enzyme

  5. Characterization of an extracellular serine protease of Leishmania (Leishmania) amazonensis.

    PubMed

    Silva-Lopez, R E; Coelho, M G Pinto; De Simone, S G

    2005-07-01

    A serine protease was purified 942-fold from culture supernatant of L. amazonensis promastigotes using (NH4)2SO4 precipitation followed by affinity chromatography on aprotinin-agarose and continuous elution electrophoresis by Prep Cell, yielding a total recovery of 61%. The molecular mass of the active enzyme estimated by SDS-PAGE under conditions of reduction was 56 kDa and 115 kDa under conditions of non-reduction, suggesting that the protease is a dimeric protein. Additionally, it was found to be a non-glycosylated enzyme, with a pI of 5.0. The optimal pH and temperature of the enzyme were 7.5 and 28 degrees C respectively, using alpha-N-rho-tosyl-L-arginine-methyl ester (L-TAME) as substrate. Assays of thermal stability indicated that 61% of the enzyme activity was preserved after 1 h of pre-treatment at 42 degrees C. Haemoglobin, bovine serum albumin (BSA), ovalbumin, fibrinogen, collagen, gelatin and peptide substrates containing arginine in an ester bond and amide substrates containing hydrophobic residues at the P1 site were hydrolysed by this extracellular protease. The insulin beta-chain was also hydrolysed by the enzyme and many peptidic bonds were susceptible to the protease action, and 4 of them (L11-V12, E3-A14, L15-Y16 and Y16-L17) were identified. Inhibition studies suggested that the enzyme belongs to the serine protease class inhibited by calcium and manganese and activated by zinc. These findings show that this enzyme of L. amazonensis is a novel serine protease, which differs from all known flagellate proteases characterized. PMID:16038400

  6. Covalent structure of human haptoglobin: a serine protease homolog.

    PubMed Central

    Kurosky, A; Barnett, D R; Lee, T H; Touchstone, B; Hay, R E; Arnott, M S; Bowman, B H; Fitch, W M

    1980-01-01

    The complete amino acid sequences and the disulfide arrangements of the two chains of human haptoglobin 1-1 were established. The alpha 1 and beta chains of haptoglobin contain 83 and 245 residues, respectively. Comparison of the primary structure of haptoglobin with that of the chymotrypsinogen family of serine proteases revealed a significant degree of chemical similarity. The probability was less than 10(-5) that the chemical similarity of the beta chain of haptoglobin to the proteases was due to chance. The amino acid sequence of the beta chain of haptoglobin is 29--33% identical to bovine trypsin, bovine chymotrypsin, porcine elastase, human thrombin, or human plasmin. Comparison of haptoglobin alpha 1 chain to activation peptide regions of the zymogens revealed an identity of 25% to the fifth "kringle" region of the activation peptide of plasminogen. The probability was less than 0.014 that this similarity was due to chance. These results strongly indicate haptoglobin to be a homolog of the chymotrypsinogen family of serine proteases. Alignment of the beta-chain sequence of haptoglobin to the serine proteases is remarkably consistent except for an insertion of 16 residues in the region corresponding to the methionyl loop of the serine proteases. The active-site residues typical of the serine proteases, histidine-57 and serine-195, are replaced in haptoglobin by lysine and alanine, respectively; however, aspartic acid-102 and the trypsin specificity, residue, aspartic acid-189, do occur in haptoglobin. Haptoglobin and the serine proteases represent a striking example of homologous proteins with different biological functions. PMID:6997877

  7. Salt stress represses production of extracellular proteases in Bacillus pumilus.

    PubMed

    Liu, R F; Huang, C L; Feng, H

    2015-01-01

    Bacillus pumilus is able to secrete subtilisin-like prote-ases, one of which has been purified and characterized biochemically, demonstrating great potential for use in industrial applications. In the current study, the biosynthesis and transcription of extracellular pro-teases in B. pumilus (BA06) under salt stress were investigated using various methods, including a proteolytic assay, zymogram analysis, and real-time PCR. Our results showed that total extracellular proteolytic activity, both in fermentation broth and on milk-containing agar plates, was considerably repressed by salt in a dosage-dependent manner. As Bacillus species usually secret multiple extracellular proteases, a vari-ety of individual extracellular protease encoding genes were selected for real-time PCR analysis. It was shown that proteases encoded by the aprE and aprX genes were the major proteases in the fermentation broth in terms of their transcripts in B. pumilus. Further, transcription of aprE, aprX, and epr genes was indeed repressed by salt stress. In con-trast, transcription of other genes (e.g., vpr and wprA) was not repressed or significantly affected by the salt. Conclusively, salt stress represses total extracellular proteolytic activity in B. pumilus, which can largely be ascribed to suppression of the major protease-encoding genes (aprE, aprX) at the transcriptional level. In contrast, transcription of other pro-tease-encoding genes (e.g., vpr, wprA) was not repressed by salt stress. PMID:25966269

  8. Protease nexin-1 regulates retinal vascular development.

    PubMed

    Selbonne, Sonia; Francois, Deborah; Raoul, William; Boulaftali, Yacine; Sennlaub, Florian; Jandrot-Perrus, Martine; Bouton, Marie-Christine; Arocas, Véronique

    2015-10-01

    We recently identified protease nexin-1 (PN-1) or serpinE2, as a possibly underestimated player in maintaining angiogenic balance. Here, we used the well-characterized postnatal vascular development of newborn mouse retina to further investigate the role and the mechanism of action of PN-1 in physiological angiogenesis. The development of retinal vasculature was analysed by endothelial cell staining with isolectin B4. PN-1-deficient (PN-1(-/-)) retina displayed increased vascularization in the postnatal period, with elevated capillary thickness and density, compared to their wild-type littermate (WT). Moreover, PN-1(-/-) retina presented more veins/arteries than WT retina. The kinetics of retinal vasculature development, retinal VEGF expression and overall retinal structure were similar in WT and PN-1(-/-) mice, but we observed a hyperproliferation of vascular cells in PN-1(-/-) retina. Expression of PN-1 was analysed by immunoblotting and X-Gal staining of retinas from mice expressing beta-galactosidase under a PN-1 promoter. PN-1 was highly expressed in the first week following birth and then progressively decreased to a low level in adult retina where it localized on the retinal arteries. PCR arrays performed on mouse retinal RNA identified two angiogenesis-related factors, midkine and Smad5, that were overexpressed in PN-1(-/-) newborn mice and this was confirmed by RT-PCR. Both the higher vascularization and the overexpression of midkine and Smad5 mRNA were also observed in gastrocnemius muscle of PN-1(-/-) mice, suggesting that PN-1 interferes with these pathways. Together, our results demonstrate that PN-1 strongly limits physiological angiogenesis and suggest that modulation of PN-1 expression could represent a new way to regulate angiogenesis. PMID:26109427

  9. HIV-1 protease-induced apoptosis

    PubMed Central

    2014-01-01

    Background Apoptosis is one of the presumptive causes of CD4+ T cell depletion during HIV infection and progression to AIDS. However, the precise role of HIV-1 in this process remains unexplained. HIV-1 protease (PR) has been suggested as a possible factor, but a direct link between HIV-1 PR enzymatic activity and apoptosis has not been established. Results Here, we show that expression of active HIV-1 PR induces death in HeLa and HEK-293 cells via the mitochondrial apoptotic pathway. This conclusion is based on in vivo observations of the direct localization of HIV-1 PR in mitochondria, a key player in triggering apoptosis. Moreover, we observed an HIV-1 PR concentration-dependent decrease in mitochondrial membrane potential and the role of HIV-1 PR in activation of caspase 9, PARP cleavage and DNA fragmentation. In addition, in vitro data demonstrated that HIV-1 PR mediates cleavage of mitochondrial proteins Tom22, VDAC and ANT, leading to release of AIF and Hsp60 proteins. By using yeast two-hybrid screening, we also identified a new HIV-1 PR interaction partner, breast carcinoma-associated protein 3 (BCA3). We found that BCA3 accelerates p53 transcriptional activity on the bax promoter, thus elevating the cellular level of pro-apoptotic Bax protein. Conclusion In summary, our results describe the involvement of HIV-1 PR in apoptosis, which is caused either by a direct effect of HIV-1 PR on mitochondrial membrane integrity or by its interaction with cellular protein BCA3. PMID:24886575

  10. Regulation of Extracellular Protease Production in Bacillus cereus T: Characterization of Mutants Producing Altered Amounts of Protease

    PubMed Central

    Aronson, A. I.; Angelo, N.; Holt, S. C.

    1971-01-01

    Twenty-nine mutants of Bacillus cereus T were selected on casein agar for their inability to produce large amounts of extracellular protease. They all formed spores, and 27 were also auxotrophs for purines or pyrimidines. Upon reversion to prototrophy, a large fraction regained the capacity to produce protease. Conversely, reversion to normal protease production resulted in loss of the purine or pyrimidine requirement in a large fraction of the revertants. One spontaneous low-protease-producing pyrimidine auxotroph studied in detail grew as well as the wild type and produced spores which were identical to those produced by the wild type on the basis of heat resistance, dipicolinic acid content, density, and appearance in the electron microscope. The rate of protein turnover in the mutant was the same as the wild type. The mutant did grow poorly, however, when casein was the principal carbon source. A mutant excreting 5 to 10 times as much protease as the wild type was isolated as a secondary mutation from the hypoproducer discussed above. Loss of the pyrimidine requirement in this case did not alter the regulation of protease production. Although the secondary mutant grew somewhat faster in most media than the wild type, the final cell yield was lower. The spores of this mutant appeared to have excess coat on the basis of both electron microscopic and chemical studies. There appear to be closely related but distinct catabolic controls for both extracellular protease and spore formation. These controls can be dissociated as for the hypoproducers but can also appear integrated as for the hyperprotease producer. Images PMID:4104235

  11. Protein unfolding and degradation by the AAA+ Lon protease.

    PubMed

    Gur, Eyal; Vishkautzan, Marina; Sauer, Robert T

    2012-02-01

    AAA+ proteases employ a hexameric ring that harnesses the energy of ATP binding and hydrolysis to unfold native substrates and translocate the unfolded polypeptide into an interior compartment for degradation. What determines the ability of different AAA+ enzymes to unfold and thus degrade different native protein substrates is currently uncertain. Here, we explore the ability of the E. coli Lon protease to unfold and degrade model protein substrates beginning at N-terminal, C-terminal, or internal degrons. Lon has historically been viewed as a weak unfoldase, but we demonstrate robust and processive unfolding/degradation of some substrates with very stable protein domains, including mDHFR and titin(I27) . For some native substrates, Lon is a more active unfoldase than related AAA+ proteases, including ClpXP and ClpAP. For other substrates, this relationship is reversed. Thus, unfolding activity does not appear to be an intrinsic enzymatic property. Instead, it depends on the specific protease and substrate, suggesting that evolution has diversified rather than optimized the protein unfolding activities of different AAA+ proteases. PMID:22162032

  12. Genetics of Extracellular Protease Production in SACCHAROMYCOPSIS LIPOLYTICA

    PubMed Central

    Ogrydziak, David M.; Mortimer, Robert K.

    1977-01-01

    Mutants of Saccharomycopsis lipolytica with reduced ability to produce zones of clearing on skim-milk agar plates were isolated and their properties studied. For 18 mutants it was possible to score unambiguously segregants of crosses between these mutants and wild type for extracellular protease production. These mutants all produce reduced levels of extracellular protease in liquid culture. The mutations are recessive and are in nuclear genes. The 18 mutations define 10 or 11 complementation groups, no two of which are closely linked. Mutants in four of the complementation groups also produced reduced levels of extracellular RNAse, and the reduced levels of extracellular protease and RNAse production segregate together. Five of the mutants exhibited reduced mating frequency, and one mutant was osmotic remedial for extracellular protease production. These results demonstrate that many genes can affect extracellular protease production. Besides mutations in the structural gene and in regulatory genes, mutations are likely to be in genes involved in steps common to the production of several extracellular enzymes or in genes coding for cell wall or membrane components necessary for extracellular enzyme production. PMID:17248782

  13. Natural products from Garcinia brasiliensis as Leishmania protease inhibitors.

    PubMed

    Pereira, Ivan O; Assis, Diego M; Juliano, Maria A; Cunha, Rodrigo L O R; Barbieri, Clara L; do Sacramento, Luis V S; Marques, Marcos J; dos Santos, Marcelo H

    2011-06-01

    The infections by protozoans of the genus Leishmania are a major worldwide health problem, with high endemicity in developing countries. The drugs of choice for the treatment of leishmaniasis are the pentavalent antimonials, which cause renal and cardiac toxicity. As part of a search for new drugs against leishmaniasis, we evaluated the in vitro Leishmania protease inhibition activity of extracts (hexanic, ethyl-acetate, and ethanolic) and fukugetin, a bioflavonoid purified from the ethyl-acetate extract of the pericarp of the fruit of Garcinia brasiliensis, a tree native to Brazilian forests. The isolated compound was characterized by using spectral analyses with nuclear magnetic resonance, mass spectroscopy, ultraviolet, and infrared techniques. The ethyl-acetate extract and the compound fukugetin showed significant activity as inhibitors of Leishmania's proteases, with mean (±SD) IC(50) (50% inhibition concentration of protease activity) values of 15.0±1.3 μg/mL and 3.2±0.5 μM/mL, respectively, characterizing a bioguided assay. In addition, this isolated compound showed no activity against promastigote and amastigote forms of L. (L.) amazonensis and mammalian cells. These results suggest that fukugetin is a potent protease inhibitor of L. (L.) amazonensis and does not cause toxicity in mammalian or Leishmania cells in vitro. This study provides new perspectives on the development of novel drugs that have leishmanicidal activity obtained from natural products and that target the parasite's proteases. PMID:21554130

  14. Extracellular proteases are released by ciliates in defined seawater microcosms.

    PubMed

    Thao, Ngo Vy; Nozawa, Akino; Obayashi, Yumiko; Kitamura, Shin-Ichi; Yokokawa, Taichi; Suzuki, Satoru

    2015-08-01

    The biodegradation of proteins in seawater requires various proteases which are commonly thought to be mainly derived from heterotrophic bacteria. We, however, found that protists showed a high protease activity and continuously produced trypsin-type enzymes. The free-living marine heterotrophic ciliate Paranophrys marina together with an associated bacterium was isolated and used for microcosm incubation with different concentrations of killed bacteria as food for 10 days. The results showed that the co-existence of the ciliate with its associated bacterium produced a significant protease activity in both cell-associated and cell-free fractions while that in the associated bacterium only microcosm was negligible. The protease profiles are different between cell-associated and cell-free fractions, and a trypsin-type enzyme hydrolyzing Boc-Val-Leu-Lys-MCA was detected throughout the period in the presence of ciliates. This suggests that ciliates release proteases into the surrounding environment which could play a role in protein digestion outside cells. It has been previously suggested that bacteria are the major transformers in seawater. We here present additional data which indicates that protists, or at least ciliates with their specific enzymes, are a potential player in organic matter degradation in water columns. PMID:26115436

  15. Protease Inhibitors in View of Peptide Substrate Databases

    PubMed Central

    2016-01-01

    Protease substrate profiling has nowadays almost become a routine task for experimentalists, and the knowledge on protease peptide substrates is easily accessible via the MEROPS database. We present a shape-based virtual screening workflow using vROCS that applies the information about the specificity of the proteases to find new small-molecule inhibitors. Peptide substrate sequences for three to four substrate positions of each substrate from the MEROPS database were used to build the training set. Two-dimensional substrate sequences were converted to three-dimensional conformations through mutation of a template peptide substrate. The vROCS query was built from single amino acid queries for each substrate position considering the relative frequencies of the amino acids. The peptide-substrate-based shape-based virtual screening approach gives good performance for the four proteases thrombin, factor Xa, factor VIIa, and caspase-3 with the DUD-E data set. The results show that the method works for protease targets with different specificity profiles as well as for targets with different active-site mechanisms. As no structure of the target and no information on small-molecule inhibitors are required to use our approach, the method has significant advantages in comparison with conventional structure- and ligand-based methods. PMID:27247997

  16. Revisiting rubisco as a protein substrate for insect midgut proteases.

    PubMed

    Bhardwaj, Usha; Bhardwaj, Amit; Kumar, Rakesh; Leelavathi, Sadhu; Reddy, Vanga Siva; Mazumdar-Leighton, Sudeshna

    2014-01-01

    Gene fragments encoding the large subunit (LS) of Rubisco (RBCL) were cloned from various species of host plants of phytophagous Lepidoptera and expressed as recombinant proteins in Escherichia coli. Recombinant RBCLs were compared among each other along with casein and native Rubisco as proteinaceous substrates for measuring total midgut protease activities of fourth instar larvae of Helicoverpa armigera feeding on casein, Pieris brassicae feeding on cauliflower, and Antheraea assamensis feeding on Litsea monopetala and Persea bombycina. Cognate rRBCL (from the pertinent host plant species) substrates performed similar to noncognate rRBCL reflecting the conserved nature of encoding genes and the versatile use of these recombinant proteins. Casein and recombinant RBCL generally outperformed native Rubisco as substrates, except where inclusion of a reducing agent in the enzyme assay likely unfolded the plant proteins. Levels of total midgut protease activities detected in A. assamensis larvae feeding on two primary host species were similar, suggesting that the suite(s) of digestive enzymes in these insects could hydrolyze a plant protein efficiently. Protease activities detected in the presence of protease inhibitors and the reducing agent dithiothreitol (DTT) suggested that recombinant RBCL was a suitable protein substrate for studying insect proteases using in vitro enzyme assays and substrate zymography. PMID:24338735

  17. Characterization, biomedical and agricultural applications of protease inhibitors: A review.

    PubMed

    Shamsi, Tooba Naz; Parveen, Romana; Fatima, Sadaf

    2016-10-01

    This review describes Protease Inhibitors (PIs) which target or inhibit proteases, protein digesting enzymes. These proteases play a crucial task in many biological events including digestion, blood coagulation, apoptosis etc. Regardless of their crucial roles, they need to be checked regularly by PIs as their excess may possibly damage host organism. On basis of amino acid composition of PIs where Protease-PI enzymatic reactions occur i.e. serine, cysteine, and aspartic acid, they are classified. Nowadays, various PIs are being worked upon to fight various parasitic or viral diseases including malaria, schistosomiasis, colds, flu', dengue etc. They prevent an ongoing process begun by carcinogen exposure by keeping a check on metastasis. They also possess potential to reduce carcinogen-induced, increased levels of gene amplification to almost normal levels. Some PIs can principally be used for treatment of hypertension and congestive heart failure by blocking conversion of angiotensin I to angiotensin II for example Angiotensin-converting enzyme inhibitors (ACEIs). Also PIs target amyloid β-peptide (Aβ) level in brain which is prime responsible for development of Alzheimer's Disease (AD). Also, PIs inhibit enzymatic activity of HIV-1 Protease Receptor (PR) by preventing cleavage events in Gag and Gag-Pol that result in production of non-virulent virus particles. PMID:26955746

  18. Mitochondrial cereblon functions as a Lon-type protease

    PubMed Central

    Kataoka, Kosuke; Nakamura, China; Asahi, Toru; Sawamura, Naoya

    2016-01-01

    Lon protease plays a major role in the protein quality control system in mammalian cell mitochondria. It is present in the mitochondrial matrix, and degrades oxidized and misfolded proteins, thereby protecting the cell from various extracellular stresses, including oxidative stress. The intellectual disability-associated and thalidomide-binding protein cereblon (CRBN) contains a large, highly conserved Lon domain. However, whether CRBN has Lon protease-like function remains unknown. Here, we determined if CRBN has a protective function against oxidative stress, similar to Lon protease. We report that CRBN partially distributes in mitochondria, suggesting it has a mitochondrial function. To specify the mitochondrial role of CRBN, we mitochondrially expressed CRBN in human neuroblastoma SH-SY5Y cells. The resulting stable SH-SY5Y cell line showed no apparent effect on the mitochondrial functions of fusion, fission, and membrane potential. However, mitochondrially expressed CRBN exhibited protease activity, and was induced by oxidative stress. In addition, stably expressed cells exhibited suppressed neuronal cell death induced by hydrogen peroxide. These results suggest that CRBN functions specifically as a Lon-type protease in mitochondria. PMID:27417535

  19. Alkaline protease from Thermoactinomyces sp. RS1 mitigates industrial pollution.

    PubMed

    Verma, Amit; Ansari, Mohammad W; Anwar, Mohmmad S; Agrawal, Ruchi; Agrawal, Sanjeev

    2014-05-01

    Proteases have found a wide application in the several industrial processes, such as laundry detergents, protein recovery or solubilization, prion degradation, meat tenderizations, and in bating of hides and skins in leather industries. But the main hurdle in industrial application of proteases is their economical production on a large scale. The present investigation aimed to exploit the locally available inexpensive agricultural and household wastes for alkaline protease production using Thermoactinomyces sp. RS1 via solid-state fermentation (SSF) technique. The alkaline enzyme is potentially useful as an additive in commercial detergents to mitigate pollution load due to extensive use of caustic soda-based detergents. Thermoactinomyces sp. RS1 showed good protease production under SSF conditions of 55 °C, pH 9, and 50 % moisture content with potato peels as solid substrate. The presented findings revealed that crude alkaline protease produced by Thermoactinomyces sp. RS1 via SSF is of potential application in silver recovery from used X-ray films. PMID:24122212

  20. Protease Inhibitors in View of Peptide Substrate Databases.

    PubMed

    Waldner, Birgit J; Fuchs, Julian E; Schauperl, Michael; Kramer, Christian; Liedl, Klaus R

    2016-06-27

    Protease substrate profiling has nowadays almost become a routine task for experimentalists, and the knowledge on protease peptide substrates is easily accessible via the MEROPS database. We present a shape-based virtual screening workflow using vROCS that applies the information about the specificity of the proteases to find new small-molecule inhibitors. Peptide substrate sequences for three to four substrate positions of each substrate from the MEROPS database were used to build the training set. Two-dimensional substrate sequences were converted to three-dimensional conformations through mutation of a template peptide substrate. The vROCS query was built from single amino acid queries for each substrate position considering the relative frequencies of the amino acids. The peptide-substrate-based shape-based virtual screening approach gives good performance for the four proteases thrombin, factor Xa, factor VIIa, and caspase-3 with the DUD-E data set. The results show that the method works for protease targets with different specificity profiles as well as for targets with different active-site mechanisms. As no structure of the target and no information on small-molecule inhibitors are required to use our approach, the method has significant advantages in comparison with conventional structure- and ligand-based methods. PMID:27247997

  1. Hydrophobic Core Flexibility Modulates Enzyme Activity in HIV-1 Protease

    SciTech Connect

    Mittal, Seema; Cai, Yufeng; Nalam, Madhavi N.L.; Bolon, Daniel N.A.; Schiffer, Celia A.

    2012-09-11

    Human immunodeficiency virus Type-1 (HIV-1) protease is crucial for viral maturation and infectivity. Studies of protease dynamics suggest that the rearrangement of the hydrophobic core is essential for enzyme activity. Many mutations in the hydrophobic core are also associated with drug resistance and may modulate the core flexibility. To test the role of flexibility in protease activity, pairs of cysteines were introduced at the interfaces of flexible regions remote from the active site. Disulfide bond formation was confirmed by crystal structures and by alkylation of free cysteines and mass spectrometry. Oxidized and reduced crystal structures of these variants show the overall structure of the protease is retained. However, cross-linking the cysteines led to drastic loss in enzyme activity, which was regained upon reducing the disulfide cross-links. Molecular dynamics simulations showed that altered dynamics propagated throughout the enzyme from the engineered disulfide. Thus, altered flexibility within the hydrophobic core can modulate HIV-1 protease activity, supporting the hypothesis that drug resistant mutations distal from the active site can alter the balance between substrate turnover and inhibitor binding by modulating enzyme activity.

  2. Streptomyces serine protease (SAM-P20): recombinant production, characterization, and interaction with endogenous protease inhibitor.

    PubMed Central

    Taguchi, S; Suzuki, M; Kojima, S; Miura, K; Momose, H

    1995-01-01

    Previously, we isolated a candidate for an endogenous target enzyme(s) of the Streptomyces subtilisin inhibitor (SSI), termed SAM-P20, from a non-SSI-producing mutant strain (S. Taguchi, A. Odaka, Y. Watanabe, and H. Momose, Appl. Environ. Microbiol. 61:180-186, 1995). In this study, in order to investigate the detailed enzymatic properties of this protease, an overproduction system of recombinant SAM-P20 was established in Streptomyces coelicolor with the SSI gene promoter. The recombinant SAM-P20 was purified by salting out and by two successive ion-exchange chromatographies to give a homogeneous band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Partial peptide mapping and amino acid composition analysis revealed that the recombinant SAM-P20 was identical to natural SAM-P20. From the results for substrate specificity and inhibitor sensitivity, SAM-P20 could be categorized as a chymotrypsin-like protease with an arginine-cleavable activity, i.e., a serine protease with broad substrate specificity. For proteolytic activity, the optimal pH was 10.0 and the optimal temperature was shifted from 50 to 80 degrees C by the addition of 10 mM calcium ion. The strong stoichiometric inhibition of SAM-P20 activity by SSI dimer protein occurred in a subunit molar ratio of these two proteins of about 1, and an inhibitor constant of SSI toward SAM-P20 was estimated to be 8.0 x 10(-10) M. The complex formation of SAM-P20 and SSI was monitored by analytical gel filtration, and a complex composed of two molecules of SAM-P20 and one dimer molecule of SSI was detected, in addition to a complex of one molecule of SAM-P20 bound to one dimer molecule of SSI. The reactive site of SSI toward SAM-P20 was identified as Met-73-Val-74 by sequence analysis of the modified form of SSI, which was produced by the acidification of the complex of SSI and SAM-P20. This reactive site is the same that toward an exogenous target enzyme, subtilisin BPN'. PMID:7592444

  3. Human mast cell tryptase: Multiple cDNAs and genes reveal a multigene serine protease family

    SciTech Connect

    Vanderslice, P.; Ballinger, S.M., Tam, E.K.; Goldstein, S.M.; Craik, C.S.; Caughey, G.H. )

    1990-05-01

    Three different cDNAs and a gene encoding human skin mast cell tryptase have been cloned and sequenced in their entirety. The deduced amino acid sequences reveal a 30-amino acid prepropeptide followed by a 245-amino acid catalytic domain. The C-terminal undecapeptide of the human preprosequence is identical in dog tryptase and appears to be part of a prosequence unique among serine proteases. The differences among the three human tryptase catalytic domains include the loss of a consensus N-glycosylation site in one cDNA, which may explain some of the heterogeneity in size and susceptibility to deglycosylation seen in tryptase preparations. All three tryptase cDNAs are distinct from a recently reported cDNA obtained from a human lung mast cell library. A skin tryptase cDNA was used to isolate a human tryptase gene, the exons of which match one of the skin-derived cDNAs. The organization of the {approx}1.8-kilobase-pair tryptase gene is unique and is not closely related to that of any other mast cell or leukocyte serine protease. The 5{prime} regulatory regions of the gene share features with those of other serine proteases, including mast cell chymase, but are unusual in being separated from the protein-coding sequence by an intron. High-stringency hybridization of a human genomic DNA blot with a fragment of the tryptase gene confirms the presence of multiple tryptase genes. These findings provide genetic evidence that human mast cell tryptases are the products of a multigene family.

  4. REASSESSING THE ROLE OF THE SECRETED PROTEASE CPAF IN CHLAMYDIA TRACHOMATIS INFECTION THROUGH GENETIC APPROACHES

    PubMed Central

    Snavely, Emily A.; Kokes, Marcela; Dunn, Joe D.; Saka, Hector A.; Nguyen, Bidong D.; Bastidas, Robert J.; McCafferty, Dewey G.; Valdivia, Raphael H.

    2014-01-01

    The secreted Chlamydia protease CPAF cleaves a defined set of mammalian and Chlamydia proteins in vitro. As a result, this protease has been proposed to modulate a range of bacterial and host cellular functions. However, it has recently come into question the extent to which many of its identified substrates constitute bona fide targets of proteolysis in infected host cell rather than artifacts of post lysis degradation. Here we clarify the role played by CPAF in cellular models of infection by analyzing Chlamydia trachomatis mutants deficient for CPAF activity. Using reverse genetic approaches, we identified two C. trachomatis strains possessing nonsense, loss-of-function mutations in cpa (CT858), and a third strain containing a mutation in Type II secretion (T2S) machinery that inhibited CPAF activity by blocking zymogen secretion and subsequent proteolytic maturation into the active hydrolase. HeLa cells infected with T2S− or CPAF− C. trachomatis mutants lacked detectable in vitro CPAF proteolytic activity, and were not defective for cellular traits that have been previously attributed to CPAF activity, including resistance to staurosporine-induced apoptosis, Golgi fragmentation, altered NFκB-dependent gene expression, and resistance to reinfection. However, CPAF-deficient mutants did display impaired generation of infectious elementary bodies (EBs), indicating an important role for this protease in the full replicative potential of C. trachomatis. In addition, we provide compelling evidence in live cells that CPAF-mediated protein processing of at least two host protein targets, vimentin filaments and the nuclear envelope protein Lamin-associated protein 1 (LAP1), occurs rapidly after the loss of the inclusion membrane integrity, but before loss of plasma membrane permeability and cell lysis. CPAF-dependent processing of host proteins correlates with a loss of inclusion membrane integrity, and so we propose that CPAF plays a role late in infection

  5. Massive accumulation of luminal protease-deficient axonal lysosomes at Alzheimer’s disease amyloid plaques

    PubMed Central

    Gowrishankar, Swetha; Yuan, Peng; Wu, Yumei; Schrag, Matthew; Paradise, Summer; Grutzendler, Jaime; De Camilli, Pietro; Ferguson, Shawn M.

    2015-01-01

    Through a comprehensive analysis of organellar markers in mouse models of Alzheimer’s disease, we document a massive accumulation of lysosome-like organelles at amyloid plaques and establish that the majority of these organelles reside within swollen axons that contact the amyloid deposits. This close spatial relationship between axonal lysosome accumulation and extracellular amyloid aggregates was observed from the earliest stages of β-amyloid deposition. Notably, we discovered that lysosomes that accumulate in such axons are lacking in multiple soluble luminal proteases and thus are predicted to be unable to efficiently degrade proteinaceous cargos. Of relevance to Alzheimer’s disease, β-secretase (BACE1), the protein that initiates amyloidogenic processing of the amyloid precursor protein and which is a substrate for these proteases, builds up at these sites. Furthermore, through a comparison between the axonal lysosome accumulations at amyloid plaques and neuronal lysosomes of the wild-type brain, we identified a similar, naturally occurring population of lysosome-like organelles in neuronal processes that is also defined by its low luminal protease content. In conjunction with emerging evidence that the lysosomal maturation of endosomes and autophagosomes is coupled to their retrograde transport, our results suggest that extracellular β-amyloid deposits cause a local impairment in the retrograde axonal transport of lysosome precursors, leading to their accumulation and a blockade in their further maturation. This study both advances understanding of Alzheimer’s disease brain pathology and provides new insights into the subcellular organization of neuronal lysosomes that may have broader relevance to other neurodegenerative diseases with a lysosomal component to their pathology. PMID:26124111

  6. Diversity of cultivable protease-producing bacteria in sediments of Jiaozhou Bay, China

    PubMed Central

    Zhang, Xi-Ying; Han, Xiao-Xu; Chen, Xiu-Lan; Dang, Hong-Yue; Xie, Bin-Bin; Qin, Qi-Long; Shi, Mei; Zhou, Bai-Cheng; Zhang, Yu-Zhong

    2015-01-01

    Although protease-producing bacteria are key players in the degradation of organic nitrogen and essential for the nitrogen recycling in marine sediments, diversity of both these bacteria and their extracellular proteases is still largely unknown. This study investigated the diversity of the cultivable protease-producing bacteria and their extracellular proteases in the sediments of the eutrophied Jiaozhou Bay, China through phylogenetic analysis and protease inhibitor tests. The abundance of the cultivable protease-producing bacteria was up to 104 cells/g in all six sediment samples. The cultivated protease-producing bacteria mostly belonged to the phyla Proteobacteria and Firmicutes with the predominant genera being Photobacterium (39.4%), Bacillus (25.8%), and Vibrio (19.7%). Protease inhibitor tests revealed that extracellular proteases secreted by the bacteria were mainly serine proteases and/or metalloproteases with relatively low proportions of cysteine proteases. This study represents the first comprehensive analysis on the diversity of protease-producing bacteria and their extracellular proteases in sediments of a eutrophic bay. PMID:26441943

  7. Lvserpin3 is involved in shrimp innate immunity via the inhibition of bacterial proteases and proteases involved in prophenoloxidase system.

    PubMed

    Liu, Yongjie; Liu, Tao; Hou, Fujun; Wang, Xianzong; Liu, Xiaolin

    2016-01-01

    Serine protease inhibitor, represented by serpin, plays an important inhibitory role on proteases involved in the immune responses. To clarify the immune characterizations of serpin, a novel serpin (Lvserpin3) encoding for 410 amino acids with a 23-amino acid signal peptide and a serpin domain was identified from the Pacific white shrimp Litopenaeus vannamei. Lvserpin3 expressed strongest in hepatopancreas, and was significantly up-regulated in the early stage upon Vibrio anguillarum, Micrococcus lysodeikticus or White Spot Syndrome Virus (WSSV) infection. Suppression of Lvserpin3 by dsRNA led to a significant increase in the transcripts of LvPPAF, LvproPO and phenoloxidase (PO) activity, and also led to the high cumulative mortality. The recombinant Lvserpin3 protein (rLvserpin3) inhibited the proteases secreted by M. lysodeikticus and Bacillus subtilis, and further exhibited inhibitory role on the growth of B. subtilis and M. lysodeikticu. Moreover, rLvserpin3 was found to be able to block the activation of prophenoloxidase system. Taken together, the results imply that Lvserpin3 may be involved in shrimp innate immunity via the inhibition of bacterial proteases and proteases involved in prophenoloxidase system. PMID:26432049

  8. (Processing and targeting of the thiol protease aleurain)

    SciTech Connect

    Rogers, J.C.

    1990-01-01

    Our goal for work during the past two years under this Grant was to characterize the barley thiol protease, aleurain, to determine if it is secreted or retained intracellularly in aleurone cells, and to begin to elucidate structural features that might control targeting of the protein to its final destination. We have shown that aleurain is synthesized as a proenzyme with two N-linked oligosaccharide chains, one high mannose-type and one complex-type. Aleurain undergoes processing to mature form by removal of an Nterminal prosegment, and is retained intracellularly; it cannot be detected among proteins secreted from aleurone cells. Treatment of aleurone cells with tunicamycin to prevent glycosylation of aleurain does not prevent processing of the unglycosylated form. The N-terminal portion of aleurain's prosegment is homologous to the comparable region in two yeast vacuolar proteases, where that region is known to contain the signal necessary for targeting the proteases to the vacuole. 18 refs., 7 figs.

  9. Acid phosphatase and protease activities in immobilized rat skeletal muscles

    NASA Technical Reports Server (NTRS)

    Witzmann, F. A.; Troup, J. P.; Fitts, R. H.

    1982-01-01

    The effect of hind-limb immobilization on selected Iysosomal enzyme activities was studied in rat hing-limb muscles composed primarily of type 1. 2A, or 2B fibers. Following immobilization, acid protease and acid phosphatase both exhibited signifcant increases in their activity per unit weight in all three fiber types. Acid phosphatase activity increased at day 14 of immobilization in the three muscles and returned to control levels by day 21. Acid protease activity also changed biphasically, displaying a higher and earlier rise than acid phosphatase. The pattern of change in acid protease, but not acid phosphatase, closely parallels observed muscle wasting. The present data therefore demonstrate enhanced proteolytic capacity of all three fiber types early during muscular atrophy. In addition, the data suggest a dependence of basal hydrolytic and proteolytic activities and their adaptive response to immobilization on muscle fiber composition.

  10. The emerging roles of serine protease cascades in the epidermis.

    PubMed

    Ovaere, Petra; Lippens, Saskia; Vandenabeele, Peter; Declercq, Wim

    2009-09-01

    It has become clear in recent years that serine proteases have an important role in epidermal homeostasis, and the signaling cascades are gradually being identified. For example, matriptase, prostasin and furin are implicated in a cascade that could activate ENaC, leading to epidermal barrier formation and hydration, probably in part through their involvement in filaggrin processing. Kallikreins can form a signaling cascade to coordinate corneocyte desquamation. Knowledge is also emerging about how endogenous inhibitors, calcium and pH control these cascades. It is becoming clear that some skin pathologies are associated with deregulated serine protease activity. Therefore, a deeper knowledge of the regulation of these serine protease cascades could form the basis for development of appropriate treatments for skin disorders such as Netherton syndrome. PMID:19726197

  11. Cleaning protocols for crystallization robots: preventing protease contamination.

    PubMed

    Naschberger, Andreas; Fürnrohr, Barbara G; Dunzendorfer-Matt, Theresia; Bonagura, Christopher A; Wright, David; Scheffzek, Klaus; Rupp, Bernhard

    2015-01-01

    The protease in the commonly used commercial low-foam enzyme cleaner Zymit cannot be completely blocked by EDTA, a widely used inhibitor of metalloproteases, at concentrations of up to 5 mM. Severe protein degradation was observed in crystallization drops after EDTA-containing wash steps unless residual Zymit protease was removed with NaOH at a concentration of at least 0.1 M. Wash steps with 0.1% SDS were also ineffective in completely removing the remaining Zymit activity. Protocols including wash steps with at least 0.1 M NaOH, as for example specified in the original ZENM protocol, are recommended to completely deactivate Zymit protease activity. PMID:25615978

  12. Tobacco Etch Virus protease: A shortcut across biotechnologies.

    PubMed

    Cesaratto, Francesca; Burrone, Oscar R; Petris, Gianluca

    2016-08-10

    About thirty years ago, studies on the RNA genome of Tobacco Etch Virus revealed the presence of an efficient and specific protease, called Tobacco Etch Virus protease (TEVp), that was part of the Nuclear Inclusion a (NIa) enzyme. TEVp is an efficient and specific protease of 27kDa that has become a valuable biotechnological tool. Nowadays TEVp is a unique endopeptidase largely exploited in biotechnology from industrial applications to in vitro and in vivo cellular studies. A number of TEVp mutants with different rate of cleavage, stability and specificity have been reported. Similarly, a panel of different target cleavage sites, derived from the canonical ENLYFQ-G/S site, has been established. In this review we describe these aspects of TEVp and some of its multiple applications. A particular focus is on the use and molecular biology of TEVp in living cells and organisms. PMID:27312702

  13. Fighting an enemy within: cytoplasmic inhibitors of bacterial cysteine proteases.

    PubMed

    Potempa, Jan; Golonka, Ewa; Filipek, Renata; Shaw, Lindsey N

    2005-08-01

    The genes encoding secreted, broad-spectrum activity cysteine proteases of Staphylococcus spp. (staphopains) and Streptococcus pyogenes (streptopain, SpeB) are genetically linked to genes encoding cytoplasmic inhibitors. While staphopain inhibitors have lipocalin-like folds, streptopain is inhibited by a protein bearing the scaffold of the enzyme profragment. Bioinformatic analysis of other prokaryotic genomes has revealed that two more species may utilize this same genetic arrangement to control streptopain-like proteases with lipocalin-like inhibitors, while three other species may employ a C-terminally located domain that resembles the profragment. This apparently represents a novel system that bacteria use to control the intracellular activity of their proteases. PMID:16045606

  14. Substrate specificity of the ubiquitin and Ubl proteases

    PubMed Central

    Ronau, Judith A; Beckmann, John F; Hochstrasser, Mark

    2016-01-01

    Conjugation and deconjugation of ubiquitin and ubiquitin-like proteins (Ubls) to cellular proteins are highly regulated processes integral to cellular homeostasis. Most often, the C-termini of these small polypeptides are attached to lysine side chains of target proteins by an amide (isopeptide) linkage. Deubiquitinating enzymes (DUBs) and Ubl-specific proteases (ULPs) comprise a diverse group of proteases that recognize and remove ubiquitin and Ubls from their substrates. How DUBs and ULPs distinguish among different modifiers, or different polymeric forms of these modifiers, remains poorly understood. The specificity of ubiquitin/Ubl-deconjugating enzymes for particular substrates depends on multiple factors, ranging from the topography of specific substrate features, as in different polyubiquitin chain types, to structural elements unique to each enzyme. Here we summarize recent structural and biochemical studies that provide insights into mechanisms of substrate specificity among various DUBs and ULPs. We also discuss the unexpected specificities of non-eukaryotic proteases in these families. PMID:27012468

  15. Protease-specific nanosensors for magnetic resonance imaging.

    PubMed

    Schellenberger, Eyk; Rudloff, Franziska; Warmuth, Carsten; Taupitz, Matthias; Hamm, Bernd; Schnorr, Jörg

    2008-12-01

    Imaging of enzyme activity is a central goal of molecular imaging. With the introduction of fluorescent smart probes, optical imaging has become the modality of choice for experimental in vivo detection of enzyme activity. Here, we present a novel high-relaxivity nanosensor that is suitable for in vivo imaging of protease activity by magnetic resonance imaging. Upon specific protease cleavage, the nanoparticles rapidly switch from a stable low-relaxivity stealth state to become adhesive, aggregating high-relaxivity particles. To demonstrate the principle, we chose a cleavage motif of matrix metalloproteinase 9 (MMP-9), an enzyme important in inflammation, atherosclerosis, tumor progression, and many other diseases with alterations of the extracellular matrix. On the basis of clinically tested very small iron oxide particles (VSOP), the MMP-9-activatable protease-specific iron oxide particles (PSOP) have a hydrodynamic diameter of only 25 nm. PSOP are rapidly activated, resulting in aggregation and increased T2*-relaxivity. PMID:19007261

  16. New therapeutic strategies in HCV: second-generation protease inhibitors.

    PubMed

    Clark, Virginia C; Peter, Joy A; Nelson, David R

    2013-02-01

    Telaprevir and boceprevir are the first direct-acting antiviral agents approved for use in HCV treatment and represent a significant advance in HCV therapy. However, these first-generation drugs also have significant limitations related to thrice-daily dosing, clinically challenging side-effect profiles, low barriers to resistance and a lack of pan-genotype activity. A second wave of protease inhibitors are in phase II and III trials and promise to provide a drug regimen with a better dosing schedule and improved tolerance. These second-wave protease inhibitors will probably be approved in combination with PEG-IFN and Ribavirin (RBV), as well as future all-oral regimens. The true second-generation protease inhibitors are in earlier stages of development and efficacy data are anxiously awaited as they may provide pan-genotypic antiviral activity and a high genetic barrier to resistance. PMID:23286850

  17. Evolution of Primary Protease Inhibitor Resistance Mutations during Protease Inhibitor Salvage Therapy

    PubMed Central

    Kantor, Rami; Fessel, W. Jeffrey; Zolopa, Andrew R.; Israelski, Dennis; Shulman, Nancy; Montoya, Jose G.; Harbour, Michael; Schapiro, Jonathan M.; Shafer, Robert W.

    2002-01-01

    In order to track the evolution of primary protease inhibitor (PI) resistance mutations in human immunodeficiency virus type 1 (HIV-1) isolates, baseline and follow-up protease sequences were obtained from patients undergoing salvage PI therapy who presented initially with isolates containing a single primary PI resistance mutation. Among 78 patients meeting study selection criteria, baseline primary PI resistance mutations included L90M (42% of patients), V82A/F/T (27%), D30N (21%), G48V (6%), and I84V (4%). Despite the switching of treatment to a new PI, primary PI resistance mutations present at the baseline persisted in 66 of 78 (85%) patients. D30N persisted less frequently than L90M (50% versus 100%, respectively; P < 0.001) and V82A/F/T (50% versus 81%, respectively; P = 0.05). HIV-1 isolates from 38 (49%) patients failing PI salvage therapy developed new primary PI resistance mutations including L90M, I84V, V82A, and G48V. Common combinations of primary and secondary PI resistance mutations after salvage therapy included mutations at amino acid positions 10, 82, and 46 and/or 54 in 16 patients; 10, 90, and 71 and/or 73 in 14 patients; 10, 73, 84, 90, and 46 and/or 54 in 5 patients; 10, 48, and 82 in 5 patients; and 30, 88 and 90 in 5 patients. In summary, during salvage PI therapy, most HIV-1 isolates with a single primary PI resistance mutation maintained their original mutations, and 49% developed additional primary PI resistance mutations. The persistence of L90M, V82A/F/T, G48V, and I84V during salvage therapy suggests that these mutations play a role in clinical resistance to multiple PIs. PMID:11897594

  18. HIV-1 Protease Mutations and Protease Inhibitor Cross-Resistance▿ † ‡

    PubMed Central

    Rhee, Soo-Yon; Taylor, Jonathan; Fessel, W. Jeffrey; Kaufman, David; Towner, William; Troia, Paolo; Ruane, Peter; Hellinger, James; Shirvani, Vivian; Zolopa, Andrew; Shafer, Robert W.

    2010-01-01

    The effects of many protease inhibitor (PI)-selected mutations on the susceptibility to individual PIs are unknown. We analyzed in vitro susceptibility test results on 2,725 HIV-1 protease isolates. More than 2,400 isolates had been tested for susceptibility to fosamprenavir, indinavir, nelfinavir, and saquinavir; 2,130 isolates had been tested for susceptibility to lopinavir; 1,644 isolates had been tested for susceptibility to atazanavir; 1,265 isolates had been tested for susceptibility to tipranavir; and 642 isolates had been tested for susceptibility to darunavir. We applied least-angle regression (LARS) to the 200 most common mutations in the data set and identified a set of 46 mutations associated with decreased PI susceptibility of which 40 were not polymorphic in the eight most common HIV-1 group M subtypes. We then used least-squares regression to ascertain the relative contribution of each of these 46 mutations. The median number of mutations associated with decreased susceptibility to each PI was 28 (range, 19 to 32), and the median number of mutations associated with increased susceptibility to each PI was 2.5 (range, 1 to 8). Of the mutations with the greatest effect on PI susceptibility, I84AV was associated with decreased susceptibility to eight PIs; V32I, G48V, I54ALMSTV, V82F, and L90M were associated with decreased susceptibility to six to seven PIs; I47A, G48M, I50V, L76V, V82ST, and N88S were associated with decreased susceptibility to four to five PIs; and D30N, I50L, and V82AL were associated with decreased susceptibility to fewer than four PIs. This study underscores the greater impact of nonpolymorphic mutations compared with polymorphic mutations on decreased PI susceptibility and provides a comprehensive quantitative assessment of the effects of individual mutations on susceptibility to the eight clinically available PIs. PMID:20660676

  19. Trichuris suis: thiol protease activity from adult worms.

    PubMed

    Hill, D E; Sakanari, J A

    1997-01-01

    Trichuris suis, the whipworm of swine, causes anemia, weight loss, anorexia, mucohemorrhagic diarrhea, and death in heavy infections. A zinc metalloprotease has been suggested to play a role in the severe enteric pathology associated with infection and the infiltration of opportunistic bacteria into deeper tissues in the swine colon. In this study, a thiol protease from gut extracts of adult T. suis and from excretory/secretory components (E/S) of adult worms was characterized using fluorogenic peptide substrates and protein substrate gels. The protease cleaved the fluorogenic substrate Z-Phe-Arg-AMC, and this cleavage was completely inhibited by the thiol protease inhibitors E-64, leupeptin, Z-Phe-Ala-CH2F, and Z-Phe-Arg-CH2F. Gelatin substrate gels and fluorescence assays using both the gut and the stichosome extracts and E/S revealed enhanced activity when 2 mM dithiothreitol or 5 mM cysteine was included in the incubation buffer, and optimal activity was seen over a pH range of 5.5 to 8.5. Incubation of gut extracts or E/S material with inhibitors of aspartic, serine, or metalloproteases had no effect on the cleavage of Z-Phe-Arg-AMC. Thiol protease activity was found in extracts of gut tissue but not in the extracts of stichocytes of adult worms. N-terminal amino acid sequencing of the protease revealed sequence homologies with cathepsin B-like thiol protease identified from parasitic and free-living nematodes. PMID:9024202

  20. Purification and properties of an extracellular protease from Myxococcus virescens.

    PubMed Central

    Gnosspelius, G

    1978-01-01

    An extracellular protease from Myxococcus virescens was purified by phosphate precipitation, gel exclusion, and ion-exchange chromatography. The enzyme appeared homogeneous upon disc electrophoresis. The molecular weight of the protease was estimated to be 26,000. The enzyme was rapidly inactivated by ethylenediaminetetraacetate, but the activity could be partially restored by divalent cations. Diisopropylphosphorofluoridate inhibited enzyme activity completely. Michaelis-Menten kinetics were obeyed with casein and hemoglobin as substrates. First-order kinetics were obtained with elastin as the substrate, provided trypsin was in excess. Petidolytic activity indicated that the peptide bonds hydrolyzed by the enzyme were mainly those involving amino acids with nonpolar side chains. PMID:22536

  1. CLIP proteases and Plasmodium melanization in Anopheles gambiae.

    PubMed

    Barillas-Mury, Carolina

    2007-07-01

    Melanization is a potent immune response mediated by phenoloxidase (PO). Multiple Clip-domain serine proteases (CLIP) regulate PO activation as part of a complex cascade of proteases that are cleaved sequentially. The role of several CLIP as key activators or suppressors of the melanization responses of Anopheles gambiae to Plasmodium berghei (murine malaria) has been established recently using a genome-wide reverse genetics approach. Important differences in regulation of PO activation between An. gambiae strains were also identified. This review summarizes these findings and discusses our current understanding of the An. gambiae melanization responses to Plasmodium. PMID:17512801

  2. Optimization of some additives to improve protease production under SSF.

    PubMed

    Tunga, R; Banerjee, R; Bhattacharyya, B C

    2001-11-01

    In a locally isolated Rhizopus oryzae strain highest-production of protease (388.54/g wheat bran) was observed in presence of Tween-80 and dioctyl sodium sulfosuccinate individually at 40mg/g wheat bran concentration. Under solid state fermentation biotin (0.0025mg/g wheat bran); Ca2+ (0.05mg/g wheat bran) and 1-Naphthyl acetic acid (0.01mg/g wheat bran) also showed some inducing effect on the synthesis of the enzyme protease by solid state fermentation. PMID:11906108

  3. Schistosome serine protease inhibitors: parasite defense or homeostasis?

    PubMed Central

    Lopez Quezada, Landys A.; McKerrow, James H.

    2016-01-01

    Serpins are a structurally conserved family of macromolecular inhibitors found in numerous biological systems. The completion and annotation of the genomes of Schistosoma mansoni and Schistosoma japonicum has enabled the identification by phylogenetic analysis of two major serpin clades. S. mansoni shows a greater multiplicity of serpin genes, perhaps reflecting adaptation to infection of a human host. Putative targets of schistosome serpins can be predicted from the sequence of the reactive center loop (RCL). Schistosome serpins may play important roles in both post-translational regulation of schistosome-derived proteases, as well as parasite defense mechanisms against the action of host proteases. PMID:21670886

  4. Surface location of a Bacteroides gingivalis glycylprolyl protease.

    PubMed Central

    Grenier, D; McBride, B C

    1989-01-01

    Various immunological methods were used to localize a glycylprolyl protease previously isolated from Bacteroides gingivalis ATCC 33277. The results obtained by enzyme-linked immunosorbent assay, indirect immunofluorescence staining, and indirect immunogold labeling suggest that the glycylprolyl protease is present on the surface of the cell outer membrane and is specific to B. gingivalis strains. The enzyme was removed from the cell envelope by treatment of the whole cells with sodium dodecyl sulfate, Triton X-100, sodium deoxycholate, and proteinase K. Images PMID:2807524

  5. Recombinant expression, refolding, purification and characterization of Pseudomonas aeruginosa protease IV in Escherichia coli.

    PubMed

    Zhao, Mingzhi; Cai, Man; Wu, Feilin; Zhang, Yao; Xiong, Zhi; Xu, Ping

    2016-10-01

    Several protease IV enzymes are widely used in proteomic research. Specifically, protease IV from Pseudomonas aeruginosa has lysyl endopeptidase activity. Here, we report the recombinant expression, refolding, activation, and purification of this protease in Escherichia coli. Proteolytic instability of the activated intermediate, a major obstacle for efficient production, is controlled through ammonium sulfate precipitation. The purified protease IV exhibits superior lysyl endopeptidase activity compared to a commercial product. PMID:27260967

  6. Gastrointestinal absorption and biological activities of serine and cysteine proteases of animal and plant origin: review on absorption of serine and cysteine proteases

    PubMed Central

    Lorkowski, Gerhard

    2012-01-01

    Research has confirmed that peptides and larger protein molecules pass through the mucosal barrier of the gastrointestinal tract. Orally administered serine and cysteine proteases of plant and animal origin also reach blood and lymph as intact, high molecular weight and physiologically active protein molecules. Their absorption may be supported by a self-enhanced paracellular transport mechanism resulting in sub-nanomolar concentration of transiently free protease molecules or, in a complex with anti-proteases, at higher concentrations. Data from pharmacokinetic investigations reveals dose linearity for maximum plasma levels of free proteases not unusual for body proteases and a high inter-individual variability. There is no interference with each other after oral administration of protease combinations, and absorption follows an unusual invasion and elimination kinetic due to slow velocity of absorption and a fast 100% protein binding to anti-proteases. Oral application of proteases leads to increased proteolytic serum activity and increased plasma concentrations of the corresponding anti-proteases. Their biological activity is determined by their proteolytic activity as free proteases on soluble peptides/proteins or cell surface receptors (e.g. protease activated receptors) and their activity in the complex formed with their specific and/or unspecific anti-proteases. The anti-protease-complexes, during immune reaction and injuries often loaded with different cytokines, are cleared from body fluids and tissue by receptor mediated endocytosis on hepatocytes and/or blood cells. Oral administration of enteric coated tablets containing proteolytic enzymes of plant and animal origin may be a safe method to stabilize, positively influence or enhance physiological and immunological processes during disease processes and in healthy consumers. PMID:22461953

  7. Regulation of distinct pools of amyloid β-protein by multiple cellular proteases.

    PubMed

    Leissring, Malcolm A; Turner, Anthony J

    2013-01-01

    Alzheimer's disease (AD) is a progressive, age-related neurodegenerative disorder characterized by extracellular and intracellular deposition of the amyloid β-protein (Aβ). The study of rare, familial forms of AD has shown that sustained elevations in the production of Aβ (either all forms or specific pathogenic variants thereof) are sufficient to trigger the full spectrum of cognitive and histopathological features of the disease. Although the exact cause or causes remain unknown, emerging evidence suggests that impairments in the clearance of Aβ, after it is produced, may underlie the vast majority of sporadic AD cases. This review focuses on Aβ-degrading proteases (AβDPs), which have emerged as particularly important mediators of Aβ clearance. A wide variety of proteases that - by virtue of their particular regional and subcellular localization profiles - define distinct pools of Aβ have been identified. Different pools of Aβ, in turn, may contribute differentially to the pathogenesis of the disease. The study of individual AβDPs, therefore, promises to offer new insights into the mechanistic basis of AD pathogenesis and, ultimately, may facilitate the development of effective methods for its prevention or treatment or both. PMID:23953275

  8. Regulation of distinct pools of amyloid β-protein by multiple cellular proteases

    PubMed Central

    2013-01-01

    Alzheimer’s disease (AD) is a progressive, age-related neurodegenerative disorder characterized by extracellular and intracellular deposition of the amyloid β-protein (Aβ). The study of rare, familial forms of AD has shown that sustained elevations in the production of Aβ (either all forms or specific pathogenic variants thereof) are sufficient to trigger the full spectrum of cognitive and histopathological features of the disease. Although the exact cause or causes remain unknown, emerging evidence suggests that impairments in the clearance of Aβ, after it is produced, may underlie the vast majority of sporadic AD cases. This review focuses on Aβ-degrading proteases (AβDPs), which have emerged as particularly important mediators of Aβ clearance. A wide variety of proteases that – by virtue of their particular regional and subcellular localization profiles – define distinct pools of Aβ have been identified. Different pools of Aβ, in turn, may contribute differentially to the pathogenesis of the disease. The study of individual AβDPs, therefore, promises to offer new insights into the mechanistic basis of AD pathogenesis and, ultimately, may facilitate the development of effective methods for its prevention or treatment or both. PMID:23953275

  9. PEGylation of Bacterial Cocaine Esterase for Protection against Protease Digestion and Immunogenicity

    PubMed Central

    Park, Jun-Beom; Kwon, Young Min; Lee, Tien-Yi; Brim, Remy; Ko, Mei-Chuan; Sunahara, Roger K.; Woods, James H.; Yang, Victor C.

    2009-01-01

    Enhancing cocaine metabolism by administration of cocaine esterase (CocE) has been considered as a promising treatment strategy for cocaine overdose and addiction, as CocE is the most efficient native enzyme yet identified for metabolizing the naturally occurring cocaine. A major obstacle to the clinical application of CocE, however, lies in its thermo-instability, rapid degradation by circulating proteases, and potential immunogenicity. PEGylation, namely by modifying a protein or peptide compound via attachment of polyethylene glycol (PEG) chains, has been proven to overcome such problems and was therefore exploited in this CocE investigation. The PEG-CocE conjugates prepared in this study showed a purity of greater than 93.5 %. Attachment of PEG to CocE apparently inhibited the binding of anti-CocE antibodies to the conjugate, as demonstrated by the enzyme-linked immunosorbent assay (ELISA) assay. In addition, PEGylation yielded protection to CocE against thermal degradation and protease digestion. Furthermore, preliminary in vivo results suggested that, similarly to native CocE, the PEG-CocE conjugates were able to protect animals from cocaine-induced toxic effects. Overall, this study provides evidence that the PEGylation may serve as a tool to prolong CocE functionality in the circulation and reduce its potential immunogenicity. PMID:19857534

  10. The role of protease-activated receptor type 2 in nociceptive signaling and pain.

    PubMed

    Mrozkova, P; Palecek, J; Spicarova, D

    2016-07-18

    Protease-activated receptors (PARs) belong to the G-protein-coupled receptor family, that are expressed in many body tissues especially in different epithelial cells, mast cells and also in neurons and astrocytes. PARs play different physiological roles according to the location of their expression. Increased evidence supports the importance of PARs activation during nociceptive signaling and in the development of chronic pain states. This short review focuses on the role of PAR2 receptors in nociceptive transmission with the emphasis on the modulation at the spinal cord level. PAR2 are cleaved and subsequently activated by endogenous proteases such as tryptase and trypsin. In vivo, peripheral and intrathecal administration of PAR2 agonists induces thermal and mechanical hypersensitivity that is thought to be mediated by PAR2-induced release of pronociceptive neuropeptides and modulation of different receptors. PAR2 activation leads also to sensitization of transient receptor potential channels (TRP) that are crucial for nociceptive signaling and modulation. PAR2 receptors may play an important modulatory role in the development and maintenance of different pathological pain states and could represent a potential target for new analgesic treatments. PMID:27070742

  11. Co-lethality studied as an asset against viral drug escape: the HIV protease case

    PubMed Central

    2010-01-01

    Background Co-lethality, or synthetic lethality is the documented genetic situation where two, separately non-lethal mutations, become lethal when combined in one genome. Each mutation is called a "synthetic lethal" (SL) or a co-lethal. Like invariant positions, SL sets (SL linked couples) are choice targets for drug design against fast-escaping RNA viruses: mutational viral escape by loss of affinity to the drug may induce (synthetic) lethality. Results From an amino acid sequence alignment of the HIV protease, we detected the potential SL couples, potential SL sets, and invariant positions. From the 3D structure of the same protein we focused on the ones that were close to each other and accessible on the protein surface, to possibly bind putative drugs. We aligned 24,155 HIV protease amino acid sequences and identified 290 potential SL couples and 25 invariant positions. After applying the distance and accessibility filter, three candidate drug design targets of respectively 7 (under the flap), 4 (in the cantilever) and 5 (in the fulcrum) amino acid positions were found. Conclusions These three replication-critical targets, located outside of the active site, are key to our anti-escape strategy. Indeed, biological evidence shows that 2/3 of those target positions perform essential biological functions. Their mutational variations to escape antiviral medication could be lethal, thus limiting the apparition of drug-resistant strains. Reviewers This article was reviewed by Arcady Mushegian, Shamil Sunyaev and Claus Wilke. PMID:20565756

  12. A Mycobacterium avium subsp. paratuberculosis Predicted Serine Protease Is Associated with Acid Stress and Intraphagosomal Survival.

    PubMed

    Kugadas, Abirami; Lamont, Elise A; Bannantine, John P; Shoyama, Fernanda M; Brenner, Evan; Janagama, Harish K; Sreevatsan, Srinand

    2016-01-01

    The ability to maintain intra-cellular pH is crucial for bacteria and other microbes to survive in diverse environments, particularly those that undergo fluctuations in pH. Mechanisms of acid resistance remain poorly understood in mycobacteria. Although, studies investigating acid stress in M. tuberculosis are gaining traction, few center on Mycobacterium avium subsp. paratuberculosis (MAP), the etiological agent of chronic enteritis in ruminants. We identified a MAP acid stress response network involved in macrophage infection. The central node of this network was MAP0403, a predicted serine protease that shared an 86% amino acid identity with MarP in M. tuberculosis. Previous studies confirmed MarP as a serine protease integral to maintaining intra-bacterial pH and survival in acid in vitro and in vivo. We show that MAP0403 is upregulated in infected macrophages and MAC-T cells that coincided with phagosome acidification. Treatment of mammalian cells with bafilomcyin A1, a potent inhibitor of phagosomal vATPases, diminished MAP0403 transcription. MAP0403 expression was also noted in acidic medium. A surrogate host, M. smegmatis mc(2) 155, was designed to express MAP0403 and when exposed to either macrophages or in vitro acid stress had increased bacterial cell viability, which corresponds to maintenance of intra-bacterial pH in acidic (pH = 5) conditions, compared to the parent strain. These data suggest that MAP0403 may be the equivalent of MarP in MAP. Future studies confirming MAP0403 as a serine protease and exploring its structure and possible substrates are warranted. PMID:27597934

  13. A Mycobacterium avium subsp. paratuberculosis Predicted Serine Protease Is Associated with Acid Stress and Intraphagosomal Survival

    PubMed Central

    Kugadas, Abirami; Lamont, Elise A.; Bannantine, John P.; Shoyama, Fernanda M.; Brenner, Evan; Janagama, Harish K.; Sreevatsan, Srinand

    2016-01-01

    The ability to maintain intra-cellular pH is crucial for bacteria and other microbes to survive in diverse environments, particularly those that undergo fluctuations in pH. Mechanisms of acid resistance remain poorly understood in mycobacteria. Although, studies investigating acid stress in M. tuberculosis are gaining traction, few center on Mycobacterium avium subsp. paratuberculosis (MAP), the etiological agent of chronic enteritis in ruminants. We identified a MAP acid stress response network involved in macrophage infection. The central node of this network was MAP0403, a predicted serine protease that shared an 86% amino acid identity with MarP in M. tuberculosis. Previous studies confirmed MarP as a serine protease integral to maintaining intra-bacterial pH and survival in acid in vitro and in vivo. We show that MAP0403 is upregulated in infected macrophages and MAC-T cells that coincided with phagosome acidification. Treatment of mammalian cells with bafilomcyin A1, a potent inhibitor of phagosomal vATPases, diminished MAP0403 transcription. MAP0403 expression was also noted in acidic medium. A surrogate host, M. smegmatis mc2 155, was designed to express MAP0403 and when exposed to either macrophages or in vitro acid stress had increased bacterial cell viability, which corresponds to maintenance of intra-bacterial pH in acidic (pH = 5) conditions, compared to the parent strain. These data suggest that MAP0403 may be the equivalent of MarP in MAP. Future studies confirming MAP0403 as a serine protease and exploring its structure and possible substrates are warranted. PMID:27597934

  14. Antibody Responses to Norovirus Genogroup GI.1 and GII.4 Proteases in Volunteers Administered Norwalk Virus

    PubMed Central

    Ajami, Nadim J.; Barry, Meagan A.; Carrillo, Berenice; Muhaxhiri, Zana; Neill, Frederick H.; Prasad, B. V. Venkataram; Opekun, Antone R.; Gilger, Mark A.; Graham, David Y.; Atmar, Robert L.

    2012-01-01

    An assay was developed to detect antibodies against two norovirus proteases among participants in a Norwalk virus (GI.1) challenge study. Prechallenge seroprevalence was lower against the protease from the homologous GI.1 virus than against protease from a heterologous GII.4 strain. Seroresponses were detected for 14 of 19 (74%) infected persons. PMID:23035177

  15. 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. PMID:26178876

  16. AAA proteases in mitochondria: diverse functions of membrane-bound proteolytic machines.

    PubMed

    Tatsuta, Takashi; Langer, Thomas

    2009-11-01

    FtsH/AAA proteases comprise a distinct family of membrane-bound, ATP-dependent proteases present in eubacteria and eukaryotic cells, where they are confined to mitochondria and chloroplasts. Here, we will summarize versatile functions of AAA proteases within mitochondria, which ensure mitochondrial integrity and cell survival, acting both as quality control and processing enzymes. PMID:19781639

  17. Teaching Foundational Topics and Scientific Skills in Biochemistry within the Conceptual Framework of HIV Protease

    ERIC Educational Resources Information Center

    Johnson, R. Jeremy

    2014-01-01

    HIV protease has served as a model protein for understanding protein structure, enzyme kinetics, structure-based drug design, and protein evolution. Inhibitors of HIV protease are also an essential part of effective HIV/AIDS treatment and have provided great societal benefits. The broad applications for HIV protease and its inhibitors make it a…

  18. Development of a high-throughput pyrosequencing assay for monitoring temporal evolution and resistance associated variant emergence in the Hepatitis C virus protease coding-region.

    PubMed

    Irving, William L; Rupp, Daniel; McClure, C Patrick; Than, Lwin Mar; Titman, Andrew; Ball, Jonathan K; Steinmann, Eike; Bartenschlager, Ralf; Pietschmann, Thomas; Brown, Richard J P

    2014-10-01

    A new generation of drugs targeting the non-structural (NS) proteins of the Hepatitis C virus (HCV) will substantially increase treatment success rates, reducing global infections. Amongst the NS proteins, the NS3 protease represents an important drug target, responsible for liberation of mature NS proteins from the nascent HCV polyprotein and suppression of host innate immunity. Despite this, the evolutionary stability of the genomic locus encoding the NS3 protease is poorly characterized in chronic HCV infection. To address this shortfall, we developed a high-throughput amplicon pyrosequencing protocol and utilised it to monitor NS3 protease coding-sequence evolution for over a decade in two patients. Although patient-specific evolutionary trends were apparent, the protease amino acid population consensus remained stable with a massive excess of synonymous mutations observed, confirming this locus is under strong purifying selection during chronic infection within individual patients. No evidence for continuous immune escape was detected. Additionally, both patients failed protease inhibitor (PI) therapy and protease sequence diversity pre- and post-therapy were also assessed. No baseline resistance associated variants (RAVs) contributed to treatment failure. Significant reductions in viral diversity were observed post-PI therapy, indicating a population bottleneck occurred. The genetic vestiges of this bottleneck were still detectable 18months after therapy discontinuation. Although significant enrichment of the Q80L mutation was observed in one patient, genetic and phenotypic data reveal no detectable RAV persistence post-therapy failure. Together this investigation provides a sensitive and reproducible high-throughput framework to interrogate viral sequence diversity at high-resolution, with potential applications for routine monitoring of treatment regimens. This study also reveals novel insights into the evolutionary processes that shape NS3 sequence

  19. Inhibitors of HGFA, Matriptase, and Hepsin Serine Proteases: A Nonkinase Strategy to Block Cell Signaling in Cancer.

    PubMed

    Han, Zhenfu; Harris, Peter K W; Jones, Darin E; Chugani, Ryan; Kim, Tommy; Agarwal, Manjula; Shen, Wei; Wildman, Scott A; Janetka, James W

    2014-11-13

    Hepatocyte growth factor activators (HGFA), matriptase, and hepsin are S1 family trypsin-like serine proteases. These proteases proteolytically cleave the single-chain zymogen precursors, pro-HGF (hepatocyte growth factor), and pro-MSP (macrophage stimulating protein) into active heterodimeric forms. HGF and MSP are activating ligands for the oncogenic receptor tyrosine kinases (RTKs), c-MET and RON, respectively. We have discovered the first substrate-based ketothiazole inhibitors of HGFA, matriptase and hepsin. The compounds were synthesized using a combination of solution and solid-phase peptide synthesis (SPPS). Compounds were tested for protease inhibition using a kinetic enzyme assay employing fluorogenic peptide substrates. Highlighted HGFA inhibitors are Ac-KRLR-kt (5g), Ac-SKFR-kt (6c), and Ac-SWLR-kt (6g) with K is = 12, 57, and 63 nM, respectively. We demonstrated that inhibitors block the conversion of native pro-HGF and pro-MSP by HGFA with equivalent potency. Finally, we show that inhibition causes a dose-dependent decrease of c-MET signaling in MDA-MB-231 breast cancer cells. This preliminary investigation provides evidence that HGFA is a promising therapeutic target in breast cancer and other tumor types driven by c-MET and RON. PMID:25408834

  20. Inhibitors of HGFA, Matriptase, and Hepsin Serine Proteases: A Nonkinase Strategy to Block Cell Signaling in Cancer

    PubMed Central

    2014-01-01

    Hepatocyte growth factor activators (HGFA), matriptase, and hepsin are S1 family trypsin-like serine proteases. These proteases proteolytically cleave the single-chain zymogen precursors, pro-HGF (hepatocyte growth factor), and pro-MSP (macrophage stimulating protein) into active heterodimeric forms. HGF and MSP are activating ligands for the oncogenic receptor tyrosine kinases (RTKs), c-MET and RON, respectively. We have discovered the first substrate-based ketothiazole inhibitors of HGFA, matriptase and hepsin. The compounds were synthesized using a combination of solution and solid-phase peptide synthesis (SPPS). Compounds were tested for protease inhibition using a kinetic enzyme assay employing fluorogenic peptide substrates. Highlighted HGFA inhibitors are Ac-KRLR-kt (5g), Ac-SKFR-kt (6c), and Ac-SWLR-kt (6g) with Kis = 12, 57, and 63 nM, respectively. We demonstrated that inhibitors block the conversion of native pro-HGF and pro-MSP by HGFA with equivalent potency. Finally, we show that inhibition causes a dose-dependent decrease of c-MET signaling in MDA-MB-231 breast cancer cells. This preliminary investigation provides evidence that HGFA is a promising therapeutic target in breast cancer and other tumor types driven by c-MET and RON. PMID:25408834

  1. An alternative pathway for fibrinolysis. I. The cleavage of fibrinogen by leukocyte proteases at physiologic pH.

    PubMed Central

    Plow, E F; Edgington, T S

    1975-01-01

    An alternative fibrinolytic system, active at physiological pH, is present in peripheral blood leukocytes. The fibrinolytic proteases localized predominantly in the leukocyte granules are capable of degrading both fibrinogen and fibrin, and plasmin activity does not contribute significantly to this proteolytic event. The specificity of the alternative fibrinolytic proteases for fibrinogen and the characteristics of the derivative cleavage fragments are clearly distinguishable from the classical plasmin system. The high molecular weight derivatives of fibrinogen, generated by the alternative system, under physiological conditions, are larger than the plasmin-generated X fragment, exhibit immunoelectrophoretic mobility comparable to native fibrinogen, and are not coagulable by thrombin. Analysis of the constituent polypeptide chains of the fragments reveals cleavage of the Aalpha, Bbeta, and gamma chains of fibrinogen. The lower molecular weight derivatives of fibrinogen, generated by the alternative system, are structurally distinct from previously described fibrinogen degradation products and exhibit potent anticoagulant activity. This anticoagulant activity can be attributed to interference with normal fibrin polymerization. The proteases of the alternative fibrinolytic systems are actively secreted by leukocytes when stimulated to undergo a nonlytic release reaction. These results provide direct evidence for a fibrinolytic system resident in leukocyte granules that is associated with the leukocyte release reaction and is capable of generating unique fibrinogen cleavage fragments. Images PMID:237938

  2. SUMO proteases ULP1c and ULP1d are required for development and osmotic stress responses in Arabidopsis thaliana.

    PubMed

    Castro, Pedro Humberto; Couto, Daniel; Freitas, Sara; Verde, Nuno; Macho, Alberto P; Huguet, Stéphanie; Botella, Miguel Angel; Ruiz-Albert, Javier; Tavares, Rui Manuel; Bejarano, Eduardo Rodríguez; Azevedo, Herlânder

    2016-09-01

    Sumoylation is an essential post-translational regulator of plant development and the response to environmental stimuli. SUMO conjugation occurs via an E1-E2-E3 cascade, and can be removed by SUMO proteases (ULPs). ULPs are numerous and likely to function as sources of specificity within the pathway, yet most ULPs remain functionally unresolved. In this report we used loss-of-function reverse genetics and transcriptomics to functionally characterize Arabidopsis thaliana ULP1c and ULP1d SUMO proteases. GUS reporter assays implicated ULP1c/d in various developmental stages, and subsequent defects in growth and germination were uncovered using loss-of-function mutants. Microarray analysis evidenced not only a deregulation of genes involved in development, but also in genes controlled by various drought-associated transcriptional regulators. We demonstrated that ulp1c ulp1d displayed diminished in vitro root growth under low water potential and higher stomatal aperture, yet leaf transpirational water loss and whole drought tolerance were not significantly altered. Generation of a triple siz1 ulp1c ulp1d mutant suggests that ULP1c/d and the SUMO E3 ligase SIZ1 may display separate functions in development yet operate epistatically in response to water deficit. We provide experimental evidence that Arabidopsis ULP1c and ULP1d proteases act redundantly as positive regulators of growth, and operate mainly as isopeptidases downstream of SIZ1 in the control of water deficit responses. PMID:27325215

  3. Hydrogen peroxide induces lysosomal protease alterations in PC12 cells.

    PubMed

    Lee, Daniel C; Mason, Ceceile W; Goodman, Carl B; Holder, Maurice S; Kirksey, Otis W; Womble, Tracy A; Severs, Walter B; Palm, Donald E

    2007-09-01

    Alterations in lysosomal proteases have been implicated in many neurodegenerative diseases. The current study demonstrates a concentration-dependent decrease in PC12 cell viability and transient changes in cystatin C (CYSC), cathepsin B (CATB), cathepsin D (CATD) and caspase-3 following exposure to H2O2. Furthermore, activation of CATD occurred following exposure to H2O2 and cysteine protease suppression, while inhibition of CATD with pepstatin A significantly improved cell viability. Additionally, significant PARP cleavage, suggestive of caspase-3-like activity, was observed following H2O2 exposure, while inhibition of caspase-3 significantly increased cell viability compared to H2O2 administration alone. Collectively, our data suggest that H2O2 induced cell death is regulated at least in part by caspase-3 and CATD. Furthermore, cysteine protease suppression increases CATD expression and activity. These studies provide insight for alternate pathways and potential therapeutic targets of cell death associated with oxidative stress and lysosomal protease alterations. PMID:17440810

  4. Design, synthesis, and activity of nanocellulosic protease sensors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Here we contrast the molecular assembly, and biochemical utility of nanocellulosic materials prepared from cotton and wood as protease sensors. The cotton-based nanocellulosic substrates were prepared in a variety of ways to produce nanocrystals, films and aerogels, which were derivatized with eithe...

  5. Processing and targeting of the thiol protease aleurain: Progress report

    SciTech Connect

    Rogers, J.C.

    1988-01-01

    This study addresses the processing and targeting of the thiol protease aleurain in monocots. A probe derived from the aleurain cDNA specific for the 5'-most 400 bp (a region encoding the first 140 amino acids of the preprotein hybridized to at least 3 separate elements in the barley genome; only one represented the aleurain gene. In contrast, a probe specific for the remaining 2/23 of the cDNA (representing the protease domain) hybridized to only a single copy sequence. To know if this pattern pertained in other, closely related, monocots, we probed Southern blots of genomic DNA from maize, rye, oats, sorghum, and pearl millet with each probe. In each instance except for maize DNA, the 5' domain probe hybridizes to several fragments in addition to those identified by the protease domain probe. Presumable the darkest hybridization in each represents the fragment carrying the sequences homologous to barley aleurain. The fragments from a given restriction enzyme identified by the protease domain probe in sorghum, millet, and maize, were indistinguishable in size indicating that the gene sequences, as well as flanking DNA, are so well conserved among the group that the location of the hexanucleotide sequences have not diverged. (3 refs., 3 figs.)

  6. Botulinum neurotoxin: a deadly protease with applications to human medicine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Botulinum neurotoxins (BoNTs) are some of the most potent biological toxins to humans. They are synthesized by the gram-positive, spore-forming bacterium Clostridium botulinum. BoNT is secreted from the bacterium as a ~150 kDa polypeptide which is cleaved by bacterial or host proteases into a ~50 kD...

  7. Protease determination using an optimized alcohol enzyme electrode.

    PubMed

    Bardeletti, G; Carillon, C

    1993-12-01

    A new method for the determination of protease activities is described. In this large family, trypsin is used as a protease model that cleaves the ethyl or methyl ester of artificial substrates producing ethanol or methanol. Alcohol is detected using an alcohol oxidase enzyme electrode. The H2O2 production that occurs is measured amperometrically. At 30 degrees C, in a 0.1M phosphate buffer, pH 7.5, the enzyme electrode response for ethanol was calibrated at 3.10(-6)-3.10(-3)M and for methanol from 3.10(-7) to 4.10(-4)M in the cell measurement. Trypsin levels as determined by the proposed method and by a conventional spectrophotometric method are in good agreement when using the same measurement conditions. A detection limit of 10 U.L-1 and a linear calibration curve of 10-100,000 U.L-1 in the sample were obtained. Measuring time for the required trypsin solution concentration was from 4 min (for the most dilute samples) to 1 min (for the most concentrate samples). In a typical experiment, protease measurements did not inactivate the alcohol oxidase on the probe, nor did a more classical use for alcohol detection. The procedure developed could permit any protease estimation on the condition that they hydrolyze ester bonds from synthetic substrate. PMID:8109959

  8. Hepatitis C Virus NS3/4A Protease Inhibitors.

    PubMed

    López-Labrador, Francesc-Xavier

    2008-11-01

    Chronic hepatitis C virus infection is a global problem worldwide due to the lack of an effective therapy (the current standard of care treatment is effective in about 40-50% of the cases), and the difficulties in developing a protective vaccine. Chronic infection progresses to end-stage liver disease and liver failure in a considerable number of infected individuals. Once liver function is compromised, the only reliable therapeutic intervention is liver transplantation. Unfortunately, re-infection of the graft is unavoidable, and a new chronic hepatitis is early established in transplant recipients, that can result in graft loss. Thus, there is an urgent need for new, specifically targeted therapies for the treatment of HCV chronic infection. Among the viral proteins, the NS3/4A protease and the NS5b RNA-dependent RNA-polymerase, essential for the virus life cycle, have concentrated the efforts in the development of new antivirals, and some promising ones have already entered clinical trials. In particular, inhibitors of the HCV NS3/4A protease are the most advanced in clinical development. This review summarizes the available data for the most important HCV NS3/4A protease inhibitors in development, the most recent patents of these type of compounds, the envisioned options for future HCV therapies, and the eventual impact of HCV genetic variability on resistance to new NS3/4A protease inhibitors. PMID:18991798

  9. Flap Dynamics in Aspartic Proteases: A Computational Perspective.

    PubMed

    Mahanti, Mukul; Bhakat, Soumendranath; Nilsson, Ulf J; Söderhjelm, Pär

    2016-08-01

    Recent advances in biochemistry and drug design have placed proteases as one of the critical target groups for developing novel small-molecule inhibitors. Among all proteases, aspartic proteases have gained significant attention due to their role in HIV/AIDS, malaria, Alzheimer's disease, etc. The binding cleft is covered by one or two β-hairpins (flaps) which need to be opened before a ligand can bind. After binding, the flaps close to retain the ligand in the active site. Development of computational tools has improved our understanding of flap dynamics and its role in ligand recognition. In the past decade, several computational approaches, for example molecular dynamics (MD) simulations, coarse-grained simulations, replica-exchange molecular dynamics (REMD) and metadynamics, have been used to understand flap dynamics and conformational motions associated with flap movements. This review is intended to summarize the computational progress towards understanding the flap dynamics of proteases and to be a reference for future studies in this field. PMID:26872937

  10. THE ROLE OF CYSTEINE PROTEASE IN ALZHEIMER DISEASE

    PubMed Central

    Hasanbasic, Samra; Jahic, Alma; Karahmet, Emina; Sejranic, Asja; Prnjavorac, Besim

    2016-01-01

    Introduction: Cysteine protease are biological catalysts which play a pivotal role in numerous biological reactions in organism. Much of the literature is inscribed to their biochemical significance, distribution and mechanism of action. Many diseases, e.g. Alzheimer’s disease, develop due to enzyme balance disruption. Understanding of cysteine protease’s disbalance is therefor a key to unravel the new possibilities of treatment. Cysteine protease are one of the most important enzymes for protein disruption during programmed cell death. Whether protein disruption is part of cell deaths is not enough clear in any cases. Thereafter, any tissue disruption, including proteolysis, generate more or less inflammation appearance. Review: This review briefly summarizes the current knowledge about pathological mechanism’s that results in AD, with significant reference to the role of cysteine protease in it. Based on the summary, new pharmacological approach and development of novel potent drugs with selective toxicity targeting cysteine protease will be a major challenge in years to come. PMID:27482169

  11. Post-translational control of genetic circuits using Potyvirus proteases.

    PubMed

    Fernandez-Rodriguez, Jesus; Voigt, Christopher A

    2016-07-27

    Genetic engineering projects often require control over when a protein is degraded. To this end, we use a fusion between a degron and an inactivating peptide that can be added to the N-terminus of a protein. When the corresponding protease is expressed, it cleaves the peptide and the protein is degraded. Three protease:cleavage site pairs from Potyvirus are shown to be orthogonal and active in exposing degrons, releasing inhibitory domains and cleaving polyproteins. This toolbox is applied to the design of genetic circuits as a means to control regulator activity and degradation. First, we demonstrate that a gate can be constructed by constitutively expressing an inactivated repressor and having an input promoter drive the expression of the protease. It is also shown that the proteolytic release of an inhibitory domain can improve the dynamic range of a transcriptional gate (200-fold repression). Next, we design polyproteins containing multiple repressors and show that their cleavage can be used to control multiple outputs. Finally, we demonstrate that the dynamic range of an output can be improved (8-fold to 190-fold) with the addition of a protease-cleaved degron. Thus, controllable proteolysis offers a powerful tool for modulating and expanding the function of synthetic gene circuits. PMID:27298256

  12. Lon protease: A key enzyme controlling mitochondrial bioenergetics in cancer

    PubMed Central

    Quirós, Pedro M; Bárcena, Clea; López-Otín, Carlos

    2014-01-01

    We have recently explored the in vivo functional and oncologic relevance of Lon protease (LONP1), an enzyme involved in mitochondrial quality control. We found that LONP1 is an essential protein for life and that it also performs a critical function in tumorigenesis by regulating the bioenergetics of cancer cells. PMID:27308364

  13. Protease Inhibitors Do Not Affect Antibody Responses to Pneumococcal Vaccination.

    PubMed

    De La Rosa, Indhira; Munjal, Iona M; Rodriguez-Barradas, Maria; Yu, Xiaoying; Pirofski, Liise-Anne; Mendoza, Daniel

    2016-06-01

    HIV(+) subjects on optimal antiretroviral therapy have persistently impaired antibody responses to pneumococcal vaccination. We explored the possibility that this effect may be due to HIV protease inhibitors (PIs). We found that in humans and mice, PIs do not affect antibody production in response to pneumococcal vaccination. PMID:27074938

  14. Local and spatial factors determining HIV-1 protease substrate recognition.

    PubMed Central

    Hazebrouck, S; Machtelinckx-Delmas, V; Kupiec, J J; Sonigo, P

    2001-01-01

    Insertional mutagenesis of the Escherichia coli thymidylate synthase (TS) was used to address substrate recognition of HIV-1 protease in a well characterized structural context. By modifying the TS conformation while maintaining its enzymic activity, we investigated the influence of protein folding on protease-substrate recognition. A slight destabilization of the TS structure permitted the cleavage of a target site, which was resistant in the native TS. This result supports a dynamic interpretation of HIV-1 protease specificity. Exposure time of the potential cleavage site, which depends on the stability of the global conformation, must be compatible with the cleavage kinetics, which are determined by the local sequence. Cleavage specificity has been described as the consequence of cumulative interactions, globally favourable, between at least six amino acids around the cleavage site. To investigate influence of local sequence, we introduced insertions of variable lengths in two exposed loops of the TS. In both environments, insertion of only two amino acids could determine specific cleavage. We then inserted libraries of dipeptides naturally cleaved by the HIV-1 protease in order to assess the limitations of established classifications of substrates in different conformational contexts. PMID:11513751

  15. Protease inhibitors interfere with the necessary factors of carcinogenesis.

    PubMed Central

    Troll, W

    1989-01-01

    Many tumor promoters are inflammatory agents that stimulate the formation of oxygen radicals (.O2-) and hydrogen peroxide (H2O2) in phagocytic neutrophils. The neutrophils use the oxygen radicals to kill bacteria, which are recognized by the cell membrane of phagocytic cells causing a signal to mount the oxygen response. The tumor promoter isolated from croton oil, 12-O-tetradecanoylphorbol-13-acetate (TPA), mimics the signal, causing an oxygen radical release that is intended to kill bacteria; instead, it injures cells in the host. Oxygen radicals cause single strand breaks in DNA and modify DNA bases. These damaging reactions appear to be related to tumor promotion, as three types of chemopreventive agents, retinoids, onion oil, and protease inhibitors, suppress the induction of oxygen radicals in phagocytic neutrophils and suppress tumor promotion in skin cancer in mice. Protease inhibitors also suppress breast and colon cancers in mice. Protease inhibitors capable of inhibiting chymotrypsin show a greater suppression of the oxygen effect and are better suppressors of tumor promotion. In addition, oxygen radicals may be one of the many agents that cause activation of oncogenes. Since retinoids and protease inhibitors suppress the expression of the ras oncogene in NIH 3T3 cells, NIH 3T3 cells may serve as a relatively facile model for finding and measuring chemopreventive agents that interfere with the carcinogenic process. PMID:2667986

  16. Protease-activated-receptor-2 affects protease-activated-receptor-1-driven breast cancer.

    PubMed

    Jaber, Mohammad; Maoz, Miriam; Kancharla, Arun; Agranovich, Daniel; Peretz, Tamar; Grisaru-Granovsky, Sorina; Uziely, Beatrice; Bar-Shavit, Rachel

    2014-07-01

    Mammalian protease-activated-receptor-1 and -2 (PAR1 and PAR2) are activated by proteases found in the flexible microenvironment of a tumor and play a central role in breast cancer. We propose in the present study that PAR1 and PAR2 act together as a functional unit during malignant and physiological invasion processes. This notion is supported by assessing pro-tumor functions in the presence of short hairpin; shRNA knocked-down hPar2 or by the use of a truncated PAR2 devoid of the entire cytoplasmic tail. Silencing of hPar2 by shRNA-attenuated thrombin induced PAR1 signaling as recapitulated by inhibiting the assembly of Etk/Bmx or Akt onto PAR1-C-tail, by thrombin-instigated colony formation and invasion. Strikingly, shRNA-hPar2 also inhibited the TFLLRN selective PAR1 pro-tumor functions. In addition, while evaluating the physiological invasion process of placenta extravillous trophoblast (EVT) organ culture, we observed inhibition of both thrombin or the selective PAR1 ligand; TFLLRNPNDK induced EVT invasion by shRNA-hPar2 but not by scrambled shRNA-hPar2. In parallel, when a truncated PAR2 was utilized in a xenograft mouse model, it inhibited PAR1-PAR2-driven tumor growth in vivo. Similarly, it also attenuated the interaction of Etk/Bmx with the PAR1-C-tail in vitro and decreased markedly selective PAR1-induced Matrigel invasion. Confocal images demonstrated co-localization of PAR1 and PAR2 in HEK293T cells over-expressing YFP-hPar2 and HA-hPar1. Co-immuno-precipitation analyses revealed PAR1-PAR2 complex formation but no PAR1-CXCR4 complex was formed. Taken together, our observations show that PAR1 and PAR2 act as a functional unit in tumor development and placenta-uterus interactions. This conclusion may have significant consequences on future breast cancer therapeutic modalities and improved late pregnancy outcome. PMID:24177339

  17. Enhanced Long-Term and Impaired Short-Term Spatial Memory in GluA1 AMPA Receptor Subunit Knockout Mice: Evidence for a Dual-Process Memory Model

    ERIC Educational Resources Information Center

    Sanderson, David J.; Good, Mark A.; Skelton, Kathryn; Sprengel, Rolf; Seeburg, Peter H.; Rawlins, J. Nicholas P.; Bannerman, David M.

    2009-01-01

    The GluA1 AMPA receptor subunit is a key mediator of hippocampal synaptic plasticity and is especially important for a rapidly-induced, short-lasting form of potentiation. GluA1 gene deletion impairs hippocampus-dependent, spatial working memory, but spares hippocampus-dependent spatial reference memory. These findings may reflect the necessity of…

  18. A Spider-Derived Kunitz-Type Serine Protease Inhibitor That Acts as a Plasmin Inhibitor and an Elastase Inhibitor

    PubMed Central

    Wan, Hu; Lee, Kwang Sik; Kim, Bo Yeon; Zou, Feng Ming; Yoon, Hyung Joo; Je, Yeon Ho; Li, Jianhong; Jin, Byung Rae

    2013-01-01

    Kunitz-type serine protease inhibitors are involved in various physiological processes, such as ion channel blocking, blood coagulation, fibrinolysis, and inflammation. While spider-derived Kunitz-type proteins show activity in trypsin or chymotrypsin inhibition and K+ channel blocking, no additional role for these proteins has been elucidated. In this study, we identified the first spider (Araneus ventricosus) Kunitz-type serine protease inhibitor (AvKTI) that acts as a plasmin inhibitor and an elastase inhibitor. AvKTI possesses a Kunitz domain consisting of a 57-amino-acid mature peptide that displays features consistent with Kunitz-type inhibitors, including six conserved cysteine residues and a P1 lysine residue. Recombinant AvKTI, expressed in baculovirus-infected insect cells, showed a dual inhibitory activity against trypsin (Ki 7.34 nM) and chymotrypsin (Ki 37.75 nM), defining a role for AvKTI as a spider-derived Kunitz-type serine protease inhibitor. Additionally, AvKTI showed no detectable inhibitory effects on factor Xa, thrombin, or tissue plasminogen activator; however, AvKTI inhibited plasmin (Ki 4.89 nM) and neutrophil elastase (Ki 169.07 nM), indicating that it acts as an antifibrinolytic factor and an antielastolytic factor. These findings constitute molecular evidence that AvKTI acts as a plasmin inhibitor and an elastase inhibitor and also provide a novel view of the functions of a spider-derived Kunitz-type serine protease inhibitor. PMID:23308198

  19. Highly efficient and easy protease-mediated protein purification.

    PubMed

    Last, Daniel; Müller, Janett; Dawood, Ayad W H; Moldenhauer, Eva J; Pavlidis, Ioannis V; Bornscheuer, Uwe T

    2016-02-01

    As both research on and application of proteins are rarely focused on the resistance towards nonspecific proteases, this property remained widely unnoticed, in particular in terms of protein purification and related fields. In the present study, diverse aspects of protease-mediated protein purification (PMPP) were explored on the basis of the complementary proteases trypsin and proteinase K as well as the model proteins green fluorescent protein (GFP) from Aequorea victoria, lipase A from Candida antarctica (CAL-A), a transaminase from Aspergillus fumigatus (AspFum), quorum quenching lactonase AiiA from Bacillus sp., and an alanine dehydrogenase from Thermus thermophilus (AlaDH). While GFP and AiiA were already known to be protease resistant, the thermostable enzymes CAL-A, AspFum, and AlaDH were selected due to the documented correlation between thermostability and protease resistance. As proof of principle for PMPP, recombinant GFP remained unaffected whereas most Escherichia coli (E. coli) host proteins were degraded by trypsin. PMPP was highly advantageous compared to the widely used heat-mediated purification of commercial CAL-A. The resistance of AspFum towards trypsin was improved by rational protein design introducing point mutation R20Q. Trypsin also served as economical and efficient substitute for site-specific endopeptidases for the removal of a His-tag fused to AiiA. Moreover, proteolysis of host enzymes with interfering properties led to a strongly improved sensitivity and accuracy of the NADH assay in E. coli cell lysate for AlaDH activity measurements. Thus, PMPP is an attractive alternative to common protein purification methods and facilitates also enzyme characterization in cell lysate. PMID:26671615

  20. Nematicidal bacteria associated to pinewood nematode produce extracellular proteases.

    PubMed

    Paiva, Gabriel; Proença, Diogo Neves; Francisco, Romeu; Verissimo, Paula; Santos, Susana S; Fonseca, Luís; Abrantes, Isabel M O; Morais, Paula V

    2013-01-01

    Bacteria associated with the nematode Bursaphelenchus xylophilus, a pathogen of trees and the causal agent of pine wilt disease (PWD) may play a role in the disease. In order to evaluate their role (positive or negative to the tree), strains isolated from the track of nematodes from infected Pinus pinaster trees were screened, in vitro, for their nematicidal potential. The bacterial products, from strains more active in killing nematodes, were screened in order to identify and characterize the nematicidal agent. Forty-seven strains were tested and, of these, 21 strains showed capacity to produce extracellular products with nematicidal activity. All Burkholderia strains were non-toxic. In contrast, all Serratia strains except one exhibited high toxicity. Nematodes incubated with Serratia strains showed, by SEM observation, deposits of bacteria on the nematode cuticle. The most nematicidal strain, Serratia sp. A88copa13, produced proteases in the supernatant. The use of selective inhibitors revealed that a serine protease with 70 kDa was majorly responsible for the toxicity of the supernatant. This extracellular serine protease is different phylogenetically, in size and biochemically from previously described proteases. Nematicidal assays revealed differences in nematicidal activity of the proteases to different species of Bursaphelenchus, suggesting its usefulness in a primary screen of the nematodes. This study offers the basis for further investigation of PWD and brings new insights on the role bacteria play in the defense of pine trees against B. xylophilus. Understanding all the factors involved is important in order to develop strategies to control B. xylophilus dispersion. PMID:24244546

  1. Structural Mechanisms of Inactivation in Scabies Mite Serine Protease Paralogues

    SciTech Connect

    Fischer, Katja; Langendorf, Christopher G.; Irving, James A.; Reynolds, Simone; Willis, Charlene; Beckham, Simone; Law, Ruby H.P.; Yang, Sundy; Bashtannyk-Puhalovich, Tanya A.; McGowan, Sheena; Whisstock, James C.; Pike, Robert N.; Kemp, David J.; Buckle, Ashley M.

    2009-08-07

    The scabies mite (Sarcoptes scabiei) is a parasite responsible for major morbidity in disadvantaged communities and immuno-compromised patients worldwide. In addition to the physical discomfort caused by the disease, scabies infestations facilitate infection by Streptococcal species via skin lesions, resulting in a high prevalence of rheumatic fever/heart disease in affected communities. The scabies mite produces 33 proteins that are closely related to those in the dust mite group 3 allergen and belong to the S1-like protease family (chymotrypsin-like). However, all but one of these molecules contain mutations in the conserved active-site catalytic triad that are predicted to render them catalytically inactive. These molecules are thus termed scabies mite inactivated protease paralogues (SMIPPs). The precise function of SMIPPs is unclear; however, it has been suggested that these proteins might function by binding and protecting target substrates from cleavage by host immune proteases, thus preventing the host from mounting an effective immune challenge. In order to begin to understand the structural basis for SMIPP function, we solved the crystal structures of SMIPP-S-I1 and SMIPP-S-D1 at 1.85 {angstrom} and 2.0 {angstrom} resolution, respectively. Both structures adopt the characteristic serine protease fold, albeit with large structural variations over much of the molecule. In both structures, mutations in the catalytic triad together with occlusion of the S1 subsite by a conserved Tyr200 residue is predicted to block substrate ingress. Accordingly, we show that both proteases lack catalytic function. Attempts to restore function (via site-directed mutagenesis of catalytic residues as well as Tyr200) were unsuccessful. Taken together, these data suggest that SMIPPs have lost the ability to bind substrates in a classical 'canonical' fashion, and instead have evolved alternative functions in the lifecycle of the scabies mite.

  2. Nematicidal Bacteria Associated to Pinewood Nematode Produce Extracellular Proteases

    PubMed Central

    Francisco, Romeu; Verissimo, Paula; Santos, Susana S.; Fonseca, Luís; Abrantes, Isabel M. O.; Morais, Paula V.

    2013-01-01

    Bacteria associated with the nematode Bursaphelenchus xylophilus, a pathogen of trees and the causal agent of pine wilt disease (PWD) may play a role in the disease. In order to evaluate their role (positive or negative to the tree), strains isolated from the track of nematodes from infected Pinus pinaster trees were screened, in vitro, for their nematicidal potential. The bacterial products, from strains more active in killing nematodes, were screened in order to identify and characterize the nematicidal agent. Forty-seven strains were tested and, of these, 21 strains showed capacity to produce extracellular products with nematicidal activity. All Burkholderia strains were non-toxic. In contrast, all Serratia strains except one exhibited high toxicity. Nematodes incubated with Serratia strains showed, by SEM observation, deposits of bacteria on the nematode cuticle. The most nematicidal strain, Serratia sp. A88copa13, produced proteases in the supernatant. The use of selective inhibitors revealed that a serine protease with 70 kDa was majorly responsible for the toxicity of the supernatant. This extracellular serine protease is different phylogenetically, in size and biochemically from previously described proteases. Nematicidal assays revealed differences in nematicidal activity of the proteases to different species of Bursaphelenchus, suggesting its usefulness in a primary screen of the nematodes. This study offers the basis for further investigation of PWD and brings new insights on the role bacteria play in the defense of pine trees against B. xylophilus. Understanding all the factors involved is important in order to develop strategies to control B. xylophilus dispersion. PMID:24244546

  3. Properties of Hemolysin and Protease Produced by Aeromonas trota

    PubMed Central

    Takahashi, Eizo; Ozaki, Haruka; Fujii, Yoshio; Kobayashi, Hidetomo; Yamanaka, Hiroyasu; Arimoto, Sakae; Negishi, Tomoe; Okamoto, Keinosuke

    2014-01-01

    We examined the properties of exotoxins produced by Aeromonas trota (A. enteropelogenes), one of the diarrheagenic species of Aeromonadaceae. Nine of 19 A. trota isolates that grew on solid media containing erythrocytes showed hemolytic activity. However, the hemolytic activities of the culture supernatants of these hemolytic strains of A. trota were markedly lower than those of A. sobria when cultured in liquid medium, and the amount of hemolysin detected by immunoblotting using antiserum against the hemolysin produced by A. sobria was also low. A mouse intestine loop assay using living bacterial cells showed that A. trota 701 caused the significant accumulation of fluid, and antiserum against the hemolysin produced suppressed the enterotoxic action of A. trota 701. These results indicated that A. trota 701 was diarrheagenic and the hemolysin produced was the causative agent of the enterotoxic activity of A. trota. The hemolysin in A. sobria was previously shown to be secreted in a preform (inactive form) and be activated when the carboxy-terminal domain was cleaved off by proteases in the culture supernatant. Since mature hemolysin was detected in the culture supernatants of A. trota, we analyzed the extracellular protease produced by A. trota. Fifteen of 19 A. trota isolates that grew on solid media containing skim milk showed proteolytic activity. We subsequently found that most A. trota isolates possessed the serine protease gene, but not the metalloprotease gene. Therefore, we determined the nucleotide sequence of the serine protease gene and its chaperone A. trota gene. The results obtained revealed that the deduced amino acid sequences of serine protease and the chaperone were homologous to those of A. sobria with identities of 83.0% and 75.8%, respectively. PMID:24633045

  4. Plasma levels of mannan-binding lectin-associated serine proteases MASP-1 and MASP-2 are elevated in type 1 diabetes and correlate with glycaemic control

    PubMed Central

    Jenny, L; Ajjan, R; King, R; Thiel, S; Schroeder, V

    2015-01-01

    There is increasing evidence that the complement system plays an important role in diabetes and the development of diabetic vascular complications. In particular, mannan-binding lectin (MBL) levels are elevated in diabetes patients, and diabetes patients with diabetic nephropathy have higher MBL levels than diabetes patients with normal renal function. The MBL-associated serine proteases (MASPs) MASP-1, MASP-2 and MASP-3 and MBL-associated protein MAp44 have not yet been studied in diabetes patients. We therefore measured plasma levels of MASP-1, MASP-2, MASP-3 and MAp44 in 30 children with type 1 diabetes mellitus (T1DM) and 17 matched control subjects, and in 45 adults with T1DM and 31 matched control subjects. MASP-1 and MASP-2 levels were significantly higher in children and adults with T1DM than in their respective control groups, whereas MASP-3 and MAp44 levels did not differ between patients and controls. MASP-1 and MASP-2 levels correlated with HbA1c, and MASP levels decreased when glycaemic control improved. Because MASP-1 and MASP-2 have been shown to interact directly with blood coagulation, elevated levels of these proteins may play a role in the enhanced thrombotic environment and consequent vascular complications in diabetes. PMID:25533914

  5. IgA Protease Activity in Haemophilus parasuis in the Absence of a Recognizable IgA Protease Gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background. Haemophilus parasuis, the bacterium responsible for Glasser’s disease, is a pathogen of significant concern in modern high-health swine production systems. Little is known regarding the molecular mechanisms of H. parasuis infection. In some Pasteurellaceae species, IgA proteases aid in d...

  6. Characterization of the Protease Activity of Detergents: Laboratory Practicals for Studying the Protease Profile and Activity of Various Commercial Detergents

    ERIC Educational Resources Information Center

    Valls, Cristina; Pujadas, Gerard; Garcia-Vallve, Santi; Mulero, Miquel

    2011-01-01

    Detergent enzymes account for about 30% of the total worldwide production of enzymes and are one of the largest and most successful applications of modern industrial biotechnology. Proteases can improve the wash performance of household, industrial, and institutional laundry detergents used to remove protein-based stains such as blood, grass, body…

  7. The Role of Calcium Activated Protease Calpain in Experimental Retinal Pathology

    PubMed Central

    Azuma, M.; Shearer, T.R.

    2008-01-01

    The purpose of this review is to present the recent evidence linking the family of ubiquitous proteases called calpains (EC 3.4.22.17) to neuropathologies of the retina. The hypothesis being tested in such studies is that over-activation of calpains by elevated intracellular calcium contributes to retinal cell death produced by conditions such as elevated intraocular pressure and hypoxia. Recent x-ray diffraction studies have provided insight into the molecular events causing calpain activation. Further, x-ray diffraction data has provided details on how side chains on calpain inhibitors affect docking into the active site of calpain 1. This opens the possibility of testing calpain-specific inhibitors, such as SJA6017 and SNJ1945, for human safety and as a site-directed form of treatment for retinal pathologies. PMID:18348880

  8. Role of mitochondrial processing peptidase and AAA proteases in processing of the yeast acetohydroxyacid synthase precursor.

    PubMed

    Dasari, Suvarna; Kölling, Ralf

    2016-07-01

    We studied presequence processing of the mitochondrial-matrix targeted acetohydroxyacid synthase (Ilv2). C-terminal 3HA-tagging altered the cleavage pattern from a single step to sequential two-step cleavage, giving rise to two Ilv2-3HA forms (A and B). Both cleavage events were dependent on the mitochondrial processing peptidase (MPP). We present evidence for the involvement of three AAA ATPases, m- and i-AAA proteases, and Mcx1, in Ilv2-3HA processing. Both, precursor to A-form and A-form to B-form cleavage were strongly affected in a ∆yme1 mutant. These defects could be suppressed by overexpression of MPP, suggesting that MPP activity is limiting in the ∆yme1 mutant. Our data suggest that for some substrates AAA ATPases could play an active role in the translocation of matrix-targeted proteins. PMID:27398316

  9. Interaction of trypsin-like protease from Streptomyces griseus with an immobilized inhibitor from kidney bean.

    PubMed

    Mosolov, V V; Fedurkina, N V; Valueva, T A

    1978-01-12

    An immobilized double-headed inhibitor from Phaseolus vulgaris L. selectively binds the trypsin-like enzyme produced by Streptomyces griseus. Binding takes place at pH 8.0, and at pH 2.0 the protease can be quantitatively released from the complex. Purified by affinity chromatography, the trypsin-like enzyme is homogeneous according to polyacrylamide gel electrophoresis and ultracentrifugation data. Physico-chemical and enzymic properties of the enzyme are identical to those exhibited by the enzyme purified by ion-exchange chromatography. Chymoelastases from Str. griseus as well as the subtilisin-like enzyme do not interact with an immobilized inhibitor. In solution, the inhibitor from P. vulgaris gives a stable ternary complex with bovine trypsin and chymotrypsin, whereas with an immobilized inhibitor the trypsin, if present, tends to displace chymotrypsin in an chymotrypsin inhibitor complex. This evidence suggests that immobilization results in considerable changes in inhibitor properties. PMID:413581

  10. Purification and biochemical characterization of an alkaline protease from marine bacteria Pseudoalteromonas sp. 129-1.

    PubMed

    Wu, Shimei; Liu, Ge; Zhang, Dechao; Li, Chaoxu; Sun, Chaomin

    2015-12-01

    An extracellular alkaline protease produced by marine bacteria strain Pseudoalteromonas sp. 129-1 was purified by ammonium sulphate precipitation, anion exchange chromatography, and gel filtration. The purity of the protease was confirmed by SDS-PAGE and molecular mass was estimated to be 35 kDa. The protease maintained considerable activity and stability at a wide temperature range of 10-60 °C and pH range of 6-11, and optimum activity was detected at temperature of 50 °C and pH of 8. Metallo-protease inhibitor, EDTA, had no inhibitory effect on protease activity even at concentration up to 15 mM, whereas 15 mM PMSF, a common serine protease inhibitor, greatly inactivated the protease. The high stability of the protease in the presence of surfactants (SDS, Tween 80, and Triton X-100), oxidizing agent H(2)O(2), and commercial detergents was observed. Moreover, the protease was tolerant to most of the tested organic solvents, and saline tolerant up to 30%. Interestingly, biofilm of Pseudomonas aeruginosa PAO1 was greatly reduced by 0.01 mg ml(-1) of the protease, and nearly completely abolished with the concentration of 1 mg ml(-1). Collectively, the protease showed valuable feathers as an additive in laundry detergent and non-toxic anti-biofilm agent. PMID:26213994

  11. Characterization of up-regulated proteases in an industrial recombinant Escherichia coli fermentation.

    PubMed

    Jordan, G L; Harcum, S W

    2002-02-01

    Proteolytic degradation of recombinant proteins is an industry-wide challenge in host organisms such as Escherichia coli. These proteases have been linked to stresses, such as the stringent and heat-shock responses. This study reports the dramatic up-regulation of protease activity in an industrial recombinant E. coli fermentation upon induction. The objective of this project was to detect and characterize up-regulated proteases due to recombinant AXOKINE overexpression upon IPTG induction. AXOKINE is a 22-kDa protein currently in clinical trials as a therapeutic for obesity associated with diabetes. AXOKINE was expressed in both the soluble and inclusion body fractions in E. coli. Sodium dodecyl sulfate gelatin-polyacrylamide gel electrophoresis (SDS-GPAGE) was used to analyze the up-regulated protease activity. Western blot analysis showed degraded AXOKINE in both the soluble and insoluble fractions. Protease inhibitors were used to characterize the proteases. The proteases were ethylenediaminetetraacetic acid (EDTA) sensitive. The protease activity increased in the presence of phenyl-methyl sulfonyl-fluoride (PMSF), a serine protease inhibitor. The incubation buffer composition was varied with respect to Mg2+ and ATP, and the protease activity was ATP independent and Mg2+ dependent. A two-dimensional electrophoresis technique was used to estimate the pI of the proteases to be between 2.9 and 4.0. PMID:12074055

  12. 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.

  13. Proteases of Wood Rot Fungi with Emphasis on the Genus Pleurotus.

    PubMed

    Inácio, Fabíola Dorneles; Ferreira, Roselene Oliveira; de Araujo, Caroline Aparecida Vaz; Brugnari, Tatiane; Castoldi, Rafael; Peralta, Rosane Marina; de Souza, Cristina Giatti Marques

    2015-01-01

    Proteases are present in all living organisms and they play an important role in physiological conditions. Cell growth and death, blood clotting, and immune defense are all examples of the importance of proteases in maintaining homeostasis. There is growing interest in proteases due to their use for industrial purposes. The search for proteases with specific characteristics is designed to reduce production costs and to find suitable properties for certain industrial sectors, as well as good producing organisms. Ninety percent of commercialized proteases are obtained from microbial sources and proteases from macromycetes have recently gained prominence in the search for new enzymes with specific characteristics. The production of proteases from saprophytic basidiomycetes has led to the identification of various classes of proteases. The genus Pleurotus has been extensively studied because of its ligninolytic enzymes. The characteristics of this genus are easy cultivation techniques, high yield, low nutrient requirements, and excellent adaptation. There are few studies in the literature about proteases of Pleurotus spp. This review gathers together information about proteases, especially those derived from basidiomycetes, and aims at stimulating further research about fungal proteases because of their physiological importance and their application in various industries such as biotechnology and medicine. PMID:26180792

  14. Copper inhibits the HIV-1 protease by both oxygen-dependent and oxygen-independent mechanisms

    SciTech Connect

    Karlstroem, A.R.; Levine, R.L. )

    1991-03-11

    The protease encoded by HIV-1 is essential for the processing of the viral polyproteins encoded by the gag and pol genes into mature viral proteins. Mutation or deletion of the protease gene blocks replication of the virus, making the protease an attractive target for antiviral therapy. The authors found that the HIV-1 protease is inhibited by micromolar concentrations of Cu{sup 2+}. Protease was 50% inhibited by exposure to 5 {mu}M copper for 5 min while exposure to 25 {mu}M caused complete inhibition. This inhibition was not oxygen-dependent and was not reversed by treatment with EDTA, presumably due to the slow off-rate of copper from the protease. Consistent with this interpretation, enzyme activity was recovered after denaturation and refolding of the copper exposed protease. Titration of the inactivated enzyme with Ellman's reagent demonstrated a loss of one of the two sulfhydryl groups present in the molecule, suggesting that copper inhibition was mediated through binding to a cysteine. This was confirmed in studies with a chemically synthesize, mutant protease in which the two cysteine residues were replaced by {alpha}-amino butyrate: The mutant protease was not inhibited by copper. However, both the wild-type and mutant protease were inactivated when exposed to copper, oxygen, and dithiothreitol. This inactivation required oxygen. Thus, the protease can also be inactivated by metal catalyzed oxidation (MCO), a presumably irreversible covalent modification.

  15. Proteases of Wood Rot Fungi with Emphasis on the Genus Pleurotus

    PubMed Central

    Inácio, Fabíola Dorneles; Ferreira, Roselene Oliveira; de Araujo, Caroline Aparecida Vaz; Brugnari, Tatiane; Castoldi, Rafael; Peralta, Rosane Marina; de Souza, Cristina Giatti Marques

    2015-01-01

    Proteases are present in all living organisms and they play an important role in physiological conditions. Cell growth and death, blood clotting, and immune defense are all examples of the importance of proteases in maintaining homeostasis. There is growing interest in proteases due to their use for industrial purposes. The search for proteases with specific characteristics is designed to reduce production costs and to find suitable properties for certain industrial sectors, as well as good producing organisms. Ninety percent of commercialized proteases are obtained from microbial sources and proteases from macromycetes have recently gained prominence in the search for new enzymes with specific characteristics. The production of proteases from saprophytic basidiomycetes has led to the identification of various classes of proteases. The genus Pleurotus has been extensively studied because of its ligninolytic enzymes. The characteristics of this genus are easy cultivation techniques, high yield, low nutrient requirements, and excellent adaptation. There are few studies in the literature about proteases of Pleurotus spp. This review gathers together information about proteases, especially those derived from basidiomycetes, and aims at stimulating further research about fungal proteases because of their physiological importance and their application in various industries such as biotechnology and medicine. PMID:26180792

  16. Redefining the concept of protease-activated receptors: cathepsin S evokes itch via activation of Mrgprs

    PubMed Central

    Reddy, Vemuri B.; Sun, Shuohao; Azimi, Ehsan; Elmariah, Sarina B.; Dong, Xinzhong; Lerner, Ethan A.

    2015-01-01

    Sensory neurons expressing Mas-related G protein coupled receptors (Mrgprs) mediate histamine-independent itch. We show that the cysteine protease cathepsin S activates MrgprC11 and evokes receptor-dependent scratching in mice. In contrast to its activation of conventional protease-activated receptors, cathepsin S mediated activation of MrgprC11 did not involve the generation of a tethered ligand. We demonstrate further that different cysteine proteases selectively activate specific mouse and human Mrgpr family members. This expansion of our understanding by which proteases interact with GPCRs redefines the concept of what constitutes a protease-activated receptor. The findings also implicate proteases as ligands to members of this orphan receptor family while providing new insights into how cysteine proteases contribute to itch. PMID:26216096

  17. Development of marine biotechnology as a resource for novel proteases and their role in modern biotechnology.

    PubMed

    Homaei, Ahmad; Lavajoo, Fatemeh; Sariri, Reyhaneh

    2016-07-01

    Marine environment consists of the largest sources diversified genetic pool of material with an enormous potential for a wide variety of enzymes including proteases. A protease hydrolyzes the peptide bond and most of proteases possess many industrial applications. Marine proteases differ considerably from those found in internal or external organs of invertebrates and vertebrates. In common with all enzymes, external factors such as temperature, pH and type of media are important for the activity, catalytic efficiency, stability and proper functioning of proteases. In this review valuable characteristics of proteases in marine organisms and their applications are gathered from a wide literature survey. Considering their biochemical significance and their increasing importance in biotechnology, a thorough understanding of marine proteases functioning could be of prime importance. PMID:27086293

  18. Short communication: molecular epidemiology of HIV type 1 infection in northern Greece (2009-2010): evidence of a transmission cluster of HIV type 1 subtype A1 drug-resistant strains among men who have sex with men.

    PubMed

    Antoniadou, Zoi-Anna; Kousiappa, Ioanna; Skoura, Lemonia; Pilalas, Dimitris; Metallidis, Simeon; Nicolaidis, Pavlos; Malisiovas, Nicolaos; Kostrikis, Leondios G

    2014-03-01

    A prospective molecular epidemiology study of HIV-1 infection was conducted in newly diagnosed and antiretroviral-naive patients in Northern Greece between 2009 and 2010 using a predefined enrolling strategy. Phylogenetic trees of the pol sequences obtained in this study with reference sequences indicated that subtypes B and A1 were the most common subtypes present and accounted for 44.9% and 42.9%, respectively, followed by subtype C (3.1%), CRF02_AG (4.1%), CRF04_cpx (2.0%), and subtypes CRF01_01, F1, and G (1.0%). A high rate of clustered transmission of subtype A1-resistant strains to reverse transcriptase (RT) inhibitors was observed among men having sex with men. Indeed, 15 out of 17 study subjects (88.2%) infected with transmitted drug resistance (TDR) strains were implicated in transmission clusters, 10 of whom (66.7%) were men who have sex with men (MSM), and were also infected with subsubtype A1 strains. The main cluster within subtype A1 (I) included eight men reporting having sex with men from Thessaloniki infected with dual-class RT-resistant strains carrying both T215C and Y181C mutations. PMID:24059291

  19. Short Communication: Molecular Epidemiology of HIV Type 1 Infection in Northern Greece (2009–2010): Evidence of a Transmission Cluster of HIV Type 1 Subtype A1 Drug-Resistant Strains Among Men Who Have Sex with Men

    PubMed Central

    Antoniadou, Zoi-Anna; Kousiappa, Ioanna; Skoura, Lemonia; Pilalas, Dimitris; Metallidis, Simeon; Nicolaidis, Pavlos; Malisiovas, Nicolaos

    2014-01-01

    Abstract A prospective molecular epidemiology study of HIV-1 infection was conducted in newly diagnosed and antiretroviral-naive patients in Northern Greece between 2009 and 2010 using a predefined enrolling strategy. Phylogenetic trees of the pol sequences obtained in this study with reference sequences indicated that subtypes B and A1 were the most common subtypes present and accounted for 44.9% and 42.9%, respectively, followed by subtype C (3.1%), CRF02_AG (4.1%), CRF04_cpx (2.0%), and subtypes CRF01_01, F1, and G (1.0%). A high rate of clustered transmission of subtype A1-resistant strains to reverse transcriptase (RT) inhibitors was observed among men having sex with men. Indeed, 15 out of 17 study subjects (88.2%) infected with transmitted drug resistance (TDR) strains were implicated in transmission clusters, 10 of whom (66.7%) were men who have sex with men (MSM), and were also infected with subsubtype A1 strains. The main cluster within subtype A1 (I) included eight men reporting having sex with men from Thessaloniki infected with dual-class RT-resistant strains carrying both T215C and Y181C mutations. PMID:24059291

  20. Quality control of a molybdoenzyme by the Lon protease.

    PubMed

    Redelberger, David; Genest, Olivier; Arabet, Dallel; Méjean, Vincent; Ilbert, Marianne; Iobbi-Nivol, Chantal

    2013-12-11

    Molybdoenzymes contain a molybdenum cofactor in their active site to catalyze various redox reactions in all domains of life. To decipher crucial steps during their biogenesis, the TorA molybdoenzyme of Escherichia coli had played a major role to understand molybdoenzyme maturation process driven by specific chaperones. TorD, the specific chaperone of TorA, is also involved in TorA protection. Here, we show that immature TorA (apoTorA) is degraded in vivo and in vitro by the Lon protease. Lon interacts with apoTorA but not with holoTorA. Lon and TorD compete for apoTorA binding but TorD binding protects apoTorA against degradation. Lon is the first protease shown to eliminate an immature or misfolded molybdoenzyme probably by targeting its inactive catalytic site. PMID:24211448

  1. Occurrence of aspartyl proteases in brine after herring marinating.

    PubMed

    Szymczak, Mariusz; Lepczyński, Adam

    2016-03-01

    Herrings are marinated in a brine consisting of salt and acetic acid. During marinating, various nitrogen fractions diffuse from fish flesh to the brine, causing significant nutritional quality losses of the raw material. In this study, it has been demonstrated for the first time that proteases diffuse from the fish to the marinating brine. Using ammonium sulphate precipitation and affinity chromatography on pepstatin-A agarose bed the aspartyl proteases were purified and concentrated over 2600-fold from a marinating brine. Pepstatin-A completely inhibited the activity of the purified preparation. The preparation was active against fluorogenic substrates specific for cathepsin D and E and inactive against substrates specific for cysteine cathepsins. Depending on incubation time, the preparation showed pH-optimum at 2.0 or 4.5. The 2D SDS-PAGE separation demonstrated the presence of a few proteins with molecular weights and pI values typical of cathepsin D, E and pepsin. PMID:26471581

  2. Cloning, heterologous expression and antigenicity of a schistosome cercarial protease.

    PubMed

    Price, H P; Doenhoff, M J; Sayers, J R

    1997-05-01

    A gene coding for the 30 kDa Schistosoma mansoni cercarial protease was amplified using the polymerase chain reaction (PCR) from genomic DNA templates. Cloning and sequencing of several independent PCR clones revealed the presence of an intron additional to the one described in the original cloning of the gene. The 3 exons were cloned into expression vectors so that they could be expressed as separate glutathione-S-transferase (GST) translational fusions. Recombinant bacteria carrying these expression plasmids expressed the fusion proteins at high levels. Western blotting of bacterial lysates with sera raised against the native S. mansoni cercarial protease showed that all 3 exons were recognized. Thus we have produced recombinant bacteria capable of providing large amounts of an S. mansoni antigen for immunological studies and evaluation as a candidate vaccine. PMID:9149415

  3. Bacterial proteases: production, isolation and utilization in animal nutrition.

    PubMed

    Michalík, I; Szabová, E; Poláková, A; Urminská, D

    1997-01-01

    Under conditions of submerged fermentation of Bacillus licheniformis strain L-3 in 15-L MBR-Schulzer bioreactor, the maximum production of proteolytic enzymes was achieved in the nutrient medium which contained 1% milk powder, 0.3% yeast autolysate, 0.5% corn starch and malt (20 ml per 100 ml of the medium). The preparation obtained of extracellular alkaline bacterial Ser-protease of the subtilisin type is characterized by optimum pH 9.8-10.2 good up to a temperature stability (65 degrees C) and has molecular weight cca of 26 kDa. The use of chemical mutagens (HNO2, 5-bromouracil) has enabled to select new strains (L-3N and L-3U) whose protease activity is 1.8-2.2 times higher as compared to the original Bacillus licheniformis strain L-3. The addition of these enzymes to the fodder has a positive effect on the retention of nitrogen substances. PMID:9505358

  4. Protease-dependent mechanisms of complement evasion by bacterial pathogens

    PubMed Central

    Potempa, Michal; Potempa, Jan

    2012-01-01

    The human immune system has evolved a variety of mechanisms for the primary task of neutralizing and eliminating microbial intruders. As the first line of defense, the complement system is responsible for rapid recognition and opsonisation of bacteria, presentation to phagocytes and bacterial cell killing by direct lysis. All successful human pathogens have mechanisms of circumventing the antibacterial activity of the complement system and escaping this stage of the immune response. One of the ways in which pathogens achieve this is the deployment of proteases. Based on the increasing number of recent publications in this area, it appears that proteolytic inactivation of the antibacterial activities of the complement system is a common strategy of avoiding targeting by this arm of host innate immune defense. In this review, we focus on those bacteria that deploy proteases capable of degrading complement system components into non-functional fragments, thus impairing complement-dependent antibacterial activity and facilitating pathogen survival inside the host. PMID:22944688

  5. Intestinal proteases of free-living and parasitic astigmatid mites.

    PubMed

    Holt, Deborah C; Burgess, Stewart T G; Reynolds, Simone L; Mahmood, Wajahat; Fischer, Katja

    2013-02-01

    Among arthropod pests, mites are responsible for considerable damage to crops, humans and other animals. However, detailed physiological data on these organisms remain sparse, mainly because of their small size but possibly also because of their extreme diversity. Focusing on intestinal proteases, we draw together information from three distinct mite species that all feed on skin but have separately adapted to a free-living, a strictly ecto-parasitic and a parasitic lifestyle. A wide range of studies involving immunohistology, molecular biology, X-ray crystallography and enzyme biochemistry of mite gut proteases suggests that these creatures have diverged considerably as house dust mites, sheep scab mites and scabies mites. Each species has evolved a particular variation of a presumably ancestral repertoire of digestive enzymes that have become specifically adapted to their individual environmental requirements. PMID:22427061

  6. Stabilization of proteases by entrapment in a new composite hydrogel.

    PubMed

    Markvicheva, E A; Tkachuk, N E; Kuptsova, S V; Dugina, T N; Strukova, S M; Kirsh YuE; Zubov, V P; Rumsh, L D

    1996-01-01

    A new one-step procedure for entrapping proteases into a polymeric composite calcium alginate-poly(N-vinyl caprolactam) hydrogel was developed that provided 75-90% retention of the activity of entrapped enzymes compared to soluble ones. Properties of entrapped carboxypeptidase B, trypsin, and thrombin were investigated. The immobilized enzymes were active within a wide pH range. The temperature optima of entrapped trypsin and carboxypeptidase B were approx 25 degrees C higher than that of the soluble enzymes, and the resistance to heating was also increased. The effects of various polar and nonpolar organic solvents on the entrapped proteases were investigated. The immobilized enzymes retained their activity within a wide concentration range (up to 90%) of organic solvents. Gel-entrapped trypsin and carboxypeptidase (CPB) were successfully used for obtaining human insulin from recombinant proinsulin. The developed stabilization method can be used to catalyze various reactions proceeding within wide pH and temperature ranges. PMID:9100346

  7. Characterization of a membrane-associated serine protease in Escherichia coli

    SciTech Connect

    Palmer, S.M.; St. John, A.C.

    1987-04-01

    Three membrane-associated proteolytic activities in Escherichia coli were resolved by DEAE-cellulose chromatography from detergent extracts of the total envelope fraction. On the basis of substrate specificity for the hydrolysis of chromogenic amino acid ester substrates, the first two eluting activities were determined previously to be protease V and protease IV, respectively. The third proteolytic activity eluting from the DEAE-cellulose column was further purified by affinity chromatography on benzamidine-Sepharose 6B. They termed this enzyme protease VI. Protease VI did not hydrolyze any of the chromogenic substrates used in the detection of protease IV and protease V. However, all three enzymes generated acid-soluble fragments from a mixture of E. coli membrane proteins which were biosynthetically labeled with radioactive amino acids. The activity of protease VI was sensitive to serine protease inhibitors. Using (/sup 3/H)diisopropylfluorophosphate as an active-site labeling reagent, they determined that protease VI has an apparent molecular weight of 43,000 in polyacrylamide gels. All three membrane-associated serine proteases were insensitive to inhibition by Ecotin, an endogenous, periplasmic inhibitor of trypsin.

  8. Proteases as Markers for Differentiation of Pathogenic and Nonpathogenic Species of Acanthamoeba

    PubMed Central

    Khan, Naveed A.; Jarroll, Edward L.; Panjwani, Noorjahan; Cao, Zhiyi; Paget, Timothy A.

    2000-01-01

    Acanthamoeba keratitis is a vision-threatening infection caused by pathogenic species of the genus Acanthamoeba. Although not all Acanthamoeba spp. can cause keratitis, it is important to differentiate pathogenic species and isolates from nonpathogens. Since extracellular proteases may play a role in ocular pathology, we used colorimetric, cytopathic, and zymographic assays to assess extracellular protease activity in pathogenic and nonpathogenic Acanthamoeba. Colorimetric assays, using azo-linked protein as a substrate, showed extracellular protease activity in Acanthamoeba-conditioned medium and differentiated pathogenic and nonpathogenic Acanthamoeba. Monolayers of immortalized corneal epithelial cells in four-well plates were used for cytopathic effect (CPE) assays. Pathogenic Acanthamoeba isolates exhibited marked CPE on immortalized corneal epithelial cells, while nonpathogenic isolates did not exhibit CPE. Protease zymography was performed with Acanthamoeba-conditioned medium as well as with Acanthamoeba- plus epithelial-cell-conditioned medium. The zymographic protease assays showed various banding patterns for different strains of Acanthamoeba. In pathogenic Acanthamoeba isolates, all protease bands were inhibited by phenylmethylsulfonyl fluoride (PMSF), suggesting serine type proteases, while in nonpathogenic strains only partial inhibition was observed by using PMSF. The pathogenic Acanthamoeba strains grown under typical laboratory conditions without epithelial cells exhibited one overexpressed protease band of 107 kDa in common; this protease was not observed in nonpathogenic Acanthamoeba strains. The 107-kDa protease exhibited activity over a pH range of 5 to 9.5. PMID:10921939

  9. Effects on the Qualities of Proteolysis to Beef by Non-coating and Coating Protease Treatment.

    PubMed

    Kim, Kwang-Il; Lee, Sang-Yoon; Kim, Soo-Jin; Seo, Jae-Hee; Lee, Joong-Kyu; Shin, Jung-Kue; Cho, Hyung-Yong; Choi, Mi-Jung

    2016-01-01

    This study was performed to improve the techniques used for tenderizing red meat as elderly food. Beef meat was immersed in liposome encapsulated enzyme solution and the effect of protease encapsulation on the beef properties was analyzed. The protease encapsulation properties were analyzed according to the size distribution and enzymatic activity. After enzyme reaction on the beef, the chemical properties of the meat such as pH, water holding capacity, shear rate, lipid oxidation and total volatile basic nitrogen (TVB-N) were analyzed. The pH of the beef increased during the reaction and coating protease (CP) was higher than non-coating protease (NCP). Total color differences were increased remarkably after 36 h and generally, the difference in CP was relatively lower than in NCP. WHC was significantly decreased within 24 h, and no effect from the protease coating was observed. Protease activity was significantly increased within 48 h and no differences in the enzyme coating were observed. The TVB-N value of NCP was increased within 24 h while CP was sustained for up to 36 h. The TVB-N value of protease treated meat increased after 36 h and no effect from the protease coating was detected. Consequently, liposome encapsulated protease was found to have similar properties as non-coated protease. Application of liposome seems to be an interesting option for injecting various functional materials without changing the properties of meat. PMID:27499672

  10. Effects on the Qualities of Proteolysis to Beef by Non-coating and Coating Protease Treatment

    PubMed Central

    Shin, Jung-Kue; Cho, Hyung-Yong

    2016-01-01

    This study was performed to improve the techniques used for tenderizing red meat as elderly food. Beef meat was immersed in liposome encapsulated enzyme solution and the effect of protease encapsulation on the beef properties was analyzed. The protease encapsulation properties were analyzed according to the size distribution and enzymatic activity. After enzyme reaction on the beef, the chemical properties of the meat such as pH, water holding capacity, shear rate, lipid oxidation and total volatile basic nitrogen (TVB-N) were analyzed. The pH of the beef increased during the reaction and coating protease (CP) was higher than non-coating protease (NCP). Total color differences were increased remarkably after 36 h and generally, the difference in CP was relatively lower than in NCP. WHC was significantly decreased within 24 h, and no effect from the protease coating was observed. Protease activity was significantly increased within 48 h and no differences in the enzyme coating were observed. The TVB-N value of NCP was increased within 24 h while CP was sustained for up to 36 h. The TVB-N value of protease treated meat increased after 36 h and no effect from the protease coating was detected. Consequently, liposome encapsulated protease was found to have similar properties as non-coated protease. Application of liposome seems to be an interesting option for injecting various functional materials without changing the properties of meat. PMID:27499672

  11. 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.

  12. Two mitochondrial matrix proteases act sequentially in the processing of mammalian matrix enzymes.

    PubMed

    Kalousek, F; Hendrick, J P; Rosenberg, L E

    1988-10-01

    The imported precursors of the mammalian matrix enzymes malate dehydrogenase [(S)-malate:NAD+ oxidoreductase, EC 1.1.1.37] and ornithine transcarbamylase (carbamoyl-phosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3) are cleaved to their mature subunits in two steps, each catalyzed by matrix-localized processing proteases. The number and properties of these proteases are the subjects of this report. We have identified and characterized two distinct protease activities in a crude matrix fraction from rat liver: processing protease I, which cleaves these precursors to the corresponding intermediate form; and processing protease II, which cleaves the intermediate forms to mature subunits. Protease I is insensitive to chelation by EDTA and to inactivation with N-ethylmaleimide; protease II is inhibited by 5 mM EDTA and is inactivated by treatment with N-ethylmaleimide. We have prepared from mitochondrial matrix an 800-fold-enriched protease I fraction free of protease II activity by using the following steps: ion exchange, hydroxyapatite, molecular sieving, and hydrophobic chromatography. Using similar procedures, we also have prepared an approximately 2000-fold-enriched protease II fraction, which has a trace amount of contaminating protease I. This enriched protease II fraction has little or no cleavage activity toward mitochondrial precursors but rapidly and efficiently converts intermediate forms to mature size. Finally, we show that protease I alone is sufficient to cleave the precursor of a third nuclear-encoded mitochondrial protein subunit--the beta subunit of propionyl-CoA carboxylase [propanoyl-CoA:carbon dioxide ligase (ADP-forming), EC 6.4.1.3]--to its mature size. PMID:3050998

  13. Host cell proteases: critical determinants of coronavirus tropism and pathogenesis

    PubMed Central

    Millet, Jean Kaoru; Whittaker, Gary R.

    2015-01-01

    Coronaviruses are a large group of enveloped, single-stranded positive-sense RNA viruses that infect a wide range of avian and mammalian species, including humans. The emergence of deadly human coronaviruses, severe acute respiratory syndrome coronavirus (SARS-CoV), and Middle East respiratory syndrome coronavirus (MERS-CoV) have bolstered research in these viral and often zoonotic pathogens. While coronavirus cell and tissue tropism, host range, and pathogenesis are initially controlled by interactions between the spike envelope glycoprotein and host cell receptor, it is becoming increasingly apparent that proteolytic activation of spike by host cell proteases also plays a critical role. Coronavirus spike proteins are the main determinant of entry as they possess both receptor binding and fusion functions. Whereas binding to the host cell receptor is an essential first step in establishing infection, the proteolytic activation step is often critical for the fusion function of spike, as it allows for controlled release of the fusion peptide into target cellular membranes. Coronaviruses have evolved multiple strategies for proteolytic activation of spike, and a large number of host proteases have been shown to proteolytically process the spike protein. These include, but are not limited to, endosomal cathepsins, cell surface transmembrane protease/serine (TMPRSS) proteases, furin, and trypsin. This review focuses on the diversity of strategies coronaviruses have evolved to proteolytically activate their fusion protein during spike protein biosynthesis and the critical entry step of their life cycle, and highlights important findings on how proteolytic activation of coronavirus spike influences tissue and cell tropism, host range and pathogenicity. PMID:25445340

  14. Taspase 1: A protease with many biological surprises

    PubMed Central

    Niizuma, Hidetaka; Cheng, Emily H; Hsieh, James J

    2015-01-01

    Taspase 1 (TASP1) cleaves the mixed-lineage leukemia (MLL) and transcription factor (TF) IIA families of nuclear proteins to orchestrate various biological processes. TASP1 is not a classical oncogene, but assists in cell proliferation and permits oncogenic initiation through cleavage of MLL and TFIIA. TASP1 is thus better classified as a “non-oncogene addiction” protease, and targeting TASP1 offers a novel and attractive anticancer therapeutic strategy. PMID:27308523

  15. Interaction of proteases with legume seed inhibitors. Molecular features.

    PubMed

    de Seidl, D S

    1996-12-01

    After having found that raw black beans (Phaseolus vulgaris) were toxic, while the cooked ones constitute the basic diet of the underdeveloped peoples of the world, in the sixties, our research directed by Dr. Jaffé, concentrated mainly around the detection and identification of the heat labile toxic factors in legume seeds. A micromethod for the detection of protease inhibitors (PI) in individual seeds was developed, for the purpose of establishing that the multiple trypsin inhibitors (TI) found in the Cubagua variety were expressions of single seeds and not a mixture of a non homogenous bean lot. Six isoinhibitors were isolated and purified, all of which were "double-headed" and interacted with trypsin (T) and chymotrypsin (CHT) independently and simultaneously, as shown by electrophoresis of their binary and ternary complexes with each and both enzymes. However, their affinity for the enzymes, including elastases, was rather variable, as well as their amino acid composition which consisted of 51 units for inhibitor V, the smallest, and 83 amino acids for inhibitor I, the largest. A low molecular weight protein fraction that inhibited subtilisin (S), but recognized neither T, CHT nor pancreatic elastase was detected in 63 varieties of Phaseolus vulgaris as well as in broad beans (Vicia faba), chick peas (Cicer arietinum), jack beans (Canavalia ensiformis), kidney beans (Vigna aureus), etc., It was absent though, in soybeans (Glycine max), lentils (Lens culinaris), green peas (Pisum sativum), cowpea (Vigna sinensis) and lupine seeds (Lupinus sp). Subtilisin inhibitors (SI) were isolated from black beans, broad beans, chick peas and jack beans. Their Mr is between 8-9KD and they show a rather high stability in the presence of denaturing agents. They are specific toward microbial proteases, in addition to subtilisins, Carlsberg and BPN', they inhibit the alkaline protease from Tritirachium album (Protease K), from Aspergillus oryzae and one isolated from

  16. Protease-catalysed Direct Asymmetric Mannich Reaction in Organic Solvent

    NASA Astrophysics Data System (ADS)

    Xue, Yang; Li, Ling-Po; He, Yan-Hong; Guan, Zhi

    2012-10-01

    We reported the first enzyme-catalysed, direct, three-component asymmetric Mannich reaction using protease type XIV from Streptomyces griseus (SGP) in acetonitrile. Yields of up to 92% with enantioselectivities of up to 88% e.e. and diastereoselectivities of up to 92:8 (syn:anti) were achieved under the optimised conditions. This enzyme's catalytic promiscuity expands the application of this biocatalyst and provides a potential alternative method for asymmetric Mannich reactions.

  17. ENaC regulation by proteases and shear stress

    PubMed Central

    Shi, Shujie; Carattino, Marcelo D.; Hughey, Rebecca P.; Kleyman, Thomas R.

    2013-01-01

    Epithelial Na+ channels (ENaCs) are comprised of subunits that have large extracellular regions linked to membrane spanning domains where the channel pore and gate reside. A variety of external factors modify channel activity by interacting at sites within extracellular regions that lead to conformational changes that are transmitted to the channel gate and alter channel open probability. Our review addresses two external factors that have important roles in regulating channel activity, proteases and laminar shear stress. PMID:23547932

  18. Protease-catalysed direct asymmetric Mannich reaction in organic solvent.

    PubMed

    Xue, Yang; Li, Ling-Po; He, Yan-Hong; Guan, Zhi

    2012-01-01

    We reported the first enzyme-catalysed, direct, three-component asymmetric Mannich reaction using protease type XIV from Streptomyces griseus (SGP) in acetonitrile. Yields of up to 92% with enantioselectivities of up to 88% e.e. and diastereoselectivities of up to 92:8 (syn:anti) were achieved under the optimised conditions. This enzyme's catalytic promiscuity expands the application of this biocatalyst and provides a potential alternative method for asymmetric Mannich reactions. PMID:23094136

  19. Protease-catalysed Direct Asymmetric Mannich Reaction in Organic Solvent

    PubMed Central

    Xue, Yang; Li, Ling-Po; He, Yan-Hong; Guan, Zhi

    2012-01-01

    We reported the first enzyme-catalysed, direct, three-component asymmetric Mannich reaction using protease type XIV from Streptomyces griseus (SGP) in acetonitrile. Yields of up to 92% with enantioselectivities of up to 88% e.e. and diastereoselectivities of up to 92:8 (syn:anti) were achieved under the optimised conditions. This enzyme's catalytic promiscuity expands the application of this biocatalyst and provides a potential alternative method for asymmetric Mannich reactions. PMID:23094136

  20. PROSTATE-SPECIFIC ANTIGEN IS A “CHYMOTRYPSIN-LIKE” SERINE PROTEASE WITH UNIQUE P1 SUBSTRATE SPECIFICITY

    PubMed Central

    LeBeau, Aaron M.; Singh, Pratap; Isaacs, John T.; Denmeade, Samuel R.

    2012-01-01

    Prostate-Specific Antigen (PSA), a serine protease belonging to the human kallikrein family, is best known as a prostate cancer biomarker. Emerging evidence suggests that PSA may also play a salient role in prostate cancer development and progression. With large amounts of enzymatically active PSA continuously and selectively produced by all stages of prostate cancer, PSA is an attractive target. PSA inhibitors, therefore, may represent a promising class of therapeutics and/or imaging agents. PSA displays chymotrypsin-like specificity, cleaving after hydrophobic residues, in addition to possessing a unique ability to cleave after glutamine in the P1 position. In this study, we investigated the structural motifs of the PSA S1 pocket that give it a distinct architecture and specificity when compared to the S1 pocket of chymotrypsin. Using the previously described PSA substrate Ser-Ser-Lys-Leu-Gln (SSKLQ) as a template, peptide aldehyde based inhibitors containing novel P1 aldehydes were made and tested against both proteases. Glutamine derivative aldehydes were highly specific for PSA while inhibitors with hydrophobic P1 aldehydes were potent inhibitors of both proteases with Ki values < 500 nM. The crystal structure of PSA was used to generate a model that allowed GOLD docking studies to be performed to further understand the critical interactions required for inhibitor binding to the S1 pockets of PSA and chymotrypsin. In conclusion, these results provide experimental and structural evidence that the S1 specificity pocket of PSA is distinctly different from that of chymotrypsin and that the development of highly specific PSA inhibitors is feasible. PMID:19281249

  1. Milk-clotting mechanism of Dregea sinensis Hemsl. protease.

    PubMed

    Zhang, Yali; Wang, Hongyan; Tao, Liang; Huang, Ai-xiang

    2015-12-01

    Dregea sinensis Hemsl. is used as a milk coagulant to produce goat milk cakes in Yunnan, China. However, the composition of milk-clotting compounds and the related mechanism have not been reported. Crude protease was extracted from the stem, purified, and then separated with a Millipore ultrafiltration centrifuge tube. Cysteine protease (procerain B) was identified as the main milk-clotting protein through electrospray ionization mass spectrometry, and its molecular weight was 23.8 kDa. The protease can partially degrade α-casein (CN) and completely degrade β- and κ-CN, and κ-CN degradation resulted in milk clotting. The molecular weight and AA sequence of the peptide fractions were determined through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and a peptide sequencer, respectively. The enzyme cleaved κ-CN at Ala90-Gln91 and produced deputy κ-CN and caseinomacropeptide with molecular weights of 12 and 6.9 kDa, respectively. This cleavage site differed from the majority of chymosins cleaved at Phe105-Met106. PMID:26506540

  2. Evolution of the protease-activated receptor family in vertebrates

    PubMed Central

    JIN, MIN; YANG, HAI-WEI; TAO, AI-LIN; WEI, JI-FU

    2016-01-01

    Belonging to the G protein-coupled receptor (GPcr) family, the protease-activated receptors (Pars) consist of 4 members, PAR1-4. PARs mediate the activation of cells via thrombin, serine and other proteases. Such protease-triggered signaling events are thought to be critical for hemostasis, thrombosis and other normal pathological processes. In the present study, we examined the evolution of PARs by analyzing phylogenetic trees, chromosome location, selective pressure and functional divergence based on the 169 functional gene alignment sequences from 57 vertebrate gene sequences. We found that the 4 PARs originated from 4 invertebrate ancestors by phylogenetic trees analysis. The selective pressure results revealed that only PAR1 appeared by positive selection during its evolution, while the other PAR members did not. In addition, we noticed that although these PARs evolved separately, the results of functional divergence indicated that their evolutional rates were similar and their functions did not significantly diverge. The findings of our study provide valuable insight into the evolutionary history of the vertebrate PAR family. PMID:26820116

  3. [Extracellular proteases of mycelial fungi as participants of pathogenic processes].

    PubMed

    Dunaevskiĭ, Ia E; Matveeva, A R; Fatkhullina, G N; Beliakova, G A; Kolomiets, T M; Kovalenko, E D; Belozerskiĭ, M A

    2008-01-01

    The interest in proteases secreted by mycelial fungi is due to several reasons of which one of the most important is their involvement in the initiation and development of the pathogenic process. A comparison of saprophytic and phytopathogenic mycelial fungi revealed one characteristic feature, namely, the appearance of a new trypsin-like activity in phytopathogens that is absent in saprophytes. To clear up the question of whether the degree of pathogenicity of a fungus is related to the activity of secreted trypsin-like protease, several species of Fusarium of various pathogenicity were compared. In two species, F. sporotrichioides (which causes ear fusa-riosis of rye) and F. heterosporum (the causative agent of root rot in wheat), a clear correlation between the activity and pathogenicity was revealed: the more pathogenetic F. sporotrichioides exhibited a higher extracellular trypsin-like activity than the less pathogenetic species F. heterosporum. Thus, the presence of trypsin-like activity in a saprotroph-pathogen pair may be an indicator of the pathogenicity of a fungus; in some cases, the value of this activity may indicate the degree of its pathogenicity. This suggests that trypsin-like proteases specific to phytopathogens are directly involved in the pathogenetic process, probably, through interaction with the "sentry" protein or the product of the resistance gene. PMID:18672678

  4. Humanized-VHH transbodies that inhibit HCV protease and replication.

    PubMed

    Jittavisutthikul, Surasak; Thanongsaksrikul, Jeeraphong; Thueng-In, Kanyarat; Chulanetra, Monrat; Srimanote, Potjanee; Seesuay, Watee; Malik, Aijaz Ahmad; Chaicumpa, Wanpen

    2015-04-01

    There is a need for safe and broadly effective anti-HCV agents that can cope with genetic multiplicity and mutations of the virus. In this study, humanized-camel VHHs to genotype 3a HCV serine protease were produced and were linked molecularly to a cell penetrating peptide, penetratin (PEN). Human hepatic (Huh7) cells transfected with the JFH-1 RNA of HCV genotype 2a and treated with the cell penetrable nanobodies (transbodies) had a marked reduction of the HCV RNA intracellularly and in their culture fluids, less HCV foci inside the cells and less amounts of HCV core antigen in culture supernatants compared with the infected cells cultured in the medium alone. The PEN-VHH-treated-transfected cells also had up-regulation of the genes coding for the host innate immune response (TRIF, TRAF3, IRF3, IL-28B and IFN-β), indicating that the cell penetrable nanobodies rescued the host innate immune response from the HCV mediated-suppression. Computerized intermolecular docking revealed that the VHHs bound to residues of the protease catalytic triad, oxyanion loop and/or the NS3 N-terminal portion important for non-covalent binding of the NS4A protease cofactor protein. The so-produced transbodies have high potential for testing further as a candidate for safe, broadly effective and virus mutation tolerable anti-HCV agents. PMID:25903832

  5. Pathogen-Secreted Proteases Activate a Novel Plant Immune Pathway

    PubMed Central

    Cheng, Zhenyu; Li, Jian-Feng; Niu, Yajie; Zhang, Xue-Cheng; Woody, Owen Z.; Xiong, Yan; Djonović, Slavica; Millet, Yves; Bush, Jenifer; McConkey, Brendan J.; Sheen, Jen; Ausubel, Frederick M.

    2015-01-01

    Mitogen-Activated Protein Kinase (MAPK) cascades play central roles in innate immune signaling networks in plants and animals1,2. In plants, however, the molecular mechanisms of how signal perception is transduced to MAPK activation remain elusive1. We report that pathogen-secreted proteases activate a previously unknown signaling pathway in Arabidopsis thaliana involving the Gα, Gβ and Gγ subunits of heterotrimeric G-protein complexes, which function upstream of a MAPK cascade. In this pathway, Receptor for Activated C Kinase 1 (RACK1) functions as a novel scaffold that binds to the Gβ subunit as well as to all three tiers of the MAPK cascade, thereby linking upstream G protein signaling to downstream activation of a MAPK cascade. The protease-G protein-RACK1-MAPK cascade modules identified in these studies are distinct from previously described plant immune signaling pathways such as the one elicited by bacterial flagellin, in which G proteins function downstream of or in parallel to a MAPK cascade without the involvement of the RACK1 scaffolding protein. The discovery of the novel protease-mediated immune signaling pathway described here was facilitated by the use of the broad host range, opportunistic bacterial pathogen Pseudomonas aeruginosa. The ability of P. aeruginosa to infect both plants and animals makes it an excellent model to identify novel types of immunoregulatory strategies that account for its niche adaptation to diverse host tissues and immune systems. PMID:25731164

  6. Crystal Structure of a Rhomboid Family Intramembrane Protease.

    SciTech Connect

    Wang,Y.; Zhang, Y.; Ha, Y.

    2006-01-01

    Escherichia coli GlpG is an integral membrane protein that belongs to the widespread rhomboid protease family. Rhomboid proteases, like site-2 protease (S2P) and {gamma}-secretase, are unique in that they cleave the transmembrane domain of other membrane proteins. Here we describe the 2.1 {angstrom} resolution crystal structure of the GlpG core domain. This structure contains six transmembrane segments. Residues previously shown to be involved in catalysis, including a Ser-His dyad, and several water molecules are found at the protein interior at a depth below the membrane surface. This putative active site is accessible by substrate through a large 'V-shaped' opening that faces laterally towards the lipid, but is blocked by a half-submerged loop structure. These observations indicate that, in intramembrane proteolysis, the scission of peptide bonds takes place within the hydrophobic environment of the membrane bilayer. The crystal structure also suggests a gating mechanism for GlpG that controls substrate access to its hydrophilic active site.

  7. Phytoferritin association induced by EGCG inhibits protein degradation by proteases.

    PubMed

    Wang, Aidong; Zhou, Kai; Qi, Xin; Zhao, Guanghua

    2014-12-01

    Phytoferritin is a promising resource of non-heme iron supplementation, but it is not stable against degradation by proteases in the gastrointestinal tract. Therefore, how to improve the stability of ferritin in the presence of proteases is a challenge. Since (-)-epigallocatechin-3-gallate (EGCG) is rich in phenolic-hydroxyl groups, it could interact with ferritin through hydrogen bonds, thereby preventing protein from degradation. To confirm this idea, we focus on the interaction between EGCG and phytoferritin, and the consequence of such interaction. Results demonstrated that EGCG did interact with ferritin, and such interaction induced the change in the tertiary/quaternary structure of protein but not in its secondary structure. Furthermore, stopped-flow and dynamic light scattering (DLS) results showed that EGCG could trigger ferritin association. Consequently, such protein association markedly inhibited protein digestion by pepsin at pH 4.0 and by trypsin at pH 7.5. These findings raise the possibility to improve the stability of phytoferritin in the presence of proteases. PMID:25384342

  8. Chloroplast Proteases: Updates on Proteolysis within and across Suborganellar Compartments.

    PubMed

    Nishimura, Kenji; Kato, Yusuke; Sakamoto, Wataru

    2016-08-01

    Chloroplasts originated from the endosymbiosis of ancestral cyanobacteria and maintain transcription and translation machineries for around 100 proteins. Most endosymbiont genes, however, have been transferred to the host nucleus, and the majority of the chloroplast proteome is composed of nucleus-encoded proteins that are biosynthesized in the cytosol and then imported into chloroplasts. How chloroplasts and the nucleus communicate to control the plastid proteome remains an important question. Protein-degrading machineries play key roles in chloroplast proteome biogenesis, remodeling, and maintenance. Research in the past few decades has revealed more than 20 chloroplast proteases, which are localized to specific suborganellar locations. In particular, two energy-dependent processive proteases of bacterial origin, Clp and FtsH, are central to protein homeostasis. Processing endopeptidases such as stromal processing peptidase and thylakoidal processing peptidase are involved in the maturation of precursor proteins imported into chloroplasts by cleaving off the amino-terminal transit peptides. Presequence peptidases and organellar oligopeptidase subsequently degrade the cleaved targeting peptides. Recent findings have indicated that not only intraplastidic but also extraplastidic processive protein-degrading systems participate in the regulation and quality control of protein translocation across the envelopes. In this review, we summarize current knowledge of the major chloroplast proteases in terms of type, suborganellar localization, and diversification. We present details of these degradation processes as case studies according to suborganellar compartment (envelope, stroma, and thylakoids). Key questions and future directions in this field are discussed. PMID:27288365

  9. Separation of proteases from yellowfin tuna spleen by ultrafiltration.

    PubMed

    Li, Zhenyu; Youravong, Wirote; H-Kittikun, Aran

    2006-12-01

    Separation of protease, trypsin and chymotrypsin from yellowfin tuna spleen extract by ultrafiltration (UF) using regenerated cellulose membranes with molecular weight cut off (MWCO) 30 and 100 kDa was studied. The 100 kDa membrane had a higher transmission of enzymes than that of the 30 kDa membrane. The enzyme transmission varied from 0.01 to 0.18 and from 0.6 to 0.8 for the 30 kDa membrane and 100 kDa membrane, respectively. The protein transmission was about 0.8 for both membranes. Increasing cross-flow rate and transmembrane pressure (TMP) increased permeate flux. The limiting fluxes at cross-flow rate 120, 240 and 360 L/h for the 30 kDa membrane were 17.3, 43.9 and 54.7 L/m2h, respectively and the limiting fluxes at the same flow rate for 100 kDa membrane were 34.1, 51.1 and 68.4 L/m2h, respectively. The separation of these proteases was achieved using the 30 kDa membrane. The purities of proteases were increased more than ten times at TMP 1.5 bar and cross-flow rate 360 L/h by diafiltration using 30 kDa membrane. PMID:16314093

  10. Human Immunodeficiency Virus gag and protease: partners in resistance

    PubMed Central

    2012-01-01

    Human Immunodeficiency Virus (HIV) maturation plays an essential role in the viral life cycle by enabling the generation of mature infectious virus particles through proteolytic processing of the viral Gag and GagPol precursor proteins. An impaired polyprotein processing results in the production of non-infectious virus particles. Consequently, particle maturation is an excellent drug target as exemplified by inhibitors specifically targeting the viral protease (protease inhibitors; PIs) and the experimental class of maturation inhibitors that target the precursor Gag and GagPol polyproteins. Considering the different target sites of the two drug classes, direct cross-resistance may seem unlikely. However, coevolution of protease and its substrate Gag during PI exposure has been observed both in vivo and in vitro. This review addresses in detail all mutations in Gag that are selected under PI pressure. We evaluate how polymorphisms and mutations in Gag affect PI therapy, an aspect of PI resistance that is currently not included in standard genotypic PI resistance testing. In addition, we consider the consequences of Gag mutations for the development and positioning of future maturation inhibitors. PMID:22867298

  11. Humanized-VHH Transbodies that Inhibit HCV Protease and Replication

    PubMed Central

    Jittavisutthikul, Surasak; Thanongsaksrikul, Jeeraphong; Thueng-in, Kanyarat; Chulanetra, Monrat; Srimanote, Potjanee; Seesuay, Watee; Malik, Aijaz Ahmad; Chaicumpa, Wanpen

    2015-01-01

    There is a need for safe and broadly effective anti-HCV agents that can cope with genetic multiplicity and mutations of the virus. In this study, humanized-camel VHHs to genotype 3a HCV serine protease were produced and were linked molecularly to a cell penetrating peptide, penetratin (PEN). Human hepatic (Huh7) cells transfected with the JFH-1 RNA of HCV genotype 2a and treated with the cell penetrable nanobodies (transbodies) had a marked reduction of the HCV RNA intracellularly and in their culture fluids, less HCV foci inside the cells and less amounts of HCV core antigen in culture supernatants compared with the infected cells cultured in the medium alone. The PEN-VHH-treated-transfected cells also had up-regulation of the genes coding for the host innate immune response (TRIF, TRAF3, IRF3, IL-28B and IFN-β), indicating that the cell penetrable nanobodies rescued the host innate immune response from the HCV mediated-suppression. Computerized intermolecular docking revealed that the VHHs bound to residues of the protease catalytic triad, oxyanion loop and/or the NS3 N-terminal portion important for non-covalent binding of the NS4A protease cofactor protein. The so-produced transbodies have high potential for testing further as a candidate for safe, broadly effective and virus mutation tolerable anti-HCV agents. PMID:25903832

  12. Characterization of resistance mutations against HCV ketoamide protease inhibitors.

    PubMed

    Tong, Xiao; Bogen, Stephane; Chase, Robert; Girijavallabhan, V; Guo, Zhuyan; Njoroge, F George; Prongay, Andrew; Saksena, Anil; Skelton, Angela; Xia, Ellen; Ralston, Robert

    2008-03-01

    An issue of clinical importance in the development of new antivirals for HCV is emergence of resistance. Several resistance loci to ketoamide inhibitors of the NS3/4A protease have been identified (residues V36, T54, R155, A156, and V170) by replicon and clinical studies. Using SCH 567312, a more potent protease inhibitor derived from SCH 503034 (boceprevir) series, we identified two new positions (Q41 and F43) that confer resistance to the ketoamide class. The catalytic efficiency of protease enzymes was not affected by most resistance mutations, whereas replicon fitness varied with specific mutations. SCH 503034 and another ketoamide inhibitor, VX-950 (telaprevir), showed moderate losses of activity against most resistance mutations (< or =10-fold); the highest resistance level was conferred by mutations at A156 locus. Although SCH 503034 and VX-950 bind similarly to the active site, differences in resistance level were observed with specific mutations. Changes at V36 and R155 had more severe impact on VX-950, whereas mutations at Q41, F43 and V170 conferred higher resistance to SCH 503034. Structural analysis of resistance mutations on inhibitor binding is discussed. PMID:18201776

  13. Functional domains of the human epididymal protease inhibitor, eppin.

    PubMed

    McCrudden, Maelíosa T C; Dafforn, Tim R; Houston, David F; Turkington, Philip T; Timson, David J

    2008-04-01

    Eppin has two potential protease inhibitory domains: a whey acid protein or four disulfide core domain and a Kunitz domain. The protein is also reported to have antibacterial activity against Gram-negative bacteria. Eppin and its whey acid protein and Kunitz domains were expressed in Escherichia coli and their ability to inhibit proteases and kill bacteria compared. The Kunitz domain inhibits elastase (EC 3.4.21.37) to a similar extent as intact eppin, whereas the whey acid protein domain has no such activity. None of these fragments inhibits trypsin (EC 3.4.21.4) or chymotrypsin (EC 3.4.21.1) at the concentrations tested. In a colony forming unit assay, both domains have some antibacterial activity against E. coli, but this was not to the same degree as intact eppin or the two domains together. When bacterial respiratory electron transport was measured using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide assay, eppin and its domains caused an increase in the rate of respiration. This suggests that the mechanism of cell killing may be partly through the permeablization of the bacterial inner membrane, resulting in uncoupling of respiratory electron transport and consequent collapse of the proton motive force. Thus, we conclude that although both of eppin's domains are involved in the protein's antibacterial activity, only the Kunitz domain is required for selective protease inhibition. PMID:18331357

  14. Enhanced Thermostability of a Fungal Alkaline Protease by Different Additives

    PubMed Central

    Nirmal, Nilesh P.; Laxman, R. Seeta

    2014-01-01

    A fungal strain (Conidiobolus brefeldianus MTCC 5184) isolated from plant detritus secreted a high activity alkaline protease. Thermostability studies of the fungal alkaline protease (FAP) revealed that the protease is stable up to 50°C with 40% residual activity after one hour. Effect of various additives such as sugars, sugar alcohols, polyols, and salts, on the thermostability of FAP was evaluated. Among the additives tested, glycerol, mannitol, xylitol, sorbitol, and trehalose were found to be very effective in increasing the stability of FAP, which was found to be concentration dependent. Fivefold increase in residual activity of FAP was observed in the presence of trehalose (50%) and sorbitol (50%) at 50°C for 4 h, compared to FAP without additive. Other additives like calcium at 20 mM and 10–15% ammonium sulphate showed lower stability improvement than trehalose and sorbitol. NaCl, MgCl2, K2HPO4, and glycine were found to be poor stabilizers and showed only a marginal improvement. PEG 6000 did not show any increase in stability but was found to be slightly inhibitory. PMID:25105022

  15. Identification of papain-like cysteine proteases from the bovine piroplasm Babesia bigemina and evolutionary relationship of piroplasms C1 family of cysteine proteases.

    PubMed

    Martins, Tiago M; do Rosário, Virgílio E; Domingos, Ana

    2011-01-01

    Papain-like cysteine proteases have been shown to have essential roles in parasitic protozoa and are under study as promising drug targets. Five genes were identified by sequence similarity search to be homologous to the cysteine protease family in the ongoing Babesia bigemina genome sequencing project database and were compared with the annotated genes from the complete bovine piroplasm genomes of Babesia bovis, Theileria annulata, and Theileria parva. Multiple genome alignments and sequence analysis were used to evaluate the molecular evolution events that occurred in the C1 family of cysteine proteases in these piroplasms of veterinary importance. BbiCPL1, one of the newly identified cysteine protease genes in the B. bigemina genome was expressed in Escherichia coli and shows activity against peptide substrates. Considerable differences were observed in the cysteine protease family between Babesia and Theileria genera, and this may partially explain the diverse infection mechanisms of these tick-borne diseases. PMID:20655912

  16. In vivo sequence diversity of the protease of human immunodeficiency virus type 1: presence of protease inhibitor-resistant variants in untreated subjects.

    PubMed Central

    Lech, W J; Wang, G; Yang, Y L; Chee, Y; Dorman, K; McCrae, D; Lazzeroni, L C; Erickson, J W; Sinsheimer, J S; Kaplan, A H

    1996-01-01

    We have evaluated the sequence diversity of the protease human immunodeficiency virus type 1 in vivo. Our analysis of 246 protease coding domain sequences obtained from 12 subjects indicates that amino acid substitutions predicted to give rise to protease inhibitor resistance may be present in patients who have not received protease inhibitors. In addition, we demonstrated that amino acid residues directly involved in enzyme-substrate interactions may be varied in infected individuals. Several of these substitutions occurred in combination either more or less frequently than would be expected if their appearance was independent, suggesting that one substitution may compensate for the effects of another. Taken together, our analysis indicates that the human immunodeficiency virus type 1 protease has flexibility sufficient to vary critical subsites in vivo, thereby retaining enzyme function and viral pathogenicity. PMID:8627733

  17. Cardiometabolic Risk Profiles in Patients With Impaired Fasting Glucose and/or Hemoglobin A1c 5.7% to 6.4%: Evidence for a Gradient According to Diagnostic Criteria: The PREDAPS Study.

    PubMed

    Giráldez-García, Carolina; Sangrós, F Javier; Díaz-Redondo, Alicia; Franch-Nadal, Josep; Serrano, Rosario; Díez, Javier; Buil-Cosiales, Pilar; García-Soidán, F Javier; Artola, Sara; Ezkurra, Patxi; Carrillo, Lourdes; Millaruelo, J Manuel; Seguí, Mateu; Martínez-Candela, Juan; Muñoz, Pedro; Goday, Albert; Regidor, Enrique

    2015-11-01

    It has been suggested that the early detection of individuals with prediabetes can help prevent cardiovascular diseases. The purpose of the current study was to examine the cardiometabolic risk profile in patients with prediabetes according to fasting plasma glucose (FPG) and/or hemoglobin A1c (HbA1c) criteria.Cross-sectional analysis from the 2022 patients in the Cohort study in Primary Health Care on the Evolution of Patients with Prediabetes (PREDAPS Study) was developed. Four glycemic status groups were defined based on American Diabetes Association criteria. Information about cardiovascular risk factors-body mass index, waist circumference, blood pressure, cholesterol, triglycerides, uric acid, gamma-glutamyltransferase, glomerular filtration-and metabolic syndrome components were analyzed. Mean values of clinical and biochemical characteristics and frequencies of metabolic syndrome were estimated adjusting by age, sex, educational level, and family history of diabetes.A linear trend (P < 0.001) was observed in most of the cardiovascular risk factors and in all components of metabolic syndrome. Normoglycemic individuals had the best values, individuals with both criteria of prediabetes had the worst, and individuals with only one-HbA1c or FPG-criterion had an intermediate position. Metabolic syndrome was present in 15.0% (95% confidence interval: 12.6-17.4), 59.5% (54.0-64.9), 62.0% (56.0-68.0), and 76.2% (72.8-79.6) of individuals classified in normoglycemia, isolated HbA1c, isolated FPG, and both criteria groups, respectively.In conclusion, individuals with prediabetes, especially those with both criteria, have worse cardiometabolic risk profile than normoglycemic individuals. These results suggest the need to use both criteria in the clinical practice to identify those individuals with the highest cardiovascular risk, in order to offer them special attention with intensive lifestyle intervention programs. PMID:26554799

  18. Cardiometabolic Risk Profiles in Patients With Impaired Fasting Glucose and/or Hemoglobin A1c 5.7% to 6.4%: Evidence for a Gradient According to Diagnostic Criteria

    PubMed Central

    Giráldez-García, Carolina; Sangrós, F. Javier; Díaz-Redondo, Alicia; Franch-Nadal, Josep; Serrano, Rosario; Díez, Javier; Buil-Cosiales, Pilar; García-Soidán, F. Javier; Artola, Sara; Ezkurra, Patxi; Carrillo, Lourdes; Millaruelo, J. Manuel; Seguí, Mateu; Martínez-Candela, Juan; Muñoz, Pedro; Goday, Albert; Regidor, Enrique

    2015-01-01

    Abstract It has been suggested that the early detection of individuals with prediabetes can help prevent cardiovascular diseases. The purpose of the current study was to examine the cardiometabolic risk profile in patients with prediabetes according to fasting plasma glucose (FPG) and/or hemoglobin A1c (HbA1c) criteria. Cross-sectional analysis from the 2022 patients in the Cohort study in Primary Health Care on the Evolution of Patients with Prediabetes (PREDAPS Study) was developed. Four glycemic status groups were defined based on American Diabetes Association criteria. Information about cardiovascular risk factors–body mass index, waist circumference, blood pressure, cholesterol, triglycerides, uric acid, gamma-glutamyltransferase, glomerular filtration–and metabolic syndrome components were analyzed. Mean values of clinical and biochemical characteristics and frequencies of metabolic syndrome were estimated adjusting by age, sex, educational level, and family history of diabetes. A linear trend (P < 0.001) was observed in most of the cardiovascular risk factors and in all components of metabolic syndrome. Normoglycemic individuals had the best values, individuals with both criteria of prediabetes had the worst, and individuals with only one–HbA1c or FPG–criterion had an intermediate position. Metabolic syndrome was present in 15.0% (95% confidence interval: 12.6–17.4), 59.5% (54.0–64.9), 62.0% (56.0–68.0), and 76.2% (72.8–79.6) of individuals classified in normoglycemia, isolated HbA1c, isolated FPG, and both criteria groups, respectively. In conclusion, individuals with prediabetes, especially those with both criteria, have worse cardiometabolic risk profile than normoglycemic individuals. These results suggest the need to use both criteria in the clinical practice to identify those individuals with the highest cardiovascular risk, in order to offer them special attention with intensive lifestyle intervention programs. PMID:26554799

  19. 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. PMID:22225984

  20. Exoerythrocytic Plasmodium Parasites Secrete a Cysteine Protease Inhibitor Involved in Sporozoite Invasion and Capable of Blocking Cell Death of Host Hepatocytes

    PubMed Central

    Rennenberg, Annika; Lehmann, Christine; Heitmann, Anna; Witt, Tina; Hansen, Guido; Nagarajan, Krishna; Deschermeier, Christina; Turk, Vito; Hilgenfeld, Rolf; Heussler, Volker T.

    2010-01-01

    Plasmodium parasites must control cysteine protease activity that is critical for hepatocyte invasion by sporozoites, liver stage development, host cell survival and merozoite liberation. Here we show that exoerythrocytic P. berghei parasites express a potent cysteine protease inhibitor (PbICP, P. berghei inhibitor of cysteine proteases). We provide evidence that it has an important function in sporozoite invasion and is capable of blocking hepatocyte cell death. Pre-incubation with specific anti-PbICP antiserum significantly decreased the ability of sporozoites to infect hepatocytes and expression of PbICP in mammalian cells protects them against peroxide- and camptothecin-induced cell death. PbICP is secreted by sporozoites prior to and after hepatocyte invasion, localizes to the parasitophorous vacuole as well as to the parasite cytoplasm in the schizont stage and is released into the host cell cytoplasm at the end of the liver stage. Like its homolog falstatin/PfICP in P. falciparum, PbICP consists of a classical N-terminal signal peptide, a long N-terminal extension region and a chagasin-like C-terminal domain. In exoerythrocytic parasites, PbICP is posttranslationally processed, leading to liberation of the C-terminal chagasin-like domain. Biochemical analysis has revealed that both full-length PbICP and the truncated C-terminal domain are very potent inhibitors of cathepsin L-like host and parasite cysteine proteases. The results presented in this study suggest that the inhibitor plays an important role in sporozoite invasion of host cells and in parasite survival during liver stage development by inhibiting host cell proteases involved in programmed cell death. PMID:20361051

  1. The serine protease inhibitor protease nexin-1 controls mammary cancer metastasis through LRP-1-mediated MMP-9 expression.

    PubMed

    Fayard, Bérengère; Bianchi, Fabrizio; Dey, Julien; Moreno, Eliza; Djaffer, Sabrina; Hynes, Nancy E; Monard, Denis

    2009-07-15

    Through their ability to degrade the extracellular matrix, proteases mediate cancer cell invasion and metastasis. Paradoxically, some serine protease inhibitors (serpins) are often overexpressed in human tumors. Using computational analysis, we found that the RNA level of protease nexin-1 (PN-1), a serpin that blocks numerous proteases activity, is significantly elevated in estrogen receptor-alpha-negative and in high-grade breast cancer. The in silico approach was complemented by mechanistic studies on two mammary cancer cell lines, the PN-1-negative 168FARN cells and the PN-1-positive 4T1 cells, both of which form primary mammary tumors, but only 4T1 tumors are able to metastasize to the lungs. We show that treatment of 168FARN cells with PN-1 stimulates extracellular signal-regulated kinase activation via low-density lipoprotein receptor-related protein-1 (LRP-1) binding, resulting in increased matrix metalloproteinase (MMP)-9 RNA, protein, and secreted activity. PN-1-silenced 4T1 cells express low MMP-9 levels. Moreover, injection of PN-1-silenced cells into mice did not affect 4T1 primary mammary tumor outgrowth; however, the tumors had impaired metastatic potential, which could be restored by reexpressing soluble MMP-9 in the PN-1-silenced 4T1 cells. Thus, using mammary tumor models, we describe a novel pathway whereby the serpin PN-1 by binding LRP-1 stimulates extracellular signal-regulated kinase signaling, MMP-9 expression, and metastatic spread of mammary tumors. Importantly, an analysis of 126 breast cancer patients revealed that those whose breast tumors had elevated PN-1 levels had a significantly higher probability to develop lung metastasis, but not metastasis to other sites, on relapse. These results suggest that PN-1 might become a prognostic marker in breast cancer. PMID:19584287

  2. In vitro anti-leishmanial efficacy of potato tuber extract (PTEx): leishmanial serine protease(s) as putative target.

    PubMed

    Paik, Dibyendu; Das, Partha; De, Tripti; Chakraborti, Tapati

    2014-11-01

    Leishmaniasis, a neglected tropical disease (NTD) causes major health problems in the tropical and subtropical world. Most of the antileishmanial modern therapies with different formulations of pentavalent antimonials, Miltefosine, Amphotericin B etc. are not satisfactory in recent times due to high toxicity to the host and present rising strain resistance issues. So there is an urgent need to develop new, safe and cost-effective drugs against leishmaniasis. In this regard, bioactive phytocomponents may lead to the discovery of new medicines with appropriate efficiency. The prominent roles played by Leishmania proteases in the virulence of this parasite make them very promising targets for the development of current therapeutics of leishmaniasis. As part of a search for novel drugs, we have evaluated in vitro anti-leishmanial activity of serine protease inhibitor rich fraction (PTEx) obtained from potato tuber. The extract (PTEx) was prepared by sodium bisulfite fractionation and inhibitors were identified by reverse zymography. Inhibition study of PTEx in gelatin-zymogram and spectrophotometric assay using BApNA and BTpNA as substrate reveal its strong inhibitory activity against trypsin as well as serine proteases present in cell lysate of Leishmania donovani infective strain. The in vitro MTT based colorimetric assay as well as ex vivo L. donovani infected macrophages showed reduced parasite viability and intracellular parasite load with IC50 = 312.5 ± 0.1 μg/ml and IC50 82.3 ± 0.2 μg/ml of PTEx respectively in a concentration dependent manner. This anti-leishmanial effect was also preceded by PTEx induced acute formation of ROS and prolonged NO generation. The PTEx has no significant cytotoxic effect on host macrophages. So taken together, these findings indicate that PTEx has promising leishmanicidal effect and thus this study provides a new perspective of natural serine protease inhibitor from potato tuber on the development of new drug against

  3. High Temperature Requirement A1, Transforming Growth Factor Beta 1, phosphoSmad2 and Ki67 in Eutopic and Ectopic Endometrium of Women With Endometriosis

    PubMed Central

    Goteri, G.; Altobelli, E.; Tossetta, G.; Zizzi, A.; Avellini, C.; Licini, C.; Lorenzi, T.; Castellucci, M.; Ciavattini, A.

    2015-01-01

    Increasing evidence supports the hypothesis that TGFβ1 signalling may be mediated by high temperature requirement A1 (HtrA1) serine protease, acting on important regulatory mechanisms such as cell proliferation and mobility. Evidence is now accumulating to suggest that HtrA1 is involved in the development and progression of several pathologies. The aim of this study was to evaluate: i) if HtrA1 and TGFβ1 expressions differ in eutopic and ectopic endometrium in women with endometriosis; ii) if HtrA1 correlates to TGFβ1, pSmad and Ki67. This study was carried out including 10 women with ovarian endometriosis (cases) and 10 women with non endometriotic diseases (controls). Endometrial tissue underwent immunohistochemical H-score analysis for HtrA1, TGFβ1, pSmad and Ki67 molecules. Data evaluation was performed by a nonparametric Kruskal-Wallis test and Spearman correlation was applied to evaluate the relationship among the molecules investigated in the epithelial and in the stromal compartment. The HtrA1 was significantly decreased in ectopic and eutopic endometrium of women with endometriosis when compared with control endometrium in epithelial compartment. TGFβ1was significantly increased in eutopic endometrium and decreased in ectopic endometrium in epithelial and stromal compartment. In addition, Ki67 was significantly increased and an increase, but not significant, was detected for pSMAd2 in eutopic and ectopic endometrium compared to control one. In summary, the significant direct correlation between TGFβ1 and pSmad2 as well as between HtrA1 and TGFβ1 and the very significant increase of Ki67 in stromal compartment of eutopic endometrium suggest a possible involvement of HtrA1 in the pathogenesis of endometriosis. PMID:26708185

  4. The Plasticity of the β-Trefoil Fold Constitutes an Evolutionary Platform for Protease Inhibition*

    PubMed Central

    Azarkan, Mohamed; Martinez-Rodriguez, Sergio; Buts, Lieven; Baeyens-Volant, Danielle; Garcia-Pino, Abel

    2011-01-01

    Proteases carry out a number of crucial functions inside and outside the cell. To protect the cells against the potentially lethal activities of these enzymes, specific inhibitors are produced to tightly regulate the protease activity. Independent reports suggest that the Kunitz-soybean trypsin inhibitor (STI) family has the potential to inhibit proteases with different specificities. In this study, we use a combination of biophysical methods to define the structural basis of the interaction of papaya protease inhibitor (PPI) with serine proteases. We show that PPI is a multiple-headed inhibitor; a single PPI molecule can bind two trypsin units at the same time. Based on sequence and structural analysis, we hypothesize that the inherent plasticity of the β-trefoil fold is paramount in the functional evolution of this family toward multiple protease inhibition. PMID:22027836

  5. Proteinaceous protease inhibitor from Lawsonia inermis: purification, characterization and antibacterial activity.

    PubMed

    Dabhade, Arvind; Patel, Priti; Pati, Ulhas

    2013-10-01

    A thermo-stable, proteinaceous protease inhibitor (LPI) from Lawsonia inermis is reported. The LPI was purified from Lawsonia inermis seeds by subsequent ammonium sulfate precipitation, ion exchange chromatography (DEAE-Cellulose) and gel permeation chromatography (Sephadex-50). The purified protease inhibitor is effective against a wide range of proteases viz. papain, trypsin, pepsin and metallo-protease. The apparent molecular weight of the protease inhibitor is 19 kDa, determined by SDS-PAGE electrophoresis. The protease inhibitor was found to be stable at 70 degrees C for 30 min. It was also examined for antibacterial activity against Pseudomonas aeruginosa MTCC 7926 and Staphylococcus aureus NCIM 2079; the IC50 values of the purified LPI were 11.4 microg/mL and 16.6 microg/mL respectively. PMID:24354203

  6. The plasticity of the β-trefoil fold constitutes an evolutionary platform for protease inhibition.

    PubMed

    Azarkan, Mohamed; Martinez-Rodriguez, Sergio; Buts, Lieven; Baeyens-Volant, Danielle; Garcia-Pino, Abel

    2011-12-23

    Proteases carry out a number of crucial functions inside and outside the cell. To protect the cells against the potentially lethal activities of these enzymes, specific inhibitors are produced to tightly regulate the protease activity. Independent reports suggest that the Kunitz-soybean trypsin inhibitor (STI) family has the potential to inhibit proteases with different specificities. In this study, we use a combination of biophysical methods to define the structural basis of the interaction of papaya protease inhibitor (PPI) with serine proteases. We show that PPI is a multiple-headed inhibitor; a single PPI molecule can bind two trypsin units at the same time. Based on sequence and structural analysis, we hypothesize that the inherent plasticity of the β-trefoil fold is paramount in the functional evolution of this family toward multiple protease inhibition. PMID:22027836

  7. Regulation of exocellular proteases in Neurospora crassa: metabolic requirements of the process.

    PubMed Central

    Drucker, H

    1975-01-01

    To induce exocellular proteolytic enzyme from carbon-starved exponential-phase cells of Neurospora crassa, both a protein substrate and an activating protease of certain specific properties must be present at the same time. The cells must be capable of protein synthesis, since cycloheximide inhibits the process, but cell growth, as determined by increase in cell mass, does not appear to be required. Both soluble (bovine serum albumin, myoglobin) and insoluble protein substrates (collagen, corn zein) will affect protease induction, although certain soluble, globular proteins (egg white globulin, bovine gamma globulin) will not. In most cases, rates of protease induction are proportional to protein concentration, regardless of the nature of the inducing protein. All activating proteases capable of affecting induction in a manner similar to that of N. crassa exocellular protease were of bacterial origin and were exoproteases. Mammalian proteases and peptidases had little or no effect on the induction process. PMID:125263

  8. Alkaline protease from Spilosoma obliqua: potential applications in bio-formulations.

    PubMed

    Anwar, A; Saleemuddin, M

    2000-04-01

    Some properties of the purified alkaline protease from larvae of the insect Spilosoma obliqua (Lepidoptera) and its potential application as an additive in various bio-formulations are reported. The novel feature of the present study is the use of insect protease. The protease was found to be compatible with some of the commercial detergents tested, and was also effective in cleaving various protein substrates tested, albeit to different extents, implying broader substrate specificity and effectiveness of the protease against a wide variety of stains. This property of the protease can also be exploited by using it as an active component in enzymic debriders in view of its ability to digest various protein substrates. The insect protease appears to be potentially useful as an additive in detergent, stain remover and other bio-formulations. PMID:10744951

  9. Cell-specific and developmental expression of lectican-cleaving proteases in mouse hippocampus and neocortex.

    PubMed

    Levy, C; Brooks, J M; Chen, J; Su, J; Fox, M A

    2015-03-01

    Mounting evidence has demonstrated that a specialized extracellular matrix exists in the mammalian brain and that this glycoprotein-rich matrix contributes to many aspects of brain development and function. The most prominent supramolecular assemblies of these extracellular matrix glycoproteins are perineuronal nets, specialized lattice-like structures that surround the cell bodies and proximal neurites of select classes of interneurons. Perineuronal nets are composed of lecticans, a family of chondroitin sulfate proteoglycans that includes aggrecan, brevican, neurocan, and versican. These lattice-like structures emerge late in postnatal brain development, coinciding with the ending of critical periods of brain development. Despite our knowledge of the presence of lecticans in perineuronal nets and their importance in regulating synaptic plasticity, we know little about the development or distribution of the extracellular proteases that are responsible for their cleavage and turnover. A subset of a large family of extracellular proteases (called a disintegrin and metalloproteinase with thrombospondin motifs [ADAMTS]) is responsible for endogenously cleaving lecticans. We therefore explored the expression pattern of two aggrecan-degrading ADAMTS family members, ADAMTS15 and ADAMTS4, in the hippocampus and neocortex. Here, we show that both lectican-degrading metalloproteases are present in these brain regions and that each exhibits a distinct temporal and spatial expression pattern. Adamts15 mRNA is expressed exclusively by parvalbumin-expressing interneurons during synaptogenesis, whereas Adamts4 mRNA is exclusively generated by telencephalic oligodendrocytes during myelination. Thus, ADAMTS15 and ADAMTS4 not only exhibit unique cellular expression patterns but their developmental upregulation by these cell types coincides with critical aspects of neural development. PMID:25349050

  10. Cell-specific and developmental expression of lectican-cleaving proteases in mouse hippocampus and neocortex

    PubMed Central

    Levy, C.; Brooks, J.M.; Chen, J.; Su, J.; Fox, M.A.

    2014-01-01

    Mounting evidence has demonstrated that a specialized extracellular matrix exists in the mammalian brain, and this glycoprotein-rich matrix contributes to many aspects of brain development and function. The most prominent supramolecular assembly of these ECM glycoproteins are perineuronal nets, specialized lattice-like structures that surround the cell bodies and proximal neurites of select classes of interneurons. Perineuronal nets are composed of lecticans – a family of chondroitin sulfate proteoglycans [CSPGs] that includes aggrecan, brevican, neurocan, and versican. The presence of these lattice-like structures emerge late in postnatal brain development and coincides with the ending of critical periods of brain development. Despite our knowledge of the presence of lecticans in perineuronal nets and their importance in regulating synaptic plasticity, we know little about the development or distribution of extracellular proteases that are responsible for their cleavage and turnover. A subset of the large “A Disintegrin and Metalloproteinase with Thrombospondin Motifs” (ADAMTS) family of extracellular proteases is responsible for endogenously cleaving lecticans. We therefore explored the expression pattern of 2 aggrecan-degrading ADAMTS family members, ADAMTS15 and ADAMTS4, in hippocampus and neocortex. Here, we show that both lectican-degrading metalloproteases are present in these brain regions and each exhibits a distinct temporal and spatial expression pattern. Adamts15 mRNA is expressed exclusively by Parvalbumin-expressing interneurons during synaptogenesis, whereas, Adamts4 mRNA is exclusively generated by telencephalic oligodendrocytes during myelination. Thus, ADAMTS15 and ADAMTS4 not only exhibit unique cellular expression patterns but their developmental upregulation by these cell types coincides with critical aspects of neural development. PMID:25349050

  11. Purification and characterization of organic solvent stable serine alkaline protease from newly isolated Bacillus circulans M34.

    PubMed

    Sari, Esma; Loğoğlu, Elif; Öktemer, Atilla

    2015-09-01

    A protease from newly isolated Bacillus circulans M34 was purified by Q-Sepharose anion exchange chromatography and Sepharose-bacitracin affinity chromatography followed by (NH4)2SO4 precipitation. The molecular mass of the purified enzyme was determined using SDS-PAGE. The optimum pH and temperature for protease activity were 11 and 50°C, respectively. The effect of various metal ions on protease activity was investigated. Alkaline protease from Bacillus circulans M34 wase activated by Zn(2+), Cu(2+) and Co(2+) up to 31%. The purified protease was found to be stable in the organic solvents, surfactants and oxidizing agent. The substrate specificity of purified protease was investigated towards different substrates. The protease was almost completely inhibited by the serine protease inhibitor phenylmethanesulfonyl fluoride. The kinetic parameters of the purified protease, maximum rate (Vmax) and Michaelis constant (Km), were determined using a Lineweaver-Burk plot. PMID:25677873

  12. Synthesis of homopolypeptides by aminolysis mediated by proteases encapsulated in silica nanospheres.

    PubMed

    Baker, Peter J; Patwardhan, Siddharth V; Numata, Keiji

    2014-11-01

    We report the encapsulation of three different proteases in bioinspired silica. Silica particles were formed under mild reaction conditions using cationic amine-rich ethyleneamines as initiators, which resulted in aggregations of nanoscale spheres. Following encapsulation, the proteases were characterized for their hydrolytic and aminolytic activities. The encapsulation resulted in an increase in the thermal stability of the proteases for both hydrolysis and aminolysis reactions. The enhanced thermal stability of the encapsulated proteases increased the production of poly-L-leucine by aminolysis. Furthermore, the encapsulation of papain resulted in an increase in the production of poly-L-alanine and poly-L-valine at 50 °C. PMID:25154484

  13. Immobilized protease on the magnetic nanoparticles used for the hydrolysis of rapeseed meals

    NASA Astrophysics Data System (ADS)

    Jin, Xin; Li, Ju-Fang; Huang, Ping-Ying; Dong, Xu-Yan; Guo, Lu-Lu; Yang, Liang; Cao, Yuan-Cheng; Wei, Fang; Zhao, Yuan-Di; Chen, Hong

    2010-07-01

    (3-aminopropl) triethoxysilaneand modified magnetic nanoparticles with the average diameter of 25.4 nm were synthesized in water-phase co-precipitation method. And then these nanoparticles were covalently coupled with alkaline protease as enzyme carrier by using 1,4-phenylene diisothlocyanate as coupling agent. Experiments showed that the immobilized protease can keep the catalytic bioactivity, which can reach to 47.8% when casein was served as substrate. Results showed that the catalytic activity of immobilized protease on these magnetic nanoparticles could retain 98.63±2.37% after 60 days. And it is more stable than the free protease during the shelf-life test. The enzyme reaction conditions such as optimum reaction temperature and pH are the same as free protease. Furthermore, mix-and-separate experiments showed that the immobilized protease could be recycled through the magnetic nanoparticles after the biocatalysis process. When the rapeseed meals were used as substrate, the degree of hydrolysis of immobilized alkaline protease achieved 9.86%, while it was 10.41% for the free protease. The macromolecular proteins of rapeseed meals were hydrolyzed by immobilized protease into small molecules such as polypeptides or amino acids. Thus, a novel efficient and economic way for the recycling of enzymes in the application of continuous production of active peptides was provided based on these magnetic nanoparticles.

  14. Cysteine Proteases: Modes of Activation and Future Prospects as Pharmacological Targets.

    PubMed

    Verma, Sonia; Dixit, Rajnikant; Pandey, Kailash C

    2016-01-01

    Proteolytic enzymes are crucial for a variety of biological processes in organisms ranging from lower (virus, bacteria, and parasite) to the higher organisms (mammals). Proteases cleave proteins into smaller fragments by catalyzing peptide bonds hydrolysis. Proteases are classified according to their catalytic site, and distributed into four major classes: cysteine proteases, serine proteases, aspartic proteases, and metalloproteases. This review will cover only cysteine proteases, papain family enzymes which are involved in multiple functions such as extracellular matrix turnover, antigen presentation, processing events, digestion, immune invasion, hemoglobin hydrolysis, parasite invasion, parasite egress, and processing surface proteins. Therefore, they are promising drug targets for various diseases. For preventing unwanted digestion, cysteine proteases are synthesized as zymogens, and contain a prodomain (regulatory) and a mature domain (catalytic). The prodomain acts as an endogenous inhibitor of the mature enzyme. For activation of the mature enzyme, removal of the prodomain is necessary and achieved by different modes. The pro-mature domain interaction can be categorized as protein-protein interactions (PPIs) and may be targeted in a range of diseases. Cysteine protease inhibitors are available that can block the active site but no such inhibitor available yet that can be targeted to block the pro-mature domain interactions and prevent it activation. This review specifically highlights the modes of activation (processing) of papain family enzymes, which involve auto-activation, trans-activation and also clarifies the future aspects of targeting PPIs to prevent the activation of cysteine proteases. PMID:27199750

  15. Scouring Potential of Mesophile Acidic Proteases of Pseudomonas aeruginosa for Grey Cotton Fabrics

    NASA Astrophysics Data System (ADS)

    Saravanan, D.

    2013-04-01

    Mesophile, acidic proteases were produced using the microbial source, Pseudomonas aeruginosa, with wider thermal tolerances. Process conditions of scouring treatment were optimized using Taguchi method for optimum temperature, time, pH and concentration of protease. Treatment with the protease lower weight loss values compared to the alkali scouring, however, significant improvement in the absorbency compared to the grey samples was observed. Large amounts of pectin left out in the samples resulted in higher extractable impurities, substantiated by the FTIR results. Relatively, lower reduction in the tear strengths was observed in both warp and weft directions after protease treatment of the cotton fabrics.

  16. Cysteine Proteases: Modes of Activation and Future Prospects as Pharmacological Targets

    PubMed Central

    Verma, Sonia; Dixit, Rajnikant; Pandey, Kailash C.

    2016-01-01

    Proteolytic enzymes are crucial for a variety of biological processes in organisms ranging from lower (virus, bacteria, and parasite) to the higher organisms (mammals). Proteases cleave proteins into smaller fragments by catalyzing peptide bonds hydrolysis. Proteases are classified according to their catalytic site, and distributed into four major classes: cysteine proteases, serine proteases, aspartic proteases, and metalloproteases. This review will cover only cysteine proteases, papain family enzymes which are involved in multiple functions such as extracellular matrix turnover, antigen presentation, processing events, digestion, immune invasion, hemoglobin hydrolysis, parasite invasion, parasite egress, and processing surface proteins. Therefore, they are promising drug targets for various diseases. For preventing unwanted digestion, cysteine proteases are synthesized as zymogens, and contain a prodomain (regulatory) and a mature domain (catalytic). The prodomain acts as an endogenous inhibitor of the mature enzyme. For activation of the mature enzyme, removal of the prodomain is necessary and achieved by different modes. The pro-mature domain interaction can be categorized as protein–protein interactions (PPIs) and may be targeted in a range of diseases. Cysteine protease inhibitors are available that can block the active site but no such inhibitor available yet that can be targeted to block the pro-mature domain interactions and prevent it activation. This review specifically highlights the modes of activation (processing) of papain family enzymes, which involve auto-activation, trans-activation and also clarifies the future aspects of targeting PPIs to prevent the activation of cysteine proteases. PMID:27199750

  17. Evolution of thrombin and other hemostatic proteases by survey of protochordate, hemichordate, and echinoderm genomes.

    PubMed

    Ponczek, Michal B; Bijak, Michal Z; Nowak, Pawel Z

    2012-06-01

    Protochordate genomes enable a prevalence of hemostasis evolution. Broad searches were performed for homologs of human serine proteases of hemostasis on the genomes of Branchiostoma floridae, Saccoglossus kowalevskii, and Strongylocentrotus purpuratus. Sequences were analyzed by multiple bioinformatic tools. The survey revealed numerous homologous components. Amphioxus was rich in some serine proteases not accompanied by gamma-carboxyglutamic or kringle domains similar more to thrombin than to other coagulation factors. The serine proteases found in amphioxus exhibited the attributes similar to those of thrombin by phylogeny relationships, sequence conservation, gene synteny, spatial structure, and ligand docking. A few plasminogen- and plasminogen activators-like proteases with kringles were also present. Those serine proteases demonstrated the greatest proximity rather to plasminogen or plasminogen activators than to thrombin. Searching for homologs of serine protease hemostatic factors in acorn worm and sea urchin revealed several components similar to those found in amphioxus. Hypothetically, the common ancestor of chordates had three separate serine proteases that evolved independently into immunoglobulin-like and kringle proteases in lancelets, and prothrombin, plasminogen activators, and plasminogen in vertebrates. Ancestral proteases evolved in vertebrates into hemostasis factors after merging the proper N-terminal domains and duplications. PMID:22752046

  18. Effect of Group A Streptococcal Cysteine Protease on Invasion of Epithelial Cells

    PubMed Central

    Tsai, Pei-Jane; Kuo, Chih-Feng; Lin, Kuei-Yuan; Lin, Yee-Shin; Lei, Huan-Yao; Chen, Fen-Fen; Wang, Jen-Ren; Wu, Jiunn-Jong

    1998-01-01

    Cysteine protease of group A streptococci (GAS) is considered an important virulence factor. However, its role in invasiveness of GAS has not been investigated. We demonstrated in this study that two strains of protease-producing GAS had the ability to invade A-549 human respiratory epithelial cells. Isogenic protease mutants were constructed by using integrational plasmids to disrupt the speB gene and confirmed by Southern hybridization and Western immunoblot analyses. No extracellular protease activity was produced by the mutants. The mutants had growth rates similar to those of the wild-type strains and produced normal levels of other extracellular proteins. When invading A-549 cells, the mutants had a two- to threefold decrease in activity compared to that of the wild-type strains. The invasion activity increased when the A-549 cells were incubated with purified cysteine protease and the mutant. However, blockage of the cysteine protease with a specific cysteine protease inhibitor, E-64, decreased the invasion activity of GAS. Intracellular growth of GAS was not found in A-549 cells. The presence or absence of protease activity did not affect the adhesive ability of GAS. These results suggested that streptococcal cysteine protease can enhance the invasion ability of GAS in human respiratory epithelial cells. PMID:9529068

  19. Effect of group A streptococcal cysteine protease on invasion of epithelial cells.

    PubMed

    Tsai, P J; Kuo, C F; Lin, K Y; Lin, Y S; Lei, H Y; Chen, F F; Wang, J R; Wu, J J

    1998-04-01

    Cysteine protease of group A streptococci (GAS) is considered an important virulence factor. However, its role in invasiveness of GAS has not been investigated. We demonstrated in this study that two strains of protease-producing GAS had the ability to invade A-549 human respiratory epithelial cells. Isogenic protease mutants were constructed by using integrational plasmids to disrupt the speB gene and confirmed by Southern hybridization and Western immunoblot analyses. No extracellular protease activity was produced by the mutants. The mutants had growth rates similar to those of the wild-type strains and produced normal levels of other extracellular proteins. When invading A-549 cells, the mutants had a two- to threefold decrease in activity compared to that of the wild-type strains. The invasion activity increased when the A-549 cells were incubated with purified cysteine protease and the mutant. However, blockage of the cysteine protease with a specific cysteine protease inhibitor, E-64, decreased the invasion activity of GAS. Intracellular growth of GAS was not found in A-549 cells. The presence or absence of protease activity did not affect the adhesive ability of GAS. These results suggested that streptococcal cysteine protease can enhance the invasion ability of GAS in human respiratory epithelial cells. PMID:9529068

  20. Comparative analysis on the distribution of protease activities among fruits and vegetable resources.

    PubMed

    Sun, Qian; Zhang, Bin; Yan, Qiao-Juan; Jiang, Zheng-Qiang

    2016-12-15

    In this study, a comparative analysis on the distribution of protease activities among 90 plant resources, including fruits and vegetables, has been performed. Protease activities of plant extracts were assayed at different pH values (pH 3.0, pH 7.5 and pH 10.5) using casein as a substrate. Ten fruits and thirteen vegetables show protease activities above 10U/g. Pineapple, fig and papaya, which are used for commercial protease production, exhibited high protease activities. Additionally, high protease activities were detected in kiwifruit (28.8U/g), broccoli (16.9U/g), ginger (16.6U/g), leek (32.7U/g) and red pepper (15.8U/g) at different pH values. SDS-PAGE and zymograms confirmed that various types of proteases existed in the five plant extracts and might be explored. Furthermore, five plant extracts were treated by different protease inhibitors. These results show that there are still many plant resources unexplored, which may be promising candidates for plant-derived protease production. PMID:27451238

  1. Synthesis and herbicidal evaluation of novel benzothiazole derivatives as potential inhibitors of D1 protease.

    PubMed

    Huang, Tonghui; Sun, Jie; An, Lin; Zhang, Lixian; Han, Cuiping

    2016-04-01

    D1 protease is a C-terminal processing protease that has been predicted to be an ideal herbicidal target. Three novel series of benzothiazole derivatives were synthesized and evaluated for their herbicidal activities against Brassica napus (rape) and Echinochloa crusgalli (barnyard grass). The preliminary bioassay indicated that most of the synthesized compounds possess promising D1 protease inhibitory activities and considerable herbicidal activities. Molecular docking was performed to position representative compounds into the active site of D1 protease to determine a probable binding model. PMID:26905829

  2. Nine Crystal Structures Determine the Substrate Envelope of the MDR HIV-1 Protease

    SciTech Connect

    Liu, Zhigang; Wang, Yong; Brunzelle, Joseph; Kovari, Iulia A.; Kovari, Ladislau C.

    2012-03-27

    Under drug selection pressure, emerging mutations render HIV-1 protease drug resistant, leading to the therapy failure in anti-HIV treatment. It is known that nine substrate cleavage site peptides bind to wild type (WT) HIV-1 protease in a conserved pattern. However, how the multidrug-resistant (MDR) HIV-1 protease binds to the substrate cleavage site peptides is yet to be determined. MDR769 HIV-1 protease (resistant mutations at residues 10, 36, 46, 54, 62, 63, 71, 82, 84, and 90) was selected for present study to understand the binding to its natural substrates. MDR769 HIV-1 protease was co-crystallized with nine substrate cleavage site hepta-peptides. Crystallographic studies show that MDR769 HIV-1 protease has an expanded substrate envelope with wide open flaps. Furthermore, ligand binding energy calculations indicate weaker binding in MDR769 HIV-1 protease-substrate complexes. These results help in designing the next generation of HIV-1 protease inhibitors by targeting the MDR HIV-1 protease.

  3. Decomposing the Energetic Impact of Drug-Resistant Mutations: The Example of HIV-1 Protease - DRV Binding

    PubMed Central

    Cai, Yufeng; Schiffer, Celia

    2016-01-01

    Summary HIV-1 protease is a major drug target for AIDS therapy. With the appearance of drug-resistant HIV-1 protease variants, understanding the mechanism of drug resistance becomes critical. Computational methods can provide more details about inhibitor-protease binding other than crystallography and isothermal titration calorimetry. Darunavir is the latest FDA approved HIV-1 protease inhibitor. In this context, the free energy component analysis is performed on the DRV binding to WT protease and ACT, a drug resistant variant, to evaluate contribution of each atoms of DRV to the binding affinity. This information can contribute to the rationale design of new HIV-1 protease inhibitors. PMID:22183557

  4. Cloning and chromosomal assignment of a human cDNA encoding a T cell- and natural killer cell-specific trypsin-like serine protease

    SciTech Connect

    Gershenfeld, H.K.; Hershberger, R.J.; Shows, T.B.; Weissman, I.L.

    1988-02-01

    A cDNA clone encoding a human T cell- and natural killer cell-specific serine protease was obtained by screening a phage lambdagt10 cDNA library from phytohemagglutinin-stimulated human peripheral blood lymphocytes with the mouse Hanukah factor cDNA clone. In an RNA blot-hybridization analysis, this human Hanukah factor cDNA hybridized with a 1.3-kilobase band in allogeneic-stimulated cytotoxic T cells and the Jurkat cell line, but this transcript was not detectable in normal muscle, liver, tonsil, or thymus. By dot-blot hybridization, this cDNA hybridized with RNA from three cytolytic T-cell clones and three noncytolytic T-cell clones grown in vitro as well as with purified CD16/sup +/ natural killer cells and CD3/sup +/, CD16/sup -/ T-cell large granular lymphocytes from peripheral blood lymphocytes (CD = cluster designation). The nucleotide sequence of this cDNA clone encodes a predicted serine protease of 262 amino acids. The active enzyme is 71% and 77% similar to the mouse sequence at the amino acid and DNA level, respectively. The human and mouse sequences conserve the active site residues of serine proteases--the trypsin-specific Asp-189 and all 10 cysteine residues. The gene for the human Hanukah factor serine protease is located on human chromosome 5. The authors propose that this trypsin-like serine protease may function as a common component necessary for lysis of target cells by cytotoxic T lymphocytes and natural killer cells.

  5. Mechanism of oxidative inactivation of human presequence protease (hPreP) by hydrogen peroxide

    PubMed Central

    Chen, Jue; Teixeira, Pedro Filipe; Glaser, Elzbieta; Levine, Rodney L.

    2014-01-01

    The mitochondrial presequence protease (PreP) is a member of the pitrilysin class of metalloproteases. It degrades the mitochondrial targeting presequences of mitochondria-localized proteins as well as unstructured peptides such as amyloid-β-peptide. The specific activity of PreP is reduced in Alzheimer patients and animal models of Alzheimer disease. The loss of activity can be mimicked in vitro by exposure to oxidizing conditions, and indirect evidence suggested that inactivation was due to methionine oxidation. We performed peptide mapping analyses to elucidate the mechanism of inactivation. None of the 24 methionine residues in recombinant human PreP was oxidized. We present evidence that inactivation was due to oxidation of cysteine residues and consequent oligomerization through intermolecular disulfide bonds. The most susceptible cysteine residues to oxidation are Cys34, Cys112, and Cys119. Most, but not all, of the activity loss is restored by the reducing agent, dithiothreitol. These findings elucidate a redox mechanism for regulation of PreP and also provide a rational basis for therapeutic intervention in conditions characterized by excessive oxidation of PreP. PMID:25236746

  6. Mechanism of oxidative inactivation of human presequence protease by hydrogen peroxide.

    PubMed

    Chen, Jue; Teixeira, Pedro Filipe; Glaser, Elzbieta; Levine, Rodney L

    2014-12-01

    The mitochondrial presequence protease (PreP) is a member of the pitrilysin class of metalloproteases. It degrades the mitochondrial targeting presequences of mitochondria-localized proteins as well as unstructured peptides such as amyloid-β peptide. The specific activity of PreP is reduced in Alzheimer patients and animal models of Alzheimer disease. The loss of activity can be mimicked in vitro by exposure to oxidizing conditions, and indirect evidence suggested that inactivation was due to methionine oxidation. We performed peptide mapping analyses to elucidate the mechanism of inactivation. None of the 24 methionine residues in recombinant human PreP was oxidized. We present evidence that inactivation is due to oxidation of cysteine residues and consequent oligomerization through intermolecular disulfide bonds. The most susceptible cysteine residues to oxidation are Cys34, Cys112, and Cys119. Most, but not all, of the activity loss is restored by the reducing agent dithiothreitol. These findings elucidate a redox mechanism for regulation of PreP and also provide a rational basis for therapeutic intervention in conditions characterized by excessive oxidation of PreP. PMID:25236746

  7. Endothelial Progenitor Cells in Sprouting Angiogenesis: Proteases Pave the Way.

    PubMed

    Laurenzana, A; Fibbi, G; Margheri, F; Biagioni, A; Luciani, C; Del Rosso, M; Chillà, A

    2015-01-01

    Sprouting angiogenesis consists of the expansion and remodelling of existing vessels, where the vascular sprouts connect each other to form new vascular loops. Endothelial Progenitor Cells (EPCs) are a subtype of stem cells, with high proliferative potential, able to differentiate into mature Endothelial Cells (ECs) during the neovascularization process. In addition to this direct structural role EPCs improve neovascularization, also secreting numerous pro-angiogenic factors able to enhance the proliferation, survival and function of mature ECs, and other surrounding progenitor cells. While sprouting angiogenesis by mature ECs involves resident ECs, the vasculogenic contribution of EPCs is a high hurdle race. Bone marrowmobilized EPCs have to detach from the stem cell niche, intravasate into bone marrow vessels, reach the hypoxic area or tumour site, extravasate and incorporate into the new vessel lumen, thus complementing the resident mature ECs in sprouting angiogenesis. The goal of this review is to highlight the role of the main protease systems able to control each of these steps. The pivotal protease systems here described, involved in vascular patterning in sprouting angiogenesis, are the matrix-metalloproteinases (MMPs), the serineproteinases urokinase-type plasminogen activator (uPA) associated with its receptor (uPAR) and receptorassociated plasminogen/plasmin, the neutrophil elastase and the cathepsins. Since angiogenesis plays a critical role not only in physiological but also in pathological processes, such as in tumours, controlling the contribution of EPCs to the angiogenic process, through the regulation of the protease systems involved, could yield new opportunities for the therapeutic prospect of efficient control of pathological angiogenesis. PMID:26321757

  8. From Protease to Decarboxylase: THE MOLECULAR METAMORPHOSIS OF PHOSPHATIDYLSERINE DECARBOXYLASE.

    PubMed

    Choi, Jae-Yeon; Duraisingh, Manoj T; Marti, Matthias; Ben Mamoun, Choukri; Voelker, Dennis R

    2015-04-24

    Phosphatidylserine decarboxylase (PSDs) play a central role in the synthesis of phosphatidylethanolamine in numerous species of prokaryotes and eukaryotes. PSDs are unusual decarboxylase containing a pyruvoyl prosthetic group within the active site. The covalently attached pyruvoyl moiety is formed in a concerted reaction when the PSD proenzyme undergoes an endoproteolytic cleavage into a large β-subunit, and a smaller α-subunit, which harbors the prosthetic group at its N terminus. The mechanism of PSD proenzyme cleavage has long been unclear. Using a coupled in vitro transcription/translation system with the soluble Plasmodium knowlesi enzyme (PkPSD), we demonstrate that the post-translational processing is inhibited by the serine protease inhibitor, phenylmethylsulfonyl fluoride. Comparison of PSD sequences across multiple phyla reveals a uniquely conserved aspartic acid within an FFXRX6RX12PXD motif, two uniquely conserved histidine residues within a PXXYHXXHXP motif, and a uniquely conserved serine residue within a GS(S/T) motif, suggesting that PSDs belong to the D-H-S serine protease family. The function of the conserved D-H-S residues was probed using site-directed mutagenesis of PkPSD. The results from these mutagenesis experiments reveal that Asp-139, His-198, and Ser-308 are all essential for endoproteolytic processing of PkPSD, which occurs in cis. In addition, within the GS(S/T) motif found in all PSDs, the Gly-307 residue is also essential, but the Ser/Thr-309 is non-essential. These results define the mechanism whereby PSDs begin their biochemical existence as proteases that execute one autoendoproteolytic cleavage reaction to give rise to a mature PSD harboring a pyruvoyl prosthetic group. PMID:25724650

  9. Comparison of HIV-1 protease expression in different fusion forms.

    PubMed

    Wan, M; Takagi, M; Loh, B N; Imanaka, T

    1995-06-01

    Earlier observations showed that the expression of recombinant protease of human immunodeficiency virus type-1 (HIV-1 PR) was usually in a low level, and its proteolytic activity and hydrophobicity were believed to be toxic for the host cells. Various constructs were investigated that contained an N-terminal extended HIV-1 PR gene (PR107) in order to find a system which can express this protease in high level. The constructs of PR107 gene expressed as fusion proteins either with glutathione S-transferase (GST) by pGEX-PR107 or with maltose-binding protein (MBP) by pMAL-PR107 showed that the full length of fusion protein exhibited self-cleavage in E. coli. The results from expression experiments indicated that the size of the fusion portion does not affect the self-processing of fused HIV-1 PR to release its mature form, despite the attachment of only one subunit of the dimeric protease to GST or MBP. The construct, pET-PR107, under the control of strong bacteriophage T7 promoter system, did not show clear advantages for expression of this HIV-1 PR. Comparing these three constructs, the pGEX-PR107 system showed the highest expression level. Quantitative immuno-blotting indicated that the amount of HIV-1 PR expressed by pGEX-PR107 was twice that expressed by pMAL-PR107, and thrice that expressed by pET-PR107. More than 1 mg of pure HIV-1 PR from per liter culture of E. coli. DH5 alpha containing pGEX-PR107 can be obtained via the purification procedures [Biochem. Mol. Biol. International, (1995) 35:899-912].(ABSTRACT TRUNCATED AT 250 WORDS)