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Sample records for putative host protease

  1. 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

  2. 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.

  3. KLIKK proteases of Tannerella forsythia: putative virulence factors with a unique domain structure.

    PubMed

    Ksiazek, Miroslaw; Mizgalska, Danuta; Eick, Sigrum; Thøgersen, Ida B; Enghild, Jan J; Potempa, Jan

    2015-01-01

    Comparative genomics of virulent Tannerella forsythia ATCC 43037 and a close health-associated relative, Tannerella BU063, revealed, in the latter, the absence of an entire array of genes encoding putative secretory proteases that possess a nearly identical C-terminal domain (CTD) that ends with a -Lys-Leu-Ile-Lys-Lys motif. This observation suggests that these proteins, referred to as KLIKK proteases, may function as virulence factors. Re-sequencing of the loci of the KLIKK proteases found only six genes grouped in two clusters. All six genes were expressed by T. forsythia in routine culture conditions, although at different levels. More importantly, a transcript of each gene was detected in gingival crevicular fluid (GCF) from periodontitis sites infected with T. forsythia indicating that the proteases are expressed in vivo. In each protein, a protease domain was flanked by a unique N-terminal profragment and a C-terminal extension ending with the CTD. Partially purified recombinant proteases showed variable levels of proteolytic activity in zymography gels and toward protein substrates, including collagen, gelatin, elastin, and casein. Taken together, these results indicate that the pathogenic strain of T. forsythia secretes active proteases capable of degrading an array of host proteins, which likely represents an important pathogenic feature of this bacterium.

  4. Conformational study of a putative HLTV-1 retroviral protease inhibitor.

    PubMed

    Llido, S; d'Estaintot, B L; Dautant, A; Geoffre, S; Picard, P; Precigoux, G

    1993-05-01

    The crystal structure of prolyl-glutaminyl-valyl-statyl-alanyl-leucine (Pro-Gln-Val-Sta-Ala-Leu, C(32)H(57)N(7)0(9).5H(2)0, M(r) = 683.9 + 90.1), a putative HTLV-1 protease inhibitor based on one of the consensus retroviral protease cleavage sequences, and containing the statine residue [(4S,3S)-4-amino-3-hydroxy-6-methylheptanoic acid], has been determined by X-ray diffraction. The same molecule has been modelled in the active site of the HTLV-1 protease and both conformations have been compared. The peptide crystallizes as a pentahydrate in space group P2(1) with a = 10.874(2), b = 9.501(2), c = 21.062(5) A, beta = 103.68 (1) degrees, Z = 2, V= 2114.3 A(3), D(x) = 1.21 g cm(-3), micro = 8.02 cm(-1), T= 293 K, lambda(Cu Kalpha) = 1.5418 A. The structure has been refined to an R value of 0.070 for 2152 observed reflections. The peptide main chain can be described as extended and adopts the usual zigzag conformation from the prolyl to the statyl residue. The main difference in conformation between the individual observed and modelled molecules is located on the Sta, Ala and Leu residues with the main chain of the modelled molecule rotated by about 180 degrees as compared to the observed conformation in the crystal state.

  5. Midgut serine proteases and alternative host plant utilization in Pieris brassicae L.

    PubMed Central

    Kumar, Rakesh; Bhardwaj, Usha; Kumar, Pawan; Mazumdar-Leighton, Sudeshna

    2015-01-01

    Pieris brassicae L. is a serious pest of cultivated crucifers in several parts of the world. Larvae of P. brassicae also feed prolifically on garden nasturtium (Tropaeolum majus L., of the family Tropaeolaceae). Proteolytic digestion was studied in larvae feeding on multiple hosts. Fourth instars were collected from cauliflower fields before transfer onto detached, aerial tissues of selected host plants in the lab. Variable levels of midgut proteases were detected in larvae fed on different hosts using protein substrates (casein and recombinant RBCL cloned from cauliflower) and diagnostic, synthetic substrates. Qualitative changes in midgut trypsin activities and quantitative changes in midgut chymotrypsin activities were implicated in physiological adaptation of larvae transferred to T. majus. Midgut proteolytic activities were inhibited to different extents by serine protease inhibitors, including putative trypsin inhibitors isolated from herbivore-attacked and herbivore-free leaves of cauliflower (CfTI) and T. majus (TpTI). Transfer of larvae to T. majus significantly influenced feeding parameters but not necessarily when transferred to different tissues of the same host. Results obtained are relevant for devising sustainable pest management strategies, including transgenic approaches using genes encoding plant protease inhibitors. PMID:25873901

  6. Cyt2Ba of Bacillus thuringiensis israelensis: activation by putative endogenous protease.

    PubMed

    Nisnevitch, Marina; Cohen, Shmuel; Ben-Dov, Eitan; Zaritsky, Arieh; Sofer, Yossef; Cahan, Rivka

    2006-05-26

    The gene cyt2Ba of Bacillus thuringiensis subsp. israelensis was cloned for expression, together with p20, in an acrystalliferous strain. The large hexagonal crystals formed were composed of Cyt2Ba, which facilitated its purification. Crystal solubilization in the presence of endogenous proteases (with spores and cell debris) enabled quick and simple procedure to obtain rather pure and active toxin species by cleavage between amino acid residues 34 and 35, most likely by a camelysin-like protease that was discovered in association with activated Cyt2Ba. The product of this cleavage displayed haemolytic activity comparable to that of exogenously activated Cyt2Ba. The sequence of this putative protease shares high homology with the cell envelope-bound metalloprotease (camelysin) of the closely related species Bacillus cereus.

  7. Identification of a novel host-specific IgG protease in Streptococcus phocae subsp. phocae.

    PubMed

    Rungelrath, Viktoria; Wohlsein, Jan Christian; Siebert, Ursula; Stott, Jeffrey; Prenger-Berninghoff, Ellen; von Pawel-Rammingen, Ulrich; Valentin-Weigand, Peter; Baums, Christoph G; Seele, Jana

    2017-03-01

    Streptococcus (S.) phocae subsp. phocae causes bronchopneumonia and septicemia in a variety of marine mammals. Especially in harbor seals infected with phocine distemper virus it plays an important role as an opportunistic pathogen. This study was initiated by the detection of IgG cleavage products in Western blot analysis after incubation of bacterial supernatant with harbor seal serum. Hence, the objectives of this study were the identification and characterization of a secreted IgG cleaving protease in S. phocae subsp. phocae isolated from marine mammals. To further identify the responsible factor of IgG cleavage a protease inhibitor profile was generated. Inhibition of the IgG cleaving activity by iodoacetamide and Z-LVG-CHN2 indicated that a cysteine protease is involved. Moreover, an anti-IdeS antibody directed against the IgG endopeptidase IdeS of S. pyogenes showed cross reactivity with the putative IgG protease of S. phocae subsp. phocae. The IgG cleaving factor of S. phocae subsp. phocae was identified through an inverse PCR approach and designated IdeP (Immunoglobulin G degrading enzyme of S. phocae subsp. phocae) in analogy to the cysteine protease IdeS. Notably, recombinant (r) IdeP is a host and substrate specific protease as it cleaves IgG from grey and harbor seals but not IgG from harbor porpoises or non-marine mammals. The identification of IdeP represents the first description of a protein in S. phocae subsp. phocae involved in immune evasion. Furthermore, the fact that IdeP cleaves solely IgG of certain marine mammals reflects functional adaption of S. phocae subsp. phocae to grey and harbor seals as its main hosts. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Rice tungro spherical virus polyprotein processing: identification of a virus-encoded protease and mutational analysis of putative cleavage sites.

    PubMed

    Thole, V; Hull, R

    1998-07-20

    Rice tungro spherical virus encodes a large polyprotein containing motifs with sequence similarity to viral serine-like proteases and RNA polymerases. Polyclonal antisera raised against domains of the putative protease and polymerase in fusion with glutathione S-transferase detected a protein of about 35 kDa and, in very low amounts, a protein of about 70 kDa, respectively, in extracts from infected plants. In in vitro transcription/translation systems and in Escherichia coli we demonstrated a proteolytic activity in the C-terminal region of the polyprotein. This protease rapidly cleaved its polyprotein precursors in vitro. Mutating a potential cleavage site located N-terminal to the protease domain, Gln2526-Asp2527, diminished processing. The transversion mutation at the putative C-terminal cleavage site of the protease, at Gln2852-Ala2853, led to a delayed and partial processing.

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

    PubMed

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

    2016-04-01

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

  10. The putative serine protease inhibitor Api m 6 from Apis mellifera venom: recombinant and structural evaluation.

    PubMed

    Michel, Y; McIntyre, M; Ginglinger, H; Ollert, M; Cifuentes, L; Blank, S; Spillner, E

    2012-01-01

    Immunoglobulin (Ig) E-mediated reactions to honeybee venom can cause severe anaphylaxis, sometimes with fatal consequences. Detailed knowledge of the allergic potential of all venom components is necessary to ensure proper diagnosis and treatment of allergy and to gain a better understanding of the allergological mechanisms of insect venoms. Our objective was to undertake an immunochemical and structural evaluation of the putative low-molecular-weight serine protease inhibitor Api m 6, a component of honeybee venom. We recombinantly produced Api m 6 as a soluble protein in Escherichia coli and in Spodoptera frugiperda (Sf9) insect cells.We also assessed specific IgE reactivity of venom-sensitized patients with 2 prokaryotically produced Api m 6 variants using enzyme-linked immunosorbent assay. Moreover, we built a structural model ofApi m 6 and compared it with other protease inhibitor structures to gain insights into the function of Api m 6. In a population of 31 honeybee venom-allergic patients, 26% showed specific IgE reactivity with prokaryotically produced Api m 6, showing it to be a minor but relevant allergen. Molecular modeling of Api m 6 revealed a typical fold of canonical protease inhibitors, supporting the putative function of this venom allergen. Although Api m 6 has a highly variant surface charge, its epitope distribution appears to be similar to that of related proteins. Api m 6 is a honeybee venom component with IgE-sensitizing potential in a fraction of venom-allergic patients. Recombinant Api m 6 can help elucidate individual component-resolved reactivity profiles and increase our understanding of immune responses to low-molecular-weight allergens

  11. Putative Serine Protease Effectors of Clavibacter michiganensis Induce a Hypersensitive Response in the Apoplast of Nicotiana Species.

    PubMed

    Lu, You; Hatsugai, Noriyuki; Katagiri, Fumiaki; Ishimaru, Carol A; Glazebrook, Jane

    2015-11-01

    Clavibacter michiganensis subspp. michiganensis and sepedonicus cause diseases on solanaceous crops. The genomes of both subspecies encode members of the pat-1 family of putative serine proteases known to function in virulence on host plants and induction of hypersensitive responses (HR) on nonhosts. One gene of this family in C. michiganensis subsp. sepedonicus, chp-7, is required for triggering HR in Nicotiana tabacum. Here, further investigation revealed that mutation of the putative catalytic serine residue at position 232 to threonine abolished the HR induction activity of Chp-7, suggesting that enzymatic activity is required. Purified Chp-7 triggered an HR in N. tabacum leaves in the absence of the pathogen, indicating Chp-7 itself is the HR elicitor from C. michiganensis subsp. sepedonicus. Ectopic expression of chp-7 constructs in N. tabacum leaves revealed that Chp-7 targeted to the apoplast triggered an HR while cytoplasmic Chp-7 did not, indicating that Chp-7 induces the HR in the apoplast of N. tabacum leaves. Chp-7 also induced HR in N. sylvestris, a progenitor of N. tabacum, but not in other Nicotiana species tested. ChpG, a related protein from C. michiganensis subsp. michiganensis, also triggered HR in N. tabacum and N. sylvestris. Unlike Chp-7, ChpG triggered HR in N. clevelandii and N. glutinosa.

  12. A Trichomonas vaginalis Rhomboid Protease and Its Substrate Modulate Parasite Attachment and Cytolysis of Host Cells

    PubMed Central

    Riestra, Angelica M.; Gandhi, Shiv; Sweredoski, Michael J.; Moradian, Annie; Hess, Sonja; Urban, Sinisa; Johnson, Patricia J.

    2015-01-01

    Trichomonas vaginalis is an extracellular eukaryotic parasite that causes the most common, non-viral sexually transmitted infection worldwide. Although disease burden is high, molecular mechanisms underlying T. vaginalis pathogenesis are poorly understood. Here, we identify a family of putative T. vaginalis rhomboid proteases and demonstrate catalytic activity for two, TvROM1 and TvROM3, using a heterologous cell cleavage assay. The two T. vaginalis intramembrane serine proteases display different subcellular localization and substrate specificities. TvROM1 is a cell surface membrane protein and cleaves atypical model rhomboid protease substrates, whereas TvROM3 appears to localize to the Golgi apparatus and recognizes a typical model substrate. To identify TvROM substrates, we interrogated the T. vaginalis surface proteome using both quantitative proteomic and bioinformatic approaches. Of the nine candidates identified, TVAG_166850 and TVAG_280090 were shown to be cleaved by TvROM1. Comparison of amino acid residues surrounding the predicted cleavage sites of TvROM1 substrates revealed a preference for small amino acids in the predicted transmembrane domain. Over-expression of TvROM1 increased attachment to and cytolysis of host ectocervical cells. Similarly, mutations that block the cleavage of a TvROM1 substrate lead to its accumulation on the cell surface and increased parasite adherence to host cells. Together, these data indicate a role for TvROM1 and its substrate(s) in modulating attachment to and lysis of host cells, which are key processes in T. vaginalis pathogenesis. PMID:26684303

  13. A Trichomonas vaginalis Rhomboid Protease and Its Substrate Modulate Parasite Attachment and Cytolysis of Host Cells.

    PubMed

    Riestra, Angelica M; Gandhi, Shiv; Sweredoski, Michael J; Moradian, Annie; Hess, Sonja; Urban, Sinisa; Johnson, Patricia J

    2015-12-01

    Trichomonas vaginalis is an extracellular eukaryotic parasite that causes the most common, non-viral sexually transmitted infection worldwide. Although disease burden is high, molecular mechanisms underlying T. vaginalis pathogenesis are poorly understood. Here, we identify a family of putative T. vaginalis rhomboid proteases and demonstrate catalytic activity for two, TvROM1 and TvROM3, using a heterologous cell cleavage assay. The two T. vaginalis intramembrane serine proteases display different subcellular localization and substrate specificities. TvROM1 is a cell surface membrane protein and cleaves atypical model rhomboid protease substrates, whereas TvROM3 appears to localize to the Golgi apparatus and recognizes a typical model substrate. To identify TvROM substrates, we interrogated the T. vaginalis surface proteome using both quantitative proteomic and bioinformatic approaches. Of the nine candidates identified, TVAG_166850 and TVAG_280090 were shown to be cleaved by TvROM1. Comparison of amino acid residues surrounding the predicted cleavage sites of TvROM1 substrates revealed a preference for small amino acids in the predicted transmembrane domain. Over-expression of TvROM1 increased attachment to and cytolysis of host ectocervical cells. Similarly, mutations that block the cleavage of a TvROM1 substrate lead to its accumulation on the cell surface and increased parasite adherence to host cells. Together, these data indicate a role for TvROM1 and its substrate(s) in modulating attachment to and lysis of host cells, which are key processes in T. vaginalis pathogenesis.

  14. Lectin Activation in Giardia lamblia by Host Protease: A Novel Host-Parasite Interaction

    NASA Astrophysics Data System (ADS)

    Lev, Boaz; Ward, Honorine; Keusch, Gerald T.; Pereira, Miercio E. A.

    1986-04-01

    A lectin in Giardia lamblia was activated by secretions from the human duodenum, the environment where the parasite lives. Incubation of the secretions with trypsin inhibitors prevented the appearance of lectin activity, implicating proteases as the activating agent. Accordingly, lectin activation was also produced by crystalline trypsin and Pronase; other proteases tested were ineffective. When activated, the lectin agglutinated intestinal cells to which the parasite adheres in vivo. The lectin was most specific to mannose-6-phosphate and apparently was bound to the plasma membrane. Activation of a parasite lectin by a host protease represents a novel mechanism of hostparasite interaction and may contribute to the affinity of Giardia lamblia to the infection site.

  15. Novel IgG-Degrading Enzymes of the IgdE Protease Family Link Substrate Specificity to Host Tropism of Streptococcus Species.

    PubMed

    Spoerry, Christian; Hessle, Pontus; Lewis, Melanie J; Paton, Lois; Woof, Jenny M; von Pawel-Rammingen, Ulrich

    2016-01-01

    Recently we have discovered an IgG degrading enzyme of the endemic pig pathogen S. suis designated IgdE that is highly specific for porcine IgG. This protease is the founding member of a novel cysteine protease family assigned C113 in the MEROPS peptidase database. Bioinformatical analyses revealed putative members of the IgdE protease family in eight other Streptococcus species. The genes of the putative IgdE family proteases of S. agalactiae, S. porcinus, S. pseudoporcinus and S. equi subsp. zooepidemicus were cloned for production of recombinant protein into expression vectors. Recombinant proteins of all four IgdE family proteases were proteolytically active against IgG of the respective Streptococcus species hosts, but not against IgG from other tested species or other classes of immunoglobulins, thereby linking the substrate specificity to the known host tropism. The novel IgdE family proteases of S. agalactiae, S. pseudoporcinus and S. equi showed IgG subtype specificity, i.e. IgdE from S. agalactiae and S. pseudoporcinus cleaved human IgG1, while IgdE from S. equi was subtype specific for equine IgG7. Porcine IgG subtype specificities of the IgdE family proteases of S. porcinus and S. pseudoporcinus remain to be determined. Cleavage of porcine IgG by IgdE of S. pseudoporcinus is suggested to be an evolutionary remaining activity reflecting ancestry of the human pathogen to the porcine pathogen S. porcinus. The IgG subtype specificity of bacterial proteases indicates the special importance of these IgG subtypes in counteracting infection or colonization and opportunistic streptococci neutralize such antibodies through expression of IgdE family proteases as putative immune evasion factors. We suggest that IgdE family proteases might be valid vaccine targets against streptococci of both human and veterinary medical concerns and could also be of therapeutic as well as biotechnological use.

  16. Flamingo cadherin: a putative host receptor for Streptococcus pneumoniae.

    PubMed

    Blau, Karin; Portnoi, Maxim; Shagan, Marilou; Kaganovich, Antonina; Rom, Slava; Kafka, Daniel; Chalifa Caspi, Vered; Porgador, Angel; Givon-Lavi, Noga; Gershoni, Jonathan M; Dagan, Ron; Mizrachi Nebenzahl, Yaffa

    2007-06-15

    Streptococcus pneumoniae fructose bisphosphate aldolase (FBA) is a cell wall-localized lectin. We demonstrate that recombinant (r) FBA and anti-rFBA antibodies inhibit encapsulated and unencapsulated S. pneumoniae serotype 3 adherence to A549 type II lung carcinoma epithelial cells. A random combinatorial peptide library expressed by filamentous phage was screened with rFBA. Eleven of 30 rFBA-binding phages inhibited 90% of S. pneumoniae adhesion to A549 cells. The insert peptide sequence of 9 of these phages matched the Flamingo cadherin receptor (FCR) when aligned against the human genome. A peptide comprising a putative FBA-binding region of FCR (FCRP) inhibited 2 genetically and capsularly unrelated pairs of encapsulated and unencapsulated S. pneumoniae strains from binding to A549 cells. Moreover, FCRP inhibited S. pneumoniae nasopharyngeal and lung colonization and, possibly, pneumonia development in the mouse intranasal inoculation model system. These data indicate that FBA is an S. pneumoniae adhesin and that FCR is its host receptor.

  17. Chlamydia-secreted protease CPAF degrades host antimicrobial peptides.

    PubMed

    Tang, Lingli; Chen, Jianlin; Zhou, Zhiguang; Yu, Ping; Yang, Zhangsheng; Zhong, Guangming

    2015-06-01

    Chlamydia trachomatis infection in the lower genital tract, if untreated, can ascend to the upper genital tract, potentially leading to complications such as tubal factor infertility. The ascension involves cell-to-cell spreading, which may require C. trachomatis organisms to overcome mucosal extracellular effectors such as antimicrobial peptides. We found that among the 8 antimicrobial peptides tested, the cathelicidin LL-37 that is produced by both urogenital epithelial cells and the recruited neutrophils possessed a most potent antichlamydial activity. Interestingly, this antichlamydial activity was completely inhibited by CPAF, a C. trachomatis-secreted serine protease. The inhibition was dependent on CPAF's proteolytic activity. CPAF selectively degraded LL-37 and other antimicrobial peptides with an antichlamydial activity. CPAF is known to secrete into and accumulate in the infected host cell cytoplasm at the late stage of chlamydial intracellular growth and may be released to confront the extracellular antimicrobial peptides before the intra-inclusion organisms are exposed to extracellular environments during host cell lysis and chlamydial spreading. Thus, the finding that CPAF selectively targets host antimicrobial peptides that possess antichlamydial activities for proteolysis suggests that CPAF may contribute to C. trachomatis pathogenicity by aiding in ascending infection.

  18. Active subtilisin-like protease from a hyperthermophilic archaeon in a form with a putative prosequence.

    PubMed

    Kannan, Y; Koga, Y; Inoue, Y; Haruki, M; Takagi, M; Imanaka, T; Morikawa, M; Kanaya, S

    2001-06-01

    The gene encoding subtilisin-like protease T. kodakaraensis subtilisin was cloned from a hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. T. kodakaraensis subtilisin is a member of the subtilisin family and composed of 422 amino acid residues with a molecular weight of 43,783. It consists of a putative presequence, prosequence, and catalytic domain. Like bacterial subtilisins, T. kodakaraensis subtilisin was overproduced in Escherichia coli in a form with a putative prosequence in inclusion bodies, solubilized in the presence of 8 M urea, and refolded and converted to an active molecule. However, unlike bacterial subtilisins, in which the prosequence was removed from the catalytic domain by autoprocessing upon refolding, T. kodakaraensis subtilisin was refolded in a form with a putative prosequence. This refolded protein of recombinant T. kodakaraensis subtilisin which is composed of 398 amino acid residues (Gly(-82) to Gly(316)), was purified to give a single band on a sodium dodecyl sulfate (SDS)-polyacrylamide gel and characterized for biochemical and enzymatic properties. The good agreement of the molecular weights estimated by SDS-polyacrylamide gel electrophoresis (44,000) and gel filtration (40,000) suggests that T. kodakaraensis subtilisin exists in a monomeric form. T. kodakaraensis subtilisin hydrolyzed the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide only in the presence of the Ca(2+) ion with an optimal pH and temperature of pH 9.5 and 80 degrees C. Like bacterial subtilisins, it showed a broad substrate specificity, with a preference for aromatic or large nonpolar P1 substrate residues. However, it was much more stable than bacterial subtilisins against heat inactivation and lost activity with half-lives of >60 min at 80 degrees C, 20 min at 90 degrees C, and 7 min at 100 degrees C.

  19. Pseudomonas aeruginosa protease IV degrades surfactant proteins and inhibits surfactant host defense and biophysical functions.

    PubMed

    Malloy, Jaret L; Veldhuizen, Ruud A W; Thibodeaux, Brett A; O'Callaghan, Richard J; Wright, Jo Rae

    2005-02-01

    Pulmonary surfactant has two distinct functions within the lung: reduction of surface tension at the air-liquid interface and participation in innate host defense. Both functions are dependent on surfactant-associated proteins. Pseudomonas aeruginosa is primarily responsible for respiratory dysfunction and death in cystic fibrosis patients and is also a leading pathogen in nosocomial pneumonia. P. aeruginosa secretes a number of proteases that contribute to its virulence. We hypothesized that P. aeruginosa protease IV degrades surfactant proteins and results in a reduction in pulmonary surfactant host defense and biophysical functions. Protease IV was isolated from cultured supernatant of P. aeruginosa by gel chromatography. Incubation of cell-free bronchoalveolar lavage fluid with protease IV resulted in degradation of surfactant proteins (SP)-A, -D, and -B. SPs were degraded in a time- and dose-dependent fashion by protease IV, and degradation was inhibited by the trypsin-like serine protease inhibitor Nalpha-p-tosyl-L-lysine-chloromethyl ketone (TLCK). Degradation by protease IV inhibited SP-A- and SP-D-mediated bacterial aggregation and uptake by macrophages. Surfactant treated with protease IV was unable to reduce surface tension as effectively as untreated surfactant, and this effect was inhibited by TLCK. We speculate that protease IV may be an important contributing factor to the development and propagation of acute lung injury associated with P. aeruginosa via loss of surfactant function within the lung.

  20. Activation of influenza viruses by proteases from host cells and bacteria in the human airway epithelium.

    PubMed

    Böttcher-Friebertshäuser, Eva; Klenk, Hans-Dieter; Garten, Wolfgang

    2013-11-01

    Influenza is an acute infection of the respiratory tract, which affects each year millions of people. Influenza virus infection is initiated by the surface glycoprotein hemagglutinin (HA) through receptor binding and fusion of viral and endosomal membranes. HA is synthesized as a precursor protein and requires cleavage by host cell proteases to gain its fusion capacity. Although cleavage of HA is crucial for virus infectivity, little was known about relevant proteases in the human airways for a long time. Recent progress in the identification and characterization of HA-activating host cell proteases has been considerable however and supports the idea of targeting HA cleavage as a novel approach for influenza treatment. Interestingly, certain bacteria have been demonstrated to support HA activation either by secreting proteases that cleave HA or due to activation of cellular proteases and thereby may contribute to virus spread and enhanced pathogenicity. In this review, we give an overview on activation of influenza viruses by proteases from host cells and bacteria with the main focus on recent progress on HA cleavage by proteases HAT and TMPRSS2 in the human airway epithelium. In addition, we outline investigations of HA-activating proteases as potential drug targets for influenza treatment.

  1. Staphylococcus aureus Manipulates Innate Immunity through Own and Host-Expressed Proteases

    PubMed Central

    Pietrocola, Giampiero; Nobile, Giulia; Rindi, Simonetta; Speziale, Pietro

    2017-01-01

    Neutrophils, complement system and skin collectively represent the main elements of the innate immune system, the first line of defense of the host against many common microorganisms. Bacterial pathogens have evolved strategies to counteract all these defense activities. Specifically, Staphylococcus aureus, a major human pathogen, secretes a variety of immune evasion molecules including proteases, which cleave components of the innate immune system or disrupt the integrity of extracellular matrix and intercellular connections of tissues. Additionally, S. aureus secretes proteins that can activate host zymogens which, in turn, target specific defense components. Secreted proteins can also inhibit the anti-bacterial function of neutrophils or complement system proteases, potentiating S. aureus chances of survival. Here, we review the current understanding of these proteases and modulators of host proteases in the functioning of innate immunity and describe the importance of these mechanisms in the pathology of staphylococcal diseases. PMID:28529927

  2. 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.

  3. 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

  4. Proteases.

    PubMed

    Barrett, A J

    2001-05-01

    The processes of growth and remodeling of cells and tissues in multicellular organisms require the breakdown of old protein molecules, in concert with the synthesis of new ones. For example, many newly-synthesized molecules require proteolytic processing to convert them to biologically active forms. Proteolysis can terminate the activity of a protein--e.g., capsases mediate apoptosis, which is a vital step in the life cycle of the cell. Proteolysis contributes to defense systems too, as the recognition of peptide fragments of foreign proteins triggers the immune response. Proteases are the class of enzymes involved in these important reactions. This unit discusses the general categories of proteases, and sets the stage for addition of overview units on cysteine proteases, aspartic proteases, and metalloproteases, as well as protocol units featuring techniques for analyzing mammalian and yeast proteasomes and protease inhibitors, among other topics.

  5. Purification and structural characterization of the putative gag-pol protease of human immunodeficiency virus.

    PubMed Central

    Lillehoj, E P; Salazar, F H; Mervis, R J; Raum, M G; Chan, H W; Ahmad, N; Venkatesan, S

    1988-01-01

    We have purified a 10,774-dalton protein from human immunodeficiency virus (HIV) type 1 that is encoded in the protease domain of the pol open reading frame (ORF). Radiochemical amino acid microsequencing identified 12 amino acids from the stretch of 39 N-terminal residues of this protein, beginning with a PQITLW sequence at position 69 of the pol ORF. Radiosequencing of selected tryptic peptides of the protein identified 11 additional residues (Leu-9 and Val-2) in six peptides encompassing the entire molecule of 99 residues. A protein of similar size and identical N-terminal sequence (determined through the first 39 residues) was present among the processed HIV pol gene products in Escherichia coli which expressed the entire HIV pol ORF. The C terminus of both the viral and E. coli-expressed proteins was inferred to be contiguous with the N terminus of the p64-p51 reverse transcriptase on the basis of tryptic mapping and specific immunoreactivity with an antiserum against a dodecapeptide located upstream of the reverse transcriptase. Thus, the initial processing of the pol precursor that generates the native protease is apparently preserved across phylogenetic barriers. Although the purified viral protease lacked measurable proteolytic activity, the bacterial extracts were capable of processing an HIV gag precursor protein synthesized in E. coli. Images PMID:3292793

  6. The Protease Locus of Francisella tularensis LVS Is Required for Stress Tolerance and Infection in the Mammalian Host

    PubMed Central

    He, Lihong; Nair, Manoj Kumar Mohan; Chen, Yuling; Liu, Xue; Zhang, Mengyun; Hazlett, Karsten R. O.

    2016-01-01

    Francisella tularensis is the causative agent of tularemia and a category A potential agent of bioterrorism, but the pathogenic mechanisms of F. tularensis are largely unknown. Our previous transposon mutagenesis screen identified 95 lung infectivity-associated F. tularensis genes, including those encoding the Lon and ClpP proteases. The present study validates the importance of Lon and ClpP in intramacrophage growth and infection of the mammalian host by using unmarked deletion mutants of the F. tularensis live vaccine strain (LVS). Further experiments revealed that lon and clpP are also required for F. tularensis tolerance to stressful conditions. A quantitative proteomic comparison between heat-stressed LVS and the isogenic Lon-deficient mutant identified 29 putative Lon substrate proteins. The follow-up protein degradation experiments identified five substrates of the F. tularensis Lon protease (FTL578, FTL663, FTL1217, FTL1228, and FTL1957). FTL578 (ornithine cyclodeaminase), FTL663 (heat shock protein), and FTL1228 (iron-sulfur activator complex subunit SufD) have been previously described as virulence-associated factors in F. tularensis. Identification of these Lon substrates has thus provided important clues for further understanding of the F. tularensis stress response and pathogenesis. The high-throughput approach developed in this study can be used for systematic identification of the Lon substrates in other prokaryotic and eukaryotic organisms. PMID:26902724

  7. Trypanosomatid cysteine protease activity may be enhanced by a kininogen-like moiety from host serum.

    PubMed Central

    Lonsdale-Eccles, J D; Mpimbaza, G W; Nkhungulu, Z R; Olobo, J; Smith, L; Tosomba, O M; Grab, D J

    1995-01-01

    observed when this putative cysteine protease inhibitor was used under these conditions. The activation of parasite cysteine proteases by commonly accepted cysteine protease inhibitors is unexpected and may have important pathological repercussions. Images Figure 1 Figure 3 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 PMID:7832773

  8. Viral protease cleavage of inhibitor of κBα triggers host cell apoptosis

    PubMed Central

    Zaragoza, Carlos; Saura, Marta; Padalko, Elizaveta Y.; Lopez-Rivera, Ester; Lizarbe, Tania R.; Lamas, Santiago; Lowenstein, Charles J.

    2006-01-01

    Apoptosis is an innate immune response to viral infection that limits viral replication. However, the mechanisms by which cells detect viral infection and activate apoptosis are not completely understood. We now show that during Coxsackievirus infection, the viral protease 3Cpro cleaves inhibitor of κBα (IκBα). A proteolytic fragment of IκBα then forms a stable complex with NF-κB, translocates to the nucleus, and inhibits NF-κB transactivation, increasing apoptosis and decreasing viral replication. In contrast, cells with reduced IκBα expression are more susceptible to viral infection, with less apoptosis and more viral replication. IκBα thus acts as a sensor of viral infection. Cleavage of host proteins by pathogen proteases is a novel mechanism by which the host recognizes and responds to viral infection. PMID:17138672

  9. A family of serine proteases of Clavibacter michiganensis subsp. michiganensis: chpC plays a role in colonization of the host plant tomato.

    PubMed

    Stork, Ines; Gartemann, Karl-Heinz; Burger, Annette; Eichenlaub, Rudolf

    2008-09-01

    Genes for seven putative serine proteases (ChpA-ChpG) belonging to the trypsin subfamily and homologous to the virulence factor pat-1 were identified on the chromosome of Clavibacter michiganensis subsp. michiganensis (Cmm) NCPPB382. All proteases have signal peptides indicating export of these proteins. Their putative function is suggested by two motifs and an aspartate residue typical for serine proteases. Furthermore, six cysteine residues are located at conserved positions. The genes are clustered in a chromosomal region of about 50 kb with a significantly lower G + C content than common for Cmm. The genes chpA, chpB and chpD are pseudogenes as they contain frame shifts and/or in-frame stop codons. The genes chpC and chpG were inactivated by the insertion of an antibiotic resistance cassette. The chpG mutant was not impaired in virulence. However, in planta the titre of the chpC mutant was drastically reduced and only weak disease symptoms were observed. Complementation of the chpC mutant by the wild-type allele restored full virulence. ChpC is the first chromosomal gene of Cmm identified so far that affects the interaction of the pathogen with the host plant.

  10. hpaR, a putative marR family transcriptional regulator, is positively controlled by HrpG and HrpX and involved in the pathogenesis, hypersensitive response, and extracellular protease production of Xanthomonas campestris pathovar campestris.

    PubMed

    Wei, Ke; Tang, Dong-Jie; He, Yong-Qiang; Feng, Jia-Xun; Jiang, Bo-Le; Lu, Guang-Tao; Chen, Baoshan; Tang, Ji-Liang

    2007-03-01

    The MarR family of transcriptional regulators of bacteria are involved in the regulation of many cellular processes, including pathogenesis. In this work, we have demonstrated genetically that hpaR (hpa, hrp associated), which encodes a putative MarR family regulator, is involved in the hypersensitive response (HR), pathogenicity, and extracellular protease production of the phytopathogenic bacterium Xanthomonas campestris pathovar campestris. A mutation in hpaR resulted in complete loss of virulence in the host plant cabbage, a delayed and weakened HR in the nonhost plant pepper ECW-10R, and an increase in extracellular protease production. Detection of the beta-glucuronidase activity of a plasmid-driven hpaR promoter-gusA reporter revealed that the expression of hpaR is positively controlled by HrpG and HrpX and is suppressed in rich medium while being strongly induced in minimal and hrp-inducing media and inside the host. These findings indicate that hpaR belongs to the hrpG and hrpX regulon and that HrpX regulates the extracellular protease production via hpaR in X. campestris pv. campestris.

  11. Significance of Cuscutain, a cysteine protease from Cuscuta reflexa, in host-parasite interactions

    PubMed Central

    2010-01-01

    Background Plant infestation with parasitic weeds like Cuscuta reflexa induces morphological as well as biochemical changes in the host and the parasite. These modifications could be caused by a change in protein or gene activity. Using a comparative macroarray approach Cuscuta genes specifically upregulated at the host attachment site were identified. Results One of the infestation specific Cuscuta genes encodes a cysteine protease. The protein and its intrinsic inhibitory peptide were heterologously expressed, purified and biochemically characterized. The haustoria specific enzyme was named cuscutain in accordance with similar proteins from other plants, e.g. papaya. The role of cuscutain and its inhibitor during the host parasite interaction was studied by external application of an inhibitor suspension, which induced a significant reduction of successful infection events. Conclusions The study provides new information about molecular events during the parasitic plant - host interaction. Inhibition of cuscutain cysteine proteinase could provide means for antagonizing parasitic plants. PMID:20964874

  12. Role of host cellular proteases in the pathogenesis of influenza and influenza-induced multiple organ failure.

    PubMed

    Kido, Hiroshi; Okumura, Yuushi; Takahashi, Etsuhisa; Pan, Hai-Yan; Wang, Siye; Yao, Dengbing; Yao, Min; Chida, Junji; Yano, Mihiro

    2012-01-01

    Influenza A virus (IAV) is one of the most common infectious pathogens in humans. Since the IVA genome does not have the processing protease for the viral hemagglutinin (HA) envelope glycoprotein precursors, entry of this virus into cells and infectious organ tropism of IAV are primarily determined by host cellular trypsin-type HA processing proteases. Several secretion-type HA processing proteases for seasonal IAV in the airway, and ubiquitously expressed furin and pro-protein convertases for highly pathogenic avian influenza (HPAI) virus, have been reported. Recently, other HA-processing proteases for seasonal IAV and HPAI have been identified in the membrane fraction. These proteases proteolytically activate viral multiplication at the time of viral entry and budding. In addition to the role of host cellular proteases in IAV pathogenicity, IAV infection results in marked upregulation of cellular trypsins and matrix metalloproteinase-9 in various organs and cells, particularly endothelial cells, through induced pro-inflammatory cytokines. These host cellular factors interact with each other as the influenza virus-cytokine-protease cycle, which is the major mechanism that induces vascular hyperpermeability and multiorgan failure in severe influenza. This mini-review discusses the roles of cellular proteases in the pathogenesis of IAV and highlights the molecular mechanisms of upregulation of trypsins as effective targets for the control of IAV infection. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome. Copyright © 2011 Elsevier B.V. All rights reserved.

  13. A putative vacuolar processing protease is regulated by ethylene and also during fruit ripening in Citrus fruit.

    PubMed Central

    Alonso, J M; Granell, A

    1995-01-01

    A putative citrus vacuolar processing thiolprotease cDNA (Cit-vac) was isolated from a cDNA library of Citrus fruits (Citrus sinensis L. Osbeck var Washington navel). The cDNA is 58 and 57% identical with vacuolar processing seed proteases from castor bean and soybean, respectively. The Citvac sequence shows a typical signal peptide for entering into the endoplasmic reticulum and two glycosylation signals. Using an in vitro transcription-translation system, we show that the Citvac precursor is able to enter a microsomal fraction and to undergo proteolytic processing and glycosylation. Transcript levels for the Citvac are developmentally regulated in the flavedo (outer colored part of the fruit peel) and increase during fruit ripening and in the flower during opening. Exogenous treatment with ethylene induces Citvac mRNA expression in both fruits and leaves. Citvac is encoded by one or two genes in the Citrus genome. The possible role of the Citvac gene product during fruit ripening and other ethylene-mediated processes is discussed. PMID:7480346

  14. Host-Parasite Interaction: Parasite-Derived and -Induced Proteases That Degrade Human Extracellular Matrix

    PubMed Central

    Piña-Vázquez, Carolina; Reyes-López, Magda; Ortíz-Estrada, Guillermo; de la Garza, Mireya; Serrano-Luna, Jesús

    2012-01-01

    Parasitic protozoa are among the most important pathogens worldwide. Diseases such as malaria, leishmaniasis, amoebiasis, giardiasis, trichomoniasis, and trypanosomiasis affect millions of people. Humans are constantly threatened by infections caused by these pathogens. Parasites engage a plethora of surface and secreted molecules to attach to and enter mammalian cells. The secretion of lytic enzymes by parasites into host organs mediates critical interactions because of the invasion and destruction of interstitial tissues, enabling parasite migration to other sites within the hosts. Extracellular matrix is a complex, cross-linked structure that holds cells together in an organized assembly and that forms the basement membrane lining (basal lamina). The extracellular matrix represents a major barrier to parasites. Therefore, the evolution of mechanisms for connective-tissue degradation may be of great importance for parasite survival. Recent advances have been achieved in our understanding of the biochemistry and molecular biology of proteases from parasitic protozoa. The focus of this paper is to discuss the role of protozoan parasitic proteases in the degradation of host ECM proteins and the participation of these molecules as virulence factors. We divide the paper into two sections, extracellular and intracellular protozoa. PMID:22792442

  15. Coral bleaching under thermal stress: putative involvement of host/symbiont recognition mechanisms

    PubMed Central

    Vidal-Dupiol, Jeremie; Adjeroud, Mehdi; Roger, Emmanuel; Foure, Laurent; Duval, David; Mone, Yves; Ferrier-Pages, Christine; Tambutte, Eric; Tambutte, Sylvie; Zoccola, Didier; Allemand, Denis; Mitta, Guillaume

    2009-01-01

    Background Coral bleaching can be defined as the loss of symbiotic zooxanthellae and/or their photosynthetic pigments from their cnidarian host. This major disturbance of reef ecosystems is principally induced by increases in water temperature. Since the beginning of the 1980s and the onset of global climate change, this phenomenon has been occurring at increasing rates and scales, and with increasing severity. Several studies have been undertaken in the last few years to better understand the cellular and molecular mechanisms of coral bleaching but the jigsaw puzzle is far from being complete, especially concerning the early events leading to symbiosis breakdown. The aim of the present study was to find molecular actors involved early in the mechanism leading to symbiosis collapse. Results In our experimental procedure, one set of Pocillopora damicornis nubbins was subjected to a gradual increase of water temperature from 28°C to 32°C over 15 days. A second control set kept at constant temperature (28°C). The differentially expressed mRNA between the stressed states (sampled just before the onset of bleaching) and the non stressed states (control) were isolated by Suppression Subtractive Hybridization. Transcription rates of the most interesting genes (considering their putative function) were quantified by Q-RT-PCR, which revealed a significant decrease in transcription of two candidates six days before bleaching. RACE-PCR experiments showed that one of them (PdC-Lectin) contained a C-Type-Lectin domain specific for mannose. Immunolocalisation demonstrated that this host gene mediates molecular interactions between the host and the symbionts suggesting a putative role in zooxanthellae acquisition and/or sequestration. The second gene corresponds to a gene putatively involved in calcification processes (Pdcyst-rich). Its down-regulation could reflect a trade-off mechanism leading to the arrest of the mineralization process under stress. Conclusion Under thermal

  16. Coral bleaching under thermal stress: putative involvement of host/symbiont recognition mechanisms.

    PubMed

    Vidal-Dupiol, Jeremie; Adjeroud, Mehdi; Roger, Emmanuel; Foure, Laurent; Duval, David; Mone, Yves; Ferrier-Pages, Christine; Tambutte, Eric; Tambutte, Sylvie; Zoccola, Didier; Allemand, Denis; Mitta, Guillaume

    2009-08-04

    Coral bleaching can be defined as the loss of symbiotic zooxanthellae and/or their photosynthetic pigments from their cnidarian host. This major disturbance of reef ecosystems is principally induced by increases in water temperature. Since the beginning of the 1980s and the onset of global climate change, this phenomenon has been occurring at increasing rates and scales, and with increasing severity. Several studies have been undertaken in the last few years to better understand the cellular and molecular mechanisms of coral bleaching but the jigsaw puzzle is far from being complete, especially concerning the early events leading to symbiosis breakdown. The aim of the present study was to find molecular actors involved early in the mechanism leading to symbiosis collapse. In our experimental procedure, one set of Pocillopora damicornis nubbins was subjected to a gradual increase of water temperature from 28 degrees C to 32 degrees C over 15 days. A second control set kept at constant temperature (28 degrees C). The differentially expressed mRNA between the stressed states (sampled just before the onset of bleaching) and the non stressed states (control) were isolated by Suppression Subtractive Hybridization. Transcription rates of the most interesting genes (considering their putative function) were quantified by Q-RT-PCR, which revealed a significant decrease in transcription of two candidates six days before bleaching. RACE-PCR experiments showed that one of them (PdC-Lectin) contained a C-Type-Lectin domain specific for mannose. Immunolocalisation demonstrated that this host gene mediates molecular interactions between the host and the symbionts suggesting a putative role in zooxanthellae acquisition and/or sequestration. The second gene corresponds to a gene putatively involved in calcification processes (Pdcyst-rich). Its down-regulation could reflect a trade-off mechanism leading to the arrest of the mineralization process under stress. Under thermal stress

  17. An aspartyl protease defines a novel pathway for export of Toxoplasma proteins into the host cell

    PubMed Central

    Coffey, Michael J; Sleebs, Brad E; Uboldi, Alessandro D; Garnham, Alexandra; Franco, Magdalena; Marino, Nicole D; Panas, Michael W; Ferguson, David JP; Enciso, Marta; O'Neill, Matthew T; Lopaticki, Sash; Stewart, Rebecca J; Dewson, Grant; Smyth, Gordon K; Smith, Brian J; Masters, Seth L; Boothroyd, John C; Boddey, Justin A; Tonkin, Christopher J

    2015-01-01

    Infection by Toxoplasma gondii leads to massive changes to the host cell. Here, we identify a novel host cell effector export pathway that requires the Golgi-resident aspartyl protease 5 (ASP5). We demonstrate that ASP5 cleaves a highly constrained amino acid motif that has similarity to the PEXEL-motif of Plasmodium parasites. We show that ASP5 matures substrates at both the N- and C-terminal ends of proteins and also controls trafficking of effectors without this motif. Furthermore, ASP5 controls establishment of the nanotubular network and is required for the efficient recruitment of host mitochondria to the vacuole. Assessment of host gene expression reveals that the ASP5-dependent pathway influences thousands of the transcriptional changes that Toxoplasma imparts on its host cell. All these changes result in attenuation of virulence of Δasp5 tachyzoites in vivo. This work characterizes the first identified machinery required for export of Toxoplasma effectors into the infected host cell. DOI: http://dx.doi.org/10.7554/eLife.10809.001 PMID:26576949

  18. An aspartyl protease defines a novel pathway for export of Toxoplasma proteins into the host cell.

    PubMed

    Coffey, Michael J; Sleebs, Brad E; Uboldi, Alessandro D; Garnham, Alexandra; Franco, Magdalena; Marino, Nicole D; Panas, Michael W; Ferguson, David Jp; Enciso, Marta; O'Neill, Matthew T; Lopaticki, Sash; Stewart, Rebecca J; Dewson, Grant; Smyth, Gordon K; Smith, Brian J; Masters, Seth L; Boothroyd, John C; Boddey, Justin A; Tonkin, Christopher J

    2015-11-18

    Infection by Toxoplasma gondii leads to massive changes to the host cell. Here, we identify a novel host cell effector export pathway that requires the Golgi-resident aspartyl protease 5 (ASP5). We demonstrate that ASP5 cleaves a highly constrained amino acid motif that has similarity to the PEXEL-motif of Plasmodium parasites. We show that ASP5 matures substrates at both the N- and C-terminal ends of proteins and also controls trafficking of effectors without this motif. Furthermore, ASP5 controls establishment of the nanotubular network and is required for the efficient recruitment of host mitochondria to the vacuole. Assessment of host gene expression reveals that the ASP5-dependent pathway influences thousands of the transcriptional changes that Toxoplasma imparts on its host cell. All these changes result in attenuation of virulence of Δasp5 tachyzoites in vivo. This work characterizes the first identified machinery required for export of Toxoplasma effectors into the infected host cell.

  19. Putative protease inhibitor gene discovery and transcript profiling during fruit development and leaf damage in grapefruit (Citrus paradisi Macf.).

    PubMed

    Shatters, Robert G; Bausher, Michael G; Hunter, Wayne B; Chaparro, José X; Dang, Phat M; Niedz, Randall P; Mayer, Richard T; McCollum, T Greg; Sinisterra, Xiomara

    2004-02-04

    Seven putative protease inhibitor (PPI) cDNAs, representing four protein families, were isolated from a grapefruit (Citrus paradisi Macf. Cv. Marsh) immature fruit flavedo cDNA library. Cloned open reading frames encoded proteins with similarity to, and protein signatures for: legume Kuntiz inhibitors (lkiL-1, lkiL-2, lkiL-3), potato trypsin inhibitor I (ptiIL-1), serpins (serpL-1), cystatins (cystL-1), and gamma thionins (gthL-1). Response of transcript abundance to fruit development and leaf wounding was determined for all but lkiL-1 using real-time RT-PCR. Immature leaves had the highest transcript levels for all PPIs. The gthL-1 transcript in immature leaves was the most abundant transcript but was absent from healthy mature leaves. In fruit flavedo, transcripts for all PPIs were most abundant in youngest fruit (<15 mm dia. fruit), and declined during development, but displayed different patterns of developmental change. Mechanical or Diaprepes root weevil (DRW) feeding damage to leaves caused a <10-fold reduction or had no effect on transcript level with the exception of gthL-1 which, as a result of damage, increased >50-fold in mature leaves and decreased >1400-fold in immature leaves. This developmental control of transcript response to wounding in a woody perennial is opposite of what has been observed for defensive proteinase inhibitors (PIs) in other plants (typically herbaceous and/or annual plants), where younger leaves typically invoke a higher defensive proteinase inhibitor transcript accumulation than older tissues. Except for gthL-1, the PPI transcripts were minimally responsive or unresponsive to wounding. Changes in PPI transcript levels suggest diverse roles for the products of these genes in citrus, with only gthL-1 responding in a defense-like manner.

  20. A computational module assembled from different protease family motifs identifies PI PLC from Bacillus cereus as a putative prolyl peptidase with a serine protease scaffold.

    PubMed

    Rendón-Ramírez, Adela; Shukla, Manish; Oda, Masataka; Chakraborty, Sandeep; Minda, Renu; Dandekar, Abhaya M; Ásgeirsson, Bjarni; Goñi, Félix M; Rao, Basuthkar J

    2013-01-01

    Proteolytic enzymes have evolved several mechanisms to cleave peptide bonds. These distinct types have been systematically categorized in the MEROPS database. While a BLAST search on these proteases identifies homologous proteins, sequence alignment methods often fail to identify relationships arising from convergent evolution, exon shuffling, and modular reuse of catalytic units. We have previously established a computational method to detect functions in proteins based on the spatial and electrostatic properties of the catalytic residues (CLASP). CLASP identified a promiscuous serine protease scaffold in alkaline phosphatases (AP) and a scaffold recognizing a β-lactam (imipenem) in a cold-active Vibrio AP. Subsequently, we defined a methodology to quantify promiscuous activities in a wide range of proteins. Here, we assemble a module which encapsulates the multifarious motifs used by protease families listed in the MEROPS database. Since APs and proteases are an integral component of outer membrane vesicles (OMV), we sought to query other OMV proteins, like phospholipase C (PLC), using this search module. Our analysis indicated that phosphoinositide-specific PLC from Bacillus cereus is a serine protease. This was validated by protease assays, mass spectrometry and by inhibition of the native phospholipase activity of PI-PLC by the well-known serine protease inhibitor AEBSF (IC50 = 0.018 mM). Edman degradation analysis linked the specificity of the protease activity to a proline in the amino terminal, suggesting that the PI-PLC is a prolyl peptidase. Thus, we propose a computational method of extending protein families based on the spatial and electrostatic congruence of active site residues.

  1. The Lon protease from the haloalkaliphilic archaeon Natrialba magadii is transcriptionally linked to a cluster of putative membrane proteases and displays DNA-binding activity.

    PubMed

    Sastre, Diego E; Paggi, Roberto A; De Castro, Rosana E

    2011-05-20

    The ATP-dependent Lon protease is universally distributed in bacteria, eukaryotic organelles and archaea. In comparison with bacterial and eukaryal Lon proteases, the biology of the archaeal Lon has been studied to a limited extent. In this study, the gene encoding the Lon protease of the alkaliphilic haloarchaeon Natrialba magadii (Nmlon) was cloned and sequenced, and the genetic organization of Nmlon was examined at the transcriptional level. Nmlon encodes a 84 kDa polypeptide with a pI of 4.42 which contains the ATPase, protease and membrane targeting domains of the archaeal-type LonB proteases. Nmlon is part of an operon that encodes membrane proteases and it is transcribed as a polycistronic mRNA in N. magadii cells at different growth stages. Accordingly, NmLon was detected in cell membranes of N. magadii throughout growth by Western blot analysis using specific anti-NmLon antibodies. Interestingly, in electrophoretic mobility shift assays, purified NmLon bound double stranded as well as single stranded DNA in the presence of elevated salt concentrations. This finding shows that DNA-binding is conserved in the LonA and LonB subfamilies and suggests that Lon-DNA interaction may be relevant for its function in haloarchaea.

  2. An aspartic protease of the scabies mite Sarcoptes scabiei is involved in the digestion of host skin and blood macromolecules.

    PubMed

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

    2013-11-01

    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. 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. 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.

  3. 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

  4. Enabling Low Cost Biopharmaceuticals: A Systematic Approach to Delete Proteases from a Well-Known Protein Production Host Trichoderma reesei

    PubMed Central

    Landowski, Christopher P.; Huuskonen, Anne; Wahl, Ramon; Westerholm-Parvinen, Ann; Kanerva, Anne; Hänninen, Anna-Liisa; Salovuori, Noora; Penttilä, Merja; Natunen, Jari; Ostermeier, Christian; Helk, Bernhard; Saarinen, Juhani; Saloheimo, Markku

    2015-01-01

    The filamentous fungus Trichoderma reesei has tremendous capability to secrete proteins. Therefore, it would be an excellent host for producing high levels of therapeutic proteins at low cost. Developing a filamentous fungus to produce sensitive therapeutic proteins requires that protease secretion is drastically reduced. We have identified 13 major secreted proteases that are related to degradation of therapeutic antibodies, interferon alpha 2b, and insulin like growth factor. The major proteases observed were aspartic, glutamic, subtilisin-like, and trypsin-like proteases. The seven most problematic proteases were sequentially removed from a strain to develop it for producing therapeutic proteins. After this the protease activity in the supernatant was dramatically reduced down to 4% of the original level based upon a casein substrate. When antibody was incubated in the six protease deletion strain supernatant, the heavy chain remained fully intact and no degradation products were observed. Interferon alpha 2b and insulin like growth factor were less stable in the same supernatant, but full length proteins remained when incubated overnight, in contrast to the original strain. As additional benefits, the multiple protease deletions have led to faster strain growth and higher levels of total protein in the culture supernatant. PMID:26309247

  5. Enabling Low Cost Biopharmaceuticals: A Systematic Approach to Delete Proteases from a Well-Known Protein Production Host Trichoderma reesei.

    PubMed

    Landowski, Christopher P; Huuskonen, Anne; Wahl, Ramon; Westerholm-Parvinen, Ann; Kanerva, Anne; Hänninen, Anna-Liisa; Salovuori, Noora; Penttilä, Merja; Natunen, Jari; Ostermeier, Christian; Helk, Bernhard; Saarinen, Juhani; Saloheimo, Markku

    2015-01-01

    The filamentous fungus Trichoderma reesei has tremendous capability to secrete proteins. Therefore, it would be an excellent host for producing high levels of therapeutic proteins at low cost. Developing a filamentous fungus to produce sensitive therapeutic proteins requires that protease secretion is drastically reduced. We have identified 13 major secreted proteases that are related to degradation of therapeutic antibodies, interferon alpha 2b, and insulin like growth factor. The major proteases observed were aspartic, glutamic, subtilisin-like, and trypsin-like proteases. The seven most problematic proteases were sequentially removed from a strain to develop it for producing therapeutic proteins. After this the protease activity in the supernatant was dramatically reduced down to 4% of the original level based upon a casein substrate. When antibody was incubated in the six protease deletion strain supernatant, the heavy chain remained fully intact and no degradation products were observed. Interferon alpha 2b and insulin like growth factor were less stable in the same supernatant, but full length proteins remained when incubated overnight, in contrast to the original strain. As additional benefits, the multiple protease deletions have led to faster strain growth and higher levels of total protein in the culture supernatant.

  6. Induction of human host cell apoptosis by Trichomonas vaginalis cysteine proteases is modulated by parasite exposure to iron.

    PubMed

    Kummer, Shelley; Hayes, Gary R; Gilbert, Robert O; Beach, David H; Lucas, John J; Singh, Bibhuti N

    2008-03-01

    Trichomonas vaginalis is an understudied parasitic organism whose mechanisms of pathogenesis remain unclear. The adherence to host cells, the induction of host cell cytotoxicity and protease activity are all, however, thought to be contributing factors towards the development of the disease. T. vaginalis CP30 is an extracellular fraction containing four cysteine proteases, CP2, CP3, CP4 and CPT that induce apoptosis in primary human vaginal epithelial cells (HVECs) [Sommer U, Costello CE, Hayes GR, Beach DH, Gilbert RO, Lucas JJ, Singh BN. Identification of Trichomonas vaginalis cysteine proteases that induce apoptosis in human vaginal epithelial cells. J Biol Chem 2005; 280: 23853-60]. We now show that CP30, and the induction of HVEC apoptosis are modulated by iron availability in the parasite growth medium. Growth of parasites under high iron conditions results in a decrease in levels of CP30 found in an extracellular soluble fraction (SF), a concomitant decline in protease activity, and a decreased ability of SF to induce host cell death. Conversely, iron restriction leads to an increase in CP30 levels, an increase in CP30 protease activity, and an increased ability to induce HVEC cell death. Iron-loaded lactoferrin, but not transferrin is an effective iron source for parasites. We hypothesize that CP30 induction of host cell apoptosis is crucial for the development of a persistent infection, and that iron plays a determining role in parasite pathogenesis.

  7. Regulation of cuticle-degrading subtilisin proteases from the entomopathogenic fungi, Lecanicillium spp: implications for host specificity.

    PubMed

    Bye, Natasha J; Charnley, A Keith

    2008-01-01

    The ability to produce cuticle-degrading proteases to facilitate host penetration does not distinguish per se entomopathogenic fungi from saprophytes. However, adapted pathogens may produce host-protein specific enzymes in response to cues. This possibility prompted an investigation of the regulation of isoforms of the subtilisin Pr1-like proteases from five aphid-pathogenic isolates of Lecanicillium spp. Significant differences were found in substrate specificity and regulation of Pr1-like proteases between isoforms of the same isolate and between different isolates. For example, the pI 8.6 isoform from KV71 was considerably more active against aphid than locust cuticle and was induced specifically by N-acetylglucosamine (NAG). Isoform pI 9.1 from the same isolate was only produced on insect cuticle while most other isoforms were more prominent on chitin containing substrates but not induced by NAG. The ability to regulate isoforms independently may allow production at critical points in host penetration. Appearance of proteases (not subtilisins) with pI 4.2 and 4.4 only on aphid cuticle was a possible link with host specificity of KV71. The absence of C or N metabolite repression in subtilisins from KV42 is unusual for pathogen proteases and may help to account for differences in virulence strategy between aphid-pathogenic isolates of Lecanicillium longisporum (unpublished data).

  8. Identification of a Novel Host-Specific IgM Protease in Streptococcus suis

    PubMed Central

    Seele, Jana; Singpiel, Alena; Spoerry, Christian; von Pawel-Rammingen, Ulrich; Valentin-Weigand, Peter

    2013-01-01

    Streptococcus suis serotype 2 is a highly invasive, extracellular pathogen in pigs with the capacity to cause severe infections in humans. This study was initiated by the finding that IgM degradation products are released after opsonization of S. suis. The objective of this work was to identify the bacterial factor responsible for IgM degradation. The results of this study showed that a member of the IdeS family, designated IdeSsuis (Immunoglobulin M-degrading enzyme of S. suis), is responsible and sufficient for IgM cleavage. Recombinant IdeSsuis was found to degrade only IgM but neither IgG nor IgA. Interestingly, Western blot analysis revealed that IdeSsuis is host specific, as it exclusively cleaves porcine IgM but not IgM from six other species, including a closely related member of the Suidae family. As demonstrated by flow cytometry and immunofluorescence microscopy, IdeSsuis modulates binding of IgM to the bacterial surface. IdeSsuis is the first prokaryotic IgM-specific protease described, indicating that this enzyme is involved in a so-far-unknown mechanism of host-pathogen interaction at an early stage of the host immune response. Furthermore, cleavage of porcine IgM by IdeSsuis is the first identified phenotype reflecting functional adaptation of S. suis to pigs as the main host. PMID:23243300

  9. The Pla Protease of Yersinia pestis Degrades Fas Ligand to Manipulate Host Cell Death and Inflammation

    PubMed Central

    Caulfield, Adam J.; Walker, Margaret E.; Gielda, Lindsay M.; Lathem, Wyndham W.

    2014-01-01

    SUMMARY Pneumonic plague is a deadly respiratory disease caused by Yersinia pestis. The bacterial protease Pla contributes to disease progression and manipulation of host immunity, but the mechanisms by which this occurs are largely unknown. Here we show that Pla degrades the apoptotic signaling molecule Fas ligand (FasL) to prevent host cell apoptosis and inflammation. Wild-type Y. pestis, but not a Pla mutant (Δpla), degrades FasL, which results in decreased downstream caspase-3/7 activation and reduced apoptosis. Similarly, lungs of mice challenged with wild-type Y. pestis show reduced levels of FasL and activated caspase-3/7 compared to Δpla infection. Consistent with a role for FasL in regulating immune responses, Δpla infection results in aberrant pro-inflammatory cytokine levels. The loss of FasL or inhibition of caspase activity alters host inflammatory responses and enables enhanced Y. pestis outgrowth in the lungs. Thus, by degrading FasL, Y. pestis manipulates host cell death pathways to facilitate infection. PMID:24721571

  10. Host Plants Indirectly Influence Plant Virus Transmission by Altering Gut Cysteine Protease Activity of Aphid Vectors.

    PubMed

    Pinheiro, Patricia V; Ghanim, Murad; Alexander, Mariko; Rebelo, Ana Rita; Santos, Rogerio S; Orsburn, Benjamin C; Gray, Stewart; Cilia, Michelle

    2017-04-01

    The green peach aphid, Myzus persicae, is a vector of the Potato leafroll virus (PLRV, Luteoviridae), transmitted exclusively by aphids in a circulative manner. PLRV transmission efficiency was significantly reduced when a clonal lineage of M. persicae was reared on turnip as compared with the weed physalis, and this was a transient effect caused by a host-switch response. A trend of higher PLRV titer in physalis-reared aphids as compared with turnip-reared aphids was observed at 24 h and 72 h after virus acquisition. The major difference in the proteomes of these aphids was the up-regulation of predicted lysosomal enzymes, in particular the cysteine protease cathepsin B (cathB), in aphids reared on turnip. The aphid midgut is the site of PLRV acquisition, and cathB and PLRV localization were starkly different in midguts of the aphids reared on the two host plants. In viruliferous aphids that were reared on turnip, there was near complete colocalization of cathB and PLRV at the cell membranes, which was not observed in physalis-reared aphids. Chemical inhibition of cathB restored the ability of aphids reared on turnip to transmit PLRV in a dose-dependent manner, showing that the increased activity of cathB and other cysteine proteases at the cell membrane indirectly decreased virus transmission by aphids. Understanding how the host plant influences virus transmission by aphids is critical for growers to manage the spread of virus among field crops. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Targeted Deletion of a Plasmodium Site-2 Protease Impairs Life Cycle Progression in the Mammalian Host

    PubMed Central

    Goulielmaki, Evi; Chalari, Anna; Withers-Martinez, Chrislaine; Siden-Kiamos, Inga; Matuschewski, Kai

    2017-01-01

    Site-2 proteases (S2P) belong to the M50 family of metalloproteases, which typically perform essential roles by mediating activation of membrane–bound transcription factors through regulated intramembrane proteolysis (RIP). Protease-dependent liberation of dormant transcription factors triggers diverse cellular responses, such as sterol regulation, Notch signalling and the unfolded protein response. Plasmodium parasites rely on regulated proteolysis for controlling essential pathways throughout the life cycle. In this study we examine the Plasmodium-encoded S2P in a murine malaria model and show that it is expressed in all stages of Plasmodium development. Localisation studies by endogenous gene tagging revealed that in all invasive stages the protein is in close proximity to the nucleus. Ablation of PbS2P by reverse genetics leads to reduced growth rates during liver and blood infection and, hence, virulence attenuation. Strikingly, absence of PbS2P was compatible with parasite life cycle progression in the mosquito and mammalian hosts under physiological conditions, suggesting redundant or dispensable roles in vivo. PMID:28107409

  12. Targeted Deletion of a Plasmodium Site-2 Protease Impairs Life Cycle Progression in the Mammalian Host.

    PubMed

    Koussis, Konstantinos; Goulielmaki, Evi; Chalari, Anna; Withers-Martinez, Chrislaine; Siden-Kiamos, Inga; Matuschewski, Kai; Loukeris, Thanasis G

    2017-01-01

    Site-2 proteases (S2P) belong to the M50 family of metalloproteases, which typically perform essential roles by mediating activation of membrane-bound transcription factors through regulated intramembrane proteolysis (RIP). Protease-dependent liberation of dormant transcription factors triggers diverse cellular responses, such as sterol regulation, Notch signalling and the unfolded protein response. Plasmodium parasites rely on regulated proteolysis for controlling essential pathways throughout the life cycle. In this study we examine the Plasmodium-encoded S2P in a murine malaria model and show that it is expressed in all stages of Plasmodium development. Localisation studies by endogenous gene tagging revealed that in all invasive stages the protein is in close proximity to the nucleus. Ablation of PbS2P by reverse genetics leads to reduced growth rates during liver and blood infection and, hence, virulence attenuation. Strikingly, absence of PbS2P was compatible with parasite life cycle progression in the mosquito and mammalian hosts under physiological conditions, suggesting redundant or dispensable roles in vivo.

  13. 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

  14. Proteolytic Activation of the Essential Parasitophorous Vacuole Cysteine Protease SERA6 Accompanies Malaria Parasite Egress from Its Host Erythrocyte*

    PubMed Central

    Ruecker, Andrea; Shea, Michael; Hackett, Fiona; Suarez, Catherine; Hirst, Elizabeth M. A.; Milutinovic, Katarina; Withers-Martinez, Chrislaine; Blackman, Michael J.

    2012-01-01

    The malaria parasite replicates within an intraerythrocytic parasitophorous vacuole (PV). The PV and host cell membranes eventually rupture, releasing merozoites in a process called egress. Certain inhibitors of serine and cysteine proteases block egress, indicating a crucial role for proteases. The Plasmodium falciparum genome encodes nine serine-repeat antigens (SERAs), each of which contains a central domain homologous to the papain-like (clan CA, family C1) protease family. SERA5 and SERA6 are indispensable in blood-stage parasites, but the function of neither is known. Here we show that SERA6 localizes to the PV where it is precisely cleaved just prior to egress by an essential serine protease called PfSUB1. Mutations that replace the predicted catalytic Cys of SERA6, or that block SERA6 processing by PfSUB1, could not be stably introduced into the parasite genomic sera6 locus, indicating that SERA6 is an essential enzyme and that processing is important for its function. We demonstrate that cleavage of SERA6 by PfSUB1 converts it to an active cysteine protease. Our observations reveal a proteolytic activation step in the malarial PV that may be required for release of the parasite from its host erythrocyte. PMID:22984267

  15. Serine proteases and metalloproteases associated with pathogenesis but not host specificity in the Entomophthoralean fungus Zoophthora radicans.

    PubMed

    Xu, J; Baldwin, D; Kindrachuk, C; Hegedus, D D

    2006-06-01

    The protease activity of a Zoophthora radicans strain that was highly infective toward Pieris brassicae (cabbage butterfly) larvae was compared with that of isogenic strains that were adapted to Plutella xylostella (diamondback moth) larvae through serial passage. All strains produced three distinct serine proteases ranging in size from 25 to 37 kDa; however, the original strain from P. brassicae also produced large amounts of an approximately 46 kDa metalloprotease. Subsequently, a cDNA encoding a 43 kDa (mature enzyme) zinc-dependent metalloprotease, ZrMEP1, was isolated from the original fungal strain and most likely corresponds to the 46 kDa protease observed with in-gel assays. ZrMEP1 possessed characteristics of both the fungalysin and thermolysin metalloprotease families found in some pulmonary and dermal pathogens. This is the first report of this type of metalloprotease from an entomo pathogenic fungus. A cDNA encoding a trypsin-like serine protease, ZrSP1, was also identified and was most similar to a serine protease from the plant pathogen Verticillium dahliae. In artificial media, ZrMEP1 and ZrSP1 were found to be differentially responsive to gelatin and catabolite repression in the fungal strains adapted to P. brassicae and P. xylostella, but their expression patterns within infected larvae were the same. It appears that while these proteases likely play a role in the infection process, they may not be major host specificity determinants.

  16. Discovery of Reactive Microbiota-Derived Metabolites that Inhibit Host Proteases.

    PubMed

    Guo, Chun-Jun; Chang, Fang-Yuan; Wyche, Thomas P; Backus, Keriann M; Acker, Timothy M; Funabashi, Masanori; Taketani, Mao; Donia, Mohamed S; Nayfach, Stephen; Pollard, Katherine S; Craik, Charles S; Cravatt, Benjamin F; Clardy, Jon; Voigt, Christopher A; Fischbach, Michael A

    2017-01-26

    The gut microbiota modulate host biology in numerous ways, but little is known about the molecular mediators of these interactions. Previously, we found a widely distributed family of nonribosomal peptide synthetase gene clusters in gut bacteria. Here, by expressing a subset of these clusters in Escherichia coli or Bacillus subtilis, we show that they encode pyrazinones and dihydropyrazinones. At least one of the 47 clusters is present in 88% of the National Institutes of Health Human Microbiome Project (NIH HMP) stool samples, and they are transcribed under conditions of host colonization. We present evidence that the active form of these molecules is the initially released peptide aldehyde, which bears potent protease inhibitory activity and selectively targets a subset of cathepsins in human cell proteomes. Our findings show that an approach combining bioinformatics, synthetic biology, and heterologous gene cluster expression can rapidly expand our knowledge of the metabolic potential of the microbiota while avoiding the challenges of cultivating fastidious commensals. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Functional and structural characterization of a novel putative cysteine protease cell wall-modifying multi-domain enzyme selected from a microbial metagenome

    PubMed Central

    Faheem, Muhammad; Martins-de-Sa, Diogo; Vidal, Julia F. D.; Álvares, Alice C. M.; Brandão-Neto, José; Bird, Louise E.; Tully, Mark D.; von Delft, Frank; Souto, Betulia M.; Quirino, Betania F.; Freitas, Sonia M.; Barbosa, João Alexandre R. G.

    2016-01-01

    A current metagenomics focus is to interpret and transform collected genomic data into biological information. By combining structural, functional and genomic data we have assessed a novel bacterial protein selected from a carbohydrate-related activity screen in a microbial metagenomic library from Capra hircus (domestic goat) gut. This uncharacterized protein was predicted as a bacterial cell wall-modifying enzyme (CWME) and shown to contain four domains: an N-terminal, a cysteine protease, a peptidoglycan-binding and an SH3 bacterial domain. We successfully cloned, expressed and purified this putative cysteine protease (PCP), which presented autoproteolytic activity and inhibition by protease inhibitors. We observed cell wall hydrolytic activity and ampicillin binding capacity, a characteristic of most bacterial CWME. Fluorimetric binding analysis yielded a Kb of 1.8 × 105 M−1 for ampicillin. Small-angle X-ray scattering (SAXS) showed a maximum particle dimension of 95 Å with a real-space Rg of 28.35 Å. The elongated molecular envelope corroborates the dynamic light scattering (DLS) estimated size. Furthermore, homology modeling and SAXS allowed the construction of a model that explains the stability and secondary structural changes observed by circular dichroism (CD). In short, we report a novel cell wall-modifying autoproteolytic PCP with insight into its biochemical, biophysical and structural features. PMID:27934875

  18. 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.

  19. Predominant role of host proteases in myocardial damage associated with infectious endocarditis induced by Enterococcus faecalis in a rat model.

    PubMed

    Augustin, Pascal; Alsalih, Ghada; Launey, Yoann; Delbosc, Sandrine; Louedec, Liliane; Ollivier, Véronique; Chau, Françoise; Montravers, Philippe; Duval, Xavier; Michel, Jean-Baptiste; Meilhac, Olivier

    2013-05-01

    Infective endocarditis (IE) remains a life-threatening infectious disease with high morbidity and mortality. The objectives of the present study are to assess the host proteolytic activities of the vegetations and their cytotoxic potential in a rat model of experimental IE. Rats were infected with a strain of Enterococcus faecalis of particularly low virulence and weak protease expression. We tested the presence of proteases released by infiltrated leukocytes (matrix metalloproteinases and elastase) or produced in situ within the septic vegetation, such as those linked to the fibrinolytic system (plasmin and plasminogen activators). We also assessed the tissue damage induced by the infective thrombus in vitro and ex vivo. The model of IE was characterized by larger and more extensive vegetations in infected than in nonseptic rats and by an intense neutrophil infiltrate interfacing with the injured underlying tissue. Neutrophil extracellular DNA was shown to trap bacteria and to produce increased levels of cell-free DNA in plasma. Matrix metalloproteinase-9, elastase, and plasminogen activators were increased in septic versus nonseptic vegetations (as shown by zymography and immunohistology). Finally, proteolysis of the extracellular matrix and apoptosis were shown to be associated with host proteases. Bacteria exhibited no detectable proteolytic activity or direct cytotoxic effects. Bacterial membranes/dead bacteria were sufficient to induce leukocyte recruitment and activation that could promote vegetation formation and growth. Our results suggest that, despite the lack of bacterial proteases, the continuous attractant signals coming from bacterial colonies may lead to a chronic and deleterious aggression toward myocardial/valvular tissues by host proteases.

  20. Molecular Mechanisms of Viral and Host Cell Substrate Recognition by Hepatitis C Virus NS3/4A Protease

    SciTech Connect

    Romano, Keith P.; Laine, Jennifer M.; Deveau, Laura M.; Cao, Hong; Massi, Francesca; Schiffer, Celia A.

    2011-08-16

    Hepatitis C NS3/4A protease is a prime therapeutic target that is responsible for cleaving the viral polyprotein at junctions 3-4A, 4A4B, 4B5A, and 5A5B and two host cell adaptor proteins of the innate immune response, TRIF and MAVS. In this study, NS3/4A crystal structures of both host cell cleavage sites were determined and compared to the crystal structures of viral substrates. Two distinct protease conformations were observed and correlated with substrate specificity: (i) 3-4A, 4A4B, 5A5B, and MAVS, which are processed more efficiently by the protease, form extensive electrostatic networks when in complex with the protease, and (ii) TRIF and 4B5A, which contain polyproline motifs in their full-length sequences, do not form electrostatic networks in their crystal complexes. These findings provide mechanistic insights into NS3/4A substrate recognition, which may assist in a more rational approach to inhibitor design in the face of the rapid acquisition of resistance.

  1. Angiostrongylus cantonensis cathepsin B-like protease (Ac-cathB-1) is involved in host gut penetration

    PubMed Central

    Long, Ying; Cao, Binbin; Yu, Liang; Tukayo, Meks; Feng, Chonglv; Wang, Yinan; Luo, Damin

    2015-01-01

    Although the global spread of the emerging zoonosis, human angiostrongyliasis, has attracted increasing attention, understanding of specific gene function has been impeded by the inaccessibility of genetic manipulation of the pathogen nematode causing this disease, Angiostrongylus cantonensis. Many parasitic proteases play key roles in host-parasite interactions, but those of A. cantonensis are always expressed as the inactive form in prokaryotic expression systems, thereby impeding functional studies. Hence, a lentiviral system that drives secreted expression of target genes fused to a Myc-His tag was used to obtain recombinant Ac-cathB-1 with biological activity. Although this class of proteases was always reported to function in nutrition and immune evasion in parasitic nematodes, recombinant Ac-cathB-1 was capable of hydrolysis of fibronectin and laminin as well as the extracellular matrix of IEC-6 monolayer, so that the intercellular space of the IEC-6 monolayer increased 5.15 times as compared to the control, while the shape of the adherent cells partly rounded up. This suggests a probable role for this protease in intestinal epithelial penetration. The inhibition of Ac-cathB-1 enzymatic activity with antiserum partly suppressed larval penetration ability in the isolated intestine. Thus, an effective system for heterologous expression of parasite proteases is presented for studying gene function in A. cantonensis; and Ac-cathB-1 was related to larval penetration ability in the host small intestine. PMID:26682577

  2. Further characterization of a putative serine protease contributing to the γ-secretase cleavage of β-amyloid precursor protein.

    PubMed

    Peuchmaur, Marine; Lacour, Marie-Agnès; Sévalle, Jean; Lisowski, Vincent; Touati-Jallabe, Youness; Rodier, Fabien; Martinez, Jean; Checler, Frédéric; Hernandez, Jean-François

    2013-02-15

    The 3-alkoxy-7-amino-4-chloro-isocoumarins JLK-6 and JLK-2 have been shown to markedly reduce the production of Amyloid β-peptide (Aβ) by Amyloid-β Precursor Protein (APP) expressing HEK293 cells by affecting the γ-secretase cleavage of APP, with no effect on the cleavage of the Notch receptor. This suggested that these compounds do not directly inhibit the presenilin-dependent γ-secretase complex but more likely interfere with an upstream target involved in γ-secretase-associated pathway. The mechanism of action of these compounds is unknown and there are high fundamental and therapeutical interests to unravel their target. Isocoumarin compounds were previously shown to behave as potent mechanism-based irreversible inhibitors of serine proteases, suggesting that the JLK-directed target could belong to such enzyme family. To get further insight into structure-activity relationships and to develop more potent isocoumarin derivatives, we have synthesized and evaluated a series of isocoumarin analogues with modifications at positions 3, 4 and 7. In particular, the 7-amino group was substituted with either acyl, urethane, alkyl or aryl groups, which could represent additional interaction sites. Altogether, the results highlighted the essential integrity of the 3-alkoxy-7-amino-4-chloro-isocoumarin scaffold for Aβ-lowering activity and supported the involvement of a serine protease, or may be more generally, a serine hydrolase. The newly reported 7-N-alkyl series produced the most active compounds with an IC(50) between 10 and 30μM. Finally, we also explored peptide boronates, a series of reversible serine protease inhibitors, previously shown to also lower cellular Aβ production. The presented data suggested they could act on the same target or interfere with the same pathway as isocoumarins derivatives.

  3. Fundamental Roles of the Golgi-Associated Toxoplasma Aspartyl Protease, ASP5, at the Host-Parasite Interface.

    PubMed

    Hammoudi, Pierre-Mehdi; Jacot, Damien; Mueller, Christina; Di Cristina, Manlio; Dogga, Sunil Kumar; Marq, Jean-Baptiste; Romano, Julia; Tosetti, Nicolò; Dubrot, Juan; Emre, Yalin; Lunghi, Matteo; Coppens, Isabelle; Yamamoto, Masahiro; Sojka, Daniel; Pino, Paco; Soldati-Favre, Dominique

    2015-10-01

    Toxoplasma gondii possesses sets of dense granule proteins (GRAs) that either assemble at, or cross the parasitophorous vacuole membrane (PVM) and exhibit motifs resembling the HT/PEXEL previously identified in a repertoire of exported Plasmodium proteins. Within Plasmodium spp., cleavage of the HT/PEXEL motif by the endoplasmic reticulum-resident protease Plasmepsin V precedes trafficking to and export across the PVM of proteins involved in pathogenicity and host cell remodelling. Here, we have functionally characterized the T. gondii aspartyl protease 5 (ASP5), a Golgi-resident protease that is phylogenetically related to Plasmepsin V. We show that deletion of ASP5 causes a significant loss in parasite fitness in vitro and an altered virulence in vivo. Furthermore, we reveal that ASP5 is necessary for the cleavage of GRA16, GRA19 and GRA20 at the PEXEL-like motif. In the absence of ASP5, the intravacuolar nanotubular network disappears and several GRAs fail to localize to the PVM, while GRA16 and GRA24, both known to be targeted to the host cell nucleus, are retained within the vacuolar space. Additionally, hypermigration of dendritic cells and bradyzoite cyst wall formation are impaired, critically impacting on parasite dissemination and persistence. Overall, the absence of ASP5 dramatically compromises the parasite's ability to modulate host signalling pathways and immune responses.

  4. Fundamental Roles of the Golgi-Associated Toxoplasma Aspartyl Protease, ASP5, at the Host-Parasite Interface

    PubMed Central

    Hammoudi, Pierre-Mehdi; Jacot, Damien; Mueller, Christina; Di Cristina, Manlio; Dogga, Sunil Kumar; Marq, Jean-Baptiste; Romano, Julia; Tosetti, Nicolò; Dubrot, Juan; Emre, Yalin; Lunghi, Matteo; Coppens, Isabelle; Yamamoto, Masahiro; Sojka, Daniel; Pino, Paco; Soldati-Favre, Dominique

    2015-01-01

    Toxoplasma gondii possesses sets of dense granule proteins (GRAs) that either assemble at, or cross the parasitophorous vacuole membrane (PVM) and exhibit motifs resembling the HT/PEXEL previously identified in a repertoire of exported Plasmodium proteins. Within Plasmodium spp., cleavage of the HT/PEXEL motif by the endoplasmic reticulum-resident protease Plasmepsin V precedes trafficking to and export across the PVM of proteins involved in pathogenicity and host cell remodelling. Here, we have functionally characterized the T. gondii aspartyl protease 5 (ASP5), a Golgi-resident protease that is phylogenetically related to Plasmepsin V. We show that deletion of ASP5 causes a significant loss in parasite fitness in vitro and an altered virulence in vivo. Furthermore, we reveal that ASP5 is necessary for the cleavage of GRA16, GRA19 and GRA20 at the PEXEL-like motif. In the absence of ASP5, the intravacuolar nanotubular network disappears and several GRAs fail to localize to the PVM, while GRA16 and GRA24, both known to be targeted to the host cell nucleus, are retained within the vacuolar space. Additionally, hypermigration of dendritic cells and bradyzoite cyst wall formation are impaired, critically impacting on parasite dissemination and persistence. Overall, the absence of ASP5 dramatically compromises the parasite’s ability to modulate host signalling pathways and immune responses. PMID:26473595

  5. A higher plant mitochondrial homologue of the yeast m-AAA protease. Molecular cloning, localization, and putative function.

    PubMed

    Kolodziejczak, Marta; Kolaczkowska, Anna; Szczesny, Bartosz; Urantowka, Adam; Knorpp, Carina; Kieleczawa, Jan; Janska, Hanna

    2002-11-15

    Mitochondrial AAA metalloproteases play a fundamental role in mitochondrial biogenesis and function. They have been identified in yeast and animals but not yet in plants. This work describes the isolation and sequence analysis of the full-length cDNA from the pea (Pisum sativum) with significant homology to the yeast matrix AAA (m-AAA) protease. The product of this clone was imported into isolated pea mitochondria where it was processed to its mature form (PsFtsH). We have shown that the central region of PsFtsH containing the chaperone domain is exposed to the matrix space. Furthermore, we have demonstrated that the pea protease can complement respiration deficiency in the yta10 and/or yta12 null yeast mutants, indicating that the plant protein can compensate for the loss of at least some of the important m-AAA functions in yeast. Based on biochemical experiments using isolated pea mitochondria, we propose that PsFtsH-like m-AAA is involved in the accumulation of the subunit 9 of the ATP synthase in the mitochondrial membrane.

  6. Identification of homologues to the pathogenicity factor Pat-1, a putative serine protease of Clavibacter michiganensis subsp. michiganensis.

    PubMed

    Burger, Annette; Gräfen, Ines; Engemann, Jutta; Niermann, Erik; Pieper, Martina; Kirchner, Oliver; Gartemann, Karl-Heinz; Eichenlaub, Rudolf

    2005-01-01

    Hybridization of Clavibacter michiganensis subsp. michiganensis total DNA against the pathogenicity gene pat-1 indicated the presence of pat-1 homologous nucleotide sequences on the chromosome and on plasmid pCM2. Isolation of the corresponding DNA fragments and nucleotide sequence determination showed that there are three pat-1 homologous genes: chpA (chromosome) and phpA and phpB (plasmid pCM2). The gene products share common characteristics, i.e. a signal sequence for Sec-dependent secretion, a serine protease motif, and six cysteine residues at conserved positions. Gene chpA located on the chromosome is a pseudogene since it contains a translational stop codon after 97 of 280 amino acids. In contrast to pat-1, cloning of the plasmid encoded homologs phpA and phpB into the avirulent plasmid free Cmm strain CMM100 did not result in a virulent phenotype. So far, no proteolytic activity could be demonstrated for Pat-1, however, site specific mutagenesis of pat-1 showed that the serine residue in the motif GDSGG is required for the virulent phenotype of pat-1 and thus Pat-1 could be a functional protease.

  7. The Serine Protease Autotransporter Pic Modulates Citrobacter rodentium Pathogenesis and Its Innate Recognition by the Host.

    PubMed

    Bhullar, Kirandeep; Zarepour, Maryam; Yu, Hongbing; Yang, Hong; Croxen, Matthew; Stahl, Martin; Finlay, B Brett; Turvey, Stuart E; Vallance, Bruce A

    2015-07-01

    Bacterial pathogens produce a number of autotransporters that possess diverse functions. These include the family of serine protease autotransporters of Enterobacteriaceae (SPATEs) produced by enteric pathogens such as Shigella flexneri and enteroaggregative Escherichia coli. Of these SPATEs, one termed "protein involved in colonization," or Pic, has been shown to possess mucinase activity in vitro, but to date, its role in in vivo enteric pathogenesis is unknown. Testing a pic null (ΔpicC) mutant in Citrobacter rodentium, a natural mouse pathogen, found that the C. rodentium ΔpicC strain was impaired in its ability to degrade mucin in vitro compared to the wild type. Upon infection of mice, the ΔpicC mutant exhibited a hypervirulent phenotype with dramatically heavier pathogen burdens found in intestinal crypts. ΔpicC mutant-infected mice suffered greater barrier disruption and more severe colitis and weight loss, necessitating their euthanization between 10 and 14 days postinfection. Notably, the virulence of the ΔpicC mutant was normalized when the picC gene was restored; however, a PicC point mutant causing loss of mucinase activity did not replicate the ΔpicC phenotype. Exploring other aspects of PicC function, the ΔpicC mutant was found to aggregate to higher levels in vivo than wild-type C. rodentium. Moreover, unlike the wild type, the C. rodentium ΔpicC mutant had a red, dry, and rough (RDAR) morphology in vitro and showed increased activation of the innate receptor Toll-like receptor 2 (TLR2). Interestingly, the C. rodentium ΔpicC mutant caused a degree of pathology similar to that of wild-type C. rodentium when infecting TLR2-deficient mice, showing that despite its mucinase activity, PicC's major role in vivo may be to limit C. rodentium's stimulation of the host's innate immune system.

  8. The Serine Protease Autotransporter Pic Modulates Citrobacter rodentium Pathogenesis and Its Innate Recognition by the Host

    PubMed Central

    Bhullar, Kirandeep; Zarepour, Maryam; Yu, Hongbing; Yang, Hong; Croxen, Matthew; Stahl, Martin; Finlay, B. Brett; Turvey, Stuart E.

    2015-01-01

    Bacterial pathogens produce a number of autotransporters that possess diverse functions. These include the family of serine protease autotransporters of Enterobacteriaceae (SPATEs) produced by enteric pathogens such as Shigella flexneri and enteroaggregative Escherichia coli. Of these SPATEs, one termed “protein involved in colonization,” or Pic, has been shown to possess mucinase activity in vitro, but to date, its role in in vivo enteric pathogenesis is unknown. Testing a pic null (ΔpicC) mutant in Citrobacter rodentium, a natural mouse pathogen, found that the C. rodentium ΔpicC strain was impaired in its ability to degrade mucin in vitro compared to the wild type. Upon infection of mice, the ΔpicC mutant exhibited a hypervirulent phenotype with dramatically heavier pathogen burdens found in intestinal crypts. ΔpicC mutant-infected mice suffered greater barrier disruption and more severe colitis and weight loss, necessitating their euthanization between 10 and 14 days postinfection. Notably, the virulence of the ΔpicC mutant was normalized when the picC gene was restored; however, a PicC point mutant causing loss of mucinase activity did not replicate the ΔpicC phenotype. Exploring other aspects of PicC function, the ΔpicC mutant was found to aggregate to higher levels in vivo than wild-type C. rodentium. Moreover, unlike the wild type, the C. rodentium ΔpicC mutant had a red, dry, and rough (RDAR) morphology in vitro and showed increased activation of the innate receptor Toll-like receptor 2 (TLR2). Interestingly, the C. rodentium ΔpicC mutant caused a degree of pathology similar to that of wild-type C. rodentium when infecting TLR2-deficient mice, showing that despite its mucinase activity, PicC's major role in vivo may be to limit C. rodentium's stimulation of the host's innate immune system. PMID:25895966

  9. Putative Bioalteration Textures Hosted Within Impact Melt Glasses From the Ries Crater, Germany

    NASA Astrophysics Data System (ADS)

    Sapers, H. M.; Osinski, G. R.; Banerjee, N. R.

    2009-05-01

    Impact cratering is a ubiquitous geological process on solid bodies. Any hypervelocity impact into a H2O- rich target has the potential to generate hydrothermal systems [1]. Recent research has suggested that such impact-induced environments may be conducive to microbial colonization [e.g., 2]. Bioalteration of terrestrial basaltic glasses produces characteristic tubular and granular aggregate textures. Such bioalteration textures preserved in Archean greenstone belts constitute one of the oldest records of life on Earth [3]. Our examination of glasses from the Ries crater in Germany has revealed tubular textures with remarkably similar morphologies to those seen in volcanic glasses. The hyperthermophilic root of the 16S phylogenic tree of life suggests an essential role for thermophilic environments in the origin or the early evolutionary history of life on Earth. Previous work has associated primitive life on Earth with submarine volcanic activity suggesting that submarine hydrothermal settings may have played an essential role in the origin of life [e.g., 4]. Impact-induced hydrothermal systems share many characteristics with submarine volcanic hydrothermal systems including the presence of chemical and thermal energy for microbial metabolism. Interestingly, the Late Heavy Bombardment period, during which life purportedly arose on Earth, was characterized by a high impact flux. Thus, impact-generated habitats were likely much more common on Earth than submarine hydrothermal systems suggesting the former as a more statistically probable habitat for the origin of life. Here we present preliminary data characterizing the putative bioalteration structures hosted within the Ries impact glasses. Establishing the biogenecity of the alteration structures observed in these glasses may have significant astrobiological implications: impact glasses share many similarities with volcanic glasses, however, fundamental differences make impact glasses unique geochemical systems

  10. Transcript Expression Analysis of Putative Trypanosoma brucei GPI-Anchored Surface Proteins during Development in the Tsetse and Mammalian Hosts

    PubMed Central

    Savage, Amy F.; Cerqueira, Gustavo C.; Regmi, Sandesh; Wu, Yineng; El Sayed, Najib M.; Aksoy, Serap

    2012-01-01

    Human African Trypanosomiasis is a devastating disease caused by the parasite Trypanosoma brucei. Trypanosomes live extracellularly in both the tsetse fly and the mammal. Trypanosome surface proteins can directly interact with the host environment, allowing parasites to effectively establish and maintain infections. Glycosylphosphatidylinositol (GPI) anchoring is a common posttranslational modification associated with eukaryotic surface proteins. In T. brucei, three GPI-anchored major surface proteins have been identified: variant surface glycoproteins (VSGs), procyclic acidic repetitive protein (PARP or procyclins), and brucei alanine rich proteins (BARP). The objective of this study was to select genes encoding predicted GPI-anchored proteins with unknown function(s) from the T. brucei genome and characterize the expression profile of a subset during cyclical development in the tsetse and mammalian hosts. An initial in silico screen of putative T. brucei proteins by Big PI algorithm identified 163 predicted GPI-anchored proteins, 106 of which had no known functions. Application of a second GPI-anchor prediction algorithm (FragAnchor), signal peptide and trans-membrane domain prediction software resulted in the identification of 25 putative hypothetical proteins. Eighty-one gene products with hypothetical functions were analyzed for stage-regulated expression using semi-quantitative RT-PCR. The expression of most of these genes were found to be upregulated in trypanosomes infecting tsetse salivary gland and proventriculus tissues, and 38% were specifically expressed only by parasites infecting salivary gland tissues. Transcripts for all of the genes specifically expressed in salivary glands were also detected in mammalian infective metacyclic trypomastigotes, suggesting a possible role for these putative proteins in invasion and/or establishment processes in the mammalian host. These results represent the first large-scale report of the differential expression of

  11. Comparative Genomics of the Zoonotic Pathogen Ehrlichia chaffeensis Reveals Candidate Type IV Effectors and Putative Host Cell Targets

    PubMed Central

    Noroy, Christophe; Meyer, Damien F.

    2017-01-01

    During infection, some intracellular pathogenic bacteria use a dedicated multiprotein complex known as the type IV secretion system to deliver type IV effector (T4E) proteins inside the host cell. These T4Es allow the bacteria to evade host defenses and to subvert host cell processes to their own advantage. Ehrlichia chaffeensis is a tick-transmitted obligate intracellular pathogenic bacterium, which causes human monocytic ehrlichiosis. Using comparative whole genome analysis, we identified the relationship between eight available E. chaffeensis genomes isolated from humans and show that these genomes are highly conserved. We identified the candidate core type IV effectome of E. chaffeensis and some conserved intracellular adaptive strategies. We assigned the West Paces strain to genetic group II and predicted the repertoires of T4Es encoded by E. chaffeensis genomes, as well as some putative host cell targets. We demonstrated that predicted T4Es are preferentially distributed in gene sparse regions of the genome. In addition to the identification of the two known type IV effectors of Anaplasmataceae, we identified two novel candidates T4Es, ECHLIB_RS02720 and ECHLIB_RS04640, which are not present in all E. chaffeensis strains and could explain some variations in inter-strain virulence. We also identified another novel candidate T4E, ECHLIB_RS02720, a hypothetical protein exhibiting EPIYA, and NLS domains as well as a classical type IV secretion signal, suggesting an important role inside the host cell. Overall, our results agree with current knowledge of Ehrlichia molecular pathogenesis, and reveal novel candidate T4Es that require experimental validation. This work demonstrates that comparative effectomics enables identification of important host pathways targeted by the bacterial pathogen. Our study, which focuses on the type IV effector repertoires among several strains of E. chaffeensis species, is an original approach and provides rational putative targets

  12. Identification and Characterization of a Novel Aspergillus fumigatus Rhomboid Family Putative Protease, RbdA, Involved in Hypoxia Sensing and Virulence

    PubMed Central

    Vaknin, Yakir; Hillmann, Falk; Iannitti, Rossana; Ben Baruch, Netali; Sandovsky-Losica, Hana; Shadkchan, Yona; Romani, Luigina; Brakhage, Axel

    2016-01-01

    Aspergillus fumigatus is the most common pathogenic mold infecting humans and a significant cause of morbidity and mortality in immunocompromised patients. In invasive pulmonary aspergillosis, A. fumigatus spores are inhaled into the lungs, undergoing germination and invasive hyphal growth. The fungus occludes and disrupts the blood vessels, leading to hypoxia and eventual tissue necrosis. The ability of this mold to adapt to hypoxia is regulated in part by the sterol regulatory element binding protein (SREBP) SrbA and the DscA to DscD Golgi E3 ligase complex critical for SREBP activation by proteolytic cleavage. Loss of the genes encoding these proteins results in avirulence. To identify novel regulators of hypoxia sensing, we screened the Neurospora crassa gene deletion library under hypoxia and identified a novel rhomboid family protease essential for hypoxic growth. Deletion of the A. fumigatus rhomboid homolog rbdA resulted in an inability to grow under hypoxia, hypersensitivity to CoCl2, nikkomycin Z, fluconazole, and ferrozine, abnormal swollen tip morphology, and transcriptional dysregulation—accurately phenocopying deletion of srbA. In vivo, rbdA deletion resulted in increased sensitivity to phagocytic killing, a reduced inflammatory Th1 and Th17 response, and strongly attenuated virulence. Phenotypic rescue of the ΔrbdA mutant was achieved by expression and nuclear localization of the N terminus of SrbA, including its HLH domain, further indicating that RbdA and SrbA act in the same signaling pathway. In summary, we have identified RbdA, a novel putative rhomboid family protease in A. fumigatus that mediates hypoxia adaptation and fungal virulence and that is likely linked to SrbA cleavage and activation. PMID:27068092

  13. The weed Sorghum almum is a putative alternative host of sugarcane infecting viruses in Florida

    USDA-ARS?s Scientific Manuscript database

    Sorghum almum or Columbus grass is a common weed growing in the Everglades Agricultural Area (EAA). In recent surveys for alternative hosts of sugarcane yellow leaf virus (SCYLV), 123 out of 141 (87%) plants of S. almum tested positive for this virus by tissue blot immunoassay (TBIA) using polyclona...

  14. Basidiome formation of an edible wild, putatively ectomycorrhizal fungus, Phlebopus portentosus without host plant.

    PubMed

    Kumla, Jaturong; Bussaban, Boonsom; Suwannarach, Nakarin; Lumyong, Saisamorn; Danell, Eric

    2012-01-01

    Phlebopus portentosus is a popular wild edible ectomycorrhizal fungus in northern Thailand. In general ectomycorrhizal fungi produce basidiomes when associated with a host plant. In this paper mycelium growth and basidiome production of P. portentosus were examined in pure culture both in vitro and in pot-culture experiments. Five mycelial strains of P. portentosus were isolated from basidiomes and used in the experiments. The mycelia grew fastest on sorghum grains supplemented with fungal-host solution. The mycelia produced sclerotia-like structures after 3 wk incubation in darkness at 30 C. All strains of P. portentosus had the ability to form primordia. The primordia were formed under lowered temperature, high humidity and a 12 h photo-period. They developed to mature basidiomes after 8-12 d in in vitro. In the pot-culture primordia were found after 28-35 d incubation in the greenhouse and mature basidiomes released basidiospores within 6-8 d. Basidiospores were germinated on fungal-host medium and formed mycelial colonies. This fungus showed an ability to produce basidiomes even 2 y after the original isolation from tissues. This research provides valuable information concerning the techniques and protocols for the large scale commercial production of P. portentosus basidiomes in the absence of a host plant.

  15. Effects of Host Sex, Plant Species, and Putative Host Species on the Prevalence of Wolbachia in Natural Populations of Bemisia tabaci (Hemiptera: Aleyrodidae): A Modified Nested PCR Study.

    PubMed

    Ji, Han-Le; Qi, Lan-Da; Hong, Xiao-Yue; Xie, Hong-Fang; Li, Yuan-Xi

    2015-02-01

    Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) is a globally distributed pest. One of the key endosymbionts in B. tabaci is Wolbachia, an α-proteobacterium implicated in many important biological processes. Previous studies indicated that the infection frequency of Wolbachia in Middle East-Asia Minor 1 (MEAM1) and Mediterranean (MED) varied greatly among populations in different areas. However, little is known about the factors that influence the prevalence of Wolbachia in B. tabaci. In this paper, 25 field populations were collected from different locations in China, and 1,161 individuals were screened for the presence of Wolbachia using a nested polymerase chain reaction (PCR)-based method, which targets the wsp gene, to confirm Wolbachia infection status. The prevalence of Wolbachia ranged from 1.54 to 66.67% within the 25 field populations, and the infection frequency of Wolbachia was affected significantly by the putative species of B. tabaci. The infection frequency (51.55%) of Wolbachia was significantly greater in native species than in the MED (25.65%) and MEAM1 (14.37%). With the exception of host plant, all factors, including putative species, geographic location, and the sex of the host, affected the Wolbachia infection frequency in whiteflies. Six Wolbachia strains were found and clustered into four distinct clades upon phylogenetic analyses. Furthermore, Wolbachia in B. tabaci have close relationships with those from other host species, including Liriomyza trifolii (Burgess), Sogatella furcifera (Horvath), Nilaparvata lugens (Stål), and Culex pipiens L. The results demonstrated the variation and diversity of Wolbachia in B. tabaci field populations, and that the application of nested PCR extended our knowledge of Wolbachia infection in B. tabaci, especially in invasive whiteflies. © The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  16. A Key Role for Plasmodium Subtilisin-like SUB1 Protease in Egress of Malaria Parasites from Host Hepatocytes*

    PubMed Central

    Tawk, Lina; Lacroix, Céline; Gueirard, Pascale; Kent, Robyn; Gorgette, Olivier; Thiberge, Sabine; Mercereau-Puijalon, Odile; Ménard, Robert; Barale, Jean-Christophe

    2013-01-01

    In their mammalian host, Plasmodium parasites have two obligatory intracellular development phases, first in hepatocytes and subsequently in erythrocytes. Both involve an orchestrated process of invasion into and egress from host cells. The Plasmodium SUB1 protease plays a dual role at the blood stage by enabling egress of the progeny merozoites from the infected erythrocyte and priming merozoites for subsequent erythrocyte invasion. Here, using conditional mutagenesis in P. berghei, we show that SUB1 plays an essential role at the hepatic stage. Stage-specific sub1 invalidation during prehepatocytic development showed that SUB1-deficient parasites failed to rupture the parasitophorous vacuole membrane and to egress from hepatocytes. Furthermore, mechanically released parasites were not adequately primed and failed to establish a blood stage infection in vivo. The critical involvement of SUB1 in both pre-erythrocytic and erythrocytic developmental phases qualifies SUB1 as an attractive multistage target for prophylactic and therapeutic anti-Plasmodium intervention strategies. PMID:24089525

  17. Different host cell proteases activate the SARS-coronavirus spike-protein for cell-cell and virus-cell fusion

    PubMed Central

    Simmons, Graham; Bertram, Stephanie; Glowacka, Ilona; Steffen, Imke; Chaipan, Chawaree; Agudelo, Juliet; Lu, Kai; Rennekamp, Andrew J.; Hofmann, Heike; Bates, Paul; Pöhlmann, Stefan

    2011-01-01

    Severe acute respiratory syndrome coronavirus (SARS-CoV) poses a considerable threat to human health. Activation of the viral spike (S)-protein by host cell proteases is essential for viral infectivity. However, the cleavage sites in SARS-S and the protease(s) activating SARS-S are incompletely defined. We found that R667 was dispensable for SARS-S-driven virus-cell fusion and for SARS-S-activation by trypsin and cathepsin L in a virus-virus fusion assay. Mutation T760R, which optimizes the minimal furin consensus motif 758-RXXR-762, and furin overexpression augmented SARS-S-activity, but did not result in detectable SARS-S cleavage. Finally, SARS-S-driven cell-cell fusion was independent of cathepsin L, a protease essential for virus-cell fusion. Instead, a so far unknown leupeptin-sensitive host cell protease activated cellular SARS-S for fusion with target cells expressing high levels of ACE2. Thus, different host cell proteases activate SARS-S for virus-cell and cell-cell fusion and SARS-S cleavage at R667 and 758-RXXR-762 can be dispensable for SARS-S activation. PMID:21435673

  18. Different host cell proteases activate the SARS-coronavirus spike-protein for cell-cell and virus-cell fusion.

    PubMed

    Simmons, Graham; Bertram, Stephanie; Glowacka, Ilona; Steffen, Imke; Chaipan, Chawaree; Agudelo, Juliet; Lu, Kai; Rennekamp, Andrew J; Hofmann, Heike; Bates, Paul; Pöhlmann, Stefan

    2011-05-10

    Severe acute respiratory syndrome coronavirus (SARS-CoV) poses a considerable threat to human health. Activation of the viral spike (S)-protein by host cell proteases is essential for viral infectivity. However, the cleavage sites in SARS-S and the protease(s) activating SARS-S are incompletely defined. We found that R667 was dispensable for SARS-S-driven virus-cell fusion and for SARS-S-activation by trypsin and cathepsin L in a virus-virus fusion assay. Mutation T760R, which optimizes the minimal furin consensus motif 758-RXXR-762, and furin overexpression augmented SARS-S activity, but did not result in detectable SARS-S cleavage. Finally, SARS-S-driven cell-cell fusion was independent of cathepsin L, a protease essential for virus-cell fusion. Instead, a so far unknown leupeptin-sensitive host cell protease activated cellular SARS-S for fusion with target cells expressing high levels of ACE2. Thus, different host cell proteases activate SARS-S for virus-cell and cell-cell fusion and SARS-S cleavage at R667 and 758-RXXR-762 can be dispensable for SARS-S activation. Copyright © 2011 Elsevier Inc. All rights reserved.

  19. Different host cell proteases activate the SARS-coronavirus spike-protein for cell-cell and virus-cell fusion

    SciTech Connect

    Simmons, Graham; Bertram, Stephanie; Glowacka, Ilona; Steffen, Imke; Chaipan, Chawaree; Agudelo, Juliet; Lu Kai; Rennekamp, Andrew J.; Hofmann, Heike; Bates, Paul; Poehlmann, Stefan

    2011-05-10

    Severe acute respiratory syndrome coronavirus (SARS-CoV) poses a considerable threat to human health. Activation of the viral spike (S)-protein by host cell proteases is essential for viral infectivity. However, the cleavage sites in SARS-S and the protease(s) activating SARS-S are incompletely defined. We found that R667 was dispensable for SARS-S-driven virus-cell fusion and for SARS-S-activation by trypsin and cathepsin L in a virus-virus fusion assay. Mutation T760R, which optimizes the minimal furin consensus motif 758-RXXR-762, and furin overexpression augmented SARS-S activity, but did not result in detectable SARS-S cleavage. Finally, SARS-S-driven cell-cell fusion was independent of cathepsin L, a protease essential for virus-cell fusion. Instead, a so far unknown leupeptin-sensitive host cell protease activated cellular SARS-S for fusion with target cells expressing high levels of ACE2. Thus, different host cell proteases activate SARS-S for virus-cell and cell-cell fusion and SARS-S cleavage at R667 and 758-RXXR-762 can be dispensable for SARS-S activation.

  20. Gut microbiota of mice putatively modifies amino acid metabolism in the host brain.

    PubMed

    Kawase, Takahiro; Nagasawa, Mao; Ikeda, Hiromi; Yasuo, Shinobu; Koga, Yasuhiro; Furuse, Mitsuhiro

    2017-03-01

    Recently, it has been found that the gut microbiota influences functions of the host brain by affecting monoamine metabolism. The present study focused on the relationship between the gut microbiota and the brain amino acids. Specific pathogen-free (SPF) and germ-free (GF) mice were used as experimental models. Plasma and brain regions were sampled from mice at 7 and 16 weeks of age, and analysed for free d- and l-amino acids, which are believed to affect many physiological functions. At 7 weeks of age, plasma concentrations of d-aspartic acid (d-Asp), l-alanine (l-Ala), l-glutamine (l-Gln) and taurine were higher in SPF mice than in GF mice, but no differences were found at 16 weeks of age. Similar patterns were observed for the concentrations of l-Asp in striatum, cerebral cortex and hippocampus, and l-arginine (l-Arg), l-Ala and l-valine (l-Val) in striatum. In addition, the concentrations of l-Asp, d-Ala, l-histidine, l-isoleucine (l-Ile), l-leucine (l-Leu), l-phenylalanine and l-Val were significantly higher in plasma of SPF mice when compared with those of GF mice. The concentrations of l-Arg, l-Gln, l-Ile and l-Leu were significantly higher in SPF than in GF mice, but those of d-Asp, d-serine and l-serine were higher in some brain regions of GF mice than in those of SPF mice. In conclusion, the concentration of amino acids in the host brain seems to be dependent on presence of the gut microbiota. Amino acid metabolism in the host brain may be modified by manipulating microbiota communities.

  1. H+ channels in embryonic Biomphalaria glabrata cell membranes: Putative roles in snail host-schistosome interactions

    PubMed Central

    Wright, Brandon J.; Bickham-Wright, Utibe; Yoshino, Timothy P.; Jackson, Meyer B.

    2017-01-01

    The human blood fluke Schistosoma mansoni causes intestinal schistosomiasis, a widespread neglected tropical disease. Infection of freshwater snails Biomphalaria spp. is an essential step in the transmission of S. mansoni to humans, although the physiological interactions between the parasite and its obligate snail host that determine success or failure are still poorly understood. In the present study, the B. glabrata embryonic (Bge) cell line, a widely used in vitro model for hemocyte-like activity, was used to investigate membrane properties, and assess the impact of larval transformation proteins (LTP) on identified ion channels. Whole-cell patch clamp recordings from Bge cells demonstrated that a Zn2+-sensitive H+ channel serves as the dominant plasma membrane conductance. Moreover, treatment of Bge cells with Zn2+ significantly inhibited an otherwise robust production of reactive oxygen species (ROS), thus implicating H+ channels in the regulation of this immune function. A heat-sensitive component of LTP appears to target H+ channels, enhancing Bge cell H+ current over 2-fold. Both Bge cells and B. glabrata hemocytes express mRNA encoding a hydrogen voltage-gated channel 1 (HVCN1)-like protein, although its function in hemocytes remains to be determined. This study is the first to identify and characterize an H+ channel in non-neuronal cells of freshwater molluscs. Importantly, the involvement of these channels in ROS production and their modulation by LTP suggest that these channels may function in immune defense responses against larval S. mansoni. PMID:28319196

  2. The relationship between pericoronitis, wisdom teeth, putative periodontal pathogens and the host response.

    PubMed

    Moloney, Justin; Stassen, Leo F A

    2008-01-01

    To review the literature concerning pericoronitis, in particular the nature of the lesion and its aetiology, what factors may be used to predict if some patients would benefit from early removal of third molars, and if a scoring system can be developed for this purpose. A literature search using PubMed and the facilities of the Dublin Dental Hospital (DDH) library were used to gather the relevent information. PubMed lists all of the journals available in the DDH library and was used to identify relevent papers, which were then retrieved from the shelves and stacks with the help of library staff. The key word used was 'pericoronitis'. The studies reviewed assert that the bacteriology of pericoronitis is predominantly anaerobic in character, yet no causative species has been identified. Marker organisms for periodontitis were not generally isolated. Host factors examined in various studies were the inflammatory markers interleukin 1b and prostaglandin E2, and the immunological responses of neutrophils, macrophages, natural killer cells, T cells, helper T cells and suppressor/cytotoxic T cells. While all of these factors, with the exception of prostaglandin E2, tend to be elevated in cases of pericoronitis, both symptomatic and asymptomatic, no clearcut measurable entity has emerged that can be used as a predictive marker. A hypothesised scoring system to predict which patients would benefit from early removal of asymptomatic impacted lower third molars would be clinically advantageous in justifying prophylactic third molar surgery, but is not yet feasible or proven.

  3. Human Transbodies to HCV NS3/4A Protease Inhibit Viral Replication and Restore Host Innate Immunity

    PubMed Central

    Jittavisutthikul, Surasak; Seesuay, Watee; Thanongsaksrikul, Jeeraphong; Thueng-in, Kanyarat; Srimanote, Potjanee; Werner, Rolf G.; Chaicumpa, Wanpen

    2016-01-01

    A safe and effective direct acting anti-hepatitis C virus (HCV) agent is still needed. In this study, human single chain variable fragments of antibody (scFvs) that bound to HCV NS3/4A protein were produced by phage display technology. The engineered scFvs were linked to nonaarginines (R9) for making them cell penetrable. HCV-RNA-transfected Huh7 cells treated with the transbodies produced from four different transformed E. coli clones had reduced HCV-RNA inside the cells and in the cell spent media, as well as fewer HCV foci in the cell monolayer compared to the transfected cells in culture medium alone. The transbodies-treated transfected cells also had up-expression of the genes coding for the host innate immune response, including TRIF, TRAF3, IRF3, IL-28B, and IFN-β. Computerized homology modeling and intermolecular docking predicted that the effective transbodies interacted with several critical residues of the NS3/4A protease, including those that form catalytic triads, oxyanion loop, and S1 and S6 pockets, as well as a zinc-binding site. Although insight into molecular mechanisms of the transbodies need further laboratory investigation, it can be deduced from the current data that the transbodies blocked the HCV NS3/4A protease activities, leading to the HCV replication inhibition and restoration of the virally suppressed host innate immunity. The engineered antibodies should be tested further for treatment of HCV infection either alone, in combination with current therapeutics, or in a mixture with their cognates specific to other HCV proteins. PMID:27617013

  4. Serine Protease-mediated Host Invasion by the Parasitic Nematode Steinernema carpocapsae*

    PubMed Central

    Toubarro, Duarte; Lucena-Robles, Miguel; Nascimento, Gisela; Santos, Romana; Montiel, Rafael; Veríssimo, Paula; Pires, Euclides; Faro, Carlos; Coelho, Ana V.; Simões, Nelson

    2010-01-01

    Steinernema carpocapsae is an insect parasitic nematode used in biological control, which infects insects penetrating by mouth and anus and invading the hemocoelium through the midgut wall. Invasion has been described as a key factor in nematode virulence and suggested to be mediated by proteases. A serine protease cDNA from the parasitic stage was sequenced (sc-sp-1); the recombinant protein was produced in an Escherichia coli system, and a native protein was purified from the secreted products. Both proteins were confirmed by mass spectrometry to be encoded by the sc-sp-1 gene. Sc-SP-1 has a pI of 8.7, a molecular mass of 27.3 kDa, a catalytic efficiency of 22.2 × 104 s−1 m−1 against N-succinyl-Ala-Ala-Pro-Phe-pNA, and is inhibited by chymostatin (IC 0.07) and PMSF (IC 0.73). Sc-SP-1 belongs to the chymotrypsin family, based on sequence and biochemical analysis. Only the nematode parasitic stage expressed sc-sp-1. These nematodes in the midgut lumen, prepared to invade the insect hemocoelium, expressed higher levels than those already in the hemocoelium. Moreover, parasitic nematode sense insect peritrophic membrane and hemolymph more quickly than they do other tissues, which initiates sc-sp-1 expression. Ex vivo, Sc-SP-1 was able to bind to insect midgut epithelium and to cause cell detachment from basal lamina. In vitro, Sc-SP-1 formed holes in an artificial membrane model (Matrigel), whereas Sc-SP-1 treated with PMSF did not, very likely because it hydrolyzes matrix glycoproteins. These findings highlight the S. carpocapsae-invasive process that is a key step in the parasitism thus opening new perspectives for improving nematode virulence to use in biological control. PMID:20656686

  5. Host plants indirectly influence plant virus transmission by altering gut cysteine protease activity of aphid vectors

    USDA-ARS?s Scientific Manuscript database

    Luteoviridae), transmitted exclusively by aphids in a circulative manner. PLRV transmission efficiency was significantly reduced when a clonal lineage of M. persicae was reared on turnip as compared to the weed physalis, a transient effect caused by a host-switch response. A trend of higher PLRV tit...

  6. Hepacivirus NS3/4A Proteases Interfere with MAVS Signaling in both Their Cognate Animal Hosts and Humans: Implications for Zoonotic Transmission.

    PubMed

    Anggakusuma; Brown, Richard J P; Banda, Dominic H; Todt, Daniel; Vieyres, Gabrielle; Steinmann, Eike; Pietschmann, Thomas

    2016-12-01

    Multiple novel members of the genus Hepacivirus have recently been discovered in diverse mammalian species. However, to date, their replication mechanisms and zoonotic potential have not been explored in detail. The NS3/4A serine protease of hepatitis C virus (HCV) is critical for cleavage of the viral polyprotein. It also cleaves the cellular innate immune adaptor MAVS, thus decreasing interferon (IFN) production and contributing to HCV persistence in the human host. To investigate the conservation of fundamental aspects of the hepaciviral life cycle, we explored if MAVS cleavage and suppression of innate immune signaling represent a common mechanism employed across different clades of the genus Hepacivirus to enhance viral replication. To estimate the zoonotic potential of these nonhuman hepaciviruses, we assessed if their NS3/4A proteases were capable of cleaving human MAVS. NS3/4A proteases of viruses infecting colobus monkeys, rodents, horses, and cows cleaved the MAVS proteins of their cognate hosts and interfered with the ability of MAVS to induce the IFN-β promoter. All NS3/4A proteases from nonhuman viruses readily cleaved human MAVS. Thus, NS3/4A-dependent cleavage of MAVS is a conserved replication strategy across multiple clades within the genus Hepacivirus Human MAVS is susceptible to cleavage by these nonhuman viral proteases, indicating that it does not pose a barrier for zoonotic transmission of these viruses to humans.

  7. Endosymbiotic and Host Proteases in the Digestive Tract of the Invasive Snail Pomacea canaliculata: Diversity, Origin and Characterization

    PubMed Central

    Godoy, Martín S.; Castro-Vasquez, Alfredo; Vega, Israel A.

    2013-01-01

    Digestive proteases of the digestive tract of the apple snail Pomacea canaliculata were studied. Luminal protease activity was found in the crop, the style sac and the coiled gut and was significantly higher in the coiled gut. Several protease bands and their apparent molecular weights were identified in both tissue extracts and luminal contents by gel zymography: (1) a 125 kDa protease in salivary gland extracts and in the crop content; (2) a 30 kDa protease throughout all studied luminal contents and in extracts of the midgut gland and of the endosymbionts isolated from this gland; (3) two proteases of 145 and 198 kDa in the coiled gut content. All these proteases were inhibited by aprotinin, a serine-protease inhibitor, and showed maximum activity between 30°C and 35°C and pH between 8.5 and 9.5. Tissue L-alanine-N-aminopeptidase activity was determined in the wall of the crop, the style sac and the coiled gut and was significantly higher in the coiled gut. Our findings show that protein digestion in P. canaliculata is carried out through a battery of diverse proteases originated from the salivary glands and the endosymbionts lodged in the midgut gland and by proteases of uncertain origin that occur in the coiled gut lumen. PMID:23818959

  8. The role of endogenous and exogenous enzymes in chronic wounds: a focus on the implications of aberrant levels of both host and bacterial proteases in wound healing.

    PubMed

    McCarty, Sara M; Cochrane, Christine A; Clegg, Peter D; Percival, Steven L

    2012-01-01

    Cutaneous wound healing is orchestrated by a number of physiological pathways that ultimately lead to reformation of skin integrity and the production of functional scar tissue. The remodeling of a wound is significantly affected by matrix metalloproteinases (MMPs), which act to control the degradation of the extracellular matrix (ECM). Regulation of MMPs is imperative for wound healing as excessive levels of MMPs can lead to disproportionate destruction of the wound ECM compared to ECM deposition. In addition to human MMPs, bacterial proteases have been found to be influential in tissue breakdown and, as such, have a role to play in the healing of infected wounds. For example, the zinc-metalloproteinase, elastase, produced by Pseudomonas aeruginosa, induces degradation of fibroblast proteins and proteoglycans in chronic wounds and has also been shown to degrade host immune cell mediators. Microbial extracellular enzymes have also been shown to degrade human wound fluid and inhibit fibroblast cell growth. It is now being acknowledged that host and bacterial MMPs may act synergistically to cause tissue breakdown within the wound bed. Several studies have suggested that bacterial-derived secreted proteases may act to up-regulate the levels of MMPs produced by the host cells. Together, these findings indicate that bacterial phenotype in terms of protease producing potential of bacteria should be taken into consideration during diagnostic and clinical intervention of infected wound management. Furthermore, both host MMPs and those derived from infecting bacteria need to be targeted in order to increase the healing capacity of the injured tissue. The aim of this review is to investigate the evidence suggestive of a relationship between unregulated levels of both host and bacterial proteases and delayed wound healing. © 2012 by the Wound Healing Society.

  9. Serine protease detection in mixed lymphocyte cultures: a histochemical method for possible prediction of graft-versus-host disease.

    PubMed

    Maiocchi, M A; Nano, R; Capelli, E; Bonfichi, M; Alessandrino, E P; Bernasconi, P

    1998-08-01

    Graft-versus-host disease (GVHD) presents an important complication of allogeneic bone marrow transplantation. A method to predict GVHD might be the analysis of cytotoxic T lymphocyte precursors, but the technique requires the use of radioactive elements not suitable in all laboratories. Serine esterase (SE) activity was studied by a cytochemical method in donor-recipient mixed lymphocytes cultures (MLC). Twelve patients, affected by acute or chronic leukemia, and 20 donors were studied. MLC incubated with and without growth factors (IGF-1 or IL-2), were analyzed. The relationship between positivity of MLC and GVHD in transplanted patients was evaluated. The data obtained showed that the percentage of SE positive cells was higher in MLC compared to negative control MLC. The highest percentage of positive cells was found in the MLC obtained from unrelated subjects. These results show that serine protease expression in MLC may be a predictive marker of GVHD and could be used as an additional early test associated with cytotoxicity.

  10. Analysis of Putative Apoplastic Effectors from the Nematode, Globodera rostochiensis, and Identification of an Expansin-Like Protein That Can Induce and Suppress Host Defenses

    PubMed Central

    Ali, Shawkat; Magne, Maxime; Chen, Shiyan; Côté, Olivier; Stare, Barbara Gerič; Obradovic, Natasa; Jamshaid, Lubna; Wang, Xiaohong; Bélair, Guy; Moffett, Peter

    2015-01-01

    The potato cyst nematode, Globodera rostochiensis, is an important pest of potato. Like other pathogens, plant parasitic nematodes are presumed to employ effector proteins, secreted into the apoplast as well as the host cytoplasm, to alter plant cellular functions and successfully infect their hosts. We have generated a library of ORFs encoding putative G. rostochiensis putative apoplastic effectors in vectors for expression in planta. These clones were assessed for morphological and developmental effects on plants as well as their ability to induce or suppress plant defenses. Several CLAVATA3/ESR-like proteins induced developmental phenotypes, whereas predicted cell wall-modifying proteins induced necrosis and chlorosis, consistent with roles in cell fate alteration and tissue invasion, respectively. When directed to the apoplast with a signal peptide, two effectors, an ubiquitin extension protein (GrUBCEP12) and an expansin-like protein (GrEXPB2), suppressed defense responses including NB-LRR signaling induced in the cytoplasm. GrEXPB2 also elicited defense response in species- and sequence-specific manner. Our results are consistent with the scenario whereby potato cyst nematodes secrete effectors that modulate host cell fate and metabolism as well as modifying host cell walls. Furthermore, we show a novel role for an apoplastic expansin-like protein in suppressing intra-cellular defense responses. PMID:25606855

  11. Different residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2

    PubMed Central

    Reinke, Lennart Michel; Hartleib, Anika; Nehlmeier, Inga; Gierer, Stefanie; Hoffmann, Markus; Hofmann-Winkler, Heike; Winkler, Michael

    2017-01-01

    The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) mediates viral entry into target cells. Cleavage and activation of SARS S by a host cell protease is essential for infectious viral entry and the responsible enzymes are potential targets for antiviral intervention. The type II transmembrane serine protease TMPRSS2 cleaves and activates SARS S in cell culture and potentially also in the infected host. Here, we investigated which determinants in SARS S control cleavage and activation by TMPRSS2. We found that SARS S residue R667, a previously identified trypsin cleavage site, is also required for S protein cleavage by TMPRSS2. The cleavage fragments produced by trypsin and TMPRSS2 differed in their decoration with N-glycans, suggesting that these proteases cleave different SARS S glycoforms. Although R667 was required for SARS S cleavage by TMPRSS2, this residue was dispensable for TMPRSS2-mediated S protein activation. Conversely, residue R797, previously reported to be required for SARS S activation by trypsin, was dispensable for S protein cleavage but required for S protein activation by TMPRSS2. Collectively, these results show that different residues in SARS S control cleavage and activation by TMPRSS2, suggesting that these processes are more complex than initially appreciated. PMID:28636671

  12. Different residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2.

    PubMed

    Reinke, Lennart Michel; Spiegel, Martin; Plegge, Teresa; Hartleib, Anika; Nehlmeier, Inga; Gierer, Stefanie; Hoffmann, Markus; Hofmann-Winkler, Heike; Winkler, Michael; Pöhlmann, Stefan

    2017-01-01

    The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) mediates viral entry into target cells. Cleavage and activation of SARS S by a host cell protease is essential for infectious viral entry and the responsible enzymes are potential targets for antiviral intervention. The type II transmembrane serine protease TMPRSS2 cleaves and activates SARS S in cell culture and potentially also in the infected host. Here, we investigated which determinants in SARS S control cleavage and activation by TMPRSS2. We found that SARS S residue R667, a previously identified trypsin cleavage site, is also required for S protein cleavage by TMPRSS2. The cleavage fragments produced by trypsin and TMPRSS2 differed in their decoration with N-glycans, suggesting that these proteases cleave different SARS S glycoforms. Although R667 was required for SARS S cleavage by TMPRSS2, this residue was dispensable for TMPRSS2-mediated S protein activation. Conversely, residue R797, previously reported to be required for SARS S activation by trypsin, was dispensable for S protein cleavage but required for S protein activation by TMPRSS2. Collectively, these results show that different residues in SARS S control cleavage and activation by TMPRSS2, suggesting that these processes are more complex than initially appreciated.

  13. PARP9-DTX3L ubiquitin ligase targets host histone H2BJ and viral 3C protease to enhance interferon signaling and control viral infection

    PubMed Central

    Zhang, Yong; Mao, Dailing; Roswit, William T.; Jin, Xiaohua; Patel, Anand C.; Patel, Dhara A.; Agapov, Eugene; Wang, Zhepeng; Tidwell, Rose M.; Atkinson, Jeffrey J.; Huang, Guangming; McCarthy, Ronald; Yu, Jinsheng; Yun, Nadezhda E.; Paessler, Slobodan; Lawson, T. Glen; Omattage, Natalie S.; Brett, Tom J.; Holtzman, Michael J.

    2015-01-01

    Enhancing the response to interferon could offer an immunological advantage to the host. In support of this concept, we used a modified form of the transcription factor STAT1 to achieve interferon hyperresponsiveness without toxicity and markedly improve antiviral function in transgenic mice and transduced human cells. We found that the improvement depends on expression of a PARP9-DTX3L complex with distinct domains for interaction with STAT1 and for activity as an E3 ubiquitin ligase that acts on host histone H2BJ to promote interferon-stimulated gene expression and on viral 3C proteases to initiate their degradation via the immunoproteasome. Together, PARP9-DTX3L acts on host and pathogen to achieve a double layer of immunity within a safe reserve in the interferon signaling pathway. PMID:26479788

  14. Expression Profile of the Schistosoma japonicum Degradome Reveals Differential Protease Expression Patterns and Potential Anti-schistosomal Intervention Targets

    PubMed Central

    Liu, Shuai; Cai, Pengfei; Piao, Xianyu; Hou, Nan; Zhou, Xiaosu; Wu, Chuang; Wang, Heng; Chen, Qijun

    2014-01-01

    Blood fluke proteases play pivotal roles in the processes of invasion, nutrition acquisition, immune evasion, and other host-parasite interactions. Hundreds of genes encoding putative proteases have been identified in the recently published schistosome genomes. However, the expression profiles of these proteases in Schistosoma species have not yet been systematically analyzed. We retrieved and culled the redundant protease sequences of Schistosoma japonicum, Schistosoma mansoni, Echinococcus multilocularis, and Clonorchis sinensis from public databases utilizing bioinformatic approaches. The degradomes of the four parasitic organisms and Homo sapiens were then comparatively analyzed. A total of 262 S. japonicum protease sequences were obtained and the expression profiles generated using whole-genome microarray. Four main clusters of protease genes with different expression patterns were identified: proteases up-regulated in hepatic schistosomula and adult worms, egg-specific or predominantly expressed proteases, cercaria-specific or predominantly expressed proteases, and constantly expressed proteases. A subset of protease genes with different expression patterns were further validated using real-time quantitative PCR. The present study represents the most comprehensive analysis of a degradome in Schistosoma species to date. These results provide a firm foundation for future research on the specific function(s) of individual proteases and may help to refine anti-proteolytic strategies in blood flukes. PMID:25275570

  15. Bacterial proteases and virulence.

    PubMed

    Frees, Dorte; Brøndsted, Lone; Ingmer, Hanne

    2013-01-01

    Bacterial pathogens rely on proteolysis for variety of purposes during the infection process. In the cytosol, the main proteolytic players are the conserved Clp and Lon proteases that directly contribute to virulence through the timely degradation of virulence regulators and indirectly by providing tolerance to adverse conditions such as those experienced in the host. In the membrane, HtrA performs similar functions whereas the extracellular proteases, in close contact with host components, pave the way for spreading infections by degrading host matrix components or interfering with host cell signalling to short-circuit host cell processes. Common to both intra- and extracellular proteases is the tight control of their proteolytic activities. In general, substrate recognition by the intracellular proteases is highly selective which is, in part, attributed to the chaperone activity associated with the proteases either encoded within the same polypeptide or on separate subunits. In contrast, substrate recognition by extracellular proteases is less selective and therefore these enzymes are generally expressed as zymogens to prevent premature proteolytic activity that would be detrimental to the cell. These extracellular proteases are activated in complex cascades involving auto-processing and proteolytic maturation. Thus, proteolysis has been adopted by bacterial pathogens at multiple levels to ensure the success of the pathogen in contact with the human host.

  16. A putative marker for human pathogenic strains of Anaplasma phagocytophilum correlates with geography and host, but not human tropism.

    PubMed

    Foley, Janet; Stephenson, Nicole; Cubilla, Michelle Pires; Qurollo, Barbara; Breitschwerdt, Edward B

    2016-03-01

    Anaplasma phagocytophilum is an Ixodes species tick-transmitted bacterium that is capable of infecting a variety of host species, although there is a diversity of bacterial strains with differing host tropism. Recent analysis of A. phagocytophilum strains suggested that "drhm", a gene locus designated "distantly related to human marker" (drhm), which was predicted to be an integral membrane protein with possible transporter functions was not present in available canine and human isolates. By assessing 117 strains from 14 host species from across the US, we extended this analysis. Phylogenetic clades were associated with geography, but not host species. Additionally, a virulent clade that lacks drhm and infects dogs, horses, and humans in northeastern US was identified.

  17. Genetically based polymorphisms in morphology and life history associated with putative host races of the water lily leaf beetle, Galerucella nymphaeae.

    PubMed

    Pappers, Stephanie M; van der Velde, Gerard; Ouborg, N Joop; van Groenendael, Jan M

    2002-08-01

    A host race is a population that is partially reproductively isolated from other conspecific populations as a direct consequence of adaptation to a specific host. The initial step in host race formation is the establishment of genetically based polymorphisms in, for example, morphology, preference, or performance. In this study we investigated whether polymorphisms observed in Galerucella nymphaeae have a genetic component. Galerucella nymphaeae, the water lily leaf beetle, is a herbivore which feeds and oviposits on the plant hosts Nuphar lutea and Nymphaea alba (both Nymphaeaceae) and Rumex hydrolapathum and Polygonum amphibium (both Polygonaceae). A full reciprocal crossing scheme (16 crosses, each replicated 10 times) and subsequent transplantation of 1,001 egg clutches revealed a genetic basis for differences in body length and mandibular width. The heritability value of these traits, based on midparent-offspring regression, ranged between 0.53 and 0.83 for the different diets. Offspring from Nymphaeaceae parents were on average 12% larger and had on average 18% larger mandibles than offspring from Polygonaceae parents. Furthermore, highly significant correlations were found between feeding preference of the offspring and the feeding preference of their parents. Finally, two fitness components were measured: development time and survival. Development time was influenced by diet, survival both by cross type and diet, the latter of which suggest adaptation of the beetles. This suggestion is strengthened by a highly significant cross x diet interaction effect for development time as well as for survival, which is generally believed to indicate local adaptation. Although no absolute genetic incompatibility among putative host races was observed, survival of the between-host family offspring, on each diet separately, was lower than the survival of the within-host family offspring on that particular host. Survival of offspring of two Nymphaeaceae parents was about

  18. Adapted J6/JFH1-Based Hepatitis C Virus Recombinants with Genotype-Specific NS4A Show Similar Efficacies against Lead Protease Inhibitors, Alpha Interferon, and a Putative NS4A Inhibitor

    PubMed Central

    Gottwein, Judith M.; Jensen, Sanne B.; Serre, Stéphanie B. N.; Ghanem, Lubna; Scheel, Troels K. H.; Jensen, Tanja B.; Krarup, Henrik; Uzcategui, Nathalie; Mikkelsen, Lotte S.

    2013-01-01

    To facilitate studies of hepatitis C virus (HCV) NS4A, we aimed at developing J6/JFH1-based recombinants with genotype 1- to 7-specific NS4A proteins. We developed efficient culture systems expressing NS4A proteins of genotypes (isolates) 1a (H77 and TN), 1b (J4), 2a (J6), 4a (ED43), 5a (SA13), 6a (HK6a), and 7a (QC69), with peak infectivity titers of ∼3.5 to 4.5 log10 focus-forming units per ml. Except for genotype 2a (J6), growth depended on adaptive mutations identified in long-term culture. Genotype 1a, 1b, and 4a recombinants were adapted by amino acid substitutions F772S (p7) and V1663A (NS4A), while 5a, 6a, and 7a recombinants required additional substitutions in the NS3 protease and/or NS4A. We demonstrated applicability of the developed recombinants for study of antivirals. Genotype 1 to 7 NS4A recombinants showed similar responses to the protease inhibitors telaprevir (VX-950), boceprevir (Sch503034), simeprevir (TMC435350), danoprevir (ITMN-191), and vaniprevir (MK-7009), to alpha interferon 2b, and to the putative NS4A inhibitor ACH-806. The efficacy of ACH-806 was lower than that of protease inhibitors and was not influenced by changes at amino acids 1042 and 1065 (in the NS3 protease), which have been suggested to mediate resistance to ACH-806 in replicons. Genotype 1a, 1b, and 2a recombinants showed viral spread under long-term treatment with ACH-806, without acquisition of resistance mutations in the NS3-NS4A region. Relatively high concentrations of ACH-806 inhibited viral assembly, but not replication, in a single-cycle production assay. The developed HCV culture systems will facilitate studies benefitting from expression of genotype-specific NS4A in a constant backbone in the context of the complete viral replication cycle, including functional studies and evaluations of the efficacy of antivirals. PMID:24060868

  19. The Xenopus laevis Atg4B Protease: Insights into Substrate Recognition and Application for Tag Removal from Proteins Expressed in Pro- and Eukaryotic Hosts.

    PubMed

    Frey, Steffen; Görlich, Dirk

    2015-01-01

    During autophagy, members of the ubiquitin-like Atg8 protein family get conjugated to phosphatidylethanolamine and act as protein-recruiting scaffolds on the autophagosomal membrane. The Atg4 protease produces mature Atg8 from C-terminally extended precursors and deconjugates lipid-bound Atg8. We now found that Xenopus laevis Atg4B (xAtg4B) is ideally suited for proteolytic removal of N-terminal tags from recombinant proteins. To implement this strategy, an Atg8 cleavage module is inserted in between tag and target protein. An optimized xAtg4B protease fragment includes the so far uncharacterized C-terminus, which crucially contributes to recognition of the Xenopus Atg8 homologs xLC3B and xGATE16. xAtg4B-mediated tag cleavage is very robust in solution or on-column, efficient at 4°C and orthogonal to TEV protease and the recently introduced proteases bdSENP1, bdNEDP1 and xUsp2. Importantly, xLC3B fusions are stable in wheat germ extract or when expressed in Saccharomyces cerevisiae, but cleavable by xAtg4B during or following purification. We also found that fusions to the bdNEDP1 substrate bdNEDD8 are stable in S. cerevisiae. In combination, or findings now provide a system, where proteins and complexes fused to xLC3B or bdNEDD8 can be expressed in a eukaryotic host and purified by successive affinity capture and proteolytic release steps.

  20. The Xenopus laevis Atg4B Protease: Insights into Substrate Recognition and Application for Tag Removal from Proteins Expressed in Pro- and Eukaryotic Hosts

    PubMed Central

    Frey, Steffen; Görlich, Dirk

    2015-01-01

    During autophagy, members of the ubiquitin-like Atg8 protein family get conjugated to phosphatidylethanolamine and act as protein-recruiting scaffolds on the autophagosomal membrane. The Atg4 protease produces mature Atg8 from C-terminally extended precursors and deconjugates lipid-bound Atg8. We now found that Xenopus laevis Atg4B (xAtg4B) is ideally suited for proteolytic removal of N-terminal tags from recombinant proteins. To implement this strategy, an Atg8 cleavage module is inserted in between tag and target protein. An optimized xAtg4B protease fragment includes the so far uncharacterized C-terminus, which crucially contributes to recognition of the Xenopus Atg8 homologs xLC3B and xGATE16. xAtg4B-mediated tag cleavage is very robust in solution or on-column, efficient at 4°C and orthogonal to TEV protease and the recently introduced proteases bdSENP1, bdNEDP1 and xUsp2. Importantly, xLC3B fusions are stable in wheat germ extract or when expressed in Saccharomyces cerevisiae, but cleavable by xAtg4B during or following purification. We also found that fusions to the bdNEDP1 substrate bdNEDD8 are stable in S. cerevisiae. In combination, or findings now provide a system, where proteins and complexes fused to xLC3B or bdNEDD8 can be expressed in a eukaryotic host and purified by successive affinity capture and proteolytic release steps. PMID:25923686

  1. A comparative genomic analysis of putative pathogenicity genes in the host-specific sibling species Colletotrichum graminicola and Colletotrichum sublineola.

    PubMed

    Buiate, E A S; Xavier, K V; Moore, N; Torres, M F; Farman, M L; Schardl, C L; Vaillancourt, L J

    2017-01-10

    Colletotrichum graminicola and C. sublineola cause anthracnose leaf and stalk diseases of maize and sorghum, respectively. In spite of their close evolutionary relationship, the two species are completely host-specific. Host specificity is often attributed to pathogen virulence factors, including specialized secondary metabolites (SSM), and small-secreted protein (SSP) effectors. Genes relevant to these categories were manually annotated in two co-occurring, contemporaneous strains of C. graminicola and C. sublineola. A comparative genomic and phylogenetic analysis was performed to address the evolutionary relationships among these and other divergent gene families in the two strains. Inoculation of maize with C. sublineola, or of sorghum with C. graminicola, resulted in rapid plant cell death at, or just after, the point of penetration. The two fungal genomes were very similar. More than 50% of the assemblies could be directly aligned, and more than 80% of the gene models were syntenous. More than 90% of the predicted proteins had orthologs in both species. Genes lacking orthologs in the other species (non-conserved genes) included many predicted to encode SSM-associated proteins and SSPs. Other common groups of non-conserved proteins included transporters, transcription factors, and CAZymes. Only 32 SSP genes appeared to be specific to C. graminicola, and 21 to C. sublineola. None of the SSM-associated genes were lineage-specific. Two different strains of C. graminicola, and three strains of C. sublineola, differed in no more than 1% percent of gene sequences from one another. Efficient non-host recognition of C. sublineola by maize, and of C. graminicola by sorghum, was observed in epidermal cells as a rapid deployment of visible resistance responses and plant cell death. Numerous non-conserved SSP and SSM-associated predicted proteins that could play a role in this non-host recognition were identified. Additional categories of genes that were also highly

  2. Putative alternative polyadenylation (APA) events in the early interaction of Salmonella enterica Typhimurium and human host cells.

    PubMed

    Afonso-Grunz, Fabian

    2015-12-01

    The immune response of epithelial cells upon infection is mediated by changing activity levels of a variety of proteins along with changes in mRNA, and also ncRNA abundance. Alternative polyadenylation (APA) represents a mechanism that diversifies gene expression similar to alternative splicing. T-cell activation, neuronal activity, development and several human diseases including viral infections involve APA, but at present it remains unclear if this mechanism is also implicated in the response to bacterial infections. Our recently published study of interacting Salmonella enterica Typhimurium and human host cells includes genome-wide expression profiles of human epithelial cells prior and subsequent to infection with the invasive pathogen. The generated dataset (GEO accession number: GSE61730) covers several points of time post infection, and one of these interaction stages was additionally profiled with MACE-based dual 3'Seq, which allows for identification of polyadenylation (PA) sites. The present study features the polyadenylation landscape in early interacting cells based on this data, and provides a comparison of the identified PA sites with those of a corresponding 3P-Seq dataset of non-interacting cells. Differential PA site usage of FTL, PRDX1 and VAPA results in transcription of mRNA isoforms with distinct sets of miRNA and protein binding sites that influence processing, localization, stability, and translation of the respective mRNA. APA of these candidate genes consequently harbors the potential to modulate the host cell response to bacterial infection.

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

    PubMed Central

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

    2014-01-01

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

  4. XopD SUMO protease affects host transcription, promotes pathogen growth, and delays symptom development in xanthomonas-infected tomato leaves.

    PubMed

    Kim, Jung-Gun; Taylor, Kyle W; Hotson, Andrew; Keegan, Mark; Schmelz, Eric A; Mudgett, Mary Beth

    2008-07-01

    We demonstrate that XopD, a type III effector from Xanthomonas campestris pathovar vesicatoria (Xcv), suppresses symptom production during the late stages of infection in susceptible tomato (Solanum lycopersicum) leaves. XopD-dependent delay of tissue degeneration correlates with reduced chlorophyll loss, reduced salicylic acid levels, and changes in the mRNA abundance of senescence- and defense-associated genes despite high pathogen titers. Subsequent structure-function analyses led to the discovery that XopD is a DNA binding protein that alters host transcription. XopD contains a putative helix-loop-helix domain required for DNA binding and two conserved ERF-associated amphiphilic motifs required to repress salicylic acid- and jasmonic acid-induced gene transcription in planta. Taken together, these data reveal that XopD is a unique virulence factor in Xcv that alters host transcription, promotes pathogen multiplication, and delays the onset of leaf chlorosis and necrosis.

  5. XopD SUMO Protease Affects Host Transcription, Promotes Pathogen Growth, and Delays Symptom Development in Xanthomonas-Infected Tomato Leaves[W][OA

    PubMed Central

    Kim, Jung-Gun; Taylor, Kyle W.; Hotson, Andrew; Keegan, Mark; Schmelz, Eric A.; Mudgett, Mary Beth

    2008-01-01

    We demonstrate that XopD, a type III effector from Xanthomonas campestris pathovar vesicatoria (Xcv), suppresses symptom production during the late stages of infection in susceptible tomato (Solanum lycopersicum) leaves. XopD-dependent delay of tissue degeneration correlates with reduced chlorophyll loss, reduced salicylic acid levels, and changes in the mRNA abundance of senescence- and defense-associated genes despite high pathogen titers. Subsequent structure-function analyses led to the discovery that XopD is a DNA binding protein that alters host transcription. XopD contains a putative helix-loop-helix domain required for DNA binding and two conserved ERF-associated amphiphilic motifs required to repress salicylic acid– and jasmonic acid–induced gene transcription in planta. Taken together, these data reveal that XopD is a unique virulence factor in Xcv that alters host transcription, promotes pathogen multiplication, and delays the onset of leaf chlorosis and necrosis. PMID:18664616

  6. Expression of the Streptococcus agalactiae virulence-associated protease CspA in a soluble, active form utilizing the Gram-positive host, Lactococcus lactis.

    PubMed

    Shelver, Daniel; Bryan, Joshua D

    2008-09-10

    The heterologous expression of streptococcal genes in common Gram-negative hosts may be complicated by low-level expression, toxicity to the host, formation of inclusion bodies, and mislocalization of the encoded proteins. Biochemical study of the Streptococcus agalactiae virulence-associated cell-envelope protease (CEP) CspA, as well as other CEPs, has been limited by the lack of effective expression systems. In this study, we present a simple strategy to express cspA as a catalytically active exoprotein. A recombinant allele of cspA, cspADeltaCWA, was engineered to eliminate the dispensable cell-wall anchor. The cspADeltaCWA allele was expressed in the Gram-positive organism, Lactococcus lactis, using an established, plasmid-based, nisin-inducible expression system. After induction, nearly all of the exoprotein observable by SDS-PAGE corresponded to CspADeltaCWA. CspADeltaCWA-containing medium exhibited similar fibrinolytic activity as whole cells of GBS, indicating the recombinant protein was active. Characterization of CspADeltaCWA indicated that like some other CEPs, it is N-terminally processed, exists predominantly as a dimer, and has the ability to cleave itself at its C-terminus. Taken together, this work presents an efficient strategy for expression of cspA that could be applied to other streptococcal proteins that are not amenable to expression using common Gram-negative hosts.

  7. Deubiquitinase function of arterivirus papain-like protease 2 suppresses the innate immune response in infected host cells.

    PubMed

    van Kasteren, Puck B; Bailey-Elkin, Ben A; James, Terrence W; Ninaber, Dennis K; Beugeling, Corrine; Khajehpour, Mazdak; Snijder, Eric J; Mark, Brian L; Kikkert, Marjolein

    2013-02-26

    Protein ubiquitination regulates important innate immune responses. The discovery of viruses encoding deubiquitinating enzymes (DUBs) suggests they remove ubiquitin to evade ubiquitin-dependent antiviral responses; however, this has never been conclusively demonstrated in virus-infected cells. Arteriviruses are economically important positive-stranded RNA viruses that encode an ovarian tumor (OTU) domain DUB known as papain-like protease 2 (PLP2). This enzyme is essential for arterivirus replication by cleaving a site within the viral replicase polyproteins and also removes ubiquitin from cellular proteins. To dissect this dual specificity, which relies on a single catalytic site, we determined the crystal structure of equine arteritis virus PLP2 in complex with ubiquitin (1.45 Å). PLP2 binds ubiquitin using a zinc finger that is uniquely integrated into an exceptionally compact OTU-domain fold that represents a new subclass of zinc-dependent OTU DUBs. Notably, the ubiquitin-binding surface is distant from the catalytic site, which allowed us to mutate this surface to significantly reduce DUB activity without affecting polyprotein cleavage. Viruses harboring such mutations exhibited WT replication kinetics, confirming that PLP2-mediated polyprotein cleavage was intact, but the loss of DUB activity strikingly enhanced innate immune signaling. Compared with WT virus infection, IFN-β mRNA levels in equine cells infected with PLP2 mutants were increased by nearly an order of magnitude. Our findings not only establish PLP2 DUB activity as a critical factor in arteriviral innate immune evasion, but the selective inactivation of DUB activity also opens unique possibilities for developing improved live attenuated vaccines against arteriviruses and other viruses encoding similar dual-specificity proteases.

  8. A Systems Biology Approach to Reveal Putative Host-Derived Biomarkers of Periodontitis by Network Topology Characterization of MMP-REDOX/NO and Apoptosis Integrated Pathways.

    PubMed

    Zeidán-Chuliá, Fares; Gürsoy, Mervi; Neves de Oliveira, Ben-Hur; Özdemir, Vural; Könönen, Eija; Gürsoy, Ulvi K

    2015-01-01

    Periodontitis, a formidable global health burden, is a common chronic disease that destroys tooth-supporting tissues. Biomarkers of the early phase of this progressive disease are of utmost importance for global health. In this context, saliva represents a non-invasive biosample. By using systems biology tools, we aimed to (1) identify an integrated interactome between matrix metalloproteinase (MMP)-REDOX/nitric oxide (NO) and apoptosis upstream pathways of periodontal inflammation, and (2) characterize the attendant topological network properties to uncover putative biomarkers to be tested in saliva from patients with periodontitis. Hence, we first generated a protein-protein network model of interactions ("BIOMARK" interactome) by using the STRING 10 database, a search tool for the retrieval of interacting genes/proteins, with "Experiments" and "Databases" as input options and a confidence score of 0.400. Second, we determined the centrality values (closeness, stress, degree or connectivity, and betweenness) for the "BIOMARK" members by using the Cytoscape software. We found Ubiquitin C (UBC), Jun proto-oncogene (JUN), and matrix metalloproteinase-14 (MMP14) as the most central hub- and non-hub-bottlenecks among the 211 genes/proteins of the whole interactome. We conclude that UBC, JUN, and MMP14 are likely an optimal candidate group of host-derived biomarkers, in combination with oral pathogenic bacteria-derived proteins, for detecting periodontitis at its early phase by using salivary samples from patients. These findings therefore have broader relevance for systems medicine in global health as well.

  9. New roles for perforins and proteases in apicomplexan egress.

    PubMed

    Roiko, Marijo S; Carruthers, Vern B

    2009-10-01

    Egress is a pivotal step in the life cycle of intracellular pathogens initiating the transition from an expiring host cell to a fresh target cell. While much attention has been focused on understanding cell invasion by intracellular pathogens, recent work is providing a new appreciation of mechanisms and therapeutic potential of microbial egress. This review highlights recent insight into cell egress by apicomplexan parasites and emerging contributions of membranolytic and proteolytic secretory products, along with host proteases. New findings suggest that Toxoplasma gondii secretes a pore-forming protein, TgPLP1, during egress that facilitates parasite escape from the cell by perforating the parasitophorous membrane. Also, in a cascade of proteolytic events, Plasmodium falciparum late-stage schizonts activate and secrete a subtilisin, PfSUB1, which processes enigmatic putative proteases called serine-repeat antigens that contribute to merozoite egress. A new report also suggests that calcium-activated host proteases called calpains aid parasite exit, possibly by acting upon the host cytoskeleton. Together these discoveries reveal important new molecular players involved in the principal steps of egress by apicomplexans.

  10. The aspartyl protease TgASP5 mediates the export of the Toxoplasma GRA16 and GRA24 effectors into host cells.

    PubMed

    Curt-Varesano, Aurélie; Braun, Laurence; Ranquet, Caroline; Hakimi, Mohamed-Ali; Bougdour, Alexandre

    2016-02-01

    Toxoplasma gondii and Plasmodium species are obligatory intracellular parasites that export proteins into the infected cells in order to interfere with host-signalling pathways, acquire nutrients or evade host defense mechanisms. With regard to export mechanism, a wealth of information in Plasmodium spp. is available, while the mechanisms operating in T. gondii remain uncertain. The recent discovery of exported proteins in T. gondii, mainly represented by dense granule resident proteins, might explain this discrepancy and offers a unique opportunity to study the export mechanism in T. gondii. Here, we report that GRA16 export is mediated by two protein elements present in its N-terminal region. Because the first element contains a putative Plasmodium export element linear motif (RRLAE), we hypothesized that GRA16 export depended on a maturation process involving protein cleavage. Using both N- and C-terminal epitope tags, we provide evidence for protein proteolysis occurring in the N-terminus of GRA16. We show that TgASP5, the T. gondii homolog of Plasmodium plasmepsin V, is essential for GRA16 export and is directly responsible for its maturation in a Plasmodium export element-dependent manner. Interestingly, TgASP5 is also involved in GRA24 export, although the GRA24 maturation mechanism is TgASP5-independent. Our data reveal different modus operandi for protein export, in which TgASP5 should play multiple functions.

  11. A Systems Biology Approach to Reveal Putative Host-Derived Biomarkers of Periodontitis by Network Topology Characterization of MMP-REDOX/NO and Apoptosis Integrated Pathways

    PubMed Central

    Zeidán-Chuliá, Fares; Gürsoy, Mervi; Neves de Oliveira, Ben-Hur; Özdemir, Vural; Könönen, Eija; Gürsoy, Ulvi K.

    2016-01-01

    Periodontitis, a formidable global health burden, is a common chronic disease that destroys tooth-supporting tissues. Biomarkers of the early phase of this progressive disease are of utmost importance for global health. In this context, saliva represents a non-invasive biosample. By using systems biology tools, we aimed to (1) identify an integrated interactome between matrix metalloproteinase (MMP)-REDOX/nitric oxide (NO) and apoptosis upstream pathways of periodontal inflammation, and (2) characterize the attendant topological network properties to uncover putative biomarkers to be tested in saliva from patients with periodontitis. Hence, we first generated a protein-protein network model of interactions (“BIOMARK” interactome) by using the STRING 10 database, a search tool for the retrieval of interacting genes/proteins, with “Experiments” and “Databases” as input options and a confidence score of 0.400. Second, we determined the centrality values (closeness, stress, degree or connectivity, and betweenness) for the “BIOMARK” members by using the Cytoscape software. We found Ubiquitin C (UBC), Jun proto-oncogene (JUN), and matrix metalloproteinase-14 (MMP14) as the most central hub- and non-hub-bottlenecks among the 211 genes/proteins of the whole interactome. We conclude that UBC, JUN, and MMP14 are likely an optimal candidate group of host-derived biomarkers, in combination with oral pathogenic bacteria-derived proteins, for detecting periodontitis at its early phase by using salivary samples from patients. These findings therefore have broader relevance for systems medicine in global health as well. PMID:26793622

  12. Campylobacter jejuni serine protease HtrA plays an important role in heat tolerance, oxygen resistance, host cell adhesion, invasion, and transmigration.

    PubMed

    Boehm, Manja; Lind, Judith; Backert, Steffen; Tegtmeyer, Nicole

    2015-03-01

    Campylobacter jejuni is an important pathogen of foodborne illness. Transmigration across the intestinal epithelial barrier and invasion are considered as primary reasons for tissue damage triggered by C. jejuni. Using knockout mutants, it was shown that the serine protease HtrA may be important for stress tolerance and physiology of C. jejuni. HtrA is also secreted in the extra-cellular environment, where it can cleave junctional host cell proteins such as E-cadherin. Aim of the present study was to establish a genetic complementation system in two C. jejuni strains in order to introduce the wild-type htrA gene in trans, test known htrA phenotypes, and provide the basis to perform further mutagenesis. We confirm that reexpression of the htrA wild-type gene in ΔhtrA mutants restored the following phenotypes: 1) C. jejuni growth at high temperature (44 °C), 2) growth under high oxygen stress conditions, 3) expression of proteolytically active HtrA oligomers, 4) secretion of HtrA into the supernatant, 5) cell attachment and invasion, and 6) transmigration across polarized epithelial cells. These results establish a genetic complementation system for htrA in C. jejuni, exclude polar effects in the ΔhtrA mutants, confirm important HtrA properties, and permit the discovery and dissection of new functions.

  13. Campylobacter jejuni serine protease HtrA plays an important role in heat tolerance, oxygen resistance, host cell adhesion, invasion, and transmigration

    PubMed Central

    Lind, Judith; Backert, Steffen; Tegtmeyer, Nicole

    2015-01-01

    Campylobacter jejuni is an important pathogen of foodborne illness. Transmigration across the intestinal epithelial barrier and invasion are considered as primary reasons for tissue damage triggered by C. jejuni. Using knockout mutants, it was shown that the serine protease HtrA may be important for stress tolerance and physiology of C. jejuni. HtrA is also secreted in the extra­cellular environment, where it can cleave junctional host cell proteins such as E-cadherin. Aim of the present study was to establish a genetic complementation system in two C. jejuni strains in order to introduce the wild-type htrA gene in trans, test known htrA phenotypes, and provide the basis to perform further mutagenesis. We confirm that reexpression of the htrA wild-type gene in ΔhtrA mutants restored the following phenotypes: 1) C. jejuni growth at high temperature (44 °C), 2) growth under high oxygen stress conditions, 3) expression of proteolytically active HtrA oligomers, 4) secretion of HtrA into the supernatant, 5) cell attachment and invasion, and 6) transmigration across polarized epithelial cells. These results establish a genetic complementation system for htrA in C. jejuni, exclude polar effects in the ΔhtrA mutants, confirm important HtrA properties, and permit the discovery and dissection of new functions. PMID:25883795

  14. The S-layer Associated Serine Protease Homolog PrtX Impacts Cell Surface-Mediated Microbe-Host Interactions of Lactobacillus acidophilus NCFM.

    PubMed

    Johnson, Brant R; O'Flaherty, Sarah; Goh, Yong Jun; Carroll, Ian; Barrangou, Rodolphe; Klaenhammer, Todd R

    2017-01-01

    Health-promoting aspects attributed to probiotic microorganisms, including adhesion to intestinal epithelia and modulation of the host mucosal immune system, are mediated by proteins found on the bacterial cell surface. Notably, certain probiotic and commensal bacteria contain a surface (S-) layer as the outermost stratum of the cell wall. S-layers are non-covalently bound semi-porous, crystalline arrays of self-assembling, proteinaceous subunits called S-layer proteins (SLPs). Recent evidence has shown that multiple proteins are non-covalently co-localized within the S-layer, designated S-layer associated proteins (SLAPs). In Lactobacillus acidophilus NCFM, SLP and SLAPs have been implicated in both mucosal immunomodulation and adhesion to the host intestinal epithelium. In this study, a S-layer associated serine protease homolog, PrtX (prtX, lba1578), was deleted from the chromosome of L. acidophilus NCFM. Compared to the parent strain, the PrtX-deficient strain (ΔprtX) demonstrated increased autoaggregation, an altered cellular morphology, and pleiotropic increases in adhesion to mucin and fibronectin, in vitro. Furthermore, ΔprtX demonstrated increased in vitro immune stimulation of IL-6, IL-12, and IL-10 compared to wild-type, when exposed to mouse dendritic cells. Finally, in vivo colonization of germ-free mice with ΔprtX led to an increase in epithelial barrier integrity. The absence of PrtX within the exoproteome of a ΔprtX strain caused morphological changes, resulting in a pleiotropic increase of the organisms' immunomodulatory properties and interactions with some intestinal epithelial cell components.

  15. The S-layer Associated Serine Protease Homolog PrtX Impacts Cell Surface-Mediated Microbe-Host Interactions of Lactobacillus acidophilus NCFM

    PubMed Central

    Johnson, Brant R.; O’Flaherty, Sarah; Goh, Yong Jun; Carroll, Ian; Barrangou, Rodolphe; Klaenhammer, Todd R.

    2017-01-01

    Health-promoting aspects attributed to probiotic microorganisms, including adhesion to intestinal epithelia and modulation of the host mucosal immune system, are mediated by proteins found on the bacterial cell surface. Notably, certain probiotic and commensal bacteria contain a surface (S-) layer as the outermost stratum of the cell wall. S-layers are non-covalently bound semi-porous, crystalline arrays of self-assembling, proteinaceous subunits called S-layer proteins (SLPs). Recent evidence has shown that multiple proteins are non-covalently co-localized within the S-layer, designated S-layer associated proteins (SLAPs). In Lactobacillus acidophilus NCFM, SLP and SLAPs have been implicated in both mucosal immunomodulation and adhesion to the host intestinal epithelium. In this study, a S-layer associated serine protease homolog, PrtX (prtX, lba1578), was deleted from the chromosome of L. acidophilus NCFM. Compared to the parent strain, the PrtX-deficient strain (ΔprtX) demonstrated increased autoaggregation, an altered cellular morphology, and pleiotropic increases in adhesion to mucin and fibronectin, in vitro. Furthermore, ΔprtX demonstrated increased in vitro immune stimulation of IL-6, IL-12, and IL-10 compared to wild-type, when exposed to mouse dendritic cells. Finally, in vivo colonization of germ-free mice with ΔprtX led to an increase in epithelial barrier integrity. The absence of PrtX within the exoproteome of a ΔprtX strain caused morphological changes, resulting in a pleiotropic increase of the organisms’ immunomodulatory properties and interactions with some intestinal epithelial cell components. PMID:28713337

  16. Proteases in bacterial pathogenesis.

    PubMed

    Ingmer, Hanne; Brøndsted, Lone

    2009-11-01

    Bacterial pathogens rely on proteolysis for protein quality control under adverse conditions experienced in the host, as well as for the timely degradation of central virulence regulators. We have focused on the contribution of the conserved Lon, Clp, HtrA and FtsH proteases to pathogenesis and have highlighted common biological processes for which their activities are important for virulence.

  17. Synthesis of macrocyclic trypanosomal cysteine protease inhibitors.

    PubMed

    Chen, Yen Ting; Lira, Ricardo; Hansell, Elizabeth; McKerrow, James H; Roush, William R

    2008-11-15

    The importance of cysteine proteases in parasites, compounded with the lack of redundancy compared to their mammalian hosts makes proteases attractive targets for the development of new therapeutic agents. The binding mode of K11002 to cruzain, the major cysteine protease of Trypanosoma cruzi was used in the design of conformationally constrained inhibitors. Vinyl sulfone-containing macrocycles were synthesized via olefin ring-closing metathesis and evaluated against cruzain and the closely related cysteine protease, rhodesain.

  18. Bacterial proteases in IBD and IBS.

    PubMed

    Steck, Natalie; Mueller, Kerstin; Schemann, Michael; Haller, Dirk

    2012-11-01

    Proteases play a decisive role in health and disease. They fulfil diverse functions and have been associated with the pathology of gastrointestinal disorders such as inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS). The current knowledge focuses on host-derived proteases including matrix metalloproteinases, various serine proteases and cathepsins. The possible contribution of bacterial proteases has been largely ignored in the pathogenesis of IBD and IBS, although there is increasing evidence, especially demonstrated for proteases from pathogenic bacteria. The underlying mechanisms extend to proteases from commensal bacteria which may be relevant for disease susceptibility. The intestinal microbiota and its proteolytic capacity exhibit the potential to contribute to the pathogenesis of IBD and IBS. This review highlights the relevance of host- and bacteria-derived proteases and their signalling mechanisms.

  19. Host regulation of lysogenic decision in bacteriophage λ: Transmembrane modulation of FtsH (HflB), the cII degrading protease, by HflKC (HflA)

    PubMed Central

    Kihara, Akio; Akiyama, Yoshinori; Ito, Koreaki

    1997-01-01

    The cII gene product of bacteriophage λ is unstable and required for the establishment of lysogenization. Its intracellular amount is important for the decision between lytic growth and lysogenization. Two genetic loci of Escherichia coli are crucial for these commitments of infecting λ genome. One of them, hflA encodes the HflKC membrane protein complex, which has been believed to be a protease degrading the cII protein. However, both its absence and overproduction stabilized cII in vivo and the proposed serine protease-like sequence motif in HflC was dispensable for the lysogenization control. Moreover, the HflKC protein was found to reside on the periplasmic side of the plasma membrane. In contrast, the other host gene, ftsH (hflB) encoding an integral membrane ATPase/protease, is positively required for degradation of cII, since loss of its function stabilized cII and its overexpression accelerated the cII degradation. In vitro, purified FtsH catalyzed ATP-dependent proteolysis of cII and HflKC antagonized the FtsH action. These results, together with our previous finding that FtsH and HflKC form a complex, suggest that FtsH is the cII degrading protease and HflKC is a modulator of the FtsH function. We propose that this transmembrane modulation differentiates the FtsH actions to different substrate proteins such as the membrane-bound SecY protein and the cytosolic cII protein. This study necessitates a revision of the prevailing view about the host control over λ lysogenic decision. PMID:9159109

  20. 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)

  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. Cathepsin proteases in Toxoplasma gondii

    PubMed Central

    Dou, Zhicheng; Carruthers, Vern B.

    2014-01-01

    Cysteine proteases are important for the growth and survival of apicomplexan parasites that infect humans. The apicomplexan Toxoplasma gondii expresses five members of the C1 family of cysteine proteases, including one cathepsin L-like (TgCPL), one cathepsin B-like (TgCPB), and three cathepsin C-like (TgCPC1, 2 and 3) proteases. Recent genetic, biochemical and structural studies reveal that cathepsins function in microneme and rhoptry protein maturation, host cell invasion, replication, and nutrient acquisition.. Here, we review the key features and roles of T. gondii cathepsins and discuss the therapeutic potential for specific inhibitor development. PMID:21660658

  3. 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.

  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. Identification of a Putative Salmonella enterica Serotype Typhimurium Host Range Factor with Homology to IpaH and YopM by Signature-Tagged Mutagenesis

    PubMed Central

    Tsolis, Renée M.; Townsend, Stacy M.; Miao, Edward A.; Miller, Samuel I.; Ficht, Thomas A.; Adams, L. Garry; Bäumler, Andreas J.

    1999-01-01

    The genetic basis for the host adaptation of Salmonella serotypes is currently unknown. We have explored a new strategy to identify Salmonella enterica serotype Typhimurium (S. typhimurium) genes involved in host adaptation, by comparing the virulence of 260 randomly generated signature-tagged mutants during the oral infection of mice and calves. This screen identified four mutants, which were defective for colonization of only one of the two host species tested. One mutant, which only displayed a colonization defect during the infection of mice, was further characterized. During competitive infection experiments performed with the S. typhimurium wild type, the mutant was defective for colonization of murine Peyer's patches but colonized bovine Peyer's patches at the wild-type level. No difference in virulence between wild type and mutant was observed when calves were infected orally with 1010 CFU/animal. In contrast, the mutant possessed a sixfold increase in 50% lethal morbidity dose when mice were infected orally. The transposon in this mutant was inserted in a 2.9-kb pathogenicity islet, which is located between uvrB and yphK on the S. typhimurium chromosome. This pathogenicity islet contained a single gene, termed slrP, with homology to ipaH of Shigella flexneri and yopM of Yersinia pestis. These data show that comparative screening of signature-tagged mutants in two animal species can be used for scanning the S. typhimurium genome for genes involved in host adaptation. PMID:10569754

  6. Structural and Immunological Characteristics of a 28-Kilodalton Cruzipain-Like Cysteine Protease of Paragonimus westermani Expressed in the Definitive Host Stage

    PubMed Central

    Yun, Doo-Hee; Chung, Joon-Yong; Chung, Young-Bae; Bahk, Young-Yil; Kang, Shin-Yong; Kong, Yoon; Cho, Seung-Yull

    2000-01-01

    A complete cDNA sequence encoding a 28-kDa cruzipain-like cysteine protease of adult Paragonimus westermani, termed Pw28CCP, was isolated from an adult cDNA library. The cDNA contained a single open reading frame of 975 bp encoding 325 amino acids, which exhibited the structural motif and domain organization characteristic of cysteine proteases of non-cathepsin Bs including a hydrophobic signal sequence, an ERFNIN motif, and essential cysteine residues as well as active sites in the mature catalytic region. Analysis of its phylogenetic position revealed that this novel enzyme belonged to the cruzipain-like cysteine proteases. The sequence of the first 13 amino acids predicted from the mature domain of Pw28CCP was in accord with that determined from the native 28-kDa enzyme purified from the adult worm. Expression of Pw28CCP was observed specifically in juvenile and adult worms, with a location in the intestinal epithelium, suggesting that this enzyme could be secreted and involved in nutrient uptake and immune modulation. The recombinant protein expressed in Escherichia coli was used to assess antigenicity by immunoblotting with sera from patients with active paragonimiasis and from those with other parasitic infections. The resulting sensitivity of 86.2% (56 of 65 samples) and specificity of 98% (147 of 150 samples) suggest its potential as an antigen for use in immunodiagnosis. PMID:11063501

  7. Molecular profile and functional characterization of the ferritin H subunit from rock bream (Oplegnathus fasciatus), revealing its putative role in host antioxidant and immune defense.

    PubMed

    Elvitigala, Don Anushka Sandaruwan; Priyathilaka, Thanthrige Thiunuwan; Lim, Bong-Soo; Whang, Ilson; Yeo, Sang-Yeob; Choi, Cheol Young; Lee, Jehee

    2014-11-01

    Ferritins are iron binding proteins made out of 24 subunits, involved in iron homeostasis and metabolism in cellular environments. Here, we sought to identify and functionally characterize a one type of subunits of ferritin (ferritin H-like subunit) from rock bream (Oplegnathus fasciatus; RbFerH). The complete coding sequence of RbFerH was 531 bp in length, encoding a 177-amino acid protein with a predicted molecular mass of 20.8 kDa. The deduced protein structure possessed the domain architecture characteristic of known ferritin H subunits, including metal ligands for iron binding, a ferroxidase center, and two iron-binding region signatures. As expected, the 5' untranslated region of the RbFerH cDNA sequence contained a putative iron response element region, a characteristic regulatory element involved in its translation. The RbFerH gene comprised 5 exons and 4 introns spanning a 4195 bp region. Overexpressed recombinant RbFerH protein demonstrated prominent Fe(II) ion depriving activity, bacteriostatic properties, and protective effects against oxidative double-stranded DNA damage. Using quantitative polymerase chain reaction (qPCR), we found that RbFerH was expressed ubiquitously in the majority of physiologically important tissues in rock bream. A greater abundance of the mRNA transcripts were detected in blood and liver tissues. Upon administering different microbial pathogens and pathogen-derived mitogens, RbFerH transcription was markedly elevated in the blood of rock bream. Taken together, our findings suggest that RbFerH acts as a potent iron sequestrator in rock bream and may actively participate in antimicrobial as well as antioxidative defense. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. Stage-specific excretory-secretory small heat shock proteins from the parasitic nematode Strongyloides ratti--putative links to host's intestinal mucosal defense system.

    PubMed

    Younis, Abuelhassan Elshazly; Geisinger, Frank; Ajonina-Ekoti, Irene; Soblik, Hanns; Steen, Hanno; Mitreva, Makedonka; Erttmann, Klaus D; Perbandt, Markus; Liebau, Eva; Brattig, Norbert W

    2011-09-01

    In a search for molecules involved in the interaction between intestinal nematodes and mammalian mucosal host cells, we performed MS to identify excretory-secretory proteins from Strongyloides ratti. In the excretory-secretory proteins of the parasitic female stage, we detected, in addition to other peptides, peptides homologous with the Caenorhabditis elegans heat shock protein (HSP)-17, named Sra-HSP-17.1 (∼ 19 kDa) and Sra-HSP-17.2 (∼ 18 kDa), with 49% amino acid identity. The full-length cDNAs (483 bp and 474 bp, respectively) were identified, and the genomic organization was analyzed. To allow further characterization, the proteins were recombinantly expressed and purified. Profiling of transcription by quantitative real-time-PCR and of protein by ELISA in various developmental stages revealed parasitic female-specific expression. Sequence analyses of both the DNA and amino acid sequences showed that the two proteins share a conserved α-crystallin domain and variable N-terminals. The Sra-HSP-17s showed the highest homology with the deduced small HSP sequence of the human pathogen Strongyloides stercoralis. We observed strong immunogenicity of both proteins, leading to strong IgG responses following infection of rats. Flow cytometric analysis indicated the binding of Sra-HSP-17s to the monocyte-macrophage lineage but not to peripheral lymphocytes or neutrophils. A rat intestinal epithelial cell line showed dose-dependent binding to Sra-HSP-17.1, but not to Sra-HSP-17.2. Exposed monocytes released interleukin-10 but not tumor necrosis factor-α in response to Sra-HSP-17s, suggesting the possible involvement of secreted female proteins in host immune responses. © 2011 The Authors Journal compilation © 2011 FEBS.

  9. Expression and characterization of Coprothermobacter proteolyticus alkaline serine protease

    USDA-ARS?s Scientific Manuscript database

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

  10. Cell Death Triggered by a Putative Amphipathic Helix of Radish mosaic virus Helicase Protein Is Tightly Correlated With Host Membrane Modification.

    PubMed

    Hashimoto, Masayoshi; Komatsu, Ken; Iwai, Ryo; Keima, Takuya; Maejima, Kensaku; Shiraishi, Takuya; Ishikawa, Kazuya; Yoshida, Tetsuya; Kitazawa, Yugo; Okano, Yukari; Yamaji, Yasuyuki; Namba, Shigetou

    2015-06-01

    Systemic necrosis is one of the most severe symptoms caused by plant RNA viruses. Recently, systemic necrosis has been suggested to have similar features to a defense response referred to as the hypersensitive response (HR), a form of programmed cell death. In virus-infected plant cells, host intracellular membrane structures are changed dramatically for more efficient viral replication. However, little is known about whether this replication-associated membrane modification is the cause of the symptoms. In this study, we identified an amino-terminal amphipathic helix of the helicase encoded by Radish mosaic virus (RaMV) (genus Comovirus) as an elicitor of cell death in RaMV-infected plants. Cell death caused by the amphipathic helix had features similar to HR, such as SGT1-dependence. Mutational analyses and inhibitor assays using cerulenin demonstrated that the amphipathic helix-induced cell death was tightly correlated with dramatic alterations in endoplasmic reticulum (ER) membrane structures. Furthermore, the cell death-inducing activity of the amphipathic helix was conserved in Cowpea mosaic virus (genus Comovirus) and Tobacco ringspot virus (genus Nepovirus), both of which are classified in the family Secoviridae. Together, these results indicate that ER membrane modification associated with viral intracellular replication may be recognized to prime defense responses against plant viruses.

  11. Small-RNA deep sequencing reveals Arctium tomentosum as a natural host of Alstroemeria virus X and a new putative Emaravirus.

    PubMed

    Bi, Yaqi; Tugume, Arthur K; Valkonen, Jari P T

    2012-01-01

    Arctium species (Asteraceae) are distributed worldwide and are used as food and rich sources of secondary metabolites for the pharmaceutical industry, e.g., against avian influenza virus. RNA silencing is an antiviral defense mechanism that detects and destroys virus-derived double-stranded RNA, resulting in accumulation of virus-derived small RNAs (21-24 nucleotides) that can be used for generic detection of viruses by small-RNA deep sequencing (SRDS). SRDS was used to detect viruses in the biennial wild plant species Arctium tomentosum (woolly burdock; family Asteraceae) displaying virus-like symptoms of vein yellowing and leaf mosaic in southern Finland. Assembly of the small-RNA reads resulted in contigs homologous to Alstroemeria virus X (AlsVX), a positive/single-stranded RNA virus of genus Potexvirus (family Alphaflexiviridae), or related to negative/single-stranded RNA viruses of the genus Emaravirus. The coat protein gene of AlsVX was 81% and 89% identical to the two AlsVX isolates from Japan and Norway, respectively. The deduced, partial nucleocapsid protein amino acid sequence of the emara-like virus was only 78% or less identical to reported emaraviruses and showed no variability among the virus isolates characterized. This virus--tentatively named as Woolly burdock yellow vein virus--was exclusively associated with yellow vein and leaf mosaic symptoms in woolly burdock, whereas AlsVX was detected in only one of the 52 plants tested. These results provide novel information about natural virus infections in Acrtium species and reveal woolly burdock as the first natural host of AlsVX besides Alstroemeria (family Alstroemeriaceae). Results also revealed a new virus related to the recently emerged Emaravirus genus and demonstrated applicability of SRDS to detect negative-strand RNA viruses. SRDS potentiates virus surveys of wild plants, a research area underrepresented in plant virology, and helps reveal natural reservoirs of viruses that cause yield losses

  12. Small-RNA Deep Sequencing Reveals Arctium tomentosum as a Natural Host of Alstroemeria virus X and a New Putative Emaravirus

    PubMed Central

    Bi, Yaqi; Tugume, Arthur K.; Valkonen, Jari P. T.

    2012-01-01

    Background Arctium species (Asteraceae) are distributed worldwide and are used as food and rich sources of secondary metabolites for the pharmaceutical industry, e.g., against avian influenza virus. RNA silencing is an antiviral defense mechanism that detects and destroys virus-derived double-stranded RNA, resulting in accumulation of virus-derived small RNAs (21–24 nucleotides) that can be used for generic detection of viruses by small-RNA deep sequencing (SRDS). Methodology/Principal Findings SRDS was used to detect viruses in the biennial wild plant species Arctium tomentosum (woolly burdock; family Asteraceae) displaying virus-like symptoms of vein yellowing and leaf mosaic in southern Finland. Assembly of the small-RNA reads resulted in contigs homologous to Alstroemeria virus X (AlsVX), a positive/single-stranded RNA virus of genus Potexvirus (family Alphaflexiviridae), or related to negative/single-stranded RNA viruses of the genus Emaravirus. The coat protein gene of AlsVX was 81% and 89% identical to the two AlsVX isolates from Japan and Norway, respectively. The deduced, partial nucleocapsid protein amino acid sequence of the emara-like virus was only 78% or less identical to reported emaraviruses and showed no variability among the virus isolates characterized. This virus—tentatively named as Woolly burdock yellow vein virus—was exclusively associated with yellow vein and leaf mosaic symptoms in woolly burdock, whereas AlsVX was detected in only one of the 52 plants tested. Conclusions/Significance These results provide novel information about natural virus infections in Acrtium species and reveal woolly burdock as the first natural host of AlsVX besides Alstroemeria (family Alstroemeriaceae). Results also revealed a new virus related to the recently emerged Emaravirus genus and demonstrated applicability of SRDS to detect negative-strand RNA viruses. SRDS potentiates virus surveys of wild plants, a research area underrepresented in plant virology

  13. The nuclear protein Sam68 is cleaved by the FMDV 3C protease redistributing Sam68 to the cytoplasm during FMDV infection of host cells

    USDA-ARS?s Scientific Manuscript database

    Infection by a variety of viruses alters the nuclear-cytoplasmic trafficking of certain host cell proteins. In our continued search for interacting factors, we reported the re-localization of RNA helicase A (RHA) from the nucleus to the cytoplasm in cells infected with foot-and-mouth disease virus ...

  14. 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

  15. Establishing the Yeast Kluyveromyces lactis as an Expression Host for Production of the Saposin-Like Domain of the Aspartic Protease Cirsin

    PubMed Central

    Curto, Pedro; Lufrano, Daniela; Pinto, Cátia; Custódio, Valéria; Gomes, Ana Catarina; Trejo, Sebastián A.; Bakás, Laura; Vairo-Cavalli, Sandra; Faro, Carlos

    2014-01-01

    Typical plant aspartic protease zymogens comprise a characteristic and plant-specific insert (PSI). PSI domains can interact with membranes, and a role as a defensive weapon against pathogens has been proposed. However, the potential of PSIs as antimicrobial agents has not been fully investigated and explored yet due to problems in producing sufficient amounts of these domains in bacteria. Here, we report the development of an expression platform for the production of the PSI domain of cirsin in the generally regarded as safe (GRAS) yeast Kluyveromyces lactis. We successfully generated K. lactis transformants expressing and secreting significant amounts of correctly processed and glycosylated PSI, as well as its nonglycosylated mutant. A purification protocol with protein yields of ∼4.0 mg/liter was established for both wild-type and nonglycosylated PSIs, which represents the highest reported yield for a nontagged PSI domain. Subsequent bioactivity assays targeting phytopathogenic fungi indicated that the PSI of cirsin is produced in a biologically active form in K. lactis and provided clear evidence for its antifungal activity. This yeast expression system thereby emerges as a promising production platform for further exploring the biotechnological potential of these plant saposin-like proteins. PMID:24123748

  16. Yeast extracellular proteases.

    PubMed

    Ogrydziak, D M

    1993-01-01

    Many species of yeast secrete significant amounts of protease(s). In this article, results of numerous surveys of yeast extracellular protease production have been compiled and inconsistencies in the data and limitations of the methodology have been examined. Regulation, purification, characterization, and processing of yeast extracellular proteases are reviewed. Results obtained from the sequences of cloned genes, especially the Saccharomyces cerevisiae Bar protease, the Candida albicans acid protease, and the Yarrowia lipolytica alkaline protease, have been emphasized. Biotechnological applications and the medical relevance of yeast extracellular proteases are covered. Yeast extracellular proteases have potential in beer and wine stabilization, and they probably contribute to pathogenicity of Candida spp. Yeast extracellular protease genes also provide secretion and processing signals for yeast expression systems designed for secretion of heterologous proteins. Coverage of the secretion of foreign proteases such as prochymosin, urokinase, and tissue plasminogen activator by yeast in included.

  17. Mycoplasma hyopneumoniae Surface proteins Mhp385 and Mhp384 bind host cilia and glycosaminoglycans and are endoproteolytically processed by proteases that recognize different cleavage motifs.

    PubMed

    Deutscher, Ania T; Tacchi, Jessica L; Minion, F Chris; Padula, Matthew P; Crossett, Ben; Bogema, Daniel R; Jenkins, Cheryl; Kuit, Tracey A; Walker, Mark J; Djordjevic, Steven P

    2012-03-02

    P97 and P102 paralogues occur as endoproteolytic cleavage fragments on the surface of Mycoplasma hyopneumoniae that bind glycosaminoglycans, plasminogen, and fibronectin and perform essential roles in colonization of ciliated epithelia. We show that the P102 paralogue Mhp384 is efficiently cleaved at an S/T-X-F↓X-D/E-like site, creating P60(384) and P50(384). The P97 paralogue Mhp385 is inefficiently cleaved, with tryptic peptides from a 115 kDa protein (P115(385)) and 88 kDa (P88(385)) and 27 kDa (P27(385)) cleavage fragments identified by LC-MS/MS. This is the first time a preprotein belonging to the P97 and P102 paralogue families has been identified by mass spectrometry. The semitryptic peptide (752)IQFELEPISLNV(763) denotes the C-terminus of P88(385) and defines the novel cleavage site (761)L-N-V↓A-V-S(766) in Mhp385. P115(385), P88(385), P27(385), P60(384), and P50(384) were shown to reside extracellularly, though it is unknown how the fragments remain attached to the cell surface. Heparin- and cilium-binding sites were identified within P60(384), P50(384), and P88(385). No primary function was attributed to P27(385); however, this molecule contains four tandem R1 repeats with similarity to porcine collagen type VI (α3 chain). P97 and P102 paralogue families are adhesins targeted by several proteases with different cleavage efficiencies, and this process generates combinatorial complexity on the surface of M. hyopneumoniae.

  18. Mast Cell Proteases and Inflammation

    PubMed Central

    Dai, Hongyan; Korthuis, Ronald J.

    2011-01-01

    Mast cells are best known for their role in allergic reactions but are also now recognized for their important contributions to a number of disparate inflammatory conditions through the release of inflammatory mediators, serglycin and other proteoglycans, and proteases. Because these tissue resident inflammatory cells express proteases in such great abundance and their enzymatic activity results in cleavage of a multitude of proteins and peptides, which in turn modify tissue function, their substrate specificity, tissue distribution, and mode of action have become the subjects of great interest. Although mast cell protease-dependent proteolysis is critical to host defense against invading pathogens, regulation of these hydrolytic enzymes is essential to limiting self-induced damage as well. Indeed, dysregulated release of mast cell proteases is now recognized to contribute to the pathogenesis of a number of inflammatory conditions including asthma, abdominal aortic aneurysm formation, vessel damage in atherosclerosis and hypertension, arthritis, and ischemia/reperfusion injury. Understanding how mast cell proteases contribute to inflammation will thus help unravel molecular mechanisms that underlie such immunologic disorders and will help identify new therapeutic targets for drug development. PMID:22125569

  19. 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)

  20. 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)

  1. Identification and Characterization of IgdE, a Novel IgG-degrading Protease of Streptococcus suis with Unique Specificity for Porcine IgG*

    PubMed Central

    Spoerry, Christian; Seele, Jana; Valentin-Weigand, Peter; Baums, Christoph G.; von Pawel-Rammingen, Ulrich

    2016-01-01

    Streptococcus suis is a major endemic pathogen of pigs causing meningitis, arthritis, and other diseases. Zoonotic S. suis infections are emerging in humans causing similar pathologies as well as severe conditions such as toxic shock-like syndrome. Recently, we discovered an IdeS family protease of S. suis that exclusively cleaves porcine IgM and represents the first virulence factor described, linking S. suis to pigs as their natural host. Here we report the identification and characterization of a novel, unrelated protease of S. suis that exclusively targets porcine IgG. This enzyme, designated IgdE for immunoglobulin G-degrading enzyme of S. suis, is a cysteine protease distinct from previous characterized streptococcal immunoglobulin degrading proteases of the IdeS family and mediates efficient cleavage of the hinge region of porcine IgG with a high degree of specificity. The findings that all S. suis strains investigated possess the IgG proteolytic activity and that piglet serum samples contain specific antibodies against IgdE strongly indicate that the protease is expressed in vivo during infection and represents a novel and putative important bacterial virulence/colonization determinant, and a thus potential therapeutic target. PMID:26861873

  2. Dual Mutation Events in the Haemagglutinin-Esterase and Fusion Protein from an Infectious Salmon Anaemia Virus HPR0 Genotype Promote Viral Fusion and Activation by an Ubiquitous Host Protease.

    PubMed

    Fourrier, Mickael; Lester, Katherine; Markussen, Turhan; Falk, Knut; Secombes, Christopher J; McBeath, Alastair; Collet, Bertrand

    2015-01-01

    In Infectious salmon anaemia virus (ISAV), deletions in the highly polymorphic region (HPR) in the near membrane domain of the haemagglutinin-esterase (HE) stalk, influence viral fusion. It is suspected that selected mutations in the associated Fusion (F) protein may also be important in regulating fusion activity. To better understand the underlying mechanisms involved in ISAV fusion, several mutated F proteins were generated from the Scottish Nevis and Norwegian SK779/06 HPR0. Co-transfection with constructs encoding HE and F were performed, fusion activity assessed by content mixing assay and the degree of proteolytic cleavage by western blot. Substitutions in Nevis F demonstrated that K276 was the most likely cleavage site in the protein. Furthermore, amino acid substitutions at three sites and two insertions, all slightly upstream of K276, increased fusion activity. Co-expression with HE harbouring a full-length HPR produced high fusion activities when trypsin and low pH were applied. In comparison, under normal culture conditions, groups containing a mutated HE with an HPR deletion were able to generate moderate fusion levels, while those with a full length HPR HE could not induce fusion. This suggested that HPR length may influence how the HE primes the F protein and promotes fusion activation by an ubiquitous host protease and/or facilitate subsequent post-cleavage refolding steps. Variations in fusion activity through accumulated mutations on surface glycoproteins have also been reported in other orthomyxoviruses and paramyxoviruses. This may in part contribute to the different virulence and tissue tropism reported for HPR0 and HPR deleted ISAV genotypes.

  3. Identification of a Putative Coreceptor on Vero Cells That Participates in Dengue 4 Virus Infection

    PubMed Central

    Martínez-Barragán, José de Jesús; del Angel, Rosa M.

    2001-01-01

    Dengue virus infects target cells by attaching to a cell surface receptor through the envelope (E) glycoprotein, located on the surface of the viral membrane. On Vero and BHK cells, heparan sulfate (HS) moieties of proteoglycans are the receptors for dengue virus; however, additional proteins have also been described as putative dengue virus receptors on C6/36, HL60, and BM cells. HS can also act as a receptor for other types of viruses or as an attachment molecule for viruses that require additional host cell molecules to allow viral penetration. In this study we searched for molecules other than HS that could participate in dengue virus infection of Vero cells. Labeled dengue 4 virus bound with high affinity to two molecules of 74 and 44 kDa. Binding of dengue virus to the 74-kDa molecule was susceptible to protease and sodium periodate treatment and resistant to heparinase treatments. Lectins such as concanavalin A and wheat germ agglutinin prevented dengue virus binding to both the 74- and the 44-kDa protein in overlay assays, while phytohemagglutinin P did not affect binding, suggesting that carbohydrate residues (α-mannose or N-acetylglucosamine) are important in virus binding to host cells. Protease susceptibility, biotin labeling, and immunofluorescence with a polyclonal antibody raised against the 74-kDa protein consistently identified the protein on the surfaces of Vero cells. Moreover, the antibody against the 74-kDa protein was able to inhibit dengue virus infection. These data suggest that HS might serve as a primary receptor, probably concentrating virus particles on the surfaces of Vero cells, and then other molecules, such as the 74-kDa protein, might participate as coreceptors in viral penetration. The 74-kDa protein possibly constitutes part of a putative receptor complex for dengue virus infection of Vero cells. PMID:11483725

  4. Protease Inhibitors Targeting Coronavirus and Filovirus Entry

    PubMed Central

    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

    2016-01-01

    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

  5. Proteases as therapeutics

    PubMed Central

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

    2015-01-01

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

  6. Acid protease production in fungal root endophytes.

    PubMed

    Mayerhofer, Michael S; Fraser, Erica; Kernaghan, Gavin

    2015-01-01

    Fungal endophytes are ubiquitous in healthy root tissue, but little is known about their ecosystem functions, including their ability to utilize organic nutrient sources such as proteins. Root-associated fungi may secrete proteases to access the carbon and mineral nutrients within proteins in the soil or in the cells of their plant host. We compared the protein utilization patterns of multiple isolates of the root endophytes Phialocephala fortinii s.l., Meliniomyces variabilis and Umbelopsis isabellina with those of two ectomycorrhizal (ECM) fungi, Hebeloma incarnatulum and Laccaria bicolor, and the wood-decay fungus Irpex lacteus at pH values of 2-9 on liquid BSA media. We also assessed protease activity using a fluorescently labeled casein assay and gelatin zymography and characterized proteases using specific protease inhibitors. I. lacteus and U. isabellina utilized protein efficiently, while the ECM fungi exhibited poor protein utilization. ECM fungi secreted metallo-proteases and had pH optima above 4, while other fungi produced aspartic proteases with lower pH optima. The ascomycetous root endophytes M. variabilis and P. fortinii exhibited intermediate levels of protein utilization and M. variabilis exhibited a very low pH optimum. Comparing proteolytic profiles between fungal root endophytes and fungi with well defined ecological roles provides insight into the ecology of these cryptic root associates.

  7. Cloning and analysis of WF146 protease, a novel thermophilic subtilisin-like protease with four inserted surface loops.

    PubMed

    Wu, Jiang; Bian, Yan; Tang, Bing; Chen, Xiangdong; Shen, Ping; Peng, Zhenrong

    2004-01-30

    Cloning and sequencing of the gene encoding WF146 protease, an extracellular subtilisin-like protease from the thermophile Bacillus sp. WF146, revealed that the WF146 protease was translated as a 416-amino acid precursor consisting of a putative 18-amino acid signal peptide, a 10-kDa N-terminal propeptide and a 32-kDa mature protease region. The mature WF146 protease shares a high degree of amino acid sequence identity with two psychrophilic subtilisins, S41 (68.2%) and S39 (65.4%), and a mesophilic subtilisin, SSII (67.1%). Significantly, these closely related proteases adapted to different temperatures all had four inserted surface loops not found in other subtilisins. However, unlike those of S41, S39 and SSII, the inserted loops of the WF146 protease possessed stabilizing features, such as the introduction of Pro residues into the loop regions. Interestingly, the WF146 protease contained five of the seven mutations previously found in a hyperstable variant of subtilisin S41 obtained by directed evolution. The proform of WF146 protease (pro-WF146 protease) was overexpressed in Escherichia coli in an inactive soluble form. After heat treatment, the 42-kDa pro-WF146 protease converted to a 32-kDa active mature form by processing the N-terminal propeptide. The purified mature WF146 protease hydrolyzed casein with an optimum temperature of 85 degrees C, and lost activity with a half-life of 30 min at 80 degrees C in the presence of 10 mM CaCl2.

  8. Crystal structures of the viral protease Npro imply distinct roles for the catalytic water in catalysis.

    PubMed

    Zögg, Thomas; Sponring, Michael; Schindler, Sabrina; Koll, Maria; Schneider, Rainer; Brandstetter, Hans; Auer, Bernhard

    2013-06-04

    Npro is a key effector protein of pestiviruses such as bovine viral diarrhea virus and abolishes host cell antiviral defense mechanisms. Synthesized as the N-terminal part of the viral polyprotein, Npro releases itself via an autoproteolytic cleavage, triggering its immunological functions. However, the mechanisms of its proteolytic action and its immune escape were unclear. Here, we present the crystal structures of Npro to 1.25 Å resolution. Structures of pre- and postcleavage intermediates identify three catalytically relevant elements. The trapping of the putative catalytic water reveals its distinct roles as a base, acid, and nucleophile. The presentation of the substrate further explains the enigmatic latency of the protease, ensuring a single in cis cleavage. Additionally, we identified a zinc-free, disulfide-linked conformation of the TRASH motif, an interaction hub of immune factors. The structure opens additional opportunities in utilizing Npro as an autocleaving fusion protein and as a pharmaceutical target.

  9. Serine protease activity in developmental stages of Eimeria tenella.

    PubMed

    Fetterer, R H; Miska, K B; Lillehoj, H; Barfield, R C

    2007-04-01

    A number of complex processes are involved in Eimeria spp. survival, including control of sporulation, intracellular invasion, evasion of host immune responses, successful reproduction, and nutrition. Proteases have been implicated in many of these processes, but the occurrence and functions of serine proteases have not been characterized. Bioinformatic analysis suggests that the Eimeria tenella genome contains several serine proteases that lack homology to trypsin. Using RT-PCR, a gene encoding a subtilisin-like and a rhomboid protease-like serine protease was shown to be developmentally regulated, both being poorly expressed in sporozoites (SZ) and merozoites (MZ). Casein substrate gel electrophoresis of oocyst extracts during sporulation demonstrated bands of proteolytic activity with relative molecular weights (Mr) of 18, 25, and 45 kDa that were eliminated by coincubation with serine protease inhibitors. A protease with Mr of 25 kDa was purified from extracts of unsporulated oocysts by a combination of affinity and anion exchange chromatography. Extracts of SZ contained only a single band of inhibitor-sensitive proteolytic activity at 25 kDa, while the pattern of proteases from extracts of MZ was similar to that of oocysts except for the occurrence of a 90 kDa protease, resistant to protease inhibitors. Excretory-secretory products (ESP) from MZ contained AEBSF (4-[2-Aminoethyl] benzenesulphonyl fluoride)-sensitive protease activity with a specific activity about 10 times greater than that observed in MZ extracts. No protease activity was observed in the ESP from SZ. Pretreatment of SZ with AEBSF significantly reduced SZ invasion and the release of the microneme protein, MIC2. The current results suggest that serine proteases are present in all the developmental stages examined.

  10. Secretion of Proteases by an Opportunistic Fungal Pathogen Scedosporium aurantiacum

    PubMed Central

    Kautto, Liisa; Nevalainen, Helena

    2017-01-01

    Scedosporium aurantiacum is an opportunistic filamentous fungus increasingly isolated from the sputum of cystic fibrosis patients, and is especially prevalent in Australia. At the moment, very little is known about the infection mechanism of this fungus. Secreted proteases have been shown to contribute to fungal virulence in several studies with other fungi. Here we have compared the profiles of proteases secreted by a clinical isolate Scedosporium aurantiacum (WM 06.482) and an environmental strain (WM 10.136) grown on a synthetic cystic fibrosis sputum medium supplemented with casein or mucin. Protease activity was assessed using class-specific substrates and inhibitors. Subtilisin-like and trypsin-like serine protease activity was detected in all cultures. The greatest difference in the secretion of proteases between the two strains occurred in mucin-supplemented medium, where the activities of the elastase-like, trypsin-like and aspartic proteases were, overall, 2.5–75 fold higher in the clinical strain compared to the environmental strain. Proteases secreted by the two strains in the mucin-supplemented medium were further analyzed by mass spectrometry. Six homologs of fungal proteases were identified from the clinical strain and five from the environmental strain. Of these, three were common for both strains including a subtilisin peptidase, a putative leucine aminopeptidase and a PA-SaNapH-like protease. Trypsin-like protease was identified by mass spectrometry only in the clinical isolate even though trypsin-like activity was present in all cultures. In contrast, high elastase-like activity was measured in the culture supernatant of the clinical strain but could not be identified by mass spectrometry searching against other fungi in the NCBI database. Future availability of an annotated genome will help finalise identification of the S. aurantiacum proteases. PMID:28060882

  11. Secretion of Proteases by an Opportunistic Fungal Pathogen Scedosporium aurantiacum.

    PubMed

    Han, Zhiping; Kautto, Liisa; Nevalainen, Helena

    2017-01-01

    Scedosporium aurantiacum is an opportunistic filamentous fungus increasingly isolated from the sputum of cystic fibrosis patients, and is especially prevalent in Australia. At the moment, very little is known about the infection mechanism of this fungus. Secreted proteases have been shown to contribute to fungal virulence in several studies with other fungi. Here we have compared the profiles of proteases secreted by a clinical isolate Scedosporium aurantiacum (WM 06.482) and an environmental strain (WM 10.136) grown on a synthetic cystic fibrosis sputum medium supplemented with casein or mucin. Protease activity was assessed using class-specific substrates and inhibitors. Subtilisin-like and trypsin-like serine protease activity was detected in all cultures. The greatest difference in the secretion of proteases between the two strains occurred in mucin-supplemented medium, where the activities of the elastase-like, trypsin-like and aspartic proteases were, overall, 2.5-75 fold higher in the clinical strain compared to the environmental strain. Proteases secreted by the two strains in the mucin-supplemented medium were further analyzed by mass spectrometry. Six homologs of fungal proteases were identified from the clinical strain and five from the environmental strain. Of these, three were common for both strains including a subtilisin peptidase, a putative leucine aminopeptidase and a PA-SaNapH-like protease. Trypsin-like protease was identified by mass spectrometry only in the clinical isolate even though trypsin-like activity was present in all cultures. In contrast, high elastase-like activity was measured in the culture supernatant of the clinical strain but could not be identified by mass spectrometry searching against other fungi in the NCBI database. Future availability of an annotated genome will help finalise identification of the S. aurantiacum proteases.

  12. Serine proteases inhibiting cyanopeptides.

    PubMed

    Radau, G

    2000-08-01

    There are many compounds inhibiting serine proteases which play an important role in the human organism. This article reviews publications on the low-molecular weight, serine protease inhibitory cyanopeptides and reports on new developments in establishing structure-activity relationships.

  13. Serine Protease(s) Secreted by the Nematode Trichuris muris Degrade the Mucus Barrier

    PubMed Central

    Hasnain, Sumaira Z.; McGuckin, Michael A.; Grencis, Richard K.; Thornton, David J.

    2012-01-01

    The polymeric mucin component of the intestinal mucus barrier changes during nematode infection to provide not only physical protection but also to directly affect pathogenic nematodes and aid expulsion. Despite this, the direct interaction of the nematodes with the mucins and the mucus barrier has not previously been addressed. We used the well-established Trichuris muris nematode model to investigate the effect on mucins of the complex mixture of immunogenic proteins secreted by the nematode called excretory/secretory products (ESPs). Different regimes of T. muris infection were used to simulate chronic (low dose) or acute (high dose) infection. Mucus/mucins isolated from mice and from the human intestinal cell line, LS174T, were treated with ESPs. We demonstrate that serine protease(s) secreted by the nematode have the ability to change the properties of the mucus barrier, making it more porous by degrading the mucin component of the mucus gel. Specifically, the serine protease(s) acted on the N-terminal polymerising domain of the major intestinal mucin Muc2, resulting in depolymerisation of Muc2 polymers. Importantly, the respiratory/gastric mucin Muc5ac, which is induced in the intestine and is critical for worm expulsion, was protected from the depolymerising effect exerted by ESPs. Furthermore, serine protease inhibitors (Serpins) which may protect the mucins, in particular Muc2, from depolymerisation, were highly expressed in mice resistant to chronic infection. Thus, we demonstrate that nematodes secrete serine protease(s) to degrade mucins within the mucus barrier, which may modify the niche of the parasite to prevent clearance from the host or facilitate efficient mating and egg laying from the posterior end of the parasite that is in intimate contact with the mucus barrier. However, during a TH2-mediated worm expulsion response, serpins, Muc5ac and increased levels of Muc2 protect the barrier from degradation by the nematode secreted protease(s). PMID

  14. Neutrophil serine proteases in antibacterial defense.

    PubMed

    Stapels, Daphne A C; Geisbrecht, Brian V; Rooijakkers, Suzan H M

    2015-02-01

    Neutrophil serine proteases (NSPs) are critical for the effective functioning of neutrophils and greatly contribute to immune protection against bacterial infections. Thanks to their broad substrate specificity, these chymotrypsin-like proteases trigger multiple reactions that are detrimental to bacterial survival such as direct bacterial killing, generation of antimicrobial peptides, inactivation of bacterial virulence factors and formation of neutrophil extracellular traps. Recently, the importance of NSPs in antibacterial defenses has been further underscored by discoveries of unique bacterial evasion strategies to combat these proteases. Bacteria can indirectly disarm NSPs by protecting bacterial substrates against NSP cleavage, but also produce inhibitory molecules that potently block NSPs. Here we review recent insights in the functional contribution of NSPs in host protection against bacterial infections and the elegant strategies that bacteria use to counteract these responses. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Insect response to plant defensive protease inhibitors.

    PubMed

    Zhu-Salzman, Keyan; Zeng, Rensen

    2015-01-07

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

  16. Miropin, a novel bacterial serpin from the periodontopathogen Tannerella forsythia, inhibits a broad range of proteases by using different peptide bonds within the reactive center loop.

    PubMed

    Ksiazek, Miroslaw; Mizgalska, Danuta; Enghild, Jan J; Scavenius, Carsten; Thogersen, Ida B; Potempa, Jan

    2015-01-02

    All prokaryotic genes encoding putative serpins identified to date are found in environmental and commensal microorganisms, and only very few prokaryotic serpins have been investigated from a mechanistic standpoint. Herein, we characterized a novel serpin (miropin) from the human pathogen Tannerella forsythia, a bacterium implicated in initiation and progression of human periodontitis. In contrast to other serpins, miropin efficiently inhibited a broad range of proteases (neutrophil and pancreatic elastases, cathepsin G, subtilisin, and trypsin) with a stoichiometry of inhibition of around 3 and second-order association rate constants that ranged from 2.7 × 10(4) (cathepsin G) to 7.1 × 10(5) m(-1)s(-1) (subtilisin). Inhibition was associated with the formation of complexes that were stable during SDS-PAGE. The unusually broad specificity of miropin for target proteases is achieved through different active sites within the reactive center loop upstream of the P1-P1' site, which was predicted from an alignment of the primary structure of miropin with those of well studied human and prokaryotic serpins. Thus, miropin is unique among inhibitory serpins, and it has apparently evolved the ability to inhibit a multitude of proteases at the expense of a high stoichiometry of inhibition and a low association rate constant. These characteristics suggest that miropin arose as an adaptation to the highly proteolytic environment of subgingival plaque, which is exposed continually to an array of host proteases in the inflammatory exudate. In such an environment, miropin may function as an important virulence factor by protecting bacterium from the destructive activity of neutrophil serine proteases. Alternatively, it may act as a housekeeping protein that regulates the activity of endogenous T. forsythia serine proteases.

  17. Membrane vesicles released by Avibacterium paragallinarum contain putative virulence factors.

    PubMed

    Ramón Rocha, Marcela O; García-González, Octavio; Pérez-Méndez, Alma; Ibarra-Caballero, Jorge; Pérez-Márquez, Victor M; Vaca, Sergio; Negrete-Abascal, Erasmo

    2006-04-01

    Avibacterium paragallinarum, the causative agent of infectious coryza, releases extracellular membrane vesicles (MVs), containing immunogenic proteins, proteases, putative RTX proteins, haemagglutinin, and nucleic acids, into the medium. MVs ranging 50-300 nm in diameter were observed by electron microscopy. They contained immunogenic proteins in the range of 20-160 kDa, detected using vaccinated or experimentally infected chicken sera raised against Av. paragallinarum, but not in pooled sera from specific pathogen-free chickens. Proteolytic activity was not detected in MVs through zymograms; however, immune recognition of high molecular mass bands was observed by Western blotting using an antiprotease serum against Actinobacillus pleuropneumoniae serotype 1 purified protease, suggesting its presence. MVs agglutinated glutaraldehyde-fixed chicken red blood cells indicating the presence of haemagglutinating antigens. Nucleic acids were also detected inside MVs. Avibacterium paragallinarum releases MVs containing putative virulence factors, which could be important in the pathogenesis of infectious coryza.

  18. Putative archaeal viruses from the mesopelagic ocean.

    PubMed

    Vik, Dean R; Roux, Simon; Brum, Jennifer R; Bolduc, Ben; Emerson, Joanne B; Padilla, Cory C; Stewart, Frank J; Sullivan, Matthew B

    2017-01-01

    Oceanic viruses that infect bacteria, or phages, are known to modulate host diversity, metabolisms, and biogeochemical cycling, while the viruses that infect marine Archaea remain understudied despite the critical ecosystem roles played by their hosts. Here we introduce "MArVD", for Metagenomic Archaeal Virus Detector, an annotation tool designed to identify putative archaeal virus contigs in metagenomic datasets. MArVD is made publicly available through the online iVirus analytical platform. Benchmarking analysis of MArVD showed it to be >99% accurate and 100% sensitive in identifying the 127 known archaeal viruses among the 12,499 viruses in the VirSorter curated dataset. Application of MArVD to 10 viral metagenomes from two depth profiles in the Eastern Tropical North Pacific (ETNP) oxygen minimum zone revealed 43 new putative archaeal virus genomes and large genome fragments ranging in size from 10 to 31 kb. Network-based classifications, which were consistent with marker gene phylogenies where available, suggested that these putative archaeal virus contigs represented six novel candidate genera. Ecological analyses, via fragment recruitment and ordination, revealed that the diversity and relative abundances of these putative archaeal viruses were correlated with oxygen concentration and temperature along two OMZ-spanning depth profiles, presumably due to structuring of the host Archaea community. Peak viral diversity and abundances were found in surface waters, where Thermoplasmata 16S rRNA genes are prevalent, suggesting these archaea as hosts in the surface habitats. Together these findings provide a baseline for identifying archaeal viruses in sequence datasets, and an initial picture of the ecology of such viruses in non-extreme environments.

  19. Putative archaeal viruses from the mesopelagic ocean

    PubMed Central

    Roux, Simon; Brum, Jennifer R.; Bolduc, Ben; Emerson, Joanne B.; Padilla, Cory C.; Stewart, Frank J.; Sullivan, Matthew B.

    2017-01-01

    Oceanic viruses that infect bacteria, or phages, are known to modulate host diversity, metabolisms, and biogeochemical cycling, while the viruses that infect marine Archaea remain understudied despite the critical ecosystem roles played by their hosts. Here we introduce “MArVD”, for Metagenomic Archaeal Virus Detector, an annotation tool designed to identify putative archaeal virus contigs in metagenomic datasets. MArVD is made publicly available through the online iVirus analytical platform. Benchmarking analysis of MArVD showed it to be >99% accurate and 100% sensitive in identifying the 127 known archaeal viruses among the 12,499 viruses in the VirSorter curated dataset. Application of MArVD to 10 viral metagenomes from two depth profiles in the Eastern Tropical North Pacific (ETNP) oxygen minimum zone revealed 43 new putative archaeal virus genomes and large genome fragments ranging in size from 10 to 31 kb. Network-based classifications, which were consistent with marker gene phylogenies where available, suggested that these putative archaeal virus contigs represented six novel candidate genera. Ecological analyses, via fragment recruitment and ordination, revealed that the diversity and relative abundances of these putative archaeal viruses were correlated with oxygen concentration and temperature along two OMZ-spanning depth profiles, presumably due to structuring of the host Archaea community. Peak viral diversity and abundances were found in surface waters, where Thermoplasmata 16S rRNA genes are prevalent, suggesting these archaea as hosts in the surface habitats. Together these findings provide a baseline for identifying archaeal viruses in sequence datasets, and an initial picture of the ecology of such viruses in non-extreme environments. PMID:28630803

  20. Specificity characterization of the α-mating factor hormone by Kex2 protease.

    PubMed

    Manfredi, Marcella Araújo; Antunes, Alyne Alexandrino; Jesus, Larissa de Oliveira Passos; Juliano, Maria Aparecida; Juliano, Luiz; Judice, Wagner Alves de Souza

    2016-12-01

    Kex2 is a Ca(2+)-dependent serine protease from S. cerevisiae. Characterization of the substrate specificity of Kex2 is of particular interest because this protease serves as the prototype of a large family of eukaryotic subtilisin-related proprotein-processing proteases that cleave sites consisting of pairs or clusters of basic residues. Our goal was to study the prime region subsite S' of Kex2 because previous studies have only taken into account non-prime sites using AMC substrates but not the specificity of prime sites identified through structural modeling or predicted cleavage sites. Therefore, we used peptides derived from Abz-KR↓EADQ-EDDnp and Abz-YKR↓EADQ-EDDnp based on the pro-α-mating factor sequence. The specificity of Kex2 due to basic residues at P1' is affected by the type of residue in the P3 position. Some residues in P1' with large or bulky side chains yielded poor substrate specificity. The kcat/KM values for peptides with P2' substitutions containing Tyr in P3 were higher than those obtained for the peptides without Tyr. In fact, P' and P modifications mainly promoted changes in kcat and KM, respectively. The pH profile of Kex2 was fit to a double-sigmoidal pH-titration curve. The specificity results suggest that Kex2 might be involved in the processing of the putative cleavage sites in a polypeptide involved in cell elongation, hyphal formation and the processing of a toxin, which result in host cell lysis. In summary, the specificity of Kex2 is dependent on the set of interactions with prime and non-prime subsites, resulting in synergism. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  1. S1 Pocket of a Bacterially Derived Subtilisin-like Protease Underpins Effective Tissue Destruction*

    PubMed Central

    Wong, Wilson; Wijeyewickrema, Lakshmi C.; Kennan, Ruth M.; Reeve, Shane B.; Steer, David L.; Reboul, Cyril; Smith, A. Ian; Pike, Robert N.; Rood, Julian I.; Whisstock, James C.; Porter, Corrine J.

    2011-01-01

    The ovine footrot pathogen, Dichelobacter nodosus, secretes three subtilisin-like proteases that play an important role in the pathogenesis of footrot through their ability to mediate tissue destruction. Virulent and benign strains of D. nodosus secrete the basic proteases BprV and BprB, respectively, with the catalytic domain of these enzymes having 96% sequence identity. At present, it is not known how sequence variation between these two putative virulence factors influences their respective biological activity. We have determined the high resolution crystal structures of BprV and BprB. These data reveal that that the S1 pocket of BprV is more hydrophobic but smaller than that of BprB. We show that BprV is more effective than BprB in degrading extracellular matrix components of the host tissue. Mutation of two residues around the S1 pocket of BprB to the equivalent residues in BprV dramatically enhanced its proteolytic activity against elastin substrates. Application of a novel approach for profiling substrate specificity, the Rapid Endopeptidase Profiling Library (REPLi) method, revealed that both enzymes prefer cleaving after hydrophobic residues (and in particular P1 leucine) but that BprV has more restricted primary substrate specificity than BprB. Furthermore, for P1 Leu-containing substrates we found that BprV is a significantly more efficient enzyme than BprB. Collectively, these data illuminate how subtle changes in D. nodosus proteases may significantly influence tissue destruction as part of the ovine footrot pathogenesis process. PMID:21990366

  2. [Chloroplast Deg proteases].

    PubMed

    Grabsztunowicz, Magda; Luciński, Robert; Baranek, Małgorzata; Sikora, Bogna; Jackowski, Grzegorz

    2011-01-01

    For some chloroplast proteases ATP binding and hydrolysis is not necessary for their catalytic activity, most probably because even strongly unfolded substrates may penetrate their catalytic chamber. Deg1, 2, 5 and 8 are the best known of Arabidopsis thaliana ATP- independent chloroplast proteases, encoded by orthologues of genes coding for DegP, DegQ and DegS proteases of Escherichia coli. Current awareness in the area of structure and functions of chloroplast Degs is much more limited vs the one about their bacterial counterparts. Deg5 and Deg8 form a catalytic heterododecamer which is loosely attached to luminal side of thylakoid membrane. The complex catalyses--supported by Deg1 and one of FtsH proteases--the degradation of PsbA damaged due to plant exposition to elevated irradiance and thus these protease are of key importance for the plants' sensitivity to photoinhibition. Deg2 role in the disposal of damaged PsbA has not been elucidated. Recombinant Deg1 may degrade PsbO and plastocyanin in vitro but it is not clear whether this reaction is performed in vivo as well.

  3. Protease inhibitor studies enrolling.

    PubMed

    1995-01-01

    The protease enzyme is essential for HIV to make copies of itself. So far, research has failed to find a protease inhibitor that works against HIV. It is believed that, regardless of what type of protease inhibitor someone takes, it will need to be supplemented with other anti-HIV drugs. Three protease inhibitors have thus far been found to be safe, although long-term effects are unknown. These drugs are saquinavir, ABT-538, and L-735,524 produced by Hoffman-LaRoche, Abbott, and Merck respectively. Clinical trials of saquinavir are promising but it has not been shown to be the knock-out drug needed. ABT-538 has high bioavailability, but studies are showing it can cause liver and eye damage. L-735,524 studies are showing that resistance develops quite quickly. Future studies at higher doses are expected. To obtain information on protease studies currently looking for participants, contact The Network. Information on other approved, alternative, and experimental drugs is also available.

  4. The effects of bioprocess parameters on extracellular proteases in a recombinant Aspergillus niger B1-D.

    PubMed

    Li, Qiang; Harvey, Linda M; McNeil, Brian

    2008-02-01

    Although host proteases are often considered to have a negative impact upon heterologous protein production by filamentous fungi, relatively little is known about the pattern of their appearance in recombinant fungal bioprocesses. In the present study, we investigated extracellular proteases from a filamentous fungus, Aspergillus niger B1-D, genetically modified to secrete hen egg white lysozyme (HEWL). Our findings indicate that extracellular protease activity is only detected after the carbon source is completely utilised in batch cultures. The proteases are predominantly acid proteases and have optimal temperature for activity at around 45 degrees C. Their activity could be partially inhibited by protease inhibitors, indicating the existence of at least four kinds of proteases in these culture fluids, aspartic-, serine-, cysteine-, and metallo-proteases. Oxygen enrichment does not have any noticeable effects on extracellular protease activity except that the onset of protease activity appears earlier in oxygen enrichment runs. Oxygen enrichment stimulates HEWL production substantially, and we propose that it is related to fungal morphology. Thermal stress imposed by raising process temperature (from 25 to 30 and 35 degrees C) in early exponential phase, led to appearance of protease activity in the medium following the heat shock. Continued cultivation at high temperatures significantly reduced HEWL production, which was associated with increased activity of the extracellular proteases in these cultures.

  5. Host-pathogen interactions: A cholera surveillance system

    SciTech Connect

    Wright, Aaron T.

    2016-02-22

    Bacterial pathogen-secreted proteases may play a key role in inhibiting a potentially widespread host-pathogen interaction. Activity-based protein profiling enabled the identification of a major Vibrio cholerae serine protease that limits the ability of a host-derived intestinal lectin to bind to the bacterial pathogen in vivo.

  6. Pnserpin: A Novel Serine Protease Inhibitor from Extremophile Pyrobaculum neutrophilum

    PubMed Central

    Zhang, Huan; Fei, Rui; Xue, Baigong; Yu, Shanshan; Zhang, Zuoming; Zhong, Sheng; Gao, Yuanqi; Zhou, Xiaoli

    2017-01-01

    Serine protease inhibitors (serpins) are native inhibitors of serine proteases, constituting a large protein family with members spread over eukaryotes and prokaryotes. However, only very few prokaryotic serpins, especially from extremophiles, have been characterized to date. In this study, Pnserpin, a putative serine protease inhibitor from the thermophile Pyrobaculum neutrophilum, was overexpressed in Escherichia coli for purification and characterization. It irreversibly inhibits chymotrypsin-, trypsin-, elastase-, and subtilisin-like proteases in a temperature range from 20 to 100 °C in a concentration-dependent manner. The stoichiometry of inhibition (SI) of Pnserpin for proteases decreases as the temperature increases, indicating that the inhibitory activity of Pnserpin increases with the temperature. SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) showed that Pnserpin inhibits proteases by forming a SDS-resistant covalent complex. Homology modeling and molecular dynamic simulations predicted that Pnserpin can form a stable common serpin fold. Results of the present work will help in understanding the structural and functional characteristics of thermophilic serpin and will broaden the current knowledge about serpins from extremophiles. PMID:28067849

  7. Zika Virus Protease: An Antiviral Drug Target.

    PubMed

    Kang, CongBao; Keller, Thomas H; Luo, Dahai

    2017-10-01

    The recent outbreak of Zika virus (ZIKV) infection has caused global concern due to its link to severe damage to the brain development of foetuses and neuronal complications in adult patients. A worldwide research effort has been undertaken to identify effective and safe treatment and vaccination options. Among the proposed viral and host components, the viral NS2B-NS3 protease represents an attractive drug target due to its essential role in the virus life cycle. Here, we outline recent progress in studies on the Zika protease. Biochemical, biophysical, and structural studies on different protease constructs provide new insight into the structure and activity of the protease. The unlinked construct displays higher enzymatic activity and better mimics the native state of the enzyme and therefore is better suited for drug discovery. Furthermore, the structure of the free enzyme adopts a closed conformation and a preformed active site. The availability of a lead fragment hit and peptide inhibitors, as well as the attainability of soakable crystals, suggest that the unlinked construct is a promising tool for drug discovery. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. The site-2 protease.

    PubMed

    Rawson, Robert B

    2013-12-01

    The site-2 protease (S2P) is an unusually-hydrophobic integral membrane protease. It cleaves its substrates, which are membrane-bound transcription factors, within membrane-spanning helices. Although structural information for S2P from animals is lacking, the available data suggest that cleavage may occur at or within the lipid bilayer. In mammalian cells, S2P is essential owing to its activation of the sterol regulatory element binding proteins (SREBPs); in the absence of exogenous lipid, cells lacking S2P cannot survive. S2P is also important in the endoplasmic reticulum (ER) stress response, activating several different membrane-bound transcription factors. Human patients harboring reduction-of-function mutations in S2P exhibit an array of pathologies ranging from skin defects to neurological abnormalities. Surprisingly, Drosophila melanogaster lacking S2P are viable and fertile. This article is part of a Special Issue entitled: Intramembrane Proteases.

  9. A Genomic Analysis of Rat Proteases and Protease Inhibitors

    PubMed Central

    Puente, Xose S.; López-Otín, Carlos

    2004-01-01

    Proteases perform important roles in multiple biological and pathological processes. The availability of the rat genome sequence has facilitated the analysis of the complete protease repertoire or degradome of this model organism. The rat degradome consists of at least 626 proteases and homologs, which are distributed into 24 aspartic, 160 cysteine, 192 metallo, 221 serine, and 29 threonine proteases. This distribution is similar to that of the mouse degradome but is more complex than that of the human degradome composed of 561 proteases and homologs. This increased complexity of rat proteases mainly derives from the expansion of several families, including placental cathepsins, testases, kallikreins, and hematopoietic serine proteases, involved in reproductive or immunological functions. These protease families have also evolved differently in rat and mouse and may contribute to explain some functional differences between these closely related species. Likewise, genomic analysis of rat protease inhibitors has shown some differences with mouse protease inhibitors and the expansion of families of cysteine and serine protease inhibitors in rodents with respect to human. These comparative analyses may provide new views on the functional diversity of proteases and inhibitors and contribute to the development of innovative strategies for treating proteolysis diseases. PMID:15060002

  10. 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.

  11. Peptide synthesis in neat organic solvents with novel thermostable proteases.

    PubMed

    Toplak, Ana; Nuijens, Timo; Quaedflieg, Peter J L M; Wu, Bian; Janssen, Dick B

    2015-06-01

    Biocatalytic peptide synthesis will benefit from enzymes that are active at low water levels in organic solvent compositions that allow good substrate and product solubility. To explore the use of proteases from thermophiles for peptide synthesis under such conditions, putative protease genes of the subtilase class were cloned from Thermus aquaticus and Deinococcus geothermalis and expressed in Escherichia coli. The purified enzymes were highly thermostable and catalyzed efficient peptide bond synthesis at 80°C and 60°C in neat acetonitrile with excellent conversion (>90%). The enzymes tolerated high levels of N,N-dimethylformamide (DMF) as a cosolvent (40-50% v/v), which improved substrate solubility and gave good conversion in 5+3 peptide condensation reactions. The results suggest that proteases from thermophiles can be used for peptide synthesis under harsh reaction conditions.

  12. The serine protease autotransporter Tsh contributes to the virulence of Edwardsiella tarda.

    PubMed

    Hu, Yong-Hua; Zhou, Hai-Zhen; Jin, Qian-Wen; Zhang, Jian

    2016-06-30

    The temperature-sensitive hemagglutinin (Tsh), identified as serine protease autotransporters of the Enterobacteriaceae (SPATEs) proteins, is an important virulence factor for avian-pathogenic Escherichia coli (APEC) and uropathogenic E. coli. However, little is known about the role of Tsh as a virulence factor in Edwardsiella tarda, a severe fish pathogen. In this study, we characterized the Tsh of E. tarda (named TshEt) and examined its function and vaccine potential. TshEt is composed of 1224 residues and has three functional domains typical for autotransporters. Quantitative real-time reverse transcriptase-PCR analysis showed that expression of tshEt was upregulated under conditions of high temperature, increased cell density, high pH, and iron starvation and during the infection of host cells. A markerless tsh in-frame mutant strain, TX01Δtsh, was constructed to determine whether TshEt participates in the pathogenicity of E. tarda, Compared to the wild type TX01, TX01Δtsh exhibited (i) retarded biofilm growth, (ii) decreased resistance against serum killing, (iii) impaired ability to block the host immune response, (iv) attenuated tissue and cellular infectivity. Introduction of a trans-expressed tsh gene restored the lost virulence of TX01Δtsh. The passenger domain of TshEt contains a putative serine protease (PepS) that exhibits apparent proteolytic activity when expressed in and purified from E. coli as a recombinant protein (rPepS). When used as a subunit vaccine to immunize Japanese flounder, rPepS was able to induce effective immune protection. This is the first study of Tsh in a fish pathogen, and the results suggest that TshEt exerts pleiotropic effects on the pathogenesis of E. tarda.

  13. Metal-Mediated Modulation of Streptococcal Cysteine Protease Activity and Its Biological Implications

    PubMed Central

    Chella Krishnan, Karthickeyan; Mukundan, Santhosh; Landero Figueroa, Julio A.; Caruso, Joseph A.

    2014-01-01

    Streptococcal cysteine protease (SpeB), the major secreted protease produced by group A streptococcus (GAS), cleaves both host and bacterial proteins and contributes importantly to the pathogenesis of invasive GAS infections. Modulation of SpeB expression and/or its activity during invasive GAS infections has been shown to affect bacterial virulence and infection severity. Expression of SpeB is regulated by the GAS CovR-CovS two-component regulatory system, and we demonstrated that bacteria with mutations in the CovR-CovS two-component regulatory system are selected for during localized GAS infections and that these bacteria lack SpeB expression and exhibit a hypervirulent phenotype. Additionally, in a separate study, we showed that expression of SpeB can also be modulated by human transferrin- and/or lactoferrin-mediated iron chelation. Accordingly, the goal of this study was to investigate the possible roles of iron and other metals in modulating SpeB expression and/or activity in a manner that would potentiate bacterial virulence. Here, we report that the divalent metals zinc and copper inhibit SpeB activity at the posttranslational level. Utilizing online metal-binding site prediction servers, we identified two putative metal-binding sites in SpeB, one of which involves the catalytic-dyad residues 47Cys and 195His. Based on our findings, we propose that zinc and/or copper availability in the bacterial microenvironment can modulate the proteolytic activity of SpeB in a manner that preserves the integrity of several other virulence factors essential for bacterial survival and dissemination within the host and thereby may exacerbate the severity of invasive GAS infections. PMID:24799625

  14. The AAA+ ATPases and HflB/FtsH proteases of 'Candidatus Phytoplasma mali': phylogenetic diversity, membrane topology, and relationship to strain virulence.

    PubMed

    Seemüller, Erich; Sule, Sandor; Kube, Michael; Jelkmann, Wilhelm; Schneider, Bernd

    2013-03-01

    Previous examination revealed a correlation of phytopathogenic data of 'Candidatus Phytoplasma mali' strains and the DNA sequence variability of a type ATP00464 hflB gene fragment. To further investigate such a relationship, all distinct genes previously annotated as hflB in the genome of 'Ca. P. mali' strain AT were fully sequenced and analyzed from a number of representative mild, moderate, and severe strains. The re-annotation indicated that the sequences encode six AAA+ ATPases and six HflB proteases. Each of the nine distinct deduced AAA+ proteins that were examined formed a coherent phylogenetic cluster. However, within these groups, sequences of three ATPases and three proteases from mild and severe strains clustered distantly, according to their virulence. This grouping was supported by an association with virulence-related amino acid substitutions. Another finding was that full-length genes from ATPase AP11 could only be identified in mild and moderate strains. Prediction of the membrane topology indicated that the long ATPase- and protease-carrying C-terminal tails of approximately half of the AAA+ proteins are extracellular, putatively facing the environment of the sieve tubes. Thus, they may be involved in pathogen-host interactions and may compromise phloem function, a major effect of phytoplasma infection. All full-length genes examined appear transcriptionally active and all deduced peptides show the key positions indicative for protein function.

  15. Rapid qualitative protease microassay (RPM).

    PubMed

    Mohan, S; Ma, P W K; Luthe, D S

    2005-09-30

    A rapid qualitative protease microassay (RPM) was developed as an alternative to conventional assays of cysteine protease activity in HPLC fractions. Using this technique protease activity in samples could be visually determined within 5 min. The method was sensitive to 3.3x10(-7) U/mL of papain and detected cysteine protease activity in dilute HPLC fractions with activity of 5.4x10(-5) U/mL. Because the method monitors the decolorization of Coomassie Brilliant Blue stained substrate, it can be modified to detect other classes of proteases.

  16. Ecology of phlebotomine sandflies and putative reservoir hosts of leishmaniasis in a border area in Northeastern Mexico: implications for the risk of transmission of Leishmania mexicana in Mexico and the USA.

    PubMed

    Rodríguez-Rojas, Jorge J; Rodríguez-Moreno, Ángel; Berzunza-Cruz, Miriam; Gutiérrez-Granados, Gabriel; Becker, Ingeborg; Sánchez-Cordero, Victor; Stephens, Christopher R; Fernández-Salas, Ildefonso; Rebollar-Téllez, Eduardo A

    2017-01-01

    Leishmaniases are a group of important diseases transmitted to humans through the bite of sandfly vectors. Several forms of leishmaniases are endemic in Mexico and especially in the Southeast region. In the Northeastern region, however, there have only been isolated reports of cases and scanty records of sandfly vectors. The main objective of this study was to analyze the diversity of sandflies and potential reservoir hosts of Leishmania spp. in the states of Nuevo León and Tamaulipas. Species richness and abundances of sandflies and rodents were recorded. A fraction of the caught sandflies was analyzed by PCR to detect Leishmania spp. Tissues from captured rodents were also screened for infection. Ecological Niche Models (ENMs) were computed for species of rodent and their association with crop-growing areas. We found 13 species of sandflies, several of which are first records for this region. Medically important species such as Lutzomyia anthophora, Lutzomyia diabolica, Lutzomyia cruciata, and Lutzomyia shannoni were documented. Leishmania spp. infection was not detected in sandflies. Nine species of rodents were recorded, and Leishmania (Leishmania) mexicana infection was found in four species of Peromyscus and Sigmodon. ENMs showed that potential distribution of rodent pest species overlaps with allocated crop areas. This shows that Leishmania (L.) mexicana infection is present in the Northeastern region of Mexico, and that previously unrecorded sandfly species occur in the same areas. These findings suggest a potential risk of transmission of Leishmania (L.) mexicana. © J. Rodríguez-Rojas et al., published by EDP Sciences, 2017.

  17. Ecology of phlebotomine sandflies and putative reservoir hosts of leishmaniasis in a border area in Northeastern Mexico: implications for the risk of transmission of Leishmania mexicana in Mexico and the USA

    PubMed Central

    Rodríguez-Rojas, Jorge J.; Rodríguez-Moreno, Ángel; Berzunza-Cruz, Miriam; Gutiérrez-Granados, Gabriel; Becker, Ingeborg; Sánchez-Cordero, Victor; Stephens, Christopher R.; Fernández-Salas, Ildefonso; Rebollar-Téllez, Eduardo A.

    2017-01-01

    Leishmaniases are a group of important diseases transmitted to humans through the bite of sandfly vectors. Several forms of leishmaniases are endemic in Mexico and especially in the Southeast region. In the Northeastern region, however, there have only been isolated reports of cases and scanty records of sandfly vectors. The main objective of this study was to analyze the diversity of sandflies and potential reservoir hosts of Leishmania spp. in the states of Nuevo León and Tamaulipas. Species richness and abundances of sandflies and rodents were recorded. A fraction of the caught sandflies was analyzed by PCR to detect Leishmania spp. Tissues from captured rodents were also screened for infection. Ecological Niche Models (ENMs) were computed for species of rodent and their association with crop-growing areas. We found 13 species of sandflies, several of which are first records for this region. Medically important species such as Lutzomyia anthophora, Lutzomyia diabolica, Lutzomyia cruciata, and Lutzomyia shannoni were documented. Leishmania spp. infection was not detected in sandflies. Nine species of rodents were recorded, and Leishmania (Leishmania) mexicana infection was found in four species of Peromyscus and Sigmodon. ENMs showed that potential distribution of rodent pest species overlaps with allocated crop areas. This shows that Leishmania (L.) mexicana infection is present in the Northeastern region of Mexico, and that previously unrecorded sandfly species occur in the same areas. These findings suggest a potential risk of transmission of Leishmania (L.) mexicana. PMID:28825400

  18. A primitive enzyme for a primitive cell: the protease required for excystation of Giardia.

    PubMed

    Ward, W; Alvarado, L; Rawlings, N D; Engel, J C; Franklin, C; McKerrow, J H

    1997-05-02

    Protozoan parasites of the genus Giardia are one of the earliest lineages of eukaryotic cells. To initiate infection, trophozoites emerge from a cyst in the host. Excystation is blocked by specific cysteine protease inhibitors. Using a biotinylated inhibitor, the target protease was identified and its corresponding gene cloned. The protease was localized to vesicles that release their contents just prior to excystation. The Giardia protease is the earliest known branch of the cathepsin B family. Its phylogeny confirms that the cathepsin B lineage evolved in primitive eukaryotic cells, prior to the divergence of plant and animal kingdoms, and underscores the diversity of cellular functions that this enzyme family facilitates.

  19. Analyzing Protease Specificity and Detecting in Vivo Proteolytic Events Using Tandem Mass Spectrometry

    SciTech Connect

    Gupta, Nitin; Hixson, Kim K.; Culley, David E.; Smith, Richard D.; Pevzner, Pavel A.

    2010-07-01

    While trypsin remains the most commonly used protease in mass spectrometry, other proteases may be employed for increasing peptide-coverage or generating overlapping peptides. Knowledge of the accurate specifcity rules of these proteases is helpful for database search tools to detect peptides, and becomes crucial when mass spectrometry is used to discover in vivo proteolytic cleavages. In this study, we use tandem mass spectrometry to analyze the specifcity rules of selected proteases and describe MS- Proteolysis, a software tool for identifying putative sites of in vivo proteolytic cleavage. Our analysis suggests that the specifcity rules for some commonly used proteases can be improved, e.g., we find that V8 protease cuts not only after Asp and Glu, as currently expected, but also shows a smaller propensity to cleave after Gly for the conditions tested in this study. Finally, we show that comparative analysis of multiple proteases can be used to detect putative in vivo proteolytic sites on a proteome-wide scale.

  20. Botulinum neurotoxin: a deadly protease with applications to human medicine

    USDA-ARS?s Scientific Manuscript database

    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...

  1. Identification of dehydration-responsive cysteine proteases during post-harvest senescence of broccoli florets.

    PubMed

    Coupe, Simon A; Sinclair, Ben K; Watson, Lyn M; Heyes, Julian A; Eason, Jocelyn R

    2003-03-01

    Harvest-induced senescence of broccoli results in tissue wilting and sepal chlorosis. As senescence progresses, chlorophyll and protein levels in floret tissues decline and endo-protease activity (measured with azo-casein) increases. Protease activity increased from 24 h after harvest for tissues held in air at 20 degrees C. Activity was lower in floret tissues from branchlets that had been held in solutions of sucrose (2% w/v) or under high carbon dioxide, low oxygen (10% CO(2), 5% O(2)) conditions. Four protease-active protein bands were identified in senescing floret tissue by zymography, and the use of chemical inhibitors of protease action suggests that some 44% of protease activity in senescing floret tissue 72 h after harvest is due to the action of cysteine and serine proteases. Four putative cysteine protease cDNAs have been isolated from broccoli floret tissue (BoCP1, BoCP2, BoCP3, BoCP4). The cDNAs are most similar (73-89% at the amino acid level) to dehydration-responsive cysteine proteases previously isolated from Arabidopsis thaliana (RD19, RD21). The mRNAs encoded by the broccoli cDNAs are expressed in floret tissue during harvest-induced senescence with mRNA accumulating within 6 h of harvest for BoCP1, 12 h of harvest for BoCP4 and within 24 h of harvest for BoCP2 and BoCP3. Induction of the cDNAs is differentially delayed when broccoli branchlets are held in solutions of water or sucrose. In addition, the expression of BoCP1 and BoCP3 is inhibited in tissue held in atmospheres of high carbon dioxide/low oxygen (10% CO(2), 5% O(2)). The putative cysteine protease mRNAs are expressed before measurable increases in endo-protease activity, loss of protein, chlorophyll or tissue chlorosis.

  2. 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

  3. From proteases to proteomics.

    PubMed

    Neurath, H

    2001-04-01

    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).

  4. A Serine Protease Homolog Negatively Regulates TEP1 Consumption in Systemic Infections of the Malaria Vector Anopheles gambiae

    PubMed Central

    Yassine, Hassan; Kamareddine, Layla; Chamat, Soulaima; Christophides, George K.; Osta, Mike A.

    2016-01-01

    Clip domain serine protease homologs are widely distributed in insect genomes and play important roles in regulating insect immune responses, yet their exact functions remain poorly understood. Here, we show that CLIPA2, a clip domain serine protease homolog of Anopheles gambiae, regulates the consumption of the mosquito complement-like protein TEP1 during systemic bacterial infections. We provide evidence that CLIPA2 localizes to microbial surfaces in a TEP1-dependent manner whereby it negatively regulates the activity of a putative TEP1 convertase, which converts the full-length TEP1-F form into active TEP1cut. CLIPA2 silencing triggers an exacerbated TEP1-mediated response that significantly enhances mosquito resistance to infections with a broad class of microorganisms including Plasmodium berghei, Escherichia coli and the entomopathogenic fungus Beauveria bassiana. We also provide further evidence for the existence of a functional link between TEP1 and activation of hemolymph prophenoloxidase during systemic infections. Interestingly, the enhanced TEP1-mediated immune response in CLIPA2 knockdown mosquitoes correlated with a significant reduction in fecundity, corroborating the existence of a trade-off between immunity and reproduction. In sum, CLIPA2 is an integral regulatory component of the mosquito complement-like pathway which functions to prevent an overwhelming response by the host in response to systemic infections. PMID:25012124

  5. Identification, Characterization and Down-Regulation of Cysteine Protease Genes in Tobacco for Use in Recombinant Protein Production

    PubMed Central

    Duwadi, Kishor; Chen, Ling; Menassa, Rima; Dhaubhadel, Sangeeta

    2015-01-01

    Plants are an attractive host system for pharmaceutical protein production. Many therapeutic proteins have been produced and scaled up in plants at a low cost compared to the conventional microbial and animal-based systems. The main technical challenge during this process is to produce sufficient levels of recombinant proteins in plants. Low yield is generally caused by proteolytic degradation during expression and downstream processing of recombinant proteins. The yield of human therapeutic interleukin (IL)-10 produced in transgenic tobacco leaves was found to be below the critical level, and may be due to degradation by tobacco proteases. Here, we identified a total of 60 putative cysteine protease genes (CysP) in tobacco. Based on their predicted expression in leaf tissue, 10 candidate CysPs (CysP1-CysP10) were selected for further characterization. The effect of CysP gene silencing on IL-10 accumulation was examined in tobacco. It was found that the recombinant protein yield in tobacco could be increased by silencing CysP6. Transient expression of CysP6 silencing construct also showed an increase in IL-10 accumulation in comparison to the control. Moreover, CysP6 localizes to the endoplasmic reticulum (ER), suggesting that ER may be the site of IL-10 degradation. Overall results suggest that CysP6 is important in determining the yield of recombinant IL-10 in tobacco leaves. PMID:26148064

  6. Vibrio type III effector VPA1380 is related to the cysteine protease domain of large bacterial toxins.

    PubMed

    Calder, Thomas; Kinch, Lisa N; Fernandez, Jessie; Salomon, Dor; Grishin, Nick V; Orth, Kim

    2014-01-01

    Vibrio parahaemolyticus is a Gram-negative halophilic bacterium and one of the leading causes of food-borne gastroenteritis. Its genome harbors two Type III Secretion Systems (T3SS1 and T3SS2), but only T3SS2 is required for enterotoxicity seen in animal models. Effector proteins secreted from T3SS2 have been previously shown to promote colonization of the intestinal epithelium, invasion of host cells, and destruction of the epithelial monolayer. In this study, we identify VPA1380, a T3SS2 effector protein that is toxic when expressed in yeast. Bioinformatic analyses revealed that VPA1380 is highly similar to the inositol hexakisphosphate (IP6)-inducible cysteine protease domains of several large bacterial toxins. Mutations in conserved catalytic residues and residues in the putative IP6-binding pocket abolished toxicity in yeast. Furthermore, VPA1380 was not toxic in IP6 deficient yeast cells. Therefore, our findings suggest that VPA1380 is a cysteine protease that requires IP6 as an activator.

  7. A biochemical comparison of proteases from pathogenic naegleria fowleri and non-pathogenic Naegleria gruberi.

    PubMed

    Serrano-Luna, Jesús; Cervantes-Sandoval, Isaac; Tsutsumi, Victor; Shibayama, Mineko

    2007-01-01

    Naegleria fowleri is the etiologic agent of primary amoebic meningoencephalitis (PAM). Proteases have been suggested to be involved in tissue invasion and destruction during infection. We analyzed and compared the complete protease profiles of total crude extract and conditioned medium of both pathogenic N. fowleri and non-pathogenic Naegleria gruberi trophozoites. Using SDS-PAGE, we found differences in the number and molecular weight of proteolytic bands between the two strains. The proteases showed optimal activity at pH 7.0 and 35 degrees C for both strains. Inhibition assays showed that the main proteolytic activity in both strains is due to cysteine proteases although serine proteases were also detected. Both N. fowleri and N. gruberi have a variety of different protease activities at different pH levels and temperatures. These proteases may allow the amoebae to acquire nutrients from different sources, including those from the host. Although, the role of the amoebic proteases in the pathogenesis of PAM is not clearly defined, it seems that proteases and other molecules of the parasite as well as those from the host, could be participating in the damage to the human central nervous system.

  8. Proteases in blood clotting.

    PubMed

    Walsh, Peter N; Ahmad, Syed S

    2002-01-01

    The serine proteases, cofactors and cell-receptor molecules that comprise the haemostatic mechanism are highly conserved modular proteins that have evolved to participate in biochemical reactions in blood coagulation, anticoagulation and fibrinolysis. Blood coagulation is initiated by exposure of tissue factor, which forms a complex with factor VIIa and factor X, which results in the generation of small quantities of thrombin and is rapidly shutdown by the tissue factor pathway inhibitor. The generation of these small quantities of thrombin then activates factor XI, resulting in a sequence of events that lead to the activation of factor IX, factor X and prothrombin. Sufficient thrombin is generated to effect normal haemostasis by converting fibrinogen into fibrin. The anticoagulant pathways that regulate blood coagulation include the protein C anticoagulant mechanism, the serine protease inhibitors in plasma, and the Kunitz-like inhibitors, tissue factor pathway inhibitor and protease nexin 2. Finally, the fibrinolytic mechanism that comprises the activation of plasminogen into plasmin prevents excessive fibrin accumulation by promoting local dissolution of thrombi and promoting wound healing by reestablishment of blood flow.

  9. Multifunctional Mitochondrial AAA Proteases

    PubMed Central

    Glynn, Steven E.

    2017-01-01

    Mitochondria perform numerous functions necessary for the survival of eukaryotic cells. These activities are coordinated by a diverse complement of proteins encoded in both the nuclear and mitochondrial genomes that must be properly organized and maintained. Misregulation of mitochondrial proteostasis impairs organellar function and can result in the development of severe human diseases. ATP-driven AAA+ proteins play crucial roles in preserving mitochondrial activity by removing and remodeling protein molecules in accordance with the needs of the cell. Two mitochondrial AAA proteases, i-AAA and m-AAA, are anchored to either face of the mitochondrial inner membrane, where they engage and process an array of substrates to impact protein biogenesis, quality control, and the regulation of key metabolic pathways. The functionality of these proteases is extended through multiple substrate-dependent modes of action, including complete degradation, partial processing, or dislocation from the membrane without proteolysis. This review discusses recent advances made toward elucidating the mechanisms of substrate recognition, handling, and degradation that allow these versatile proteases to control diverse activities in this multifunctional organelle. PMID:28589125

  10. Non-proteolytic functions of microbial proteases increase pathological complexity.

    PubMed

    Jarocki, Veronica M; Tacchi, Jessica L; Djordjevic, Steven P

    2015-03-01

    Proteases are enzymes that catalyse hydrolysis of peptide bonds thereby controlling the shape, size, function, composition, turnover and degradation of other proteins. In microbes, proteases are often identified as important virulence factors and as such have been targets for novel drug design. It is emerging that some proteases possess additional non-proteolytic functions that play important roles in host epithelia adhesion, tissue invasion and in modulating immune responses. These additional "moonlighting" functions have the potential to obfuscate data interpretation and have implications for therapeutic design. Moonlighting enzymes comprise a subcategory of multifunctional proteins that possess at least two distinct biological functions on a single polypeptide chain. Presently, identifying moonlighting proteins relies heavily on serendipitous empirical data with clues arising from proteins lacking signal peptides that are localised to the cell surface. Here, we describe examples of microbial proteases with additional non-proteolytic functions, including streptococcal pyrogenic exotoxin B, PepO and C5a peptidases, mycoplasmal aminopeptidases, mycobacterial chaperones and viral papain-like proteases. We explore how these non-proteolytic functions contribute to host cell adhesion, modulate the coagulation pathway, assist in non-covalent folding of proteins, participate in cell signalling, and increase substrate repertoire. We conclude by describing how proteomics has aided in moonlighting protein discovery, focusing attention on potential moonlighters in microbial exoproteomes.

  11. Effect of lanthanides on Porphyromonas gingivalis proteases.

    PubMed

    Sunkara, Sasi K; Ciancio, Sebastian G; Sojar, Hakimuddin T

    2010-01-01

    Host and bacterial proteases play a vital role in periodontitis. Inhibitors of these proteases are necessary for control of this disease. The purpose of this study was to evaluate the effect of lanthanides on proteins from Porphyromonas gingivalis, a major pathogen in periodontitis. Benzoyl-L-Arg-p-nitroanilide (BAPNA); H-Gly-Pro-pNA x HCl and gelatin were used to evaluate the activity of P. gingivalis proteins in the presence of lanthanides. Proteins extracted from cell surfaces and culture media of P. gingivalis were assessed for activity in the presence of different lanthanides by BAPNA assay. Only gadolinium chloride was used for H-Gly-Pro-pNA x HCl assay and gelatin-zymography. Concentration-dependent reduction of absorbance was observed in the presence of lanthanides with BAPNA and a similar observation was made with gadolinium chloride using H-Gly-Pro-pNa. Collagenolytic activity in cell surface extracts and culture media-precipitated proteins was absent in the presence of gadolinium chloride. These results suggest that the lanthanide gadolinium can be a potential inhibitor of P. gingivalis proteases.

  12. Corruption of innate immunity by bacterial proteases.

    PubMed

    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.

  13. The roles of intramembrane proteases in protozoan parasites.

    PubMed

    Sibley, L David

    2013-12-01

    Intramembrane proteolysis is widely conserved throughout different forms of life, with three major types of proteases being known for their ability to cleave peptide bonds directly within the transmembrane domains of their substrates. Although intramembrane proteases have been extensively studied in humans and model organisms, they have only more recently been investigated in protozoan parasites, where they turn out to play important and sometimes unexpected roles. Signal peptide peptidases are involved in endoplasmic reticulum (ER) quality control and signal peptide degradation from exported proteins. Recent studies suggest that repurposing inhibitors developed for blocking presenilins may be useful for inhibiting the growth of Plasmodium, and possibly other protozoan parasites, by blocking signal peptide peptidases. Rhomboid proteases, originally described in the fly, are also widespread in parasites, and are especially expanded in apicomplexans. Their study in parasites has revealed novel roles that expand our understanding of how these proteases function. Within this diverse group of parasites, rhomboid proteases contribute to processing of adhesins involved in attachment, invasion, intracellular replication, phagocytosis, and immune evasion, placing them at the vertex of host-parasite interactions. This article is part of a Special Issue entitled: Intramembrane Proteases. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. Regulation of intestinal permeability: The role of proteases

    PubMed Central

    Van Spaendonk, Hanne; Ceuleers, Hannah; Witters, Leonie; Patteet, Eveline; Joossens, Jurgen; Augustyns, Koen; Lambeir, Anne-Marie; De Meester, Ingrid; De Man, Joris G; De Winter, Benedicte Y

    2017-01-01

    The gastrointestinal barrier is - with approximately 400 m2 - the human body’s largest surface separating the external environment from the internal milieu. This barrier serves a dual function: permitting the absorption of nutrients, water and electrolytes on the one hand, while limiting host contact with noxious luminal antigens on the other hand. To maintain this selective barrier, junction protein complexes seal the intercellular space between adjacent epithelial cells and regulate the paracellular transport. Increased intestinal permeability is associated with and suggested as a player in the pathophysiology of various gastrointestinal and extra-intestinal diseases such as inflammatory bowel disease, celiac disease and type 1 diabetes. The gastrointestinal tract is exposed to high levels of endogenous and exogenous proteases, both in the lumen and in the mucosa. There is increasing evidence to suggest that a dysregulation of the protease/antiprotease balance in the gut contributes to epithelial damage and increased permeability. Excessive proteolysis leads to direct cleavage of intercellular junction proteins, or to opening of the junction proteins via activation of protease activated receptors. In addition, proteases regulate the activity and availability of cytokines and growth factors, which are also known modulators of intestinal permeability. This review aims at outlining the mechanisms by which proteases alter the intestinal permeability. More knowledge on the role of proteases in mucosal homeostasis and gastrointestinal barrier function will definitely contribute to the identification of new therapeutic targets for permeability-related diseases. PMID:28405139

  15. Protease signalling: the cutting edge

    PubMed Central

    Turk, Boris; Turk, Dus̆an; Turk, Vito

    2012-01-01

    Protease research has undergone a major expansion in the last decade, largely due to the extremely rapid development of new technologies, such as quantitative proteomics and in-vivo imaging, as well as an extensive use of in-vivo models. These have led to identification of physiological substrates and resulted in a paradigm shift from the concept of proteases as protein-degrading enzymes to proteases as key signalling molecules. However, we are still at the beginning of an understanding of protease signalling pathways. We have only identified a minor subset of true physiological substrates for a limited number of proteases, and their physiological regulation is still not well understood. Similarly, links with other signalling systems are not well established. Herein, we will highlight current challenges in protease research. PMID:22367392

  16. Protease signalling: the cutting edge.

    PubMed

    Turk, Boris; Turk, Dušan; Turk, Vito

    2012-04-04

    Protease research has undergone a major expansion in the last decade, largely due to the extremely rapid development of new technologies, such as quantitative proteomics and in-vivo imaging, as well as an extensive use of in-vivo models. These have led to identification of physiological substrates and resulted in a paradigm shift from the concept of proteases as protein-degrading enzymes to proteases as key signalling molecules. However, we are still at the beginning of an understanding of protease signalling pathways. We have only identified a minor subset of true physiological substrates for a limited number of proteases, and their physiological regulation is still not well understood. Similarly, links with other signalling systems are not well established. Herein, we will highlight current challenges in protease research.

  17. Serine protease inhibitors suppress pancreatic endogenous proteases and modulate bacterial neutral proteases.

    PubMed

    Nduaguibe, Chikodili C; Bentsi-Barnes, Kwamina; Mullen, Yoko; Kandeel, Fouad; Al-Abdullah, Ismail

    2010-01-01

    Pefabloc, Trasylol and Urinary Trypsin Inhibitor (UTI) have been reported to be effective serine protease inhibitors that impair pancreatic endogenous proteases resulting in improved islet yield. Here we evaluated the effect of these inhibitors on endogenous proteases (trypsin, chymotrypsin and elastase), bacterial neutral proteases (thermolysin and neutral protease) and islet isolation digestion samples. Protease activity was measured using a fluorimetric assay and islet function was assessed by dynamic perifusion. Trypsin, chymotrypsin and elastase were significantly inhibited by Pefabloc and UTI. Trasylol showed strong inhibitory effects on trypsin and chymotrypsin but also decreased thermolysin activity. UTI was found to inhibit the activity of endogenous proteases and increase the activity of bacterial neutral proteases. Human islets exposed to Pefabloc had reduced insulin response, unlike Trasylol or UTI, which had no detrimental effect on insulin secretion. Although Trasylol was an effective inhibitor of endogenous proteases, FDA regulatory issues preclude its use in clinical application and thus in the isolation process. UTI has the greatest potential because it impairs endogenous pancreatic proteases and enhances digestion enzymes.

  18. 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.

  19. Molecular Mechanisms of Foot-and-Mouth Disease Virus Targeting the Host Antiviral Response

    PubMed Central

    Rodríguez Pulido, Miguel; Sáiz, Margarita

    2017-01-01

    Foot-and-mouth disease virus (FMDV) is the causative agent of an acute vesicular disease affecting pigs, cattle and other domestic, and wild animals worldwide. The aim of the host interferon (IFN) response is to limit viral replication and spread. Detection of the viral genome and products by specialized cellular sensors initiates a signaling cascade that leads to a rapid antiviral response involving the secretion of type I- and type III-IFNs and other antiviral cytokines with antiproliferative and immunomodulatory functions. During co-evolution with their hosts, viruses have acquired strategies to actively counteract host antiviral responses and the balance between innate response and viral antagonism may determine the outcome of disease and pathogenesis. FMDV proteases Lpro and 3C have been found to antagonize the host IFN response by a repertoire of mechanisms. Moreover, the putative role of other viral proteins in IFN antagonism is being recently unveiled, uncovering sophisticated immune evasion strategies different to those reported to date for other members of the Picornaviridae family. Here, we review the interplay between antiviral responses induced by FMDV infection and viral countermeasures to block them. Research on strategies used by viruses to modulate immunity will provide insights into the function of host pathways involved in defense against pathogens and will also lead to development of new therapeutic strategies to fight virus infections. PMID:28660175

  20. Characterization of a papain-like cysteine protease essential for the survival of Babesia ovis merozoites.

    PubMed

    Carletti, Tamara; Barreto, Carmo; Mesplet, Maria; Mira, Anabela; Weir, William; Shiels, Brian; Oliva, Abel Gonzalez; Schnittger, Leonhard; Florin-Christensen, Monica

    2016-02-01

    Babesia ovis, a tick-transmitted intraerythrocytic protozoan parasite, causes severe infections in small ruminants from Southern Europe, Middle East, and Northern Africa. With the aim of finding potential targets for the development of control methods against this parasite, sequence analysis of its genome led to the identification of four putative cysteine proteases of the C1A family. Orthology between B. ovis, B. bovis, T. annulata, and T. parva sequences showed that each B. ovis C1A peptidase sequence clustered within one of the four ortholog groups previously reported for these piroplasmids. The ortholog of bovipain-2 of B. bovis and falcipain-2 of Plasmodium falciparum, respectively, was designated "ovipain-2" and further characterized. In silico analysis showed that ovipain-2 has the typical topology of papain-like cysteine peptidases and a highly similar predicted three dimensional structure to bovipain-2 and falcipain-2, suggesting susceptibility to similar inhibitors. Immunoblotting using antibodies raised against a recombinant form of ovipain-2 (r-ovipain-2) demonstrated expression of ovipain-2 in in vitro cultured B. ovis merozoites. By immunofluorescence, these antibodies reacted with merozoites and stained the cytoplasm of infected erythrocytes. This suggests that ovipain-2 is secreted by the parasite and could be involved in intra- and extracellular digestion of hemoglobin and/or cleavage of erythrocyte proteins facilitating parasite egress. A significant reduction in the percentage of parasitized erythrocytes was obtained upon incubation of B. ovis in vitro cultures with anti-r-ovipain-2 antibodies, indicating an important functional role for ovipain-2 in the intra erythrocytic development cycle of this parasite. Finally, studies of the reactivity of sera from B. ovis-positive and negative sheep against r-ovipain-2 showed that this protease is expressed in vivo, and can be recognized by host antibodies. The results of this study suggest that ovipain-2

  1. Autocatalytic activity and substrate specificity of the pestivirus N-terminal protease N{sup pro}

    SciTech Connect

    Gottipati, Keerthi; Acholi, Sudheer; Ruggli, Nicolas; Choi, Kyung H.

    2014-03-15

    Pestivirus N{sup pro} is the first protein translated in the viral polypeptide, and cleaves itself off co-translationally generating the N-terminus of the core protein. Once released, N{sup pro} blocks the host's interferon response by inducing degradation of interferon regulatory factor-3. N{sup pro'}s intracellular autocatalytic activity and lack of trans-activity have hampered in vitro cleavage studies to establish its substrate specificity and the roles of individual residues. We constructed N{sup pro}-GFP fusion proteins that carry the authentic cleavage site and determined the autoproteolytic activities of N{sup pro} proteins containing substitutions at the predicted catalytic sites Glu22 and Cys69, at Arg100 that forms a salt bridge with Glu22, and at the cleavage site Cys168. Contrary to previous reports, we show that N{sup pro'}s catalytic activity does not involve Glu22, which may instead be involved in protein stability. Furthermore, N{sup pro} does not have specificity for Cys168 at the cleavage site even though this residue is conserved throughout the pestivirus genus. - Highlights: • N{sup pro'}s autoproteolysis is studied using N{sup pro}-GFP fusion proteins. • N-terminal 17 amino acids are dispensable without loss of protease activity. • The putative catalytic residue Glu22 is not involved in protease catalysis. • No specificity for Cys168 at the cleavage site despite evolutionary conservation. • N{sup pro} prefers small amino acids with non-branched beta carbons at the P1 position.

  2. Unraveling the in vitro secretome of the phytopathogen Botrytis cinerea to understand the interaction with its hosts

    PubMed Central

    González-Fernández, Raquel; Valero-Galván, José; Gómez-Gálvez, Francisco J.; Jorrín-Novo, Jesús V.

    2015-01-01

    Botrytis cinerea is a necrotrophic fungus with high adaptability to different environments and hosts. It secretes a large number of extracellular proteins, which favor plant tissue penetration and colonization, thus contributing to virulence. Secretomics is a proteomics sub-discipline which study the secreted proteins and their secretion mechanisms, so-called secretome. By using proteomics as experimental approach, many secreted proteins by B. cinerea have been identified from in vitro experiments, and belonging to different functional categories: (i) cell wall-degrading enzymes such as pectinesterases and endo-polygalacturonases; (ii) proteases involved in host protein degradation such as an aspartic protease; (iii) proteins related to the oxidative burst such as glyoxal oxidase; (iv) proteins which may induce the plant hypersensitive response such as a cerato-platanin domain-containing protein; and (v) proteins related to production and secretion of toxins such as malate dehydrogenase. In this mini-review, we made an overview of the proteomics contribution to the study and knowledge of the B. cinerea extracellular secreted proteins based on our current work carried out from in vitro experiments, and recent published papers both in vitro and in planta studies on this fungi. We hypothesize on the putative functions of these secreted proteins, and their connection to the biology of the B. cinerea interaction with its hosts. PMID:26500673

  3. Proteases, cystic fibrosis and the epithelial sodium channel (ENaC).

    PubMed

    Thibodeau, P H; Butterworth, M B

    2013-02-01

    Proteases perform a diverse array of biological functions. From simple peptide digestion for nutrient absorption to complex signaling cascades, proteases are found in organisms from prokaryotes to humans. In the human airway, proteases are associated with the regulation of the airway surface liquid layer, tissue remodeling, host defense and pathogenic infection and inflammation. A number of proteases are released in the airways under both physiological and pathophysiological states by both the host and invading pathogens. In airway diseases such as cystic fibrosis, proteases have been shown to be associated with increased morbidity and airway disease progression. In this review, we focus on the regulation of proteases and discuss specifically those proteases found in human airways. Attention then shifts to the epithelial sodium channel (ENaC), which is regulated by proteolytic cleavage and that is considered to be an important component of cystic fibrosis disease. Finally, we discuss bacterial proteases, in particular, those of the most prevalent bacterial pathogen found in cystic fibrosis, Pseudomonas aeruginosa.

  4. A Phytophthora infestans Cystatin-Like Protein Targets a Novel Tomato Papain-Like Apoplastic Protease1[W][OA

    PubMed Central

    Tian, Miaoying; Win, Joe; Song, Jing; van der Hoorn, Renier; van der Knaap, Esther; Kamoun, Sophien

    2007-01-01

    There is emerging evidence that the proteolytic machinery of plants plays important roles in defense against pathogens. The oomycete pathogen Phytophthora infestans, the agent of the devastating late blight disease of tomato (Lycopersicon esculentum) and potato (Solanum tuberosum), has evolved an arsenal of protease inhibitors to overcome the action of host proteases. Previously, we described a family of 14 Kazal-like extracellular serine protease inhibitors from P. infestans. Among these, EPI1 and EPI10 bind and inhibit the pathogenesis-related (PR) P69B subtilisin-like serine protease of tomato. Here, we describe EPIC1 to EPIC4, a new family of P. infestans secreted proteins with similarity to cystatin-like protease inhibitor domains. Among these, the epiC1 and epiC2 genes lacked orthologs in Phytophthora sojae and Phytophthora ramorum, were relatively fast-evolving within P. infestans, and were up-regulated during infection of tomato, suggesting a role during P. infestans-host interactions. Biochemical functional analyses revealed that EPIC2B interacts with and inhibits a novel papain-like extracellular cysteine protease, termed Phytophthora Inhibited Protease 1 (PIP1). Characterization of PIP1 revealed that it is a PR protein closely related to Rcr3, a tomato apoplastic cysteine protease that functions in fungal resistance. Altogether, this and earlier studies suggest that interplay between host proteases of diverse catalytic families and pathogen inhibitors is a general defense-counterdefense process in plant-pathogen interactions. PMID:17085509

  5. Contribution of Aspartic Proteases in Candida Virulence. Protease Inhibitors against Candida Infections.

    PubMed

    Staniszewska, Monika; Małgorzata, Bondaryk; Zbigniew, Ochal

    2016-08-09

    Candida species are the major opportunistic human pathogens accounting for 70-90% of all invasive fungal infections. Candida spp, especially C. albicans, are able to produce and secrete hydrolytic enzymes, particularly aspartic proteases (Saps). These enzymes production is an evolutionary adaptation of pathogens to utilize nutrients and survive in host. Sap1-10 are believed to contribute to the adhesion and invasion of host tissues through the degradation of cell surface structures. Aspartic proteases control several steps in innate immune evasion and they degrade proteins related to immunological defense (antibodies, complement and cytokines), allowing the fungus to escape from the first line of host defense. The existing ways to identify potential drug targets rely on specific subset like virulence genes, transcriptional and stress response factors. Candida virulence factors like Sap isoenzymes can be pivotal targets for drug development. The identification of mechanism of a non-canonical inflammasome exerted by Saps could open novel therapeutic strategies to dampen hyperinflammatory response in candidiasis.

  6. Proteases from psychrotrophs: an overview.

    PubMed

    Kasana, Ramesh Chand

    2010-05-01

    Proteases are hydrolytic enzymes which catalyze the total hydrolysis of proteins in to amino acids. Although proteolytic enzymes can be obtained from animals and plants but microorganisms are the preferred source for industrial applications in view of scientific and economical advantage. Among various groups of microbes, psychrotrophs are ideal candidates for enzymes production keeping in mind that enzymes active at low temperature and stable under alkaline condition, in presence of oxidants and detergents are in large demand as laundry additive. The proteases from psychrotrophs also find application in environmental bioremediation, food and molecular biology. During the previous two decades, proteases from psychrotrophs have received increased attention because of their wide range of applications, but the full potential of psychrotrophic proteases has not been exploited. This review focuses attention on the present status of knowledge on the production, optimization, molecular characteristics, applications, substrate specificity, and crystal structure of psychrotrophic proteases. The review will help in making strategies for exploitation of psychrotrophic protease resources and improvement of enzymes to obtain more robust proteases of industrial and biotechnological significance.

  7. Proteases in gastrointestinal neoplastic diseases.

    PubMed

    Herszényi, L; Plebani, M; Carraro, P; De Paoli, M; Roveroni, G; Cardin, R; Foschia, F; Tulassay, Z; Naccarato, R; Farinati, F

    2000-02-15

    Cysteine and serine proteases are involved in cancer invasion and metastasis. In the past few years we investigated the tissue levels of these proteases in gastric cancer (GC), gastric precancerous changes (CAG), colorectal cancer (CRC) and the plasma and serum levels of proteases in several gastrointestinal tumours, using ELISA methods. Significantly higher antigen levels were found not only in GC tissue but also in CAG with respect to the levels found normal tissue; with respect to CAG, patients with dysplasia had higher levels than patients without dysplasia. The same findings were obtained in CRC. In general protease levels correlated with the major histomorphological parameters, such as grading and histotype in GC as well as in CRC. Tissue protease levels had a strong prognostic impact in GC, in which UPA was singled out by multivariate analysis as the major prognostic factor, and CRC. The plasma levels of urokinase-type plasminogen activator (UPA) and the serum levels of cathepsin B were significantly increased in patients with gastrointestinal tumours. In conclusions, cysteine and serine proteases may have a part not only in GC and CRC invasion and metastasis, but also in the progression of gastric precancerous changes into cancer. They are strong prognostic factors in GC and CRC. These proteases may also have a role as tumour markers in the early diagnosis of gastrointestinal tract tumours.

  8. Comparative site-directed mutagenesis in the catalytic amino acid triad in calicivirus proteases.

    PubMed

    Oka, Tomoichiro; Murakami, Kosuke; Wakita, Takaji; Katayama, Kazuhiko

    2011-02-01

    Calicivirus proteases cleave the viral precursor polyprotein encoded by open reading frame 1 (ORF1) into multiple intermediate and mature proteins. These proteases have conserved histidine (His), glutamic acid (Glu) or aspartic acid (Asp), and cysteine (Cys) residues that are thought to act as a catalytic triad (i.e. general base, acid and nucleophile, respectively). However, is the triad critical for processing the polyprotein? In the present study, we examined these amino acids in viruses representing the four major genera of Caliciviridae: Norwalk virus (NoV), Rabbit hemorrhagic disease virus (RHDV), Sapporo virus (SaV) and Feline calicivirus (FCV). Using single amino-acid substitutions, we found that an acidic amino acid (Glu or Asp), as well as the His and Cys in the putative catalytic triad, cannot be replaced by Ala for normal processing activity of the ORF1 polyprotein in vitro. Similarly, normal activity is not retained if the nucleophile Cys is replaced with Ser. These results showed the calicivirus protease is a Cys protease and the catalytic triad formation is important for protease activity. Our study is the first to directly compare the proteases of the four representative calicivirus genera. Interestingly, we found that RHDV and SaV proteases critically need the acidic residues during catalysis, whereas proteolytic cleavage occurs normally at several cleavage sites in the ORF1 polyprotein without a functional acid residue in the NoV and FCV proteases. Thus, the substrate recognition mechanism may be different between the SaV and RHDV proteases and the NoV and FCV proteases. © 2011 The Societies and Blackwell Publishing Asia Pty Ltd.

  9. Pallial mucus of the oyster Crassostrea virginica regulates the expression of putative virulence genes of its pathogen Perkinsus marinus.

    PubMed

    Pales Espinosa, Emmanuelle; Corre, Erwan; Allam, Bassem

    2014-04-01

    Perkinsus marinus is a pathogen responsible for severe mortalities of the eastern oyster Crassostrea virginica along the East and Gulf coasts of the United States. When cultivated, the pathogenicity of this microorganism decreases significantly, hampering the study of its virulence factors. Recent investigations have shown a significant increase of the in vivo virulence of P. marinus exposed to oyster pallial mucus. In the current study, we investigated the effect of pallial mucus on P. marinus gene expression compared with cultures supplemented with oyster digestive extracts or with un-supplemented cultures. In parallel, parasite cells cultured under these three conditions were used to challenge oysters and to assess virulence in vivo. Perkinsus marinus mRNA sequencing was performed on an Illumina GAIIX sequencer and data were analysed using the Tuxedo RNAseq suite for mapping against the draft P. marinus genome and for differential expression analysis. Results showed that exposure of P. marinus to mucus induces significant regulation of nearly 3,600 transcripts, many of which are considered as putative virulence factors. Pallial mucus is suspected to mimic internal host conditions, thereby preparing the pathogen to overcome defense factors before invasion. This hypothesis is supported by significant regulation in several antioxidant proteins, heat shock proteins, protease inhibitors and proteasome subunits. In addition, mucus exposure induced the modulation of several genes known to affect immunity and apoptosis in vertebrates and invertebrates. Several proteases (proteolysis) and merozoite surface proteins (cell recognition) were also modulated. Overall, these results provide a baseline for targeted, in depth analysis of candidate virulence factors in P. marinus.

  10. Modulation of the epithelial sodium channel (ENaC) by bacterial metalloproteases and protease inhibitors.

    PubMed

    Butterworth, Michael B; Zhang, Liang; Liu, Xiaoning; Shanks, Robert M; Thibodeau, Patrick H

    2014-01-01

    The serralysin family of metalloproteases is associated with the virulence of multiple gram-negative human pathogens, including Pseudomonas aeruginosa and Serratia marcescens. The serralysin proteases share highly conserved catalytic domains and show evolutionary similarity to the mammalian matrix metalloproteases. Our previous studies demonstrated that alkaline protease (AP) from Pseudomonas aeruginosa is capable of activating the epithelial sodium channel (ENaC), leading to an increase in sodium absorption in airway epithelia. The serralysin proteases are often co-expressed with endogenous, intracellular or periplasmic inhibitors, which putatively protect the bacterium from unwanted or unregulated protease activities. To evaluate the potential use of these small protein inhibitors in regulating the serralysin induced activation of ENaC, proteases from Pseudomonas aeruginosa and Serratia marcescens were purified for characterization along with a high affinity inhibitor from Pseudomonas. Both proteases showed activity against in vitro substrates and could be blocked by near stoichiometric concentrations of the inhibitor. In addition, both proteases were capable of activating ENaC when added to the apical surfaces of multiple epithelial cells with similar slow activation kinetics. The high-affinity periplasmic inhibitor from Pseudomonas effectively blocked this activation. These data suggest that multiple metalloproteases are capable of activating ENaC. Further, the endogenous, periplasmic bacterial inhibitors may be useful for modulating the downstream effects of the serralysin virulence factors under physiological conditions.

  11. Modulation of the Epithelial Sodium Channel (ENaC) by Bacterial Metalloproteases and Protease Inhibitors

    PubMed Central

    Butterworth, Michael B.; Zhang, Liang; Liu, Xiaoning; Shanks, Robert M.; Thibodeau, Patrick H.

    2014-01-01

    The serralysin family of metalloproteases is associated with the virulence of multiple gram-negative human pathogens, including Pseudomonas aeruginosa and Serratia marcescens. The serralysin proteases share highly conserved catalytic domains and show evolutionary similarity to the mammalian matrix metalloproteases. Our previous studies demonstrated that alkaline protease (AP) from Pseudomonas aeruginosa is capable of activating the epithelial sodium channel (ENaC), leading to an increase in sodium absorption in airway epithelia. The serralysin proteases are often co-expressed with endogenous, intracellular or periplasmic inhibitors, which putatively protect the bacterium from unwanted or unregulated protease activities. To evaluate the potential use of these small protein inhibitors in regulating the serralysin induced activation of ENaC, proteases from Pseudomonas aeruginosa and Serratia marcescens were purified for characterization along with a high affinity inhibitor from Pseudomonas. Both proteases showed activity against in vitro substrates and could be blocked by near stoichiometric concentrations of the inhibitor. In addition, both proteases were capable of activating ENaC when added to the apical surfaces of multiple epithelial cells with similar slow activation kinetics. The high-affinity periplasmic inhibitor from Pseudomonas effectively blocked this activation. These data suggest that multiple metalloproteases are capable of activating ENaC. Further, the endogenous, periplasmic bacterial inhibitors may be useful for modulating the downstream effects of the serralysin virulence factors under physiological conditions. PMID:24963801

  12. Evolutionary optimization of peptide substrates for proteases that exhibit rapid hydrolysis kinetics.

    PubMed

    Boulware, Kevin T; Jabaiah, Abeer; Daugherty, Patrick S

    2010-06-15

    Protease cleavage site recognition motifs can be identified using protease substrate discovery methodologies, but typically exhibit non-optimal specificity and activity. To enable evolutionary optimization of substrate cleavage kinetics, a two-color cellular library of peptide substrates (CLiPS) methodology was developed. Two-color CLiPS was applied to identify peptide substrates for the tobacco etch virus (TEV) protease from a random pentapeptide library, which were then optimized by screening of a focused, extended substrate library. Quantitative library screening yielded seven amino acid substrates exhibiting rapid hydrolysis by TEV protease and high sequence similarity to the native seven-amino-acid substrate, with a strong consensus of EXLYPhiQG. Comparison of hydrolysis rates for a family of closely related substrates indicates that the native seven-residue TEV substrate co-evolved with TEV protease to facilitate highly efficient hydrolysis. Consensus motifs revealed by screening enabled database identification of a family of related, putative viral protease substrates. More generally, our results suggest that substrate evolution using CLiPS may be useful for optimizing substrate selectivity and activity to enable the design of more effective protease activity probes, molecular imaging agents, and prodrugs.

  13. Distinct contribution of Toxoplasma gondii rhomboid proteases 4 and 5 to micronemal protein protease 1 activity during invasion.

    PubMed

    Rugarabamu, George; Marq, Jean-Baptiste; Guérin, Amandine; Lebrun, Maryse; Soldati-Favre, Dominique

    2015-07-01

    Host cell entry by the Apicomplexa is associated with the sequential secretion of invasion factors from specialized apical organelles. Secretion of micronemal proteins (MICs) complexes by Toxoplasma gondii facilitates parasite gliding motility, host cell attachment and entry, as well as egress from infected cells. The shedding of MICs during these steps is mediated by micronemal protein proteases MPP1, MPP2 and MPP3. The constitutive activity of MPP1 leads to the cleavage of transmembrane MICs and is linked to the surface rhomboid protease 4 (ROM4) and possibly to rhomboid protease 5 (ROM5). To determine their importance and respective contribution to MPP1 activity, in this study ROM4 and ROM5 genes were abrogated using Cre-recombinase and CRISPR-Cas9 nuclease, respectively, and shown to be dispensable for parasite survival. Parasites lacking ROM4 predominantly engage in twirling motility and exhibit enhanced attachment and impaired invasion, whereas intracellular growth and egress is not affected. The substrates MIC2 and MIC6 are not cleaved in rom4-ko parasites, in contrast, intramembrane cleavage of AMA1 is reduced but not completely abolished. Shedding of MICs and invasion are not altered in the absence of ROM5; however, this protease responsible for the residual cleavage of AMA1 is able to cleave other AMA family members and exhibits a detectable contribution to invasion in the absence of ROM4.

  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.

  15. Application of Protease Technology in Dermatology

    PubMed Central

    Del Rosso, James Q.

    2013-01-01

    This article reviews background on proteases and their functions, their physiological significance in skin, and the potential implications of incorporating specific proteases and protease blends into dermatological products, including skin care formulations. The history of protease blend formulations used in wound model studies and for other disorders is reviewed. In vitro data with use of a specific 3-protease blend with evaluation of the impact on various skin proteins and peptides is also discussed in this article. PMID:23882305

  16. The battle in the apoplast: further insights into the roles of proteases and their inhibitors in plant–pathogen interactions

    PubMed Central

    Jashni, Mansoor Karimi; Mehrabi, Rahim; Collemare, Jérôme; Mesarich, Carl H.; de Wit, Pierre J. G. M.

    2015-01-01

    Upon host penetration, fungal pathogens secrete a plethora of effectors to promote disease, including proteases that degrade plant antimicrobial proteins, and protease inhibitors (PIs) that inhibit plant proteases with antimicrobial activity. Conversely, plants secrete proteases and PIs to protect themselves against pathogens or to mediate recognition of pathogen proteases and PIs, which leads to induction of defense responses. Many examples of proteases and PIs mediating effector-triggered immunity in host plants have been reported in the literature, but little is known about their role in compromising basal defense responses induced by microbe-associated molecular patterns. Recently, several reports appeared in literature on secreted fungal proteases that modify or degrade pathogenesis-related proteins, including plant chitinases or PIs that compromise their activities. This prompted us to review the recent advances on proteases and PIs involved in fungal virulence and plant defense. Proteases and PIs from plants and their fungal pathogens play an important role in the arms race between plants and pathogens, which has resulted in co-evolutionary diversification and adaptation shaping pathogen lifestyles. PMID:26284100

  17. Xanthomonas campestris pv. vesicatoria Secretes Proteases and Xylanases via the Xps Type II Secretion System and Outer Membrane Vesicles.

    PubMed

    Solé, Magali; Scheibner, Felix; Hoffmeister, Anne-Katrin; Hartmann, Nadine; Hause, Gerd; Rother, Annekatrin; Jordan, Michael; Lautier, Martine; Arlat, Matthieu; Büttner, Daniela

    2015-09-01

    Many plant-pathogenic bacteria utilize type II secretion (T2S) systems to secrete degradative enzymes into the extracellular milieu. T2S substrates presumably mediate the degradation of plant cell wall components during the host-pathogen interaction and thus promote bacterial virulence. Previously, the Xps-T2S system from Xanthomonas campestris pv. vesicatoria was shown to contribute to extracellular protease activity and the secretion of a virulence-associated xylanase. The identities and functions of additional T2S substrates from X. campestris pv. vesicatoria, however, are still unknown. In the present study, the analysis of 25 candidate proteins from X. campestris pv. vesicatoria led to the identification of two type II secreted predicted xylanases, a putative protease and a lipase which was previously identified as a virulence factor of X. campestris pv. vesicatoria. Studies with mutant strains revealed that the identified xylanases and the protease contribute to virulence and in planta growth of X. campestris pv. vesicatoria. When analyzed in the related pathogen X. campestris pv. campestris, several T2S substrates from X. campestris pv. vesicatoria were secreted independently of the T2S systems, presumably because of differences in the T2S substrate specificities of the two pathogens. Furthermore, in X. campestris pv. vesicatoria T2S mutants, secretion of T2S substrates was not completely absent, suggesting the contribution of additional transport systems to protein secretion. In line with this hypothesis, T2S substrates were detected in outer membrane vesicles, which were frequently observed for X. campestris pv. vesicatoria. We, therefore, propose that extracellular virulence-associated enzymes from X. campestris pv. vesicatoria are targeted to the Xps-T2S system and to outer membrane vesicles. The virulence of plant-pathogenic bacteria often depends on TS2 systems, which secrete degradative enzymes into the extracellular milieu. T2S substrates are being

  18. Xanthomonas campestris pv. vesicatoria Secretes Proteases and Xylanases via the Xps Type II Secretion System and Outer Membrane Vesicles

    PubMed Central

    Solé, Magali; Scheibner, Felix; Hoffmeister, Anne-Katrin; Hartmann, Nadine; Hause, Gerd; Rother, Annekatrin; Jordan, Michael; Lautier, Martine; Arlat, Matthieu

    2015-01-01

    ABSTRACT Many plant-pathogenic bacteria utilize type II secretion (T2S) systems to secrete degradative enzymes into the extracellular milieu. T2S substrates presumably mediate the degradation of plant cell wall components during the host-pathogen interaction and thus promote bacterial virulence. Previously, the Xps-T2S system from Xanthomonas campestris pv. vesicatoria was shown to contribute to extracellular protease activity and the secretion of a virulence-associated xylanase. The identities and functions of additional T2S substrates from X. campestris pv. vesicatoria, however, are still unknown. In the present study, the analysis of 25 candidate proteins from X. campestris pv. vesicatoria led to the identification of two type II secreted predicted xylanases, a putative protease and a lipase which was previously identified as a virulence factor of X. campestris pv. vesicatoria. Studies with mutant strains revealed that the identified xylanases and the protease contribute to virulence and in planta growth of X. campestris pv. vesicatoria. When analyzed in the related pathogen X. campestris pv. campestris, several T2S substrates from X. campestris pv. vesicatoria were secreted independently of the T2S systems, presumably because of differences in the T2S substrate specificities of the two pathogens. Furthermore, in X. campestris pv. vesicatoria T2S mutants, secretion of T2S substrates was not completely absent, suggesting the contribution of additional transport systems to protein secretion. In line with this hypothesis, T2S substrates were detected in outer membrane vesicles, which were frequently observed for X. campestris pv. vesicatoria. We, therefore, propose that extracellular virulence-associated enzymes from X. campestris pv. vesicatoria are targeted to the Xps-T2S system and to outer membrane vesicles. IMPORTANCE The virulence of plant-pathogenic bacteria often depends on TS2 systems, which secrete degradative enzymes into the extracellular milieu. T2S

  19. Serine proteases, serine protease inhibitors, and protease-activated receptors: roles in synaptic function and behavior.

    PubMed

    Almonte, Antoine G; Sweatt, J David

    2011-08-17

    Serine proteases, serine protease inhibitors, and protease-activated receptors have been intensively investigated in the periphery and their roles in a wide range of processes-coagulation, inflammation, and digestion, for example-have been well characterized (see Coughlin, 2000; Macfarlane et al., 2001; Molinari et al., 2003; Wang et al., 2008; Di Cera, 2009 for reviews). A growing number of studies demonstrate that these protein systems are widely expressed in many cell types and regions in mammalian brains. Accumulating lines of evidence suggest that the brain has co-opted the activities of these interesting proteins to regulate various processes underlying synaptic activity and behavior. In this review, we discuss emerging roles for serine proteases in the regulation of mechanisms underlying synaptic plasticity and memory formation.

  20. Serine proteases, serine protease inhibitors, and protease-activated receptors: roles in synaptic function and behavior

    PubMed Central

    Almonte, Antoine G.; Sweatt, J. David

    2011-01-01

    Serine proteases, serine protease inhibitors, and protease-activated receptors have been intensively investigated in the periphery and their roles in a wide range of processes—coagulation, inflammation, and digestion, for example—have been well characterized (see Coughlin, 2000; Macfarlane et al., 2001; Molinari et al., 2003; Wang et al., 2008; Di Cera, 2009 for reviews). A growing number of studies demonstrate that these protein systems are widely expressed in many cell types and regions in mammalian brains. Accumulating lines of evidence suggest that the brain has co-opted the activities of these interesting proteins to regulate various processes underlying synaptic activity and behavior. In this review, we discuss emerging roles for serine proteases in the regulation of mechanisms underlying synaptic plasticity and memory formation. PMID:21782155

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

    PubMed

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

    2016-09-01

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

  2. Expression profiles of putative defence-related proteins in oil palm (Elaeis guineensis) colonized by Ganoderma boninense.

    PubMed

    Tan, Yung-Chie; Yeoh, Keat-Ai; Wong, Mui-Yun; Ho, Chai-Ling

    2013-11-01

    Basal stem rot (BSR) is a major disease of oil palm caused by a pathogenic fungus, Ganoderma boninense. However, the interaction between the host plant and its pathogen is not well characterized. To better understand the response of oil palm to G. boninense, transcript profiles of eleven putative defence-related genes from oil palm were measured by quantitative reverse-transcription (qRT)-PCR in the roots of oil palms treated with G. boninense from 3 to 12 weeks post infection (wpi). These transcripts encode putative Bowman-Birk serine protease inhibitors (EgBBI1 and 2), defensin (EgDFS), dehydrin (EgDHN), early methionine-labeled polypeptides (EgEMLP1 and 2), glycine-rich RNA binding protein (EgGRRBP), isoflavone reductase (EgIFR), metallothionein-like protein (EgMT), pathogenesis-related-1 protein (EgPRP), and type 2 ribosome-inactivating protein (EgT2RIP). The transcript abundance of EgBBI2 increased in G. boninense-treated roots at 3 and 6wpi compared to those of controls; while the transcript abundance of EgBBI1, EgDFS, EgEMLP1, EgMT, and EgT2RIP increased in G. boninense-treated roots at 6 or 12wpi. Meanwhile, the gene expression of EgDHN was up-regulated at all three time points in G. boninense-treated roots. The expression profiles of the eleven transcripts were also studied in leaf samples upon inoculation of G. boninense and Trichoderma harzianum to identify potential biomarkers for early detection of BSR. Two candidate genes (EgEMLP1 and EgMT) that have different profiles in G. boninense-treated leaves compared to those infected by T. harzianum may have the potential to be developed as biomarkers for early detection of G. boninense infection. Copyright © 2013 Elsevier GmbH. All rights reserved.

  3. 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.

  4. 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.

  5. Serine proteases in rodent hippocampus.

    PubMed

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

    1998-09-04

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

  6. Genomic and exoproteomic analyses of cold- and alkaline-adapted bacteria reveal an abundance of secreted subtilisin-like proteases.

    PubMed

    Lylloff, Jeanette E; Hansen, Lea B S; Jepsen, Morten; Sanggaard, Kristian W; Vester, Jan K; Enghild, Jan J; Sørensen, Søren J; Stougaard, Peter; Glaring, Mikkel A

    2016-03-01

    Proteases active at low temperature or high pH are used in many commercial applications, including the detergent, food and feed industries, and bacteria specifically adapted to these conditions are a potential source of novel proteases. Environments combining these two extremes are very rare, but offer the promise of proteases ideally suited to work at both high pH and low temperature. In this report, bacteria from two cold and alkaline environments, the ikaite columns in Greenland and alkaline ponds in the McMurdo Dry Valley region, Antarctica, were screened for extracellular protease activity. Two isolates, Arsukibacterium ikkense from Greenland and a related strain, Arsukibacterium sp. MJ3, from Antarctica, were further characterized with respect to protease production. Genome sequencing identified a range of potential extracellular proteases including a number of putative secreted subtilisins. An extensive liquid chromatography-tandem mass spectrometry analysis of proteins secreted by A. ikkense identified six subtilisin-like proteases as abundant components of the exoproteome in addition to other peptidases potentially involved in complete degradation of extracellular protein. Screening of Arsukibacterium genome libraries in Escherichia coli identified two orthologous secreted subtilisins active at pH 10 and 20 °C, which were also present in the A. ikkense exoproteome. Recombinant production of both proteases confirmed the observed activity.

  7. The cysteine-rich domain regulates ADAM protease function in vivo.

    PubMed

    Smith, Katherine M; Gaultier, Alban; Cousin, Helene; Alfandari, Dominique; White, Judith M; DeSimone, Douglas W

    2002-12-09

    ADAMs are membrane-anchored proteases that regulate cell behavior by proteolytically modifying the cell surface and ECM. Like other membrane-anchored proteases, ADAMs contain candidate "adhesive" domains downstream of their metalloprotease domains. The mechanism by which membrane-anchored cell surface proteases utilize these putative adhesive domains to regulate protease function in vivo is not well understood. We address this important question by analyzing the relative contributions of downstream extracellular domains (disintegrin, cysteine rich, and EGF-like repeat) of the ADAM13 metalloprotease during Xenopus laevis development. When expressed in embryos, ADAM13 induces hyperplasia of the cement gland, whereas ADAM10 does not. Using chimeric constructs, we find that the metalloprotease domain of ADAM10 can substitute for that of ADAM13, but that specificity for cement gland expansion requires a downstream extracellular domain of ADAM13. Analysis of finer resolution chimeras indicates an essential role for the cysteine-rich domain and a supporting role for the disintegrin domain. These and other results reveal that the cysteine-rich domain of ADAM13 cooperates intramolecularly with the ADAM13 metalloprotease domain to regulate its function in vivo. Our findings thus provide the first evidence that a downstream extracellular adhesive domain plays an active role in regulating ADAM protease function in vivo. These findings are likely relevant to other membrane-anchored cell surface proteases.

  8. Protease inhibitors interfere with the necessary factors of carcinogenesis.

    PubMed

    Troll, W

    1989-05-01

    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.

  9. Unveiling antimicrobial peptide-generating human proteases using PROTEASIX.

    PubMed

    Bastos, Paulo; Trindade, Fábio; Ferreira, Rita; Casteleiro, Mercedes Arguello; Stevens, Robert; Klein, Julie; Vitorino, Rui

    2017-02-27

    Extracting information from peptidomics data is a major current challenge, as endogenous peptides can result from the activity of multiple enzymes. Proteolytic enzymes can display overlapping or complementary specificity. The activity spectrum of human endogenous peptide-generating proteases is not fully known. Hence, the indirect study of proteolytic enzymes through the analysis of its substrates is largely hampered. Antimicrobial peptides (AMPs) represent a primordial set of immune defense molecules generated by proteolytic cleavage of precursor proteins. These peptides can be modulated by host and microorganismal stimuli, which both dictate proteolytic enzymes' expression and activity. Peptidomics is an attractive approach to identify peptides with a biological role and to assess proteolytic activity. However, bioinformatics tools to deal with peptidomics data are lacking. PROTEASIX is an excellent choice for the prediction of AMPs-generating proteases based on the reconstitution of a substrate's cleavage sites and the crossing of such information with known proteases' specificity retrieved by several publicly available databases. Therefore, the focus of the present tutorial is to explore the potential of PROTEASIX when gather information concerning proteases involved in the generation of human AMPs and to teach the user how to make the most out of peptidomics results using PROTEASIX.

  10. Cytomegalovirus protease targeted prodrug development.

    PubMed

    Sabit, Hairat; Dahan, Arik; Sun, Jing; Provoda, Chester J; Lee, Kyung-Dall; Hilfinger, John H; Amidon, Gordon L

    2013-04-01

    Human cytomegalovirus (HCMV) is a prevalent virus that infects up to 90% of the population. The goal of this research is to determine if small molecular prodrug substrates can be developed for a specific HCMV encoded protease and thus achieve site-specific activation. HCMV encodes a 256 amino acid serine protease that is responsible for capsid assembly, an essential process for herpes virus production. The esterase activity of the more stable HCMV A143T/A144T protease mutant was evaluated with model p-nitrophenol (ONp) esters, Boc-Xaa-ONp (Ala, Leu, Ile, Val, Gln, Phe at the Xaa position). We demonstrate that the A143T/A144T mutant has esterase activity toward specific small ester compounds, e.g., Boc-L-Ala-ONp. Mono amino acid and dipeptide prodrugs of ganciclovir (GCV) were also synthesized and evaluated for hydrolysis by the A143T/A144T protease mutant in solution. Hydrolysis of these prodrugs was also evaluated in Caco-2 cell homogenates, human liver microsomes (HLMs), and rat and human plasma. For the selectivity potential of the prodrugs, the hydrolysis ratio was evaluated as a percentage of prodrug hydrolyzed by the HCMV protease over the percentages of prodrug hydrolyses by Caco-2 cell homogenates, HLMs, and human/rat plasma. A dipeptide prodrug of ganciclovir, Ac-l-Gln-l-Ala-GCV, emerged as a potential selective prodrug candidate. The results of this research demonstrate that targeting prodrugs for activation by a specific protease encoded by the infectious HCMV pathogen may be achievable.

  11. Recombinant expression and functional analysis of proteases from Streptococcus pneumoniae, Bacillus anthracis, and Yersinia pestis

    PubMed Central

    2011-01-01

    Background Uncharacterized proteases naturally expressed by bacterial pathogens represents important topic in infectious disease research, because these enzymes may have critical roles in pathogenicity and cell physiology. It has been observed that cloning, expression and purification of proteases often fail due to their catalytic functions which, in turn, cause toxicity in the E. coli heterologous host. Results In order to address this problem systematically, a modified pipeline of our high-throughput protein expression and purification platform was developed. This included the use of a specific E. coli strain, BL21(DE3) pLysS to tightly control the expression of recombinant proteins and various expression vectors encoding fusion proteins to enhance recombinant protein solubility. Proteases fused to large fusion protein domains, maltosebinding protein (MBP), SP-MBP which contains signal peptide at the N-terminus of MBP, disulfide oxidoreductase (DsbA) and Glutathione S-transferase (GST) improved expression and solubility of proteases. Overall, 86.1% of selected protease genes including hypothetical proteins were expressed and purified using a combination of five different expression vectors. To detect novel proteolytic activities, zymography and fluorescence-based assays were performed and the protease activities of more than 46% of purified proteases and 40% of hypothetical proteins that were predicted to be proteases were confirmed. Conclusions Multiple expression vectors, employing distinct fusion tags in a high throughput pipeline increased overall success rates in expression, solubility and purification of proteases. The combinatorial functional analysis of the purified proteases using fluorescence assays and zymography confirmed their function. PMID:21545736

  12. Purification and characterization of cloned alkaline protease gene of Geobacillus stearothermophilus.

    PubMed

    Iqbal, Irfana; Aftab, Muhammad Nauman; Afzal, Mohammed; Ur-Rehman, Asad; Aftab, Saima; Zafar, Asma; Ud-Din, Zia; Khuharo, Ateeque Rahman; Iqbal, Jawad; Ul-Haq, Ikram

    2015-02-01

    Thermostable alkaline serine protease gene of Geobacillus stearothermophilus B-1172 was cloned and expressed in Escherichia coli BL21 (DE3) using pET-22b(+), as an expression vector. The growth conditions were optimized for maximal production of the protease using variable fermentation parameters, i.e., pH, temperature, and addition of an inducer. Protease, thus produced, was purified by ammonium sulfate precipitation followed by ion exchange chromatography with 13.7-fold purification, with specific activity of 97.5 U mg(-1) , and a recovery of 23.6%. Molecular weight of the purified protease, 39 kDa, was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was stable at 90 °C at pH 9. The enzyme activity was steady in the presence of EDTA indicating that the protease was not a metalloprotease. No significant change in the activity of protease after addition of various metal ions further strengthened this fact. However, an addition of 1% Triton X-100 or SDS surfactants constrained the enzyme specific activity to 34 and 19%, respectively. Among organic solvents, an addition of 1-butanol (20%) augmented the enzyme activity by 29% of the original activity. With casein as a substrate, the enzyme activity under optimized conditions was found to be 73.8 U mg(-1) . The effect of protease expression on the host cells growth was also studied and found to negatively affect E. coli cells to certain extent. Catalytic domains of serine proteases from eight important thermostable organisms were analyzed through WebLogo and found to be conserved in all serine protease sequences suggesting that protease of G. stearothermophilus could be beneficially used as a biocontrol agent and in many industries including detergent industry.

  13. Coronavirus genome: prediction of putative functional domains in the non-structural polyprotein by comparative amino acid sequence analysis.

    PubMed Central

    Gorbalenya, A E; Koonin, E V; Donchenko, A P; Blinov, V M

    1989-01-01

    Amino acid sequences of 2 giant non-structural polyproteins (F1 and F2) of infectious bronchitis virus (IBV), a member of Coronaviridae, were compared, by computer-assisted methods, to sequences of a number of other positive strand RNA viral and cellular proteins. By this approach, juxtaposed putative RNA-dependent RNA polymerase, nucleic acid binding ("finger"-like) and RNA helicase domains were identified in F2. Together, these domains might constitute the core of the protein complex involved in the primer-dependent transcription, replication and recombination of coronaviruses. In F1, two cysteine protease-like domains and a growth factor-like one were revealed. One of the putative proteases of IBV is similar to 3C proteases of picornaviruses and related enzymes of como- nepo- and potyviruses. Search of IBV F1 and F2 sequences for sites similar to those cleaved by the latter proteases and intercomparison of the surrounding sequence stretches revealed 13 dipeptides Q/S(G) which are probably cleaved by the coronavirus 3C-like protease. Based on these observations, a partial tentative scheme for the functional organization and expression strategy of the non-structural polyproteins of IBV was proposed. It implies that, despite the general similarity to other positive strand RNA viruses, and particularly to potyviruses, coronaviruses possess a number of unique structural and functional features. PMID:2526320

  14. Microbial proteases: detection, production, and genetic improvement.

    PubMed

    Kasana, Ramesh Chand; Salwan, Richa; Yadav, Sudesh Kumar

    2011-08-01

    Microbial proteases are one of the important groups of industrially and commercially produced enzymes contributing approximately 2/3 of all enzyme sales. Though proteases are produced by many microorganisms, emphasis is on the microorganisms producing proteases with desired characters. As demand for novel proteases is increasing day by day the initial screening methods and assays for protease detection are of utmost importance. This review focuses attention on present status of knowledge on the various methods and protocols available for protease screening, detection, and quantification starting from plate assays to spectrophotometric, fluorometric, and nanoparticles based assays. The review will help in making strategies for exploitation of protease resources and improvement of enzymes to obtain more robust proteases.

  15. Pathogen-secreted proteases activate a novel plant immune pathway.

    PubMed

    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

    2015-05-14

    Mitogen-activated protein kinase (MAPK) cascades play central roles in innate immune signalling networks in plants and animals. In plants, however, the molecular mechanisms of how signal perception is transduced to MAPK activation remain elusive. Here we report that pathogen-secreted proteases activate a previously unknown signalling pathway in Arabidopsis thaliana involving the Gα, Gβ, and Gγ subunits of heterotrimeric G-protein complexes, which function upstream of an 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 signalling to downstream activation of an MAPK cascade. The protease-G-protein-RACK1-MAPK cascade modules identified in these studies are distinct from previously described plant immune signalling pathways such as that elicited by bacterial flagellin, in which G proteins function downstream of or in parallel to an MAPK cascade without the involvement of the RACK1 scaffolding protein. The discovery of the new protease-mediated immune signalling 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 immunoregulatory strategies that account for its niche adaptation to diverse host tissues and immune systems.

  16. Characterization of cysteine protease-like genes in the striped rice stem borer, Chilo suppressalis.

    PubMed

    Ge, Zhao-Yu; Wan, Pin-Jun; Li, Guo-Qing; Xia, Yong-Gui; Han, Zhao-Jun

    2014-02-01

    The striped rice stem borer, Chilo suppressalis (Walker), is a major pest for rice production in China and the rest of Southeast Asia. Chemical control is the main means to alleviate losses due to this pest, which causes serious environmental pollution. An effective and environmentally friendly approach is needed for the management of the striped rice stem borer. Cysteine proteases in insects could be useful targets for pest management either through engineering plant protease inhibitors, targeting insect digestive cysteine proteases, or through RNA interference-based silencing of cysteine proteases, disrupting developmental regulation of insects. In this study, eight cysteine protease-like genes were identified and partially characterized. The genes CCO2 and CCL4 were exclusively expressed in the larval gut, and their expression was affected by the state of nutrition in the insect. The expression of CCL2, CCL3, and CCO1 was significantly affected by the type of host plant, suggesting a role in host plant - insect interactions. Our initial characterization of the striped rice stem borer cysteine protease-like genes provides a foundation for further research on this important group of genes in this major insect pest of rice.

  17. Heterologous expression of the plant cysteine protease bromelain and its inhibitor in Pichia pastoris.

    PubMed

    Luniak, Nora; Meiser, Peter; Burkart, Sonja; Müller, Rolf

    2017-01-01

    Expression of proteases in heterologous hosts remains an ambitious challenge due to severe problems associated with digestion of host proteins. On the other hand, proteases are broadly used in industrial applications and resemble promising drug candidates. Bromelain is an herbal drug that is medicinally used for treatment of oedematous swellings and inflammatory conditions and consists in large part of proteolytic enzymes. Even though various experiments underline the requirement of active cysteine proteases for biological activity, so far no investigation succeeded to clearly clarify the pharmacological mode of action of bromelain. The potential role of proteases themselves and other molecules of this multi-component extract currently remain largely unknown or ill defined. Here, we set out to express several bromelain cysteine proteases as well as a bromelain inhibitor molecule in order to gain defined molecular entities for subsequent studies. After cloning the genes from its natural source Ananas comosus (pineapple plant) into Pichia pastoris and subsequent fermentation and purification, we obtained active protease and inhibitor molecules which were subsequently biochemically characterized. Employing purified bromelain fractions paves the way for further elucidation of pharmacological activities of this natural product. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:54-65, 2017. © 2016 American Institute of Chemical Engineers.

  18. Functional analysis of rhomboid proteases during Toxoplasma invasion.

    PubMed

    Shen, Bang; Buguliskis, Jeffrey S; Lee, Tobie D; Sibley, L David

    2014-10-21

    Host cell invasion by Toxoplasma gondii and other apicomplexan parasites requires transmembrane adhesins that mediate binding to receptors on the substrate and host cell to facilitate motility and invasion. Rhomboid proteases (ROMs) are thought to cleave adhesins within their transmembrane segments, thus allowing the parasite to disengage from receptors and completely enter the host cell. To examine the specific roles of individual ROMs during invasion, we generated single, double, and triple knockouts for the three ROMs expressed in T. gondii tachyzoites. Analysis of these mutants demonstrated that ROM4 is the primary protease involved in adhesin processing and host cell invasion, whereas ROM1 or ROM5 plays negligible roles in these processes. Deletion of ROM4 blocked the shedding of adhesins such as MIC2 (microneme protein 2), causing them to accumulate on the surface of extracellular parasites. Increased surface adhesins led to nonproductive attachment, altered gliding motility, impaired moving junction formation, and reduced invasion efficiency. Despite the importance of ROM4 for efficient invasion, mutants lacking all three ROMs were viable and MIC2 was still efficiently removed from the surface of invaded mutant parasites, implying the existence of ROM-independent mechanisms for adhesin removal during invasion. Collectively, these results suggest that although ROM processing of adhesins is not absolutely essential, it is important for efficient host cell invasion by T. gondii. Importance: Apicomplexan parasites such as Toxoplasma gondii express surface proteins that bind host cell receptors to aid invasion. Many of these adhesins are subject to cleavage by rhomboid proteases (ROMs) within their transmembrane segments during invasion. Previous studies have demonstrated the importance of adhesin cleavage for parasite invasion and proposed that the ROMs responsible for processing would be essential for parasite survival. In T. gondii, ROM5 was thought to be the

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

    PubMed Central

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

    2013-01-01

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

  20. Single Cell Analysis of Leukocyte Protease Activity Using Integrated Continuous-Flow Microfluidics.

    PubMed

    Jing, Tengyang; Lai, Zhangxing; Wu, Lidan; Han, Jongyoon; Lim, Chwee Teck; Chen, Chia-Hung

    2016-12-06

    Leukocytes are the essential cells of the immune system that protect the human body against bacteria, viruses, and other foreign invaders. Secretory products of individual leukocytes, such as matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase (ADAMs), are critical for regulating the inflammatory response and mediating host defense. Conventional single cell analytical methods, such as flow cytometry for cellular surface biomarker studies, are insufficient for performing functional assays of the protease activity of individual leukocytes. Here, an integrated continuous-flow microfluidic assay is developed to effectively detect secretory protease activity of individual viable leukocytes. Leukocytes in blood are first washed on-chip with defined buffer to remove background activity, followed by encapsulating individual leukocytes with protease sensors in water-in-oil droplets and incubating for 1 h to measure protease secretion. With this design, single leukocyte protease profiles under naive and phorbol 12-myristate 13-acetate (PMA)-stimulated conditions are reliably measured. It is found that PMA treatment not only elevates the average protease activity level but also reduces the cellular heterogeneity in protease secretion, which is important in understanding immune capability and the disease condition of individual patients.

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

    PubMed

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

    2015-10-01

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

  2. Curcumin derivatives as HIV-1 protease inhibitors

    SciTech Connect

    Sui, Z.; Li, J.; Craik, C.S.; Ortiz de Montellano, P.R.

    1993-12-31

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

  3. Proteases in Fas-mediated apoptosis.

    PubMed

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

    1997-01-01

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

  4. Anti-trypanosomal activity of non-peptidic nitrile-based cysteine protease inhibitors.

    PubMed

    Burtoloso, Antonio C B; de Albuquerque, Sérgio; Furber, Mark; Gomes, Juliana C; Gonçalez, Cristiana; Kenny, Peter W; Leitão, Andrei; Montanari, Carlos A; Quilles, José Carlos; Ribeiro, Jean F R; Rocha, Josmar R

    2017-02-01

    The cysteine protease cruzipain is considered to be a validated target for therapeutic intervention in the treatment of Chagas disease. Anti-trypanosomal activity against the CL Brener strain of T. cruzi was observed in the 0.1 μM to 1 μM range for three nitrile-based cysteine protease inhibitors based on two scaffolds known to be associated with cathepsin K inhibition. The two compounds showing the greatest potency against the trypanosome were characterized by EC50 values (0.12 μM and 0.25 μM) that were an order of magnitude lower than the corresponding Ki values measured against cruzain, a recombinant form of cruzipain, in an enzyme inhibition assay. This implies that the anti-trypanosomal activity of these two compounds may not be explained only by the inhibition of the cruzain enzyme, thereby triggering a putative polypharmacological profile towards cysteine proteases.

  5. Structure of RC1339/APRc from Rickettsia conorii, a retropepsin-like aspartic protease

    PubMed Central

    Li, Mi; Gustchina, Alla; Cruz, Rui; Simões, Marisa; Curto, Pedro; Martinez, Juan; Faro, Carlos; Simões, Isaura; Wlodawer, Alexander

    2015-01-01

    The crystal structures of two constructs of RC1339/APRc from Rickettsia conorii, consisting of either residues 105–231 or 110–231 followed by a His tag, have been determined in three different crystal forms. As predicted, the fold of a monomer of APRc resembles one-half of the mandatory homodimer of retroviral pepsin-like aspartic proteases (retropepsins), but the quaternary structure of the dimer of APRc differs from that of the canonical retropepsins. The observed dimer is most likely an artifact of the expression and/or crystallization conditions since it cannot support the previously reported enzymatic activity of this bacterial aspartic protease. However, the fold of the core of each monomer is very closely related to the fold of retropepsins from a variety of retroviruses and to a single domain of pepsin-like eukaryotic enzymes, and may represent a putative common ancestor of monomeric and dimeric aspartic proteases. PMID:26457434

  6. Anti-trypanosomal activity of non-peptidic nitrile-based cysteine protease inhibitors

    PubMed Central

    Burtoloso, Antonio C. B.; de Albuquerque, Sérgio; Furber, Mark; Gomes, Juliana C.; Gonçalez, Cristiana; Kenny, Peter W.; Leitão, Andrei; Quilles, José Carlos; Ribeiro, Jean F. R.; Rocha, Josmar R.

    2017-01-01

    The cysteine protease cruzipain is considered to be a validated target for therapeutic intervention in the treatment of Chagas disease. Anti-trypanosomal activity against the CL Brener strain of T. cruzi was observed in the 0.1 μM to 1 μM range for three nitrile-based cysteine protease inhibitors based on two scaffolds known to be associated with cathepsin K inhibition. The two compounds showing the greatest potency against the trypanosome were characterized by EC50 values (0.12 μM and 0.25 μM) that were an order of magnitude lower than the corresponding Ki values measured against cruzain, a recombinant form of cruzipain, in an enzyme inhibition assay. This implies that the anti-trypanosomal activity of these two compounds may not be explained only by the inhibition of the cruzain enzyme, thereby triggering a putative polypharmacological profile towards cysteine proteases. PMID:28222138

  7. Possible identity of IL-8 converting enzyme in human fibroblasts as a cysteine protease.

    PubMed

    Ohashi, Kensaku; Sano, Emiko; Nakaki, Toshio; Naruto, Masanobu

    2003-04-01

    A converting activity was characterized in human diploid fibroblasts, which secrete 72IL-8 and 77IL-8 in treatment with IFN-beta and poly I: poly C. 77IL-8 was significantly converted to 72IL-8 by a partially purified fraction of the culture supernatant of human diploid fibroblasts. The converting activity, which was temperature-dependent and optimal at pH 6, was completely inhibited by cysteine protease inhibitors, antipain dihydrochloride and E-64, but not by other types of protease inhibitors. These data clearly show that human diploid fibroblasts are capable of processing IL-8 to produce a mature IL-8 and that the putative converting enzyme appears to be a cysteine protease.

  8. Characterization of the in vitro activities of the P1 and helper component proteases of Soybean mosaic virus Strain G2 and Tobacco vein mottling virus

    USDA-ARS?s Scientific Manuscript database

    Potyviruses express their RNA genomes through the production of polyproteins that are processed in host cells by three virus-encoded proteases. Soybean plants produce large amounts of protease inhibitors during seed development and in response to wounding that could affect the activities of these pr...

  9. Cysteine Proteases Inhibitors with immunoglobulin-like fold in protozoan parasites and their role in pathogenesis.

    PubMed

    Jimenez-Sandoval, Pedro; Lopez-Castillo, Laura Margarita; Trasviña-Arenas, Carlos H; Brieba, Luis G

    2016-08-13

    The number of protein folds in nature is limited, thus is not surprising that proteins with the same fold are able to exert different functions. The cysteine protease inhibitors that adopt an immunoglobulin-like fold (Ig-ICPs) are inhibitors encoded in bacteria and protozoan parasites. Structural studies indicate that these inhibitors resemble the structure of archetypical proteins with an Ig fold, like antibodies, cadherins or cell receptors. The structure of Ig-ICPs from four different protozoan parasites clearly shows the presence of three loops that form part of a protein-ligand interaction surface that resembles the antigen binding sites of antibodies. Thus, Ig-ICPs bind to different cysteine proteases using a tripartite mechanism in which their BC, DE and FG loops are responsible for the main interactions with the target cysteine protease. Ig-ICPs from different protozoan parasites regulate the enzymatic activity of host or parasite&#039;s proteases and thus regulate virulence and pathogenesis.

  10. Excretory bladder: the source of cysteine proteases in Paragonimus westermani metacercariae

    PubMed Central

    Yang, Hyun-Jong; Kang, Shin-Yong; Kong, Yoon; Cho, Seung-Yull

    2002-01-01

    The cysteine proteases of Paragonimus westermani metacercariae are involved in metacercarial excystment, host immune modulation, and possibly in tissue penetration. In order to clarify the origin of the enzymes, 28 and 27 kDa cysteine proteases in metacercarial excretory-secretory products were purified through the FPLC system using Mono Q column chromatography. The polyclonal antibodies to the enzymes were produced in BALB/c mice. Immunolocalization studies revealed that both cysteine proteases were distributed at the linings of excretory bladder and excretory concretions of the metacercariae. It was suggested that the excretory epithelium of P. westermani undertake the secretory function of metacercarial cysteine proteases, in addition to its role as a route for eliminating waste products. PMID:12073734

  11. Protease Inhibitors of Parasitic Flukes: Emerging Roles in Parasite Survival and Immune Defence.

    PubMed

    Ranasinghe, Shiwanthi L; McManus, Donald P

    2017-05-01

    Protease inhibitors play crucial roles in parasite development and survival, counteracting the potentially damaging immune responses of their vertebrate hosts. However, limited information is currently available on protease inhibitors from schistosomes and food-borne trematodes. Future characterization of these molecules is important not only to expand knowledge on parasitic fluke biology but also to determine whether they represent novel vaccine and/or drug targets. Moreover, protease inhibitors from flukes may represent lead compounds for the development of a new range of therapeutic agents against inflammatory disorders and cancer. This review discusses already identified protease inhibitors of fluke origin, emphasizing their biological function and their possible future development as new intervention targets. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Protease digestion of hepatitis A virus: disparate effects on capsid proteins, antigenicity, and infectivity.

    PubMed Central

    Lemon, S M; Amphlett, E; Sangar, D

    1991-01-01

    High concentrations of either trypsin or chymotrypsin caused nearly complete cleavage of capsid protein VP2 of hepatitis A virus but did not significantly reduce the infectivity, thermostability, or antigenicity of the virus. Chymotrypsin also had a lesser effect on VP1. These findings indicate the presence of a protease-accessible VP2 surface site which neither contributes significantly to the dominant antigenic site nor plays a role in the attachment of the virus to putative cell receptors. Images PMID:1654460

  13. Proteases of Stored Product Insects and Their Inhibition by Specific Protease Inhibitors from Soybeans and Wheat Grain

    DTIC Science & Technology

    1989-12-15

    PROTEASES; PROTEASE INHIBITORS; STORED-PRODUCT INISECTS; TRIBOLIUM CASIANEUH; MIDGUT PROTEASES; TENEBRIO MOLITOR MIDGUT-PROTEASES; LOCUST CAECAL...separation and identification of numerous midgut proteases in Tenebrio and Tribolium . The PAGE-gelatin matrix revealed the inhibitory effect of BBI...the proteinaceous trypsin-chymotrypsin inhibitor from soybeans) on several Tribolium proteases - an effect which was not detectable in inhibition

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

    PubMed Central

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

    2017-01-01

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

  15. Role of an extracellular neutral protease in infection against nematodes by Brevibacillus laterosporus strain G4.

    PubMed

    Tian, Baoyu; Yang, Jinkui; Lian, Lihui; Wang, Chunyan; Li, Ning; Zhang, Ke-Qin

    2007-02-01

    Proteases have been proposed as virulence factors in microbial pathogenicity against nematodes. However, what kinds of extracellular proteases from these pathogens and how they contribute to the pathogenesis of infections against nematode in vivo remain largely unknown. A previous analysis using a strain with a deletion in an extracellular alkaline protease BLG4 gene from Brevibacillus laterosporus demonstrated that BLG4 was responsible for the majority of nematicidal activity by destroying host's cuticle. In recent studies, a neutral protease NPE-4, purified from the mutant BLG4-6, was found to be responsible for the majority of the remaining EDTA-inhibited protease activity. However, the purified NPE-4 and recombinant NPE-4 in a related species Bacillus subtilis showed little nematicidal activity in vitro and were unable to degrade the intact cuticle of the host. It is interesting to note that the addition of NPE-4 improved the pathogenicity of crude enzyme extract from wild-type B. laterosporus but had no effect on the BLG4-deficient mutant. This result suggests that NPE-4 functions in the presence of protease BLG4. Moreover, NPE-4 could degrade proteins from the inner layer of purified cuticles from nematode Panagrellus redivivus in vitro. These results indicated that the two different bacterial extracellular proteases might play differential roles at different stages of infection or a synthetic role in penetration of nematode cuticle in B. laterosporus. This is among the first reports to systematically evaluate and define the roles of different bacterial extracellular proteases in infection against nematodes.

  16. Function and Regulation of SUMO Proteases

    PubMed Central

    Hickey, Christopher M.; Wilson, Nicole R.; Hochstrasser, Mark

    2013-01-01

    Covalent attachment of small ubiquitin-like modifier (SUMO) to proteins is highly dynamic, and both SUMO-protein conjugation and cleavage can be regulated. Protein desumoylation is performed by SUMO proteases, which control cellular mechanisms ranging from transcription and cell division to ribosome biogenesis. Recent advances include the discovery of two novel classes of SUMO proteases, insights regarding SUMO protease specificity, and revelations of previously unappreciated SUMO protease functions in several key cellular pathways. These developments, together with new connections between SUMO proteases and the recently discovered SUMO-targeted ubiquitin ligases (STUbLs), make this an exciting period for the study of these enzymes. PMID:23175280

  17. The Multifaceted Poliovirus 2A Protease: Regulation of Gene Expression by Picornavirus Proteases

    PubMed Central

    Castelló, Alfredo; Álvarez, Enrique; Carrasco, Luis

    2011-01-01

    After entry into animal cells, most viruses hijack essential components involved in gene expression. This is the case of poliovirus, which abrogates cellular translation soon after virus internalization. Abrogation is achieved by cleavage of both eIF4GI and eIF4GII by the viral protease 2A. Apart from the interference of poliovirus with cellular protein synthesis, other gene expression steps such as RNA and protein trafficking between nucleus and cytoplasm are also altered. Poliovirus 2Apro is capable of hydrolyzing components of the nuclear pore, thus preventing an efficient antiviral response by the host cell. Here, we compare in detail poliovirus 2Apro with other viral proteins (from picornaviruses and unrelated families) as regard to their activity on key host factors that control gene expression. It is possible that future analyses to determine the cellular proteins targeted by 2Apro will uncover other cellular functions ablated by poliovirus infection. Further understanding of the cellular proteins hydrolyzed by 2Apro will add further insight into the molecular mechanism by which poliovirus and other viruses interact with the host cell. PMID:21541224

  18. 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

  19. Structural basis for the immunomodulatory function of cysteine protease inhibitor from human roundworm Ascaris lumbricoides.

    PubMed

    Mei, Guoqiang; Dong, Jianmei; Li, Zhaotao; Liu, Sanling; Liu, Yunfeng; Sun, Mingze; Liu, Guiyun; Su, Zhong; Liu, Jinsong

    2014-01-01

    Immunosuppression associated with infections of nematode parasites has been documented. Cysteine protease inhibitor (CPI) released by the nematode parasites is identified as one of the major modulators of host immune response. In this report, we demonstrated that the recombinant CPI protein of Ascaris lumbricoides (Al-CPI) strongly inhibited the activities of cathepsin L, C, S, and showed weaker effect to cathepsin B. Crystal structure of Al-CPI was determined to 2.1 Å resolution. Two segments of Al-CPI, loop 1 and loop 2, were proposed as the key structure motifs responsible for Al-CPI binding with proteases and its inhibitory activity. Mutations at loop 1 and loop 2 abrogated the protease inhibition activity to various extents. These results provide the molecular insight into the interaction between the nematode parasite and its host and will facilitate the development of anthelmintic agents or design of anti-autoimmune disease drugs.

  20. Immune evasion by pathogenic Leptospira strains: the secretion of proteases that directly cleave complement proteins.

    PubMed

    Fraga, Tatiana Rodrigues; Courrol, Daniella Dos Santos; Castiblanco-Valencia, Mónica Marcela; Hirata, Izaura Yoshico; Vasconcellos, Sílvio Arruda; Juliano, Luiz; Barbosa, Angela Silva; Isaac, Lourdes

    2014-03-01

    Leptospirosis is an infectious disease of public health importance. To successfully colonize the host, pathogens have evolved multiple strategies to escape the complement system. Here we demonstrate that the culture supernatant of pathogenic but not saprophytic Leptospira inhibit the three complement pathways. We showed that the proteolytic activity in the supernatants of pathogenic strains targets the central complement molecule C3 and specific proteins from each pathway, such as factor B, C2, and C4b. The proteases cleaved α and β chains of C3 and work in synergy with host regulators to inactivate C3b. Proteolytic activity was inhibited by 1,10-phenanthroline, suggesting the participation of metalloproteases. A recombinant leptospiral metalloprotease from the thermolysin family cleaved C3 in serum and could be one of the proteases responsible for the supernatant activity. We conclude that pathogenic leptospiral proteases can deactivate immune effector molecules and represent potential targets to the development of new therapies in leptospirosis.

  1. Degradation of immunoglobulins, protease inhibitors, and interleukin-1 by a secretory proteinase of Acanthamoeba castellanii

    PubMed Central

    Na, Byoung-Kuk; Cho, Jong-Hwa; Song, Chul-Yong; Kim, Tong-Soo

    2002-01-01

    The effect of a secretory proteinase from the pathogenic amoebae Acanthamoeba castellanii on host's defense-oriented or regulatory proteins such as immunoglobulins, interleukin-1, and protease inhibitors was investigated. The enzyme was found to degrade secretory immunoglobulin A (sIgA), IgG, and IgM. It also degraded interleukin-1α (IL-1α) and IL-1β. Its activity was not inhibited by endogenous protease inhibitors, such as α2-macroglobulin, α1-trypsin inhibitor, and α2-antiplasmin. Furthermore, the enzyme rapidly degraded those endogenous protease inhibitors as well. The degradation of host's defense-oriented or regulatory proteins by the Acanthamoeba proteinase suggested that the enzyme might be an important virulence factor in the pathogenesis of Acanthamoeba infection. PMID:12073735

  2. Molecular and functional characterisation of a stress responsive cysteine protease, EhCP6 from Entamoeba histolytica.

    PubMed

    Ghosh, Anupama; Raha, Sanghamitra

    2015-05-01

    Entamoeba histolytica cysteine protease 6 (EhCP6) is a stress responsive cysteine protease that is upregulated in response to heat shock and during pathogen invasion of the host tissue. In the present study an attempt has been made to express and purify recombinant EhCP6 in order to gain insights into its biochemical properties. The recombinant and refolded protein has been shown to undergo autoproteolysis in the presence of DTT and SDS to give rise to ∼25kDa mature form. The mature form of the protein was found to exhibit a protease activity that is sensitive to E-64, a specific cysteine protease inhibitor. In silico homology modelling of EhCP6 revealed that the protein exhibits conservation of almost all the major structural features of cathepsin-L like cysteine proteases. Further in vivo studies are needed to decipher the function of the protein in response to different stressed conditions.

  3. 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

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

    PubMed

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

    2014-06-04

    . Phylogenetic analysis using Bayes approach provided support for inferring functional divergence among regulatory cysteine and serine proteases. Numerous putative proteases were identified for the first time in T. solium, and important regulatory proteases have been predicted. This comprehensive analysis not only complements the growing knowledge base of proteolytic enzymes, but also provides a platform from which to expand knowledge of cestode proteases and to explore their biochemistry and potential as intervention targets.

  5. A serine protease from a detergent-soluble extract of Leishmania (Leishmania) amazonensis.

    PubMed

    da Silva Lopez, Raquel Elisa; De Simone, Salvatore Giovanni

    2004-01-01

    Proteases mediate important crucial functions in parasitic diseases, and their characterization contributes to the understanding of host-parasite interaction. A serine protease was purified about 43-fold with a total recovery of 60% from a detergent-soluble extract of promastigotes of Leishmania amazonensis. The purification procedures included aprotinin-agarose affinity chromatography and gel filtration high performance liquid chromatography. The molecular mass of active enzyme was 110 kDa by native gel filtration HPLC and by SDS-PAGE gelatin under non-reducing conditions. Under conditions of reduction using SDS-PAGE gelatin analyses the activity of enzyme was observed in two proteins of 60 and 45 kDa, suggesting that the enzyme may be considered as a dimer. The Leishmania protease was not glycosylated, and its isoelectric point (pI) was around 4.8. The maximal protease activity was at pH 7.0 and 28 degrees C, using a-N-o-tosyl-L-arginyl-methyl ester (L-TAME) as substrate. Assays of thermal stability indicated that this enzyme was totally denatured after pre-treatment at 42 degrees C for 12 min and preserved only 20% of its activity after pre-treatment at 37 degrees C for 24 h, in the absence of substrate. Hemoglobin, bovine serum albumin (BSA), ovalbumin and gelatin were hydrolyzed by Leishmania protease. Inhibition studies indicated that the enzyme belonged to a serine protease class because of a significant impediment by serine protease inhibitors such as benzamidine, aprotinin, and antipain. The activity of the present serine protease is negatively modulated by calcium and zinc and positively modulated by manganese ions. This is the first study that reports the purification of a protease from a detergent-soluble extract of Leishmania species.

  6. Inhibition of Melanization by a Parasitoid Serine Protease Homolog Venom Protein Requires Both the Clip and the Non-Catalytic Protease-Like Domains.

    PubMed

    Thomas, Pune; Asgari, Sassan

    2011-11-25

    Endoparasitoid wasps inject a variety of components into their host hemocoel at oviposition to facilitate successful development of their progeny. Among these are venom proteins which have been shown to play crucial roles in host regulation. A serine protease homolog (SPH)-like venom protein from Cotesia rubecula was previously shown to inhibit melanization in the host hemolymph by blocking activation of prophenoloxidase to phenoloxidase, a key enzyme in melanin formation. Similar to other SPHs, Vn50 consists of a clip and a protease-like (SPL) domain. Protein modeling demonstrated that Vn50 has a very similar structure to known SPHs and functional analysis of Vn50 domains expressed in insect cells indicated that neither of the domains on its own has an inhibitory effect on melanization.

  7. A Typhus Group-Specific Protease Defies Reductive Evolution in Rickettsiae ▿ † ‡

    PubMed Central

    Ammerman, Nicole C.; Gillespie, Joseph J.; Neuwald, Andrew F.; Sobral, Bruno W.; Azad, Abdu F.

    2009-01-01

    Phylogenomics reveals extreme gene loss in typhus group (TG) rickettsiae relative to the levels for other rickettsial lineages. We report here a curious protease-encoding gene (ppcE) that is conserved only in TG rickettsiae. As a possible determinant of host pathogenicity, ppcE warrants consideration in the development of therapeutics against epidemic and murine typhus. PMID:19820087

  8. Roles of proteases during invasion and egress by Plasmodium and Toxoplasma.

    PubMed

    Dowse, Timothy J; Koussis, Konstantinos; Blackman, Michael J; Soldati-Favre, Dominique

    2008-01-01

    Apicomplexan pathogens replicate exclusively within the confines of a host cell. Entry into (invasion) and exit from (egress) these cells requires an array of specialized parasite molecules, many of which have long been considered to have potential as targets of drug or vaccine-based therapies. In this chapter the authors discuss the current state of knowledge regarding the role of parasite proteolytic enzymes in these critical steps in the life cycle of two clinically important apicomplexan genera, Plasmodium and Toxoplasma. At least three distinct proteases of the cysteine mechanistic class have been implicated in egress of the malaria parasite from cells of its vertebrate and insect host. In contrast, the bulk of the evidence indicates a prime role for serine proteases of the subtilisin and rhomboid families in invasion by both parasites. Whereas proteases involved in egress may function predominantly to degrade host cell structures, proteases involved in invasion probably act primarily as maturases and 'sheddases', required to activate and ultimately remove ligands involved in interactions with the host cell.

  9. Fasciola hepatica Surface Tegument: Glycoproteins at the Interface of Parasite and Host.

    PubMed

    Ravidà, Alessandra; Cwiklinski, Krystyna; Aldridge, Allison M; Clarke, Paul; Thompson, Roisin; Gerlach, Jared Q; Kilcoyne, Michelle; Hokke, Cornelis H; Dalton, John P; O'Neill, Sandra M

    2016-10-01

    Fasciola hepatica, commonly known as liver fluke, is a trematode that causes Fasciolosis in ruminants and humans. The outer tegumental coat of F. hepatica (FhTeg) is a complex metabolically active biological matrix that is continually exposed to the host immune system and therefore makes a good vaccine target. F. hepatica tegumental coat is highly glycosylated and helminth-derived immunogenic oligosaccharide motifs and glycoproteins are currently being investigated as novel vaccine candidates. This report presents the first systematic characterization of FhTeg glycosylation using lectin microarrays to characterize carbohydrates motifs present, and lectin histochemistry to localize these on the F. hepatica tegument. We discovered that FhTeg glycoproteins are predominantly oligomannose oligosaccharides that are expressed on the spines, suckers and tegumental coat of F. hepatica and lectin blot analysis confirmed the abundance of N- glycosylated proteins. Although some oligosaccharides are widely distributed on the fluke surface other subsets are restricted to distinct anatomical regions. We selectively enriched for FhTeg mannosylated glycoprotein subsets using lectin affinity chromatography and identified 369 proteins by mass spectrometric analysis. Among these proteins are a number of potential vaccine candidates with known immune modulatory properties including proteases, protease inhibitors, paramyosin, Venom Allergen-like II, Enolase and two proteins, nardilysin and TRIL, that have not been previously associated with F. hepatica Furthermore, we provide a comprehensive insight regarding the putative glycosylation of FhTeg components that could highlight the importance of further studies examining glycoconjugates in host-parasite interactions in the context of F. hepatica infection and the development of an effective vaccine. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Fasciola hepatica Surface Tegument: Glycoproteins at the Interface of Parasite and Host*

    PubMed Central

    Ravidà, Alessandra; Cwiklinski, Krystyna; Aldridge, Allison M.; Clarke, Paul; Thompson, Roisin; Gerlach, Jared Q.; Kilcoyne, Michelle; Hokke, Cornelis H.; Dalton, John P.; O'Neill, Sandra M.

    2016-01-01

    Fasciola hepatica, commonly known as liver fluke, is a trematode that causes Fasciolosis in ruminants and humans. The outer tegumental coat of F. hepatica (FhTeg) is a complex metabolically active biological matrix that is continually exposed to the host immune system and therefore makes a good vaccine target. F. hepatica tegumental coat is highly glycosylated and helminth-derived immunogenic oligosaccharide motifs and glycoproteins are currently being investigated as novel vaccine candidates. This report presents the first systematic characterization of FhTeg glycosylation using lectin microarrays to characterize carbohydrates motifs present, and lectin histochemistry to localize these on the F. hepatica tegument. We discovered that FhTeg glycoproteins are predominantly oligomannose oligosaccharides that are expressed on the spines, suckers and tegumental coat of F. hepatica and lectin blot analysis confirmed the abundance of N- glycosylated proteins. Although some oligosaccharides are widely distributed on the fluke surface other subsets are restricted to distinct anatomical regions. We selectively enriched for FhTeg mannosylated glycoprotein subsets using lectin affinity chromatography and identified 369 proteins by mass spectrometric analysis. Among these proteins are a number of potential vaccine candidates with known immune modulatory properties including proteases, protease inhibitors, paramyosin, Venom Allergen-like II, Enolase and two proteins, nardilysin and TRIL, that have not been previously associated with F. hepatica. Furthermore, we provide a comprehensive insight regarding the putative glycosylation of FhTeg components that could highlight the importance of further studies examining glycoconjugates in host-parasite interactions in the context of F. hepatica infection and the development of an effective vaccine. PMID:27466253

  11. Characterization of a metagenome-derived protease from contaminated agricultural soil microorganisms and its random mutagenesis.

    PubMed

    Jiang, Chengjian; Zhang, Liang; Li, Fajia; Meng, Can; Zeng, Rong; Deng, Jie; Shen, Peihong; Ou, Qian; Wu, Bo

    2017-04-05

    Proteases are typical key enzymes that hydrolyze proteins into amino acids and peptides. Numerous proteases have been studied, but the discovery of metagenome-derived proteases is still significant for both commercial applications and basic research. An unexplored protease gene sep1A was identified by function-based screening from a plasmid metagenomic library derived from uncultured contaminated agricultural soil microorganisms. The putative protease gene was subcloned into pET-32a (+) vector and overexpressed in E. coli BL21(DE3) pLysS, then the recombinant protein was purified to homogeneity. The detailed biochemical characterization of the Sep1A protein was performed, including its molecular characterization, specific activity, pH-activity profile, metal ion-activity profile, and enzyme kinetic assays. Furthermore, the protein engineering approach of random mutagenesis via error-prone PCR was applied on the original Sep1A protein. Biochemical characterization demonstrated that the purified recombinant Ep48 protein could hydrolyze casein. Compared with the original Sep1A protein, the best variant of Ep48 in the random mutagenesis library, with the Gln307Leu and Asp391Gly changes, exhibited 2.62-fold activity at the optimal reaction conditions of 50 °C and pH 9.0. These results are the first step toward a better understanding of the properties of Sep1A protein. Protein engineering with error-prone PCR paves the way toward the metagenome-derived genes for biotechnological applications.

  12. Cysteine protease gene expression and proteolytic activity during senescence of Alstroemeria petals.

    PubMed

    Wagstaff, Carol; Leverentz, Michael K; Griffiths, Gareth; Thomas, Brian; Chanasut, Usawadee; Stead, Anthony D; Rogers, Hilary J

    2002-02-01

    The functional life of the flower is terminated by senescence and/or abscission. Multiple processes contribute to produce the visible signs of petal wilting and inrolling that typify senescence, but one of the most important is that of protein degradation and remobilization. This is mediated in many species through protein ubiquitination and the action of specific protease enzymes. This paper reports the changes in protein and protease activity during development and senescence of Alstroemeria flowers, a Liliaceous species that shows very little sensitivity to ethylene during senescence and which shows perianth abscission 8-10 d after flower opening. Partial cDNAs of ubiquitin (ALSUQ1) and a putative cysteine protease (ALSCYP1) were cloned from Alstroemeria using degenerate PCR primers and the expression pattern of these genes was determined semi-quantitatively by RT-PCR. While the levels of ALSUQ1 only fluctuated slightly during floral development and senescence, there was a dramatic increase in the expression of ALSCYP1 indicating that this gene may encode an important enzyme for the proteolytic process in this species. Three papain class cysteine protease enzymes showing different patterns of activity during flower development were identified on zymograms, one of which showed a similar expression pattern to the cysteine protease cDNA.

  13. Nelfinavir: fourth protease inhibitor approved.

    PubMed

    1997-01-01

    The Food and Drug Administration (FDA) has granted accelerated approval to nelfinavir in both adult and pediatric formulations. Agouron, the manufacturer, used innovative computerized drug design techniques to discover, design, and refine the nelfinavir molecule. Nelfinavir is marketed under the trade name Viracept, and costs $5,000 per year. Early clinical trials find it to be as powerful as the other protease inhibitors, but with a different resistance profile. The drug has relatively few drug indications; however, several compounds have been contraindicated.

  14. Inactivation of Streptococcus pyogenes extracellular cysteine protease significantly decreases mouse lethality of serotype M3 and M49 strains.

    PubMed Central

    Lukomski, S; Sreevatsan, S; Amberg, C; Reichardt, W; Woischnik, M; Podbielski, A; Musser, J M

    1997-01-01

    Cysteine proteases have been implicated as important virulence factors in a wide range of prokaryotic and eukaryotic pathogens, but little direct evidence has been presented to support this notion. Virtually all strains of the human bacterial pathogen Streptococcus pyogenes express a highly conserved extracellular cysteine protease known as streptococcal pyrogenic exotoxin B (SpeB). Two sets of isogenic strains deficient in SpeB cysteine protease activity were constructed by integrational mutagenesis using nonreplicating recombinant plasmids containing a truncated segment of the speB gene. Immunoblot analyses and enzyme assays confirmed that the mutant derivatives were deficient in expression of enzymatically active SpeB cysteine protease. To test the hypothesis that the cysteine protease participates in host mortality, we assessed the ability of serotype M3 and M49 wild-type strains and isogenic protease-negative mutants to cause death in outbred mice after intraperitoneal inoculation. Compared to wild-type parental organisms, the serotype M3 speB mutant lost virtually all ability to cause mouse death (P < 0.00001), and similarly, the virulence of the M49 mutant was detrimentally altered (P < 0.005). The data unambiguously demonstrate that the streptococcal enzyme is a virulence factor, and thereby provide additional evidence that microbial cysteine proteases are critical in host-pathogen interactions. PMID:9169486

  15. Serine Protease Activity of Calnuc

    PubMed Central

    Kanuru, Madhavi; Raman, Rajeev; Aradhyam, Gopala Krishna

    2013-01-01

    The functions of calnuc, a novel Ca2+-binding protein with multiple structural domains and diverse interacting partners, are yet unknown. We demonstrate unknown facets of calnuc, which is a serine protease in which Ser-378 of GXSXG motif, Asp-328 of DTG motif, and His-339 form the “catalytic triad,” locating the enzyme active site in the C-terminal region. Analogous to the active site of Zn2+ carboxypeptidases, calnuc has two high affinity (Kd ∼ 20 nm), well conserved Zn2+-binding sites near its N terminus, although it is inactive as a peptidase. Zn2+ binding allosterically and negatively regulates the serine protease activity of calnuc, inhibition being caused by an “open to close” change in its conformation not seen upon Ca2+ binding. Most strikingly, interaction with G protein α subunit completely inhibits the enzymatic activity of calnuc. We thus illustrate that G proteins and Zn2+ act as two “keys” that control enzymatic activity of calnuc, arresting it in “locked” state. Calnuc, therefore, exists dynamically in two different forms, (i) as a Ca2+-binding protein in Zn2+-bound form and (ii) as a protease in Zn2+-free form, commissioning it to perform multiple functions. PMID:23195954

  16. 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

  17. Dimerization and protease resistance: new insight into the function of PR-1.

    PubMed

    Lu, Shunwen; Faris, Justin D; Sherwood, Robert; Edwards, Michael C

    2013-01-01

    The group 1 pathogenesis-related (PR-1) proteins have long been considered hallmarks of hypersensitive response/defense pathways in plants, but their biochemical functions are still obscure despite resolution of the NMR/X-ray structures of several PR-1-like proteins, including P14a (the prototype PR-1). We report here the characterization of two basic PR-1 proteins (PR-1-1 and PR-1-5) recently identified from hexaploid wheat (Triticum aestivum). Both proteins were expressed in Pichia pastoris as a single major species of ∼15 kDa. Sequence identity of the expressed PR-1 proteins was verified by MALDI-TOF/TOF analysis. Accumulation of the native PR-1-5 protein in pathogen-challenged wheat was confirmed by protein gel blot analysis. Low-temperature SDS-PAGE and yeast two-hybrid assays revealed that PR-1-1 exists primarily as a monomer whereas PR-1-5 forms homodimers. Both PR-1 proteins are resistant to proteases compared to bovine serum albumin, but PR-1-1 shows resistance mainly to subtilisin and protease K (serine proteases) whereas PR-1-5 shows resistance to subtilisin, protease K and papain (a cysteine protease). Site-specific mutations at the five putative active sites in the PR-1 domain all affected dimerization, with the mutations at Glu-72 and Glu-102 (in the PR-1-5 numeration) also diminishing protease resistance. Sequence analysis revealed that the Glu-72 and Glu-102 residues are located in motif-like sequences that are conserved in both PR-1 and the human apoptosis-related caspase proteins. These findings prompt us to examine the function of PR-1 for a role in protease-mediated programmed cell death pathways in plants. Copyright © 2012 Elsevier GmbH. All rights reserved.

  18. The effect of environmental conditions on expression of Bacteroides fragilis and Bacteroides thetaiotaomicron C10 protease genes

    PubMed Central

    2012-01-01

    Background Bacteroides fragilis and Bacteroides thetaiotaomicron are members of the normal human intestinal microbiota. However, both organisms are capable of causing opportunistic infections, during which the environmental conditions to which the bacteria are exposed change dramatically. To further explore their potential for contributing to infection, we have characterized the expression in B. thetaiotaomicron of four homologues of the gene encoding the C10 cysteine protease SpeB, a potent extracellular virulence factor produced by Streptococcus pyogenes. Results We identified a paralogous set of genes (btp genes) in the B. thetaiotaomicron genome, that were related to C10 protease genes we recently identified in B. fragilis. Similar to C10 proteases found in B. fragilis, three of the B. thetaiotaomicron homologues were transcriptionally coupled to genes encoding small proteins that are similar in structural architecture to Staphostatins, protease inhibitors associated with Staphopains in Staphylococcus aureus. The expression of genes for these C10 proteases in both B. fragilis and B. thetaiotaomicron was found to be regulated by environmental stimuli, in particular by exposure to oxygen, which may be important for their contribution to the development of opportunistic infections. Conclusions Genes encoding C10 proteases are increasingly identified in operons which also contain genes encoding proteins homologous to protease inhibitors. The Bacteroides C10 protease gene expression levels are responsive to different environmental stimuli suggesting they may have distinct roles in the bacterial-host interaction. PMID:22943521

  19. The effect of environmental conditions on expression of Bacteroides fragilis and Bacteroides thetaiotaomicron C10 protease genes.

    PubMed

    Thornton, Roibeard F; Murphy, Elizabeth C; Kagawa, Todd F; O'Toole, Paul W; Cooney, Jakki C

    2012-09-03

    Bacteroides fragilis and Bacteroides thetaiotaomicron are members of the normal human intestinal microbiota. However, both organisms are capable of causing opportunistic infections, during which the environmental conditions to which the bacteria are exposed change dramatically. To further explore their potential for contributing to infection, we have characterized the expression in B. thetaiotaomicron of four homologues of the gene encoding the C10 cysteine protease SpeB, a potent extracellular virulence factor produced by Streptococcus pyogenes. We identified a paralogous set of genes (btp genes) in the B. thetaiotaomicron genome, that were related to C10 protease genes we recently identified in B. fragilis. Similar to C10 proteases found in B. fragilis, three of the B. thetaiotaomicron homologues were transcriptionally coupled to genes encoding small proteins that are similar in structural architecture to Staphostatins, protease inhibitors associated with Staphopains in Staphylococcus aureus. The expression of genes for these C10 proteases in both B. fragilis and B. thetaiotaomicron was found to be regulated by environmental stimuli, in particular by exposure to oxygen, which may be important for their contribution to the development of opportunistic infections. Genes encoding C10 proteases are increasingly identified in operons which also contain genes encoding proteins homologous to protease inhibitors. The Bacteroides C10 protease gene expression levels are responsive to different environmental stimuli suggesting they may have distinct roles in the bacterial-host interaction.

  20. Blunting the knife: development of vaccines targeting digestive proteases of blood-feeding helminth parasites.

    PubMed

    Pearson, Mark S; Ranjit, Najju; Loukas, Alex

    2010-08-01

    Proteases are pivotal to parasitism, mediating biological processes crucial to worm survival including larval migration through tissue, immune evasion/modulation and nutrient acquisition by the adult parasite. In haematophagous parasites, many of these proteolytic enzymes are secreted from the intestine (nematodes) or gastrodermis (trematodes) where they act to degrade host haemoglobin and serum proteins as part of the feeding process. These proteases are exposed to components of the immune system of the host when the worms ingest blood, and therefore present targets for the development of anti-helminth vaccines. The protective effects of current vaccine antigens against nematodes that infect humans (hookworm) and livestock (barber's pole worm) are based on haemoglobin-degrading intestinal proteases and act largely as a result of the neutralisation of these proteases by antibodies that are ingested with the blood-meal. In this review, we survey the current status of helminth proteases that show promise as vaccines and describe their vital contribution to a parasitic existence.

  1. 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. © 2016 American Society of Plant Biologists. All Rights Reserved.

  2. Proteases from Entamoeba spp. and Pathogenic Free-Living Amoebae as Virulence Factors

    PubMed Central

    Serrano-Luna, Jesús; Piña-Vázquez, Carolina; Reyes-López, Magda; Ortiz-Estrada, Guillermo

    2013-01-01

    The standard reference for pathogenic and nonpathogenic amoebae is the human parasite Entamoeba histolytica; a direct correlation between virulence and protease expression has been demonstrated for this amoeba. Traditionally, proteases are considered virulence factors, including those that produce cytopathic effects in the host or that have been implicated in manipulating the immune response. Here, we expand the scope to other amoebae, including less-pathogenic Entamoeba species and highly pathogenic free-living amoebae. In this paper, proteases that affect mucin, extracellular matrix, immune system components, and diverse tissues and cells are included, based on studies in amoebic cultures and animal models. We also include proteases used by amoebae to degrade iron-containing proteins because iron scavenger capacity is currently considered a virulence factor for pathogens. In addition, proteases that have a role in adhesion and encystation, which are essential for establishing and transmitting infection, are discussed. The study of proteases and their specific inhibitors is relevant to the search for new therapeutic targets and to increase the power of drugs used to treat the diseases caused by these complex microorganisms. PMID:23476670

  3. Proteases from Entamoeba spp. and Pathogenic Free-Living Amoebae as Virulence Factors.

    PubMed

    Serrano-Luna, Jesús; Piña-Vázquez, Carolina; Reyes-López, Magda; Ortiz-Estrada, Guillermo; de la Garza, Mireya

    2013-01-01

    The standard reference for pathogenic and nonpathogenic amoebae is the human parasite Entamoeba histolytica; a direct correlation between virulence and protease expression has been demonstrated for this amoeba. Traditionally, proteases are considered virulence factors, including those that produce cytopathic effects in the host or that have been implicated in manipulating the immune response. Here, we expand the scope to other amoebae, including less-pathogenic Entamoeba species and highly pathogenic free-living amoebae. In this paper, proteases that affect mucin, extracellular matrix, immune system components, and diverse tissues and cells are included, based on studies in amoebic cultures and animal models. We also include proteases used by amoebae to degrade iron-containing proteins because iron scavenger capacity is currently considered a virulence factor for pathogens. In addition, proteases that have a role in adhesion and encystation, which are essential for establishing and transmitting infection, are discussed. The study of proteases and their specific inhibitors is relevant to the search for new therapeutic targets and to increase the power of drugs used to treat the diseases caused by these complex microorganisms.

  4. Advances in protease engineering for laundry detergents.

    PubMed

    Vojcic, Ljubica; Pitzler, Christian; Körfer, Georgette; Jakob, Felix; Ronny Martinez; Maurer, Karl-Heinz; Schwaneberg, Ulrich

    2015-12-25

    Proteases are essential ingredients in modern laundry detergents. Over the past 30 years, subtilisin proteases employed in the laundry detergent industry have been engineered by directed evolution and rational design to tailor their properties towards industrial demands. This comprehensive review discusses recent success stories in subtilisin protease engineering. Advances in protease engineering for laundry detergents comprise simultaneous improvement of thermal resistance and activity at low temperatures, a rational strategy to modulate pH profiles, and a general hypothesis for how to increase promiscuous activity towards the production of peroxycarboxylic acids as mild bleaching agents. The three protease engineering campaigns presented provide in-depth analysis of protease properties and have identified principles that can be applied to improve or generate enzyme variants for industrial applications beyond laundry detergents. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Structure and mechanism of rhomboid protease.

    PubMed

    Ha, Ya; Akiyama, Yoshinori; Xue, Yi

    2013-05-31

    Rhomboid protease was first discovered in Drosophila. Mutation of the fly gene interfered with growth factor signaling and produced a characteristic phenotype of a pointed head skeleton. The name rhomboid has since been widely used to describe a large family of related membrane proteins that have diverse biological functions but share a common catalytic core domain composed of six membrane-spanning segments. Most rhomboid proteases cleave membrane protein substrates near the N terminus of their transmembrane domains. How these proteases function within the confines of the membrane is not completely understood. Recent progress in crystallographic analysis of the Escherichia coli rhomboid protease GlpG in complex with inhibitors has provided new insights into the catalytic mechanism of the protease and its conformational change. Improved biochemical assays have also identified a substrate sequence motif that is specifically recognized by many rhomboid proteases.

  6. Secretion of proteases from Pasteurella multocida isolates.

    PubMed

    Negrete-Abascal, E; Tenorio, V R; de la Garza, M

    1999-01-01

    The capability of Pasteurella multocida to secrete proteases to the culture medium and their characterization were studied in five animal isolates (bovine, chicken, sheep, and two from pig). All the isolates produced proteases in a wide range of molecular mass. It is suggested that they are neutral metalloproteases, since they were optimally active between pH 6 and 7, inhibited by chelating agents but not by other protease inhibitors, and reactivated by calcium. Proteases from isolates were able to degrade IgG. Several proteins from supernatants of cultures precipitated with 70% (NH4)2SO4 of all the P. multocida isolates were recognized by a polyclonal antiserum raised against a purified protease from Actinobacillus pleuropneumoniae. Protease production might play an important role during tissue colonization and in P. multocida diseases.

  7. Biofluid proteases profiling in diabetes mellitus.

    PubMed

    Trindade, Fábio; Ferreira, Rita; Amado, Francisco; Vitorino, Rui

    2015-01-01

    The investigation of protease relevance in biologic systems beyond catabolism of proteins and peptides to amino acids has stimulated interest as to their role in the pathogenesis of several disorders including diabetes mellitus (DM). Evaluation of proteases and the assessment of their activity in biofluids are fundamental to elucidate these proteolytic systems in DM and its related complications. In contrast to traditional immunoassay or substrate based approaches that targeted specific proteases and their inhibitors, the field of degradomics has provided a comprehensive approach to study these enzymes. Although the degradome contains over 500 proteases, very few have been associated with DM and its micro- and macrovascular complications. In this paper, we review these proteases and their respective inhibitors with emphasis on DM. It is likely that future research will expand these initial studies and look to develop high throughput automated technologies to identify and characterize biofluid proteases of diagnostic and prognostic value in other pathologies.

  8. Structure and Mechanism of Rhomboid Protease*

    PubMed Central

    Ha, Ya; Akiyama, Yoshinori; Xue, Yi

    2013-01-01

    Rhomboid protease was first discovered in Drosophila. Mutation of the fly gene interfered with growth factor signaling and produced a characteristic phenotype of a pointed head skeleton. The name rhomboid has since been widely used to describe a large family of related membrane proteins that have diverse biological functions but share a common catalytic core domain composed of six membrane-spanning segments. Most rhomboid proteases cleave membrane protein substrates near the N terminus of their transmembrane domains. How these proteases function within the confines of the membrane is not completely understood. Recent progress in crystallographic analysis of the Escherichia coli rhomboid protease GlpG in complex with inhibitors has provided new insights into the catalytic mechanism of the protease and its conformational change. Improved biochemical assays have also identified a substrate sequence motif that is specifically recognized by many rhomboid proteases. PMID:23585569

  9. Structural and Functional Characterization of Cleavage and Inactivation of Human Serine Protease Inhibitors by the Bacterial SPATE Protease EspPα from Enterohemorrhagic E. coli

    PubMed Central

    Weiss, André; Joerss, Hanna; Brockmeyer, Jens

    2014-01-01

    EspPα and EspI are serine protease autotransporters found in enterohemorrhagic Escherichia coli. They both belong to the SPATE autotransporter family and are believed to contribute to pathogenicity via proteolytic cleavage and inactivation of different key host proteins during infection. Here, we describe the specific cleavage and functional inactivation of serine protease inhibitors (serpins) by EspPα and compare this activity with the related SPATE EspI. Serpins are structurally related proteins that regulate vital protease cascades, such as blood coagulation and inflammatory host response. For the rapid determination of serpin cleavage sites, we applied direct MALDI-TOF-MS or ESI-FTMS analysis of coincubations of serpins and SPATE proteases and confirmed observed cleavage positions using in-gel-digest of SDS-PAGE-separated degradation products. Activities of both serpin and SPATE protease were assessed in a newly developed photometrical assay using chromogenic peptide substrates. EspPα cleaved the serpins α1-protease inhibitor (α1-PI), α1-antichymotrypsin, angiotensinogen, and α2-antiplasmin. Serpin cleavage led to loss of inhibitory function as demonstrated for α1-PI while EspPα activity was not affected. Notably, EspPα showed pronounced specificity and cleaved procoagulatory serpins such as α2-antiplasmin while the anticoagulatory antithrombin III was not affected. Together with recently published research, this underlines the interference of EspPα with hemostasis or inflammatory responses during infection, while the observed interaction of EspI with serpins is likely to be not physiologically relevant. EspPα-mediated serpin cleavage occurred always in flexible loops, indicating that this structural motif might be required for substrate recognition. PMID:25347319

  10. Cysteine protease antigens cleave CD123, the α subunit of murine IL-3 receptor, on basophils and suppress IL-3-mediated basophil expansion

    SciTech Connect

    Nishikado, Hideto; Fujimura, Tsutomu; Taka, Hikari; Mineki, Reiko; Ogawa, Hideoki; Okumura, Ko; Takai, Toshiro

    2015-05-01

    Th2 type immune responses are essential for protective immunity against parasites and play crucial roles in allergic disorders. Helminth parasites secrete a variety of proteases for their infectious cycles including for host entry, tissue migration, and suppression of host immune effector cell function. Furthermore, a number of pathogen-derived antigens, as well as allergens such as papain, belong to the family of cysteine proteases. Although the link between protease activity and Th2 type immunity is well documented, the mechanisms by which proteases regulate host immune responses are largely unknown. Here, we demonstrate that the cysteine proteases papain and bromelain selectively cleave the α subunit of the IL-3 receptor (IL-3Rα/CD123) on the surface of murine basophils. The decrease in CD123 expression on the cell surface, and the degradation of the extracellular domain of recombinant CD123 were dependent on the protease activity of papain and bromelain. Pre-treatment of murine basophils with papain resulted in inhibition of IL-3-IL-3R signaling and suppressed IL-3- but not thymic stromal lymphopoietin-induced expansion of basophils in vitro. Our unexpected findings illuminate a novel mechanism for the regulation of basophil functions by protease antigens. Because IL-3 plays pivotal roles in the activation and proliferation of basophils and in protective immunity against helminth parasites, pathogen-derived proteases might contribute to the pathogenesis of infections by regulating IL-3-mediated functions in basophils. - Highlights: • We identified the murine IL3R as a novel target of papain-family cysteine proteases. • Papain-family cysteine proteases cleaved IL3Rα/CD123 on murine basophils. • Papain suppressed IL3- but not TSLP-induced expansion of murine basophils. • The inactivation of IL3R might be a strategy for pathogens to suppress host immunity.

  11. Bacterial proteases: targets for diagnostics and therapy.

    PubMed

    Kaman, W E; Hays, J P; Endtz, H P; Bikker, F J

    2014-07-01

    Proteases are essential for the proliferation and growth of bacteria, and are also known to contribute to bacterial virulence. This makes them interesting candidates as diagnostic and therapeutic targets for infectious diseases. In this review, the authors discuss the most recent developments and potential applications for bacterial proteases in the diagnosis and treatment of bacterial infections. Current and future bacterial protease targets are described and their limitations outlined.

  12. A putative methyltransferase, mtrA, contributes to development, spore viability, protein secretion and virulence in the entomopathogenic fungus Beauveria bassiana.

    PubMed

    Qin, Yuqi; Ortiz-Urquiza, Almudena; Keyhani, Nemat O

    2014-11-01

    The filamentous fungus, Beauveria bassiana, is a ubiquitously distributed insect pathogen, currently used as an alternative to chemical pesticides for pest control. Conidiospores are the means by which the fungus disseminates in the environment, and these cells also represent the infectious agent most commonly used in field applications. Little, however, is known concerning the molecular basis for maintenance of spore viability, a critical feature for survival and persistence. Here, we report on the role of a putative methyltransferase, BbmtrA, in conidial viability, normal fungal growth and development, and virulence, via characterization of a targeted gene knockout strain. Loss of BbmtrA resulted in pleiotropic effects including reduced germination, growth and conidiation, with growing mycelia displaying greater branching than the WT parent. Conidial viability dramatically decreased over time, with <5 % of the cells remaining viable after 30 days as compared with >80 % of the WT. Reduced production of extracellular proteins was also observed for the ΔBbmtrA mutant, including protease/peptidases, glycoside hydrolases and the hyd1 hydrophobin. The latter was further confirmed by hyd1 gene expression analysis. Insect bioassays using the greater wax moth, Galleria mellonella, further revealed that the ΔBbmtrA strain was attenuated in virulence and failed to sporulate on host cadavers. These data support a global role for mtrA in fungal physiological processes. © 2014 The Authors.

  13. Identification of potential molecular associations between chikungunya virus non-structural protein 2 and human host proteins.

    PubMed

    Rana, J; Gulati, S; Rajasekharan, S; Gupta, A; Chaudhary, V; Gupta, S

    2017-01-01

    Chikungunya virus (CHIKV) non-structural protein 2 (nsP2) is considered to be the master regulator of viral RNA replication and host responses generated during viral infection. This protein has two main functional domains: an N-terminal domain which exhibits NTPase, RNA triphosphatase and helicase activities and a C-terminal protease domain. Understanding how CHIKV nsP2 interacts with its host proteins is essential for elucidating all the required processes for viral replication and pathogenesis along with the identification of potential targets for antiviral therapy. In current study yeast two-hybrid (Y2H) screening of a human fetal brain cDNA library was performed using nsP2 protein as bait. The analysis identified seven host proteins (CCDC130, CPNE6, POLR2C, MAPK9, EIF4A2, EEF1A1 and EIF3I) as putative interactors of CHIKV nsP2 which were selected for further analysis based on their roles in host cellular machinery. The gene ontology analysis indicates that these proteins are mainly involved in apoptosis, transcription and translational mechanism of host cell. Domain mapping of nsP2 revealed that these associations are not random connections but instead they have functional significance. Further studies to identify the amino acid residues and their chemical interactions that may help in opening new possibilities for preventing these interactions, thus reducing chances of chikungunya infection were performed. This study expands the understanding of CHIKV-host interactions and is important for rational approaches of discovering new antiviral agents.

  14. 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.

  15. Leishmania (Leishmania) amazonensis: purification and characterization of a promastigote serine protease.

    PubMed

    da Silva-Lopez, Raquel Elisa; Giovanni-De-Simone, Salvatore

    2004-01-01

    Pathogenic protozoan proteases play crucial roles in the host-parasite interaction, and its characterization contributes to the understanding of protozoan disease mechanisms. A Leishmania amazonensis promastigote protease was purified 36-fold, using aprotinin-agarose affinity chromatography and gel filtration high performance liquid chromatography, yielding a total recovery of 49%. The molecular mass of active enzyme obtained from native gel filtration HPLC and SDS-PAGE under conditions of reduction and non-reduction was 68 kDa, suggesting that the enzyme may exist as a monomer. The protease isoelectric point (pI) was around 4.45 and, as demonstrated by deglycosylation assay, it did not have any carbohydrate content. The optimal pH and temperature of the enzyme were 8.0 and 28 degrees C, respectively, determined using alpha-N-rho-tosyl-L-arginyl-methyl ester (L-TAME) as substrate. Assays of thermal stability indicated that 50% of the enzymatic activity was preserved after 4 min of pre-treatment at 42 degrees C and after 24 h of pre-treatment at 37 degrees C, both in the absence of substrate. Hemoglobin, bovine serum albumin (BSA), ovalbumin, and both gelatin and peptide substrates containing arginine in ester bound were hydrolyzed by 68 kDa protease. The insulin beta-chain was also hydrolyzed by the protease, and four peptidic bonds (L11-V12, E13-A14, L15-Y16, and Y16-L17) were susceptible to the 68-kDa protease action. Inhibition studies suggested that the enzyme belonged to a serine protease class inhibited by calcium ions and activated by manganese ions. These findings demonstrate that the L. amazonensis 68-kDa serine protease differs from those of other protozoan parasites.

  16. The role of the S-S bridge in retroviral protease function and virion maturation.

    PubMed

    Zábranská, Helena; Tůma, Roman; Kluh, Ivan; Svatos, Ales; Ruml, Tomás; Hrabal, Richard; Pichová, Iva

    2007-02-02

    Retroviral proteases are translated as a part of Gag-related polyproteins, and are released and activated during particle release. Mason-Pfizer monkey virus (M-PMV) Gag polyproteins assemble into immature capsids within the cytoplasm of the host cells; however, their processing occurs only after transport to the plasma membrane and subsequent release. Thus, the activity of M-PMV protease is expected to be highly regulated during the replication cycle. It has been proposed that reversible oxidation of protease cysteine residues might be responsible for such regulation. We show that cysteine residues in M-PMV protease can form an intramolecular S-S bridge. The disulfide bridge shifts the monomer/dimer equilibrium in favor of the dimer, and increases the proteolytic activity significantly. To investigate the role of this disulfide bridge in virus maturation and replication, we engineered an M-PMV clone in which both protease cysteine residues were replaced by alanine (M-PMV(PRC7A/C106A)). Surprisingly, the cysteine residues were dispensable for Gag polyprotein processing within the virus, indicating that even low levels of protease activity are sufficient for polyprotein processing during maturation. However, the long-term infectivity of M-PMV(PRC7A/C106A) was noticeably compromised. These results show clearly that the proposed redox mechanism does not rely solely on the formation of the stabilizing S-S bridge in the protease. Thus, in addition to the protease disulfide bridge, reversible oxidation of cysteine and/or methionine residues in other domains of the Gag polyprotein or in related cellular proteins must be involved in the regulation of maturation.

  17. Interference with nuclear factor kappaB signaling pathway by pathogen-encoded proteases: global and selective inhibition.

    PubMed

    Hodgson, Andrea; Wan, Fengyi

    2016-02-01

    Pathogens have evolved a myriad of ways to abrogate and manipulate the host response to infections. Of the various mechanisms involved, pathogen-encoded and sometimes host-encoded proteases are an important category of virulence factors that cause robust changes on the host response by targeting key proteins along signaling cascades. The nuclear factor kappaB (NF-κB) signaling pathway is a crucial regulatory mechanism for the cell, controlling the expression of survival, immune and proliferation genes. Proteases from pathogens of almost all types have been demonstrated to target and cleave members of the NF-κB signaling pathway at nearly every level. This review provides discussion of proteases targeting the most abundant NF-κB subunit, p65, and the impact of protease-mediated p65 cleavage on the immune responses and survival of the infected host cell. After examining various examples of protease interference, it becomes evident that the cleavage fragments produced by pathogen-driven proteolytic processing should be further characterized to determine whether they have novel and unique functions within the cell. The selective targeting of p65 and its effect on gene transcription reveals unique mechanisms by which pathogens acutely alter their microenvironment, and further research may open new opportunities for novel therapeutics to combat pathogens.

  18. 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.

  19. Efficient expression systems for cysteine proteases of malaria parasites

    PubMed Central

    Sarduy, Emir Salas; de los A. Chávez Planes, María

    2013-01-01

    Papain-like cysteine proteases of malaria parasites are considered important chemotherapeutic targets or valuable models for the evaluation of drug candidates. Consequently, many of these enzymes have been cloned and expressed in Escherichia coli for their biochemical characterization. However, their expression has been problematic, showing low yield and leading to the formation of insoluble aggregates. Given that highly-productive expression systems are required for the high-throughput evaluation of inhibitors, we analyzed the existing expression systems to identify the causes of such apparent issues. We found that significant divergences in codon and nucleotide composition from host genes are the most probable cause of expression failure, and propose several strategies to overcome these limitations. Finally we predict that yeast hosts Saccharomyces cerevisiae and Pichia pastoris may be better suited than E. coli for the efficient expression of plasmodial genes, presumably leading to soluble and active products reproducing structural and functional characteristics of the natural enzymes. PMID:23018863

  20. Complete Coding Genome Sequence of a Putative Novel Teschovirus Serotype 12 Strain

    PubMed Central

    Jiménez-Clavero, M. A.

    2016-01-01

    Porcine teschoviruses are ubiquitous and prevalent viruses generally harmless to their hosts, the suids. Here, we report the first complete coding genome sequence of a putative new serotype of porcine teschovirus (PTV-12), strain CC25, isolated from fecal material from a healthy pig in Spain. PMID:26966207

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

    PubMed

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

    2016-01-01

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

  2. 3C-like protease of rabbit hemorrhagic disease virus: identification of cleavage sites in the ORF1 polyprotein and analysis of cleavage specificity.

    PubMed Central

    Wirblich, C; Sibilia, M; Boniotti, M B; Rossi, C; Thiel, H J; Meyers, G

    1995-01-01

    Rabbit hemorrhagic disease virus, a positive-stranded RNA virus of the family Caliciviridae, encodes a trypsin-like cysteine protease as part of a large polyprotein. Upon expression in Escherichia coli, the protease releases itself from larger precursors by proteolytic cleavages at its N and C termini. Both cleavage sites were determined by N-terminal sequence analysis of the cleavage products. Cleavage at the N terminus of the protease occurred with high efficiency at an EG dipeptide at positions 1108 and 1109. Cleavage at the C terminus of the protease occurred with low efficiency at an ET dipeptide at positions 1251 and 1252. To study the cleavage specificity of the protease, amino acid substitutions were introduced at the P2, P1, and P1' positions at the cleavage site at the N-terminal boundary of the protease. This analysis showed that the amino acid at the P1 position is the most important determinant for substrate recognition. Only glutamic acid, glutamine, and aspartic acid were tolerated at this position. At the P1' position, glycine, serine, and alanine were the preferred substrates of the protease, but a number of amino acids with larger side chains were also tolerated. Substitutions at the P2 position had only little effect on the cleavage efficiency. Cell-free expression of the C-terminal half of the ORF1 polyprotein showed that the protease catalyzes cleavage at the junction of the RNA polymerase and the capsid protein. An EG dipeptide at positions 1767 and 1768 was identified as the putative cleavage site. Our data show that rabbit hemorrhagic disease virus encodes a trypsin-like cysteine protease that is similar to 3C proteases with regard to function and specificity but is more similar to 2A proteases with regard to size. PMID:7474137

  3. HupW Protease Specifically Required for Processing of the Catalytic Subunit of the Uptake Hydrogenase in the Cyanobacterium Nostoc sp. Strain PCC 7120

    PubMed Central

    Lindberg, Pia; Devine, Ellenor; Stensjö, Karin

    2012-01-01

    The maturation process of [NiFe] hydrogenases includes a proteolytic cleavage of the large subunit. We constructed a mutant of Nostoc strain PCC 7120 in which hupW, encoding a putative hydrogenase-specific protease, is inactivated. Our results indicate that the protein product of hupW selectively cleaves the uptake hydrogenase in this cyanobacterium. PMID:22020512

  4. Characterization of cDNAs encoding serine proteases and their transcriptional responses to Cry1Ab protoxin in the gut of Ostrinia nubilalis larvae

    USDA-ARS?s Scientific Manuscript database

    Serine proteases, such as trypsin and chymotrypsin, are the primary digestive enzymes in lepidopteran larvae, and are also involved in Bacillus thuringiensis (Bt) protoxin activation and protoxin/toxin degradation. We isolated and sequenced 34 cDNAs putatively encoding trypsins, chymotrypsins and th...

  5. Proteases as regulators of pathogenesis: examples from the Apicomplexa.

    PubMed

    Li, Hao; Child, Matthew A; Bogyo, Matthew

    2012-01-01

    The diverse functional roles that proteases play in basic biological processes make them essential for virtually all organisms. Not surprisingly, proteolysis is also a critical process required for many aspects of pathogenesis. In particular, obligate intracellular parasites must precisely coordinate proteolytic events during their highly regulated life cycle inside multiple host cell environments. Advances in chemical, proteomic and genetic tools that can be applied to parasite biology have led to an increased understanding of the complex events centrally regulated by proteases. In this review, we outline recent advances in our knowledge of specific proteolytic enzymes in two medically relevant apicomplexan parasites: Plasmodium falciparum and Toxoplasma gondii. Efforts over the last decade have begun to provide a map of key proteotolyic events that are essential for both parasite survival and propagation inside host cells. These advances in our molecular understanding of proteolytic events involved in parasite pathogenesis provide a foundation for the validation of new networks and enzyme targets that could be exploited for therapeutic purposes. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome. Copyright © 2011 Elsevier B.V. All rights reserved.

  6. Proteases as Regulators of Pathogenesis: Examples from the Apicomplexa

    PubMed Central

    Li, Hao; Child, Matthew A.; Bogyo, Matthew

    2011-01-01

    The diverse functional roles that proteases play in basic biological processes make them essential for virtually all organisms. Not surprisingly, proteolysis is also a critical process required for many aspects of pathogenesis. In particular, obligate intracellular parasites must precisely coordinate proteolytic events during their highly regulated life cycle inside multiple host cell environments. Advances in chemical, proteomic and genetic tools that can be applied to parasite biology have led to an increased understanding of the complex events centrally regulated by proteases. In this review, we outline recent advances in our knowledge of specific proteolytic enzymes in two medically relevant apicomplexan parasites: Plasmodium falciparum and Toxoplasma gondii. Efforts over the last decade have begun to provide a map of key proteotolyic events that are essential for both parasite survival and propagation inside host cells. These advances in our molecular understanding of proteolytic events involved in parasite pathogenesis provide a foundation for the validation of new networks and enzyme targets that could be exploited for therapeutic purposes. PMID:21683169

  7. Bacterial proteases from the intracellular vacuole niche; protease conservation and adaptation for pathogenic advantage.

    PubMed

    Huston, Wilhelmina M

    2010-06-01

    Proteases with important roles for bacterial pathogens that specifically reside within intracellular vacuoles are frequently homologous to those that have important virulence functions for other bacteria. Research has identified that some of these conserved proteases have evolved specialized functions for intracellular vacuole-residing bacteria. Unique proteases with pathogenic functions have also been described from Chlamydia, Mycobacteria, and Legionella. These findings suggest that there are further novel functions for proteases from these bacteria that remain to be described. This review summarizes the recent findings of novel protease functions from the intracellular human pathogenic bacteria that reside exclusively in vacuoles.

  8. Cleavage of the SARS coronavirus spike glycoprotein by airway proteases enhances virus entry into human bronchial epithelial cells in vitro.

    PubMed

    Kam, Yiu-Wing; Okumura, Yuushi; Kido, Hiroshi; Ng, Lisa F P; Bruzzone, Roberto; Altmeyer, Ralf

    2009-11-17

    Entry of enveloped viruses into host cells requires the activation of viral envelope glycoproteins through cleavage by either intracellular or extracellular proteases. In order to gain insight into the molecular basis of protease cleavage and its impact on the efficiency of viral entry, we investigated the susceptibility of a recombinant native full-length S-protein trimer (triSpike) of the severe acute respiratory syndrome coronavirus (SARS-CoV) to cleavage by various airway proteases. PURIFIED TRISPIKE PROTEINS WERE READILY CLEAVED IN VITRO BY THREE DIFFERENT AIRWAY PROTEASES: trypsin, plasmin and TMPRSS11a. High Performance Liquid Chromatography (HPLC) and amino acid sequencing analyses identified two arginine residues (R667 and R797) as potential protease cleavage site(s). The effect of protease-dependent enhancement of SARS-CoV infection was demonstrated with ACE2 expressing human bronchial epithelial cells 16HBE. Airway proteases regulate the infectivity of SARS-CoV in a fashion dependent on previous receptor binding. The role of arginine residues was further shown with mutant constructs (R667A, R797A or R797AR667A). Mutation of R667 or R797 did not affect the expression of S-protein but resulted in a differential efficacy of pseudotyping into SARS-CoVpp. The R667A SARS-CoVpp mutant exhibited a lack of virus entry enhancement following protease treatment. These results suggest that SARS S-protein is susceptible to airway protease cleavage and, furthermore, that protease mediated enhancement of virus entry depends on specific conformation of SARS S-protein upon ACE2 binding. These data have direct implications for the cell entry mechanism of SARS-CoV along the respiratory system and, furthermore expand the possibility of identifying potential therapeutic agents against SARS-CoV.

  9. Tissue Dissociation Enzyme Neutral Protease Assessment

    PubMed Central

    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

  10. 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.

  11. Large-Scale Structure-Based Prediction and Identification of Novel Protease Substrates Using Computational Protein Design.

    PubMed

    Pethe, Manasi A; Rubenstein, Aliza B; Khare, Sagar D

    2017-01-20

    Characterizing the substrate specificity of protease enzymes is critical for illuminating the molecular basis of their diverse and complex roles in a wide array of biological processes. Rapid and accurate prediction of their extended substrate specificity would also aid in the design of custom proteases capable of selectively and controllably cleaving biotechnologically or therapeutically relevant targets. However, current in silico approaches for protease specificity prediction, rely on, and are therefore limited by, machine learning of sequence patterns in known experimental data. Here, we describe a general approach for predicting peptidase substrates de novo using protein structure modeling and biophysical evaluation of enzyme-substrate complexes. We construct atomic resolution models of thousands of candidate substrate-enzyme complexes for each of five model proteases belonging to the four major protease mechanistic classes-serine, cysteine, aspartyl, and metallo-proteases-and develop a discriminatory scoring function using enzyme design modules from Rosetta and AMBER's MMPBSA. We rank putative substrates based on calculated interaction energy with a modeled near-attack conformation of the enzyme active site. We show that the energetic patterns obtained from these simulations can be used to robustly rank and classify known cleaved and uncleaved peptides and that these structural-energetic patterns have greater discriminatory power compared to purely sequence-based statistical inference. Combining sequence and energetic patterns using machine-learning algorithms further improves classification performance, and analysis of structural models provides physical insight into the structural basis for the observed specificities. We further tested the predictive capability of the model by designing and experimentally characterizing the cleavage of four novel substrate motifs for the hepatitis C virus NS3/4 protease using an in vivo assay. The presented structure

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

    PubMed Central

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

    2014-01-01

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

  13. Mirolase, a novel subtilisin-like serine protease from the periodontopathogen Tannerella forsythia

    PubMed Central

    Ksiazek, Miroslaw; Karim, Abdulkarim Y.; Bryzek, Danuta; Enghild, Jan J.; Thøgersen, Ida B.; Koziel, Joanna; Potempa, Jan

    2015-01-01

    The genome of Tannerella forsythia, an etiologic factor of chronic periodontitis, contains several genes encoding putative proteases. Here, we characterized a subtilisin-like serine protease of T. forsythia referred to as mirolase. Recombinant full-length latent promirolase (85 kDa, without its signal peptide) processed itself through sequential autoproteolytic cleavages into a mature enzyme of 40 kDa. Mirolase latency was driven by the N-terminal prodomain (NTP). In stark contrast to almost all known subtilases, the cleaved NTP remained non-covalently associated with mirolase, inhibiting its proteolytic, but not amidolytic, activity. Full activity was observed only after the NTP was gradually, and fully, degraded. Both activity and processing was absolutely dependent on calcium ions, which were also essential for enzyme stability. As a consequence, both serine protease inhibitors and calcium ions chelators inhibited mirolase activity. Activity assays using an array of chromogenic substrates revealed that mirolase specificity is driven not only by the substrate-binding subsite S1, but also by other subsites. Taken together mirolase is a calcium-dependent serine protease of the S8 family with the unique mechanism of activation that may contribute to T. forsythia pathogenicity by degradation of fibrinogen, hemoglobin and the antimicrobial peptide LL-37. PMID:25391881

  14. Mirolase, a novel subtilisin-like serine protease from the periodontopathogen Tannerella forsythia.

    PubMed

    Ksiazek, Miroslaw; Karim, Abdulkarim Y; Bryzek, Danuta; Enghild, Jan J; Thøgersen, Ida B; Koziel, Joanna; Potempa, Jan

    2015-03-01

    The genome of Tannerella forsythia, an etiological factor of chronic periodontitis, contains several genes encoding putative proteases. Here, we characterized a subtilisin-like serine protease of T. forsythia referred to as mirolase. Recombinant full-length latent promirolase [85 kDa, without its signal peptide (SP)] processed itself through sequential autoproteolytic cleavages into a mature enzyme of 40 kDa. Mirolase latency was driven by the N-terminal prodomain (NTP). In stark contrast to almost all known subtilases, the cleaved NTP remained non-covalently associated with mirolase, inhibiting its proteolytic, but not amidolytic, activity. Full activity was observed only after the NTP was gradually, and fully, degraded. Both activity and processing was absolutely dependent on calcium ions, which were also essential for enzyme stability. As a consequence, both serine protease inhibitors and calcium ions chelators inhibited mirolase activity. Activity assays using an array of chromogenic substrates revealed that mirolase specificity is driven not only by the substrate-binding subsite S1, but also by other subsites. Taken together, mirolase is a calcium-dependent serine protease of the S8 family with the unique mechanism of activation that may contribute to T. forsythia pathogenicity by degradation of fibrinogen, hemoglobin, and the antimicrobial peptide LL-37.

  15. Identification and functional analysis of Penicillium digitatum genes putatively involved in virulence towards citrus fruit.

    PubMed

    López-Pérez, Mario; Ballester, Ana-Rosa; González-Candelas, Luis

    2015-04-01

    The fungus Penicillium digitatum, the causal agent of green mould rot, is the most destructive post-harvest pathogen of citrus fruit in Mediterranean regions. In order to identify P. digitatum genes up-regulated during the infection of oranges that may constitute putative virulence factors, we followed a polymerase chain reaction (PCR)-based suppression subtractive hybridization and cDNA macroarray hybridization approach. The origin of expressed sequence tags (ESTs) was determined by comparison against the available genome sequences of both organisms. Genes coding for fungal proteases and plant cell wall-degrading enzymes represent the largest categories in the subtracted cDNA library. Northern blot analysis of a selection of P. digitatum genes, including those coding for proteases, cell wall-related enzymes, redox homoeostasis and detoxification processes, confirmed their up-regulation at varying time points during the infection process. Agrobacterium tumefaciens-mediated transformation was used to generate knockout mutants for two genes encoding a pectin lyase (Pnl1) and a naphthalene dioxygenase (Ndo1). Two independent P. digitatum Δndo1 mutants were as virulent as the wild-type. However, the two Δpnl1 mutants analysed were less virulent than the parental strain or an ectopic transformant. Together, these results provide a significant advance in our understanding of the putative determinants of the virulence mechanisms of P. digitatum.

  16. Gene expression and activity of digestive proteases in Daphnia: effects of cyanobacterial protease inhibitors

    PubMed Central

    2010-01-01

    Background The frequency of cyanobacterial blooms has increased worldwide, and these blooms have been claimed to be a major factor leading to the decline of the most important freshwater herbivores, i.e. representatives of the genus Daphnia. This suppression of Daphnia is partly attributed to the presence of biologically active secondary metabolites in cyanobacteria. Among these metabolites, protease inhibitors are found in almost every natural cyanobacterial bloom and have been shown to specifically inhibit Daphnia's digestive proteases in vitro, but to date no physiological responses of these serine proteases to cyanobacterial protease inhibitors in Daphnia have been reported in situ at the protein and genetic levels. Results Nine digestive proteases were detected in D. magna using activity-stained SDS-PAGE. Subsequent analyses by LC-MS/MS and database search led to the identification of respective protease genes. D. magna responded to dietary protease inhibitors by up-regulation of the expression of these respective proteases at the RNA-level and by the induction of new and less sensitive protease isoforms at the protein level. The up-regulation in response to dietary trypsin- and chymotrypsin-inhibitors ranged from 1.4-fold to 25.6-fold. These physiological responses of Daphnia, i.e. up-regulation of protease expression and the induction of isoforms, took place even after feeding on 20% cyanobacterial food for only 24 h. These physiological responses proved to be independent from microcystin effects. Conclusion Here for the first time it was shown in situ that a D. magna clone responds physiologically to dietary cyanobacterial protease inhibitors by phenotypic plasticity of the targets of these specific inhibitors, i.e. Daphnia gut proteases. These regulatory responses are adaptive for D. magna, as they increase the capacity for protein digestion in the presence of dietary protease inhibitors. The type and extent of these responses in protease expression might

  17. Cloning and Characterization of Two Potent Kunitz Type Protease Inhibitors from Echinococcus granulosus

    PubMed Central

    Ranasinghe, Shiwanthi L.; Fischer, Katja; Zhang, Wenbao; Gobert, Geoffrey N.; McManus, Donald P.

    2015-01-01

    The tapeworm Echinococcus granulosus is responsible for cystic echinococcosis (CE), a cosmopolitan disease which imposes a significant burden on the health and economy of affected communities. Little is known about the molecular mechanisms whereby E. granulosus is able to survive in the hostile mammalian host environment, avoiding attack by host enzymes and evading immune responses, but protease inhibitors released by the parasite are likely implicated. We identified two nucleotide sequences corresponding to secreted single domain Kunitz type protease inhibitors (EgKIs) in the E. granulosus genome, and their cDNAs were cloned, bacterially expressed and purified. EgKI-1 is highly expressed in the oncosphere (egg) stage and is a potent chymotrypsin and neutrophil elastase inhibitor that binds calcium and reduced neutrophil infiltration in a local inflammation model. EgKI-2 is highly expressed in adult worms and is a potent inhibitor of trypsin. As powerful inhibitors of mammalian intestinal proteases, the EgKIs may play a pivotal protective role in preventing proteolytic enzyme attack thereby ensuring survival of E. granulosus within its mammalian hosts. EgKI-1 may also be involved in the oncosphere in host immune evasion by inhibiting neutrophil elastase and cathepsin G once this stage is exposed to the mammalian blood system. In light of their key roles in protecting E. granulosus from host enzymatic attack, the EgKI proteins represent potential intervention targets to control CE. This is important as new public health measures against CE are required, given the inefficiencies of available drugs and the current difficulties in its treatment and control. In addition, being a small sized highly potent serine protease inhibitor, and an inhibitor of neutrophil chemotaxis, EgKI-1 may have clinical potential as a novel anti-inflammatory therapeutic. PMID:26645974

  18. Rapid Detection of Thrombin and Other Protease Activity Directly in Whole Blood

    NASA Astrophysics Data System (ADS)

    Yu, Johnson Chung Sing

    Thrombin is a serine protease that plays a key role in the clotting cascade to promote hemostasis following injury to the endothelium. From a clinical diagnostic perspective, in-vivo thrombin activity is linked to various blood clotting disorders, as well as cardiovascular disease (DVT, arteriosclerosis, etc). Thus, the ability to rapidly measure protease activity directly in whole blood will provide important new diagnostics, and clinical researchers with a powerful tool to further elucidate the relationship between circulating protease levels and disease. The ultimate goal is to design novel point of care (POC) diagnostic devices that are capable of monitoring protease activities directly in whole blood and biological sample. A charge-changing substrate specific to the thrombin enzyme was engineered and its functionality was confirmed by a series of experiments. This led to the preliminary design, construction, and testing of two device platforms deemed fully functional for the electrophoretic separation and focusing of charged peptide fragments. The concept of using the existing charge-changing substrate platform for bacterial protease detection was also investigated. Certain strains of E coli are associated with severe symptoms such as abdominal cramps, bloody diarrhea, and vomiting. The OmpT protease is expressed on the outer membrane of E coli and plays a role in the cleavage of antimicrobial peptides, the degradation of recombinant heterologous proteins, and the activation of plasminogen in the host. Thus, a synthetic peptide substrate specific to the OmpT protease was designed and modeled for the purpose of detecting E coli in biological sample.

  19. Protease and protease-activated receptor-2 signaling in the pathogenesis of atopic dermatitis.

    PubMed

    Lee, Sang Eun; Jeong, Se Kyoo; Lee, Seung Hun

    2010-11-01

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

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

    PubMed Central

    Lee, Sang Eun; Jeong, Se Kyoo

    2010-01-01

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

  1. Assessing the impact of long term frozen storage of faecal samples on protein concentration and protease activity

    PubMed Central

    Morris, Laura S.; Marchesi, Julian R.

    2016-01-01

    Background The proteome is the second axis of the microbiome:host interactome and proteases are a significant aspect in this interaction. They interact with a large variety of host proteins and structures and in many situations are implicated in pathogenesis. Furthermore faecal samples are commonly collected and stored frozen so they can be analysed at a later date. So we were interested to know whether long term storage affected the integrity of proteases and total protein and whether historical native faecal samples were still a viable option for answering research questions around the functional proteome. Methods Faecal samples were collected from 3 healthy volunteers (3 biological replicates) and processed in order to be stored at both − 20 °C and − 80 °C and in a variety of storage buffers. Protein extraction, protein content and protease activity were assessed at the time of collection, after 24 h, 1 week, 1 month, 3 months 6 months and finally 1 year. Results Beadbeating impacted the quantity of protein extracted, while sodium azide did not impact protease assays. Long term storage of extracted proteins showed that both total protein and protease activity were affected when they were stored as extracted protein. Intact faecal samples were shown to maintain both protein levels and protease activity regardless of time and temperature. Conclusions Beadbeating increases the protein and protease activity when extracting from a faecal sample, however, the extracted protein is not stable and activity is lost, even with a suitable storage buffer. The most robust solution is to store the proteins in an intact frozen native faecal matrix and extract at the time of assay or analysis, this approach was shown to be suitable for samples in which, there are low levels of protease activity and which had been frozen for a year. PMID:26853125

  2. 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.

  3. Activation of Bacteroides fragilis toxin by a novel bacterial protease contributes to anaerobic sepsis

    PubMed Central

    Choi, Vivian M.; Herrou, Julien; Hecht, Aaron L.; Teoh, Wei Ping; Turner, Jerrold R.; Crosson, Sean; Wardenburg, Juliane Bubeck

    2016-01-01

    Bacteroides fragilis is the leading cause of anaerobic bacteremia and sepsis 1. Enterotoxigenic strains producing B. fragilis toxin (BFT, fragilysin) contribute to colitis 2 and intestinal malignancy 3, yet are also isolated in bloodstream infection 4,5. It is not known whether these strains harbor unique genetic determinants that confer virulence in extra-intestinal disease. We demonstrate that BFT contributes to sepsis and identify a B. fragilis protease, fragipain (Fpn), which is required for endogenous activation of BFT through removal of its auto-inhibitory prodomain. Structural analysis of Fpn reveals a His-Cys catalytic dyad characteristic of C11 family cysteine proteases that are conserved in multiple pathogenic Bacteroides spp and Clostridium spp. Fpn-deficient enterotoxigenic B. fragilis is attenuated in its ability to induce sepsis, however Fpn is dispensable in B. fragilis colitis wherein host proteases mediate BFT activation. Our findings define a role for B. fragilis enterotoxin and its activating protease in the pathogenesis of bloodstream infection, indicating a greater complexity of cellular targeting and action of BFT than previously appreciated. The expression of fpn by both toxigenic and non-toxigenic strains suggests this protease may contribute to anaerobic sepsis beyond its role in toxin activation, potentially serving as a target for disease modification. PMID:27089515

  4. 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

  5. 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

  6. Chemoproteomic profiling of host and pathogen enzymes active in cholera

    PubMed Central

    Hatzios, Stavroula K.; Hubbard, Troy; Sasabe, Jumpei; Munera, Diana; Clark, Lars; Bachovchin, Daniel A.; Qadri, Firdausi; Ryan, Edward T.; Davis, Brigid M.; Weerapana, Eranthie; Waldor, Matthew K.

    2016-01-01

    Activity-based protein profiling (ABPP) is a chemoproteomic tool for detecting active enzymes in complex biological systems. We used ABPP to identify secreted bacterial and host serine hydrolases that are active in animals infected with the cholera pathogen Vibrio cholerae. Four V. cholerae proteases were consistently active in infected rabbits, and one, VC0157 (renamed IvaP), was also active in human cholera stool. Inactivation of IvaP influenced the activity of other secreted V. cholerae and rabbit enzymes in vivo, while genetic disruption of all four proteases increased the abundance and binding of an intestinal lectin—intelectin—to V. cholerae in infected rabbits. Intelectin also bound to other enteric bacterial pathogens, suggesting it may constitute a previously unrecognized mechanism of bacterial surveillance in the intestine that is inhibited by pathogen-secreted proteases. Our work demonstrates the power of activity-based proteomics to reveal host-pathogen enzymatic dialogue in an animal model of infection. PMID:26900865

  7. Chemoproteomic profiling of host and pathogen enzymes active in cholera.

    PubMed

    Hatzios, Stavroula K; Abel, Sören; Martell, Julianne; Hubbard, Troy; Sasabe, Jumpei; Munera, Diana; Clark, Lars; Bachovchin, Daniel A; Qadri, Firdausi; Ryan, Edward T; Davis, Brigid M; Weerapana, Eranthie; Waldor, Matthew K

    2016-04-01

    Activity-based protein profiling (ABPP) is a chemoproteomic tool for detecting active enzymes in complex biological systems. We used ABPP to identify secreted bacterial and host serine hydrolases that are active in animals infected with the cholera pathogen Vibrio cholerae. Four V. cholerae proteases were consistently active in infected rabbits, and one, VC0157 (renamed IvaP), was also active in human choleric stool. Inactivation of IvaP influenced the activity of other secreted V. cholerae and rabbit enzymes in vivo, and genetic disruption of all four proteases increased the abundance of intelectin, an intestinal lectin, and its binding to V. cholerae in infected rabbits. Intelectin also bound to other enteric bacterial pathogens, suggesting that it may constitute a previously unrecognized mechanism of bacterial surveillance in the intestine that is inhibited by pathogen-secreted proteases. Our work demonstrates the power of activity-based proteomics to reveal host-pathogen enzymatic dialog in an animal model of infection.

  8. Structural Insights into the Protease-like Antigen Plasmodium falciparum SERA5 and Its Noncanonical Active-Site Serine

    SciTech Connect

    Hodder, Anthony N.; Malby, Robyn L.; Clarke, Oliver B.; Fairlie, W. Douglas; Colman, Peter M.; Crabb, Brendan S.; Smith, Brian J.

    2009-08-28

    The sera genes of the malaria-causing parasite Plasmodium encode a family of unique proteins that are maximally expressed at the time of egress of parasites from infected red blood cells. These multi-domain proteins are unique, containing a central papain-like cysteine-protease fragment enclosed between the disulfide-linked N- and C-terminal domains. However, the central fragment of several members of this family, including serine repeat antigen 5 (SERA5), contains a serine (S596) in place of the active-site cysteine. Here we report the crystal structure of the central protease-like domain of Plasmodium falciparum SERA5, revealing a number of anomalies in addition to the putative nucleophilic serine: (1) the structure of the putative active site is not conducive to binding substrate in the canonical cysteine-protease manner; (2) the side chain of D594 restricts access of substrate to the putative active site; and (3) the S{sub 2} specificity pocket is occupied by the side chain of Y735, reducing this site to a small depression on the protein surface. Attempts to determine the structure in complex with known inhibitors were not successful. Thus, despite having revealed its structure, the function of the catalytic domain of SERA5 remains an enigma.

  9. The Race against Protease Activation Defines the Role of ESCRTs in HIV Budding.

    PubMed

    Bendjennat, Mourad; Saffarian, Saveez

    2016-06-01

    HIV virions assemble on the plasma membrane and bud out of infected cells using interactions with endosomal sorting complexes required for transport (ESCRTs). HIV protease activation is essential for maturation and infectivity of progeny virions, however, the precise timing of protease activation and its relationship to budding has not been well defined. We show that compromised interactions with ESCRTs result in delayed budding of virions from host cells. Specifically, we show that Gag mutants with compromised interactions with ALIX and Tsg101, two early ESCRT factors, have an average budding delay of ~75 minutes and ~10 hours, respectively. Virions with inactive proteases incorporated the full Gag-Pol and had ~60 minutes delay in budding. We demonstrate that during budding delay, activated proteases release critical HIV enzymes back to host cytosol leading to production of non-infectious progeny virions. To explain the molecular mechanism of the observed budding delay, we modulated the Pol size artificially and show that virion release delays are size-dependent and also show size-dependency in requirements for Tsg101 and ALIX. We highlight the sensitivity of HIV to budding "on-time" and suggest that budding delay is a potent mechanism for inhibition of infectious retroviral release.

  10. The Race against Protease Activation Defines the Role of ESCRTs in HIV Budding

    PubMed Central

    Bendjennat, Mourad; Saffarian, Saveez

    2016-01-01

    HIV virions assemble on the plasma membrane and bud out of infected cells using interactions with endosomal sorting complexes required for transport (ESCRTs). HIV protease activation is essential for maturation and infectivity of progeny virions, however, the precise timing of protease activation and its relationship to budding has not been well defined. We show that compromised interactions with ESCRTs result in delayed budding of virions from host cells. Specifically, we show that Gag mutants with compromised interactions with ALIX and Tsg101, two early ESCRT factors, have an average budding delay of ~75 minutes and ~10 hours, respectively. Virions with inactive proteases incorporated the full Gag-Pol and had ~60 minutes delay in budding. We demonstrate that during budding delay, activated proteases release critical HIV enzymes back to host cytosol leading to production of non-infectious progeny virions. To explain the molecular mechanism of the observed budding delay, we modulated the Pol size artificially and show that virion release delays are size-dependent and also show size-dependency in requirements for Tsg101 and ALIX. We highlight the sensitivity of HIV to budding “on-time” and suggest that budding delay is a potent mechanism for inhibition of infectious retroviral release. PMID:27280284

  11. Inhibition of outer membrane proteases of the omptin family by aprotinin.

    PubMed

    Brannon, John R; Burk, David L; Leclerc, Jean-Mathieu; Thomassin, Jenny-Lee; Portt, Andrea; Berghuis, Albert M; Gruenheid, Samantha; Le Moual, Hervé

    2015-06-01

    Bacterial proteases are important virulence factors that inactivate host defense proteins and contribute to tissue destruction and bacterial dissemination. Outer membrane proteases of the omptin family, exemplified by Escherichia coli OmpT, are found in some Gram-negative bacteria. Omptins cleave a variety of substrates at the host-pathogen interface, including plasminogen and antimicrobial peptides. Multiple omptin substrates relevant to infection have been identified; nonetheless, an effective omptin inhibitor remains to be found. Here, we purified native CroP, the OmpT ortholog in the murine pathogen Citrobacter rodentium. Purified CroP was found to readily cleave both a synthetic fluorescence resonance energy transfer substrate and the murine cathelicidin-related antimicrobial peptide. In contrast, CroP was found to poorly activate plasminogen into active plasmin. Although classical protease inhibitors were ineffective against CroP activity, we found that the serine protease inhibitor aprotinin displays inhibitory potency in the micromolar range. Aprotinin was shown to act as a competitive inhibitor of CroP activity and to interfere with the cleavage of the murine cathelicidin-related antimicrobial peptide. Importantly, aprotinin was able to inhibit not only CroP but also Yersinia pestis Pla and, to a lesser extent, E. coli OmpT. We propose a structural model of the aprotinin-omptin complex in which Lys15 of aprotinin forms salt bridges with conserved negatively charged residues of the omptin active site.

  12. Inhibition of Outer Membrane Proteases of the Omptin Family by Aprotinin

    PubMed Central

    Brannon, John R.; Burk, David L.; Leclerc, Jean-Mathieu; Thomassin, Jenny-Lee; Portt, Andrea; Berghuis, Albert M.; Gruenheid, Samantha

    2015-01-01

    Bacterial proteases are important virulence factors that inactivate host defense proteins and contribute to tissue destruction and bacterial dissemination. Outer membrane proteases of the omptin family, exemplified by Escherichia coli OmpT, are found in some Gram-negative bacteria. Omptins cleave a variety of substrates at the host-pathogen interface, including plasminogen and antimicrobial peptides. Multiple omptin substrates relevant to infection have been identified; nonetheless, an effective omptin inhibitor remains to be found. Here, we purified native CroP, the OmpT ortholog in the murine pathogen Citrobacter rodentium. Purified CroP was found to readily cleave both a synthetic fluorescence resonance energy transfer substrate and the murine cathelicidin-related antimicrobial peptide. In contrast, CroP was found to poorly activate plasminogen into active plasmin. Although classical protease inhibitors were ineffective against CroP activity, we found that the serine protease inhibitor aprotinin displays inhibitory potency in the micromolar range. Aprotinin was shown to act as a competitive inhibitor of CroP activity and to interfere with the cleavage of the murine cathelicidin-related antimicrobial peptide. Importantly, aprotinin was able to inhibit not only CroP but also Yersinia pestis Pla and, to a lesser extent, E. coli OmpT. We propose a structural model of the aprotinin-omptin complex in which Lys15 of aprotinin forms salt bridges with conserved negatively charged residues of the omptin active site. PMID:25824836

  13. Proteolytic activity of Plasmodium falciparum subtilisin-like protease 3 on parasite profilin, a multifunctional protein.

    PubMed

    Alam, Asrar; Bhatnagar, Raj K; Relan, Udbhav; Mukherjee, Paushali; Chauhan, Virander S

    2013-10-01

    Subtilisin-like proteases of malaria parasite Plasmodium falciparum (PfSUB1, 2 and 3) are expressed at late asexual blood stages. PfSUB1 and 2 are considered important drug targets due to their essentiality for parasite blood stages and role in merozoite egress and invasion of erythrocytes. We have earlier shown the in vitro serine protease activity of PfSUB3 and its localization at asexual blood stages. In this study, we attempted to identify the biological substrate(s) of PfSUB3 and found parasite profilin (PfPRF) as a substrate of the protease. Eukaryotic profilins are multifunctional proteins with primary role in regulation of actin filament assembly. PfPRF possesses biochemical features of eukaryotic profilins and its rodent ortholog is essential in blood stages. Profilin from related apicomplexan parasite Toxoplasma gondii (TgPRF) is known to be involved in parasite motility, host cell invasion, active egress from host cell, immune evasion and virulence in mice. In this study, mature PfSUB3 proteolysed recombinant PfPRF in a dose-dependent manner in in vitro assays. Recombinant PfPRF was assessed for its proinflammatory activity and found to induce high level of TNF-α and low but significant level of IL-12 from mouse bone marrow-derived dendritic cells. Proteolysis of PfPRF by PfSUB3 is suggestive of the probable role of the protease in the processes of motility, virulence and immune evasion.

  14. Cysteine Proteases from Bloodfeeding Arthropod Ectoparasites

    PubMed Central

    Sojka, Daniel; Francischetti, Ivo M. B.; Calvo, Eric; Kotsyfakis, Michalis

    2012-01-01

    Cysteine proteases have been discovered in various bloodfeeding ectoparasites. Here, we assemble the available information about the function of these peptidases and reveal their role in hematophagy and parasite development. While most of the data shed light on key proteolytic events that play a role in arthropod physiology, we also report on the association of cysteine proteases with arthropod vectorial capacity. With emphasis on ticks, specifically Ixodes ricinus, we finally propose a model about the contribution of cysteine peptidases to blood digestion, and how their concerted action with other tick midgut proteases leads to the absorbance of nutrients by the midgut epithelial cells. PMID:21660665

  15. 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.

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

    PubMed Central

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

    2012-01-01

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

  17. Identification and partial characterization of extracellular aspartic protease genes from Metschnikowia pulcherrima IWBT Y1123 and Candida apicola IWBT Y1384.

    PubMed

    Reid, Vernita J; Theron, Louwrens W; du Toit, Maret; Divol, Benoit

    2012-10-01

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

  18. 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.

  19. Reaching the melting point: Degradative enzymes and protease inhibitors involved in baculovirus infection and dissemination.

    PubMed

    Ishimwe, Egide; Hodgson, Jeffrey J; Clem, Rollie J; Passarelli, A Lorena

    2015-05-01

    Baculovirus infection of a host insect involves several steps, beginning with initiation of virus infection in the midgut, followed by dissemination of infection from the midgut to other tissues in the insect, and finally culminating in "melting" or liquefaction of the host, which allows for horizontal spread of infection to other insects. While all of the viral gene products are involved in ultimately reaching this dramatic infection endpoint, this review focuses on two particular types of baculovirus-encoded proteins: degradative enzymes and protease inhibitors. Neither of these types of proteins is commonly found in other virus families, but they both play important roles in baculovirus infection. The types of degradative enzymes and protease inhibitors encoded by baculoviruses are discussed, as are the roles of these proteins in the infection process. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Reaching the Melting Point: Degradative Enzymes and Protease Inhibitors Involved in Baculovirus Infection and Dissemination

    PubMed Central

    Ishimwe, Egide; Hodgson, Jeffrey J.; Clem, Rollie J.; Passarelli, A. Lorena

    2015-01-01

    Baculovirus infection of a host insect involves several steps, beginning with initiation of virus infection in the midgut, followed by dissemination of infection from the midgut to other tissues in the insect, and finally culminating in “melting” or liquefaction of the host, which allows for horizontal spread of infection to other insects. While all of the viral gene products are involved in ultimately reaching this dramatic infection endpoint, this review focuses on two particular types of baculovirus-encoded proteins: degradative enzymes and protease inhibitors. Neither of these types of proteins is commonly found in other virus families, but they both play important roles in baculovirus infection. The types of degradative enzymes and protease inhibitors encoded by baculoviruses are discussed, as are the roles of these proteins in the infection process. PMID:25724418

  1. Antiviral effects of a thiol protease inhibitor on foot-and-mouth disease virus.

    PubMed Central

    Kleina, L G; Grubman, M J

    1992-01-01

    The thiol protease inhibitor E-64 specifically blocks autocatalytic activity of the leader protease of foot-and-mouth disease virus (FMDV) and interferes with cleavage of the structural protein precursor in an in vitro translation assay programmed with virion RNA. Experiments with FMDV-infected cells and E-64 or a membrane-permeable analog, E-64d, have confirmed these results and demonstrated interference in virus assembly, causing a reduction in virus yield. In addition, there is a lag in the appearance of virus-induced cellular morphologic alterations, a delay in cleavage of host cell protein p220 and in shutoff of host protein synthesis, and a decrease in viral protein and RNA synthesis. The implications of using E-64-based compounds as potential antiviral agents for FMDV are discussed. Images PMID:1331517

  2. Secreted fungal aspartic proteases: A review.

    PubMed

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

    2016-01-01

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

  3. Role of cockroach proteases in allergic disease.

    PubMed

    Page, Kristen

    2012-10-01

    Allergic asthma is on the rise in developed countries, and cockroach exposure is a major risk factor for the development of asthma. In recent years, a number of studies have investigated the importance of allergen-associated proteases in modulating allergic airway inflammation. Many of the studies have suggested the importance of allergen-associated proteases as having a direct role on airway epithelial cells and dendritic cells. In most cases, activation of the protease activated receptor (PAR)-2 has been implicated as a mechanism behind the potent allergenicity associated with cockroaches. In this review, we focus on recent evidence linking cockroach proteases to activation of a variety of cells important in allergic airway inflammation and the role of PAR-2 in this process. We will highlight recent data exploring the potential mechanisms involved in the biological effects of the allergen.

  4. Putative cryptoendolithic life in Devonian pillow basalt, Rheinisches Schiefergebirge, Germany.

    PubMed

    Peckmann, J; Bach, W; Behrens, K; Reitner, J

    2008-03-01

    Middle Devonian (Givetian) pillow basalt and inter-pillow breccia from the Rheinisches Schiefergebirge in Germany were found to contain putative biogenic filaments that indicate that life once proliferated within these volcanic rocks. Mineralized filaments are found in carbonate amygdules (vesicles filled by carbonate cement) in the volcanic rock, where they started to form on the internal surface of the once water-filled vesicles. Biogenicity of the filaments is indicated by (1) their size and shape resembling modern microorganisms including a constant diameter along the length of curved filaments, (2) their independence of crystal faces or cleavage planes, (3) branching patterns reminiscent of modern microorganisms, and (4) their spatial clustering and preferential occurrence close to the margin of pillows and in the inter-pillow breccias. A time lag between the deposition of pillow basalt and the activity of endoliths is revealed by the sequence of carbonate cements filling the amygdules. The putative filamentous microorganisms thrived after the formation of early fibrous rim cement, but before later equant calcite spar filled most of the remaining porosity. Microbial clay authigenesis analogous to the encrustation of prokaryotes in modern iron-rich environments led to the preservation of filaments. The filaments predominantly consist of the clay minerals chamosite and illite. Having dwelled in water-filled vesicles, the Devonian basalt-hosted filaments apparently represent cryptoendoliths. This finding suggests that a previously unrecognized niche for life exists within volcanic rock.

  5. Regulatory Characteristics of Bacillus pumilus Protease Promoters.

    PubMed

    Toymentseva, Anna A; Mascher, Thorsten; Sharipova, Margarita R

    2017-05-01

    Expression of extracellular protease genes of Bacilli is subject to regulation by many positive and negative regulators. Here we analyzed 5' regulatory regions of genes encoding proteolytic proteases AprBp, GseBp, and MprBp from Bacillus pumilus strain 3-19. Gfp fusion constructs with upstream genomic regions of different lengths were created for all three genes to identify their natural promoters (regulatory regions). Our results suggest that the aprBp gene, encoding the major subtilisin-like protease, has the most extensive promoter region of approximately 445 bp, while the minor protease genes encoding glutamyl endopeptidase (gseBp) and metalloproteinase (mprBp) are preceded by promoters of 150 and 250 bp in length, respectively. Promoter analysis of P aprBp -gfpmu3 and P gseBp -gfpmu3 reporter fusion constructs in degU and spo0A mutants indicates a positive regulatory effect of DegU and Spo0A on protease expression, while the disruption of abrB, sinR, and scoC repressor genes did not significantly affect promoter activities of all protease genes. On the other hand, the expression of P aprBp -gfpmu3 and P gseBp -gfpmu3 reporters increased 1.6- and 3.0-fold, respectively, in sigD-deficient cells, indicating that the prevention of motility gene expression promotes protease expression. Our results indicate that all examined regulators regulated serine proteases production in B. subtilis.

  6. Protease Mediated Anti-Cancer Therapy

    DTIC Science & Technology

    2006-08-01

    anticancer therapy and focal light illumination is expected to be an effective treatment with reduced phototoxicity given the quenched state of the...to months following photodynamic therapy (PDT). Herein, we report a novel design of protease-mediated photosensitization by which phototoxicity can...W81XWH-05-1-0515 TITLE: Protease Mediated Anti-Cancer Therapy PRINCIPAL INVESTIGATOR: Ching-Hsuan Tung CONTRACTING ORGANIZATION

  7. Structural Insight into Serine Protease Rv3671c that Protects M. tuberculosis from Oxidative and Acidic Stress

    SciTech Connect

    Biswas, Tapan; Small, Jennifer; Vandal, Omar; Odaira, Toshiko; Deng, Haiteng; Ehrt, Sabine; Tsodikov, Oleg V.

    2010-11-15

    Rv3671c, a putative serine protease, is crucial for persistence of Mycobacterium tuberculosis in the hostile environment of the phagosome. We show that Rv3671c is required for M. tuberculosis resistance to oxidative stress in addition to its role in protection from acidification. Structural and biochemical analyses demonstrate that the periplasmic domain of Rv3671c is a functional serine protease of the chymotrypsin family and, remarkably, that its activity increases on oxidation. High-resolution crystal structures of this protease in an active strained state and in an inactive relaxed state reveal that a solvent-exposed disulfide bond controls the protease activity by constraining two distant regions of Rv3671c and stabilizing it in the catalytically active conformation. In vitro biochemical studies confirm that activation of the protease in an oxidative environment is dependent on this reversible disulfide bond. These results suggest that the disulfide bond modulates activity of Rv3671c depending on the oxidative environment in vivo.

  8. The Putative Son's Attractiveness Alters the Perceived Attractiveness of the Putative Father.

    PubMed

    Prokop, Pavol

    2015-08-01

    A body of literature has investigated female mate choice in the pre-mating context (pre-mating sexual selection). Humans, however, are long-living mammals forming pair-bonds which sequentially produce offspring. Post-mating evaluations of a partner's attractiveness may thus significantly influence the reproductive success of men and women. I tested herein the theory that the attractiveness of putative sons provides extra information about the genetic quality of fathers, thereby influencing fathers' attractiveness across three studies. As predicted, facially attractive boys were more frequently attributed to attractive putative fathers and vice versa (Study 1). Furthermore, priming with an attractive putative son increased the attractiveness of the putative father with the reverse being true for unattractive putative sons. When putative fathers were presented as stepfathers, the effect of the boy's attractiveness on the stepfather's attractiveness was lower and less consistent (Study 2). This suggests that the presence of an attractive boy has the strongest effect on the perceived attractiveness of putative fathers rather than on non-fathers. The generalized effect of priming with beautiful non-human objects also exists, but its effect is much weaker compared with the effects of putative biological sons (Study 3). Overall, this study highlighted the importance of post-mating sexual selection in humans and suggests that the heritable attractive traits of men are also evaluated by females after mating and/or may be used by females in mate poaching.

  9. Targeting the AKT pathway: Repositioning HIV protease inhibitors as radiosensitizers.

    PubMed

    Goda, Jayant S; Pachpor, Tejaswini; Basu, Trinanjan; Chopra, Supriya; Gota, Vikram

    2016-02-01

    Cellular resistance in tumour cells to different therapeutic approaches has been a limiting factor in the curative treatment of cancer. Resistance to therapeutic radiation is a common phenomenon which significantly reduces treatment options and impacts survival. One of the mechanisms of acquiring resistance to ionizing radiation is the overexpression or activation of various oncogenes like the EGFR (epidermal growth factor receptor), RAS (rat sarcoma) oncogene or loss of PTEN (phosphatase and tensin homologue) which in turn activates the phosphatidyl inositol 3-kinase/protein kinase B (PI3-K)/AKT pathway responsible for radiation resistance in various tumours. Blocking the pathway enhances the radiation response both in vitro and in vivo. Due to the differential activation of this pathway (constitutively activated in tumour cells and not in the normal host cells), it is an excellent candidate target for molecular targeted therapy to enhance radiation sensitivity. In this regard, HIV protease inhibitors (HPIs) known to interfere with PI3-K/AKT signaling in tumour cells, have been shown to sensitize various tumour cells to radiation both in vitro and in vivo. As a result, HPIs are now being investigated as possible radiosensitizers along with various chemotherapeutic drugs. This review describes the mechanisms by which PI3-K/AKT pathway causes radioresistance and the role of HIV protease inhibitors especially nelfinavir as a potential candidate drug to target the AKT pathway for overcoming radioresistance and its use in various clinical trials for different malignancies.

  10. Targeting the AKT pathway: Repositioning HIV protease inhibitors as radiosensitizers

    PubMed Central

    Goda, Jayant S.; Pachpor, Tejaswini; Basu, Trinanjan; Chopra, Supriya; Gota, Vikram

    2016-01-01

    Cellular resistance in tumour cells to different therapeutic approaches has been a limiting factor in the curative treatment of cancer. Resistance to therapeutic radiation is a common phenomenon which significantly reduces treatment options and impacts survival. One of the mechanisms of acquiring resistance to ionizing radiation is the overexpression or activation of various oncogenes like the EGFR (epidermal growth factor receptor), RAS (rat sarcoma) oncogene or loss of PTEN (phosphatase and tensin homologue) which in turn activates the phosphatidyl inositol 3-kinase/protein kinase B (PI3-K)/AKT pathway responsible for radiation resistance in various tumours. Blocking the pathway enhances the radiation response both in vitro and in vivo. Due to the differential activation of this pathway (constitutively activated in tumour cells and not in the normal host cells), it is an excellent candidate target for molecular targeted therapy to enhance radiation sensitivity. In this regard, HIV protease inhibitors (HPIs) known to interfere with PI3-K/AKT signaling in tumour cells, have been shown to sensitize various tumour cells to radiation both in vitro and in vivo. As a result, HPIs are now being investigated as possible radiosensitizers along with various chemotherapeutic drugs. This review describes the mechanisms by which PI3-K/AKT pathway causes radioresistance and the role of HIV protease inhibitors especially nelfinavir as a potential candidate drug to target the AKT pathway for overcoming radioresistance and its use in various clinical trials for different malignancies. PMID:27121513

  11. How proteases regulate bone morphogenesis.

    PubMed

    Ortega, Nathalie; Behonick, Danielle; Stickens, Dominique; Werb, Zena

    2003-05-01

    Matrix metalloproteinases (MMPs) degrade most components of the extracellular matrix (ECM), as well as many non-ECM molecules. MMPs participate in (1). degradation of ECM to allow cell migration; (2). alteration of the ECM microenvironment resulting in alteration in cellular behavior; (3). modulation of biologically active molecules by direct cleavage or release from ECM stores; (4). regulation of the activity of other proteases; and (5). cell attachment, proliferation, differentiation, and apoptosis. We have sought to understand the role of MMPs during development and tissue repair in transgenic mice. Endochondral bone formation presents a particularly interesting developmental challenge. During this process, an avascular tissue (cartilage) is converted into one of the most highly vascularized tissues (bone) in the vertebrate body. Ossification begins with invasion of the calcified hypertrophic cartilage by capillaries. Apoptosis of the terminal hypertrophic chondrocytes, degradation of the cartilage matrix, and deposition of bone matrix by osteoblasts accompanies neovascularization of the growth plate. Remodeling of ECM results in a cavity filled with vascular channels containing hematopoietic cells. Our results reveal that MMP9, MMP13, and vascular endothelial growth factor are key regulators for the remodeling of the skeletal tissues. They coordinate not only matrix degradation, but also the recruitment and differentiation of endothelial cells, osteoclasts, chondroclasts, and osteoprogenitors.

  12. PCSK9: an enigmatic protease.

    PubMed

    Lopez, Dayami

    2008-04-01

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a critical role in cholesterol metabolism by controlling the levels of low density lipoprotein (LDL) particles that circulate in the bloodstream. Several gain-of-function and loss-of-function mutations in the PCSK9 gene, that occur naturally, have been identified and linked to hypercholesterolemia and hypocholesterolemia, respectively. PCSK9 expression has been shown to be regulated by sterol regulatory element binding proteins (SREBPs) and statins similar to other genes involved in cholesterol homeostasis. The most critical finding concerning PCSK9 is that this protease is able to influence the number of LDL receptor molecules expressed on the cell surface. Studies have demonstrated that PCSK9 acts mainly by enhancing degradation of LDL receptor protein in the liver. Inactivation of PCSK9 in mice reduces plasma cholesterol levels primarily by increasing hepatic expression of LDL receptor protein and thereby accelerating clearance of circulating LDL cholesterol. The objective of this review is to summarize the current information related to the regulation and function of PCSK9 and to identify gaps in our present knowledge.

  13. Expression and characterization of alkaline protease from the metagenomic library of tannery activated sludge.

    PubMed

    Devi, Selvaraju Gayathri; Fathima, Anwar Aliya; Sanitha, Mary; Iyappan, Sellamuthu; Curtis, Wayne R; Ramya, Mohandass

    2016-12-01

    Metagenomics has the potential to facilitate the discovery of novel enzymes; however, to date, only a few alkaline proteases have been characterized from environmentally-sourced DNA. We report the identification and characterization of an alkaline serine protease designated as Prt1A from the metagenomic library of tannery activated sludge. Sequence analysis revealed that Prt1A is closely related to S8A family subtilisins with a catalytic triad of Asp143, His173, and Ser326. The putative protease gene (prt-1A) was subcloned in pET 28a (+) vector and overexpressed in Escherichia coli BL21(DE3)pLysS cells. This 38.8 KDa recombinant protease was purified to homogeneity by nickel affinity chromatography and exhibited optimal enzyme activity at elevated pH (11.0) and temperature (55°C). The enzyme activity was enhanced by the addition of 5 mM Ca(2+) ions, and was stable in the presence of anionic detergent, oxidizing agent and various organic solvents. The enzyme displayed high affinity and catalytic efficiency for casein under standard assay conditions (Vmax = 279 U/mg/min, Km = 1.70 mg/mL) and was also compatible with commercial detergents. These results suggest that Prt1A protease could act as an efficient enzyme in various industrial applications. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  14. A tandem Kunitz protease inhibitor (KPI106)-serine carboxypeptidase (SCP1) controls mycorrhiza establishment and arbuscule development in Medicago truncatula.

    PubMed

    Rech, Stefanie S; Heidt, Sven; Requena, Natalia

    2013-09-01

    Plant proteases and protease inhibitors are involved in plant developmental processes including those involving interactions with microbes. Here we show that a tandem between a Kunitz protease inhibitor (KPI106) and a serine carboxypeptidase (SCP1) controls arbuscular mycorrhiza development in the root cortex of Medicago truncatula. Both proteins are only induced during mycorrhiza formation and belong to large families whose members are also mycorrhiza-specific. Furthermore, the interaction between KPI106 and SCP1 analysed using the yeast two-hybrid system is specific, indicating that each family member might have a defined counterpart. In silico docking analysis predicted a putative P1 residue in KPI106 (Lys173) that fits into the catalytic pocket of SCP1, suggesting that KPI106 might inhibit the enzyme activity by mimicking the protease substrate. In vitro mutagenesis of the Lys173 showed that this residue is important in determining the strength and specificity of the interaction. The RNA interference (RNAi) inactivation of the serine carboxypeptidase SCP1 produces aberrant mycorrhizal development with an increased number of septated hyphae and degenerate arbuscules, a phenotype also observed when overexpressing KPI106. Protease and inhibitor are both secreted as observed when expressed in Nicotiana benthamiana epidermal cells. Taken together we envisage a model in which the protease SCP1 is secreted in the apoplast where it produces a peptide signal critical for proper fungal development within the root. KPI106 also at the apoplast would modulate the spatial and/or temporal activity of SCP1 by competing with the protease substrate. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

  15. 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.

  16. Cross-inhibition of pathogenic agents and the host proteins they exploit

    PubMed Central

    Zilbermintz, Leeor; Leonardi, William; Tran, Sharon H.; Zozaya, Josue; Mathew-Joseph, Alyssa; Liem, Spencer; Levitin, Anastasia; Martchenko, Mikhail

    2016-01-01

    The major limitations of pathogen-directed therapies are the emergence of drug-resistance and their narrow spectrum of coverage. A recently applied approach directs therapies against host proteins exploited by pathogens in order to circumvent these limitations. However, host-oriented drugs leave the pathogens unaffected and may result in continued pathogen dissemination. In this study we aimed to discover drugs that could simultaneously cross-inhibit pathogenic agents, as well as the host proteins that mediate their lethality. We observed that many pathogenic and host-assisting proteins belong to the same functional class. In doing so we targeted a protease component of anthrax toxin as well as host proteases exploited by this toxin. We identified two approved drugs, ascorbic acid 6-palmitate and salmon sperm protamine, that effectively inhibited anthrax cytotoxic protease and demonstrated that they also block proteolytic activities of host furin, cathepsin B, and caspases that mediate toxin’s lethality in cells. We demonstrated that these drugs are broad-spectrum and reduce cellular sensitivity to other bacterial toxins that require the same host proteases. This approach should be generally applicable to the discovery of simultaneous pathogen and host-targeting inhibitors of many additional pathogenic agents. PMID:27703274

  17. Cardiovascular considerations in patients treated with HIV protease inhibitors.

    PubMed

    Colagreco, Joseph P

    2004-01-01

    Highly active antiretroviral therapy (HAART) has dramatically reduced mortality from HIV infection, transforming it in many cases to a chronic condition. However, protease inhibitors (PIs), which are integral components of most HAART regimens, are commonly associated with a host of metabolic disturbances that may increase the risk of cardiovascular disease in patients with HIV infection, potentially counteracting some of the positive health effects of PIs. Dyslipidemia is of particular concern. The Adult AIDS Clinical Trials Group has established preliminary guidelines to evaluate and treat PI-associated dyslipidemia. A number of strategies exist for the management of PI-based dyslipidemia in HAART recipients; their advantages and disadvantages should be considered when treating patients with HIV infection.

  18. Cleavage and Activation of the Severe Acute Respiratory Syndrome Coronavirus Spike Protein by Human Airway Trypsin-Like Protease

    PubMed Central

    Bertram, Stephanie; Glowacka, Ilona; Müller, Marcel A.; Lavender, Hayley; Gnirss, Kerstin; Nehlmeier, Inga; Niemeyer, Daniela; He, Yuxian; Simmons, Graham; Drosten, Christian; Soilleux, Elizabeth J.; Jahn, Olaf; Steffen, Imke; Pöhlmann, Stefan

    2011-01-01

    The highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) poses a constant threat to human health. The viral spike protein (SARS-S) mediates host cell entry and is a potential target for antiviral intervention. Activation of SARS-S by host cell proteases is essential for SARS-CoV infectivity but remains incompletely understood. Here, we analyzed the role of the type II transmembrane serine proteases (TTSPs) human airway trypsin-like protease (HAT) and transmembrane protease, serine 2 (TMPRSS2), in SARS-S activation. We found that HAT activates SARS-S in the context of surrogate systems and authentic SARS-CoV infection and is coexpressed with the viral receptor angiotensin-converting enzyme 2 (ACE2) in bronchial epithelial cells and pneumocytes. HAT cleaved SARS-S at R667, as determined by mutagenesis and mass spectrometry, and activated SARS-S for cell-cell fusion in cis and trans, while the related pulmonary protease TMPRSS2 cleaved SARS-S at multiple sites and activated SARS-S only in trans. However, TMPRSS2 but not HAT expression rendered SARS-S-driven virus-cell fusion independent of cathepsin activity, indicating that HAT and TMPRSS2 activate SARS-S differentially. Collectively, our results show that HAT cleaves and activates SARS-S and might support viral spread in patients. PMID:21994442

  19. Screen of Non-annotated Small Secreted Proteins of Pseudomonas syringae Reveals a Virulence Factor That Inhibits Tomato Immune Proteases

    PubMed Central

    Shindo, Takayuki; Kaschani, Farnusch; Kovács, Judit; Tian, Fang; Kourelis, Jiorgos; Hong, Tram Ngoc; Colby, Tom; Shabab, Mohammed; Chawla, Rohini; Kumari, Selva; Ilyas, Muhammad; Hörger, Anja C.; Alfano, James R.; van der Hoorn, Renier A. L.

    2016-01-01

    Pseudomonas syringae pv. tomato DC3000 (PtoDC3000) is an extracellular model plant pathogen, yet its potential to produce secreted effectors that manipulate the apoplast has been under investigated. Here we identified 131 candidate small, secreted, non-annotated proteins from the PtoDC3000 genome, most of which are common to Pseudomonas species and potentially expressed during apoplastic colonization. We produced 43 of these proteins through a custom-made gateway-compatible expression system for extracellular bacterial proteins, and screened them for their ability to inhibit the secreted immune protease C14 of tomato using competitive activity-based protein profiling. This screen revealed C14-inhibiting protein-1 (Cip1), which contains motifs of the chagasin-like protease inhibitors. Cip1 mutants are less virulent on tomato, demonstrating the importance of this effector in apoplastic immunity. Cip1 also inhibits immune protease Pip1, which is known to suppress PtoDC3000 infection, but has a lower affinity for its close homolog Rcr3, explaining why this protein is not recognized in tomato plants carrying the Cf-2 resistance gene, which uses Rcr3 as a co-receptor to detect pathogen-derived protease inhibitors. Thus, this approach uncovered a protease inhibitor of P. syringae, indicating that also P. syringae secretes effectors that selectively target apoplastic host proteases of tomato, similar to tomato pathogenic fungi, oomycetes and nematodes. PMID:27603016

  20. Toddlers' Duration of Attention toward Putative Threat

    ERIC Educational Resources Information Center

    Kiel, Elizabeth J.; Buss, Kristin A.

    2011-01-01

    Although individual differences in reactions to novelty in the toddler years have been consistently linked to risk of developing anxious behavior, toddlers' attention toward a novel, putatively threatening stimulus while in the presence of other enjoyable activities has rarely been examined as a precursor to such risk. The current study examined…

  1. Toddlers' Duration of Attention toward Putative Threat

    ERIC Educational Resources Information Center

    Kiel, Elizabeth J.; Buss, Kristin A.

    2011-01-01

    Although individual differences in reactions to novelty in the toddler years have been consistently linked to risk of developing anxious behavior, toddlers' attention toward a novel, putatively threatening stimulus while in the presence of other enjoyable activities has rarely been examined as a precursor to such risk. The current study examined…

  2. Downregulation of an Entamoeba histolytica rhomboid protease reveals roles in regulating parasite adhesion and phagocytosis.

    PubMed

    Baxt, Leigh A; Rastew, Elena; Bracha, Rivka; Mirelman, David; Singh, Upinder

    2010-08-01

    Entamoeba histolytica is a deep-branching eukaryotic pathogen. Rhomboid proteases are intramembrane serine proteases, which cleave transmembrane proteins in, or in close proximity to, their transmembrane domain. We have previously shown that E. histolytica contains a single functional rhomboid protease (EhROM1) and has unique substrate specificity. EhROM1 is present on the trophozoite surface and relocalizes to internal vesicles during erythrophagocytosis and to the base of the cap during surface receptor capping. In order to further examine the biological function of EhROM1 we downregulated EhROM1 expression by >95% by utilizing the epigenetic silencing mechanism of the G3 parasite strain. Despite the observation that EhROM1 relocalized to the cap during surface receptor capping, EhROM1 knockdown [ROM(KD)] parasites had no gross changes in cap formation or complement resistance. However, ROM(KD) parasites demonstrated decreased host cell adhesion, a result recapitulated by treatment of wild-type parasites with DCI, a serine protease inhibitor with activity against rhomboid proteases. The reduced adhesion phenotype of ROM(KD) parasites was noted exclusively with healthy cells, and not with apoptotic cells. Additionally, ROM(KD) parasites had decreased phagocytic ability with reduced ingestion of healthy cells, apoptotic cells, and rice starch. Decreased phagocytic ability is thus independent of the reduced adhesion phenotype, since phagocytosis of apoptotic cells was reduced despite normal adhesion levels. The defect in host cell adhesion was not explained by altered expression or localization of the heavy subunit of the Gal/GalNAc surface lectin. These results suggest no significant role of EhROM1 in complement resistance but unexpected roles in parasite adhesion and phagocytosis.

  3. Evaluation of proteases and protease inhibitors in Heterodera glycines cysts obtained from laboratory and field populations

    USDA-ARS?s Scientific Manuscript database

    Proteases and proteases inhibitors were evaluated in a number of preparations of Heterodera glycines cysts obtained from glasshouse cultures (GH) and field (LR) populations. Using a FRET-peptide library comprising 512 peptide substrate pools that detect 4 endoprotease types (aspartic, cysteine, meta...

  4. Isolation and Identification of an Extracellular Subtilisin-Like Serine Protease Secreted by the Bat Pathogen Pseudogymnoascus destructans

    PubMed Central

    Pannkuk, Evan L.; Risch, Thomas S.; Savary, Brett J.

    2015-01-01

    White nose syndrome (WNS) is a cutaneous fungal disease of bats. WNS is responsible for unprecedented mortalities in North American cave bat populations. There have been few descriptions of enzyme activities that may function in WNS host/pathogen interactions, while no study has isolated and described secreted proteases. To address the hypothesis that Pseudogymnoascus destructans secretes extracellular proteases that function in wing necrosis during WNS infection, the object of this study was to culture P. destructans on various media, then isolate and structurally identify those proteases accumulated stably in the culture medium. We found a single dominant protease activity on minimal nutrient broth enriched with protein substrates, which was strongly inhibited by phenylmethylsulfonyl fluoride. This P. destructans serine protease (PdSP1) was isolated by preparative isoelectric focusing and concanavalin A lectin affinity chromatography. PdSP1 showed a molecular weight 27,900 (estimated by SDS-PAGE), broad pH optimum 6-8, and temperature optimum 60°C. Structural characterization of PdSP1 by MALDI-TOF MS, Orbitrap MS/MS, and Edman amino-terminal peptide sequencing matched it directly to a hypothetical protein accession from the sequenced P. destructans genome that is further identified as a MEROPS family S8A subtilisin-like serine peptidase. Two additional isoforms, PdSP2 and PdSP3, were identified in the P. destructans genome with 90% and 53% homology, respectively. P. destructans S8A serine proteases showed closer sequence conservation to P. pannorum and plant pathogenic fungi than to human pathogenic dermatophytes. Peptide-specific polyclonal antibodies developed from the PdSP1 sequence detected the protein in western blots. These subtilisin-like serine proteases are candidates for further functional studies in WNS host-pathogen interaction. PMID:25785714

  5. Isolation and identification of an extracellular subtilisin-like serine protease secreted by the bat pathogen Pseudogymnoascus destructans.

    PubMed

    Pannkuk, Evan L; Risch, Thomas S; Savary, Brett J

    2015-01-01

    White nose syndrome (WNS) is a cutaneous fungal disease of bats. WNS is responsible for unprecedented mortalities in North American cave bat populations. There have been few descriptions of enzyme activities that may function in WNS host/pathogen interactions, while no study has isolated and described secreted proteases. To address the hypothesis that Pseudogymnoascus destructans secretes extracellular proteases that function in wing necrosis during WNS infection, the object of this study was to culture P. destructans on various media, then isolate and structurally identify those proteases accumulated stably in the culture medium. We found a single dominant protease activity on minimal nutrient broth enriched with protein substrates, which was strongly inhibited by phenylmethylsulfonyl fluoride. This P. destructans serine protease (PdSP1) was isolated by preparative isoelectric focusing and concanavalin A lectin affinity chromatography. PdSP1 showed a molecular weight 27,900 (estimated by SDS-PAGE), broad pH optimum 6-8, and temperature optimum 60°C. Structural characterization of PdSP1 by MALDI-TOF MS, Orbitrap MS/MS, and Edman amino-terminal peptide sequencing matched it directly to a hypothetical protein accession from the sequenced P. destructans genome that is further identified as a MEROPS family S8A subtilisin-like serine peptidase. Two additional isoforms, PdSP2 and PdSP3, were identified in the P. destructans genome with 90% and 53% homology, respectively. P. destructans S8A serine proteases showed closer sequence conservation to P. pannorum and plant pathogenic fungi than to human pathogenic dermatophytes. Peptide-specific polyclonal antibodies developed from the PdSP1 sequence detected the protein in western blots. These subtilisin-like serine proteases are candidates for further functional studies in WNS host-pathogen interaction.

  6. Tissue factor residues that putatively interact with membrane phospholipids.

    PubMed

    Ke, Ke; Yuan, Jian; Morrissey, James H

    2014-01-01

    Blood clotting is initiated by the two-subunit enzyme consisting of the plasma protease, factor VIIa (the catalytic subunit), bound to the integral membrane protein, tissue factor (the regulatory subunit). Molecular dynamics simulations have predicted that certain residues in the tissue factor ectodomain interact with phosphatidylserine headgroups to ensure optimal positioning of the tissue factor/factor VIIa complex relative to its membrane-bound protein substrates, factors IX and X. In this study, we individually mutated to alanine all the putative phosphatidylserine-interactive residues in the tissue factor ectodomain and measured their effects on tissue factor cofactor function (activation of factors IX and X by tissue factor/factor VIIa, and clotting of plasma). Some tissue factor mutants exhibited decreased activity in all three assays, with the most profound defects observed from mutations in or near the flexible loop from Lys159 to Gly164. The decreased activity of all of these tissue factor mutants could be partially or completely overcome by increasing the phosphatidylserine content of tissue factor-liposomes. Additionally, yeast surface display was used to screen a random library of tissue factor mutants for enhanced factor VIIa binding. Surprisingly, mutations at a single amino acid (Lys165) predominated, with the Lys165→Glu mutant exhibiting a 3-fold enhancement in factor VIIa binding affinity. Our studies reveal the functional contributions of residues in the C-terminal half of the tissue factor ectodomain that are implicated in interacting with phosphatidylserine headgroups to enhance tissue factor cofactor activity, possibly by allosterically modulating the conformation of the adjacent substrate-binding exosite region of tissue factor.

  7. Engineered Protease-resistant Antibodies with Selectable Cell-killing Functions*

    PubMed Central

    Kinder, Michelle; Greenplate, Allison R.; Grugan, Katharine D.; Soring, Keri L.; Heeringa, Katharine A.; McCarthy, Stephen G.; Bannish, Gregory; Perpetua, Meredith; Lynch, Frank; Jordan, Robert E.; Strohl, William R.; Brezski, Randall J.

    2013-01-01

    Molecularly engineered antibodies with fit-for-purpose properties will differentiate next generation antibody therapeutics from traditional IgG1 scaffolds. One requirement for engineering the most appropriate properties for a particular therapeutic area is an understanding of the intricacies of the target microenvironment in which the antibody is expected to function. Our group and others have demonstrated that proteases secreted by invasive tumors and pathological microorganisms are capable of cleaving human IgG1, the most commonly adopted isotype among monoclonal antibody therapeutics. Specific cleavage in the lower hinge of IgG1 results in a loss of Fc-mediated cell-killing functions without a concomitant loss of antigen binding capability or circulating antibody half-life. Proteolytic cleavage in the hinge region by tumor-associated or microbial proteases is postulated as a means of evading host immune responses, and antibodies engineered with potent cell-killing functions that are also resistant to hinge proteolysis are of interest. Mutation of the lower hinge region of an IgG1 resulted in protease resistance but also resulted in a profound loss of Fc-mediated cell-killing functions. In the present study, we demonstrate that specific mutations of the CH2 domain in conjunction with lower hinge mutations can restore and sometimes enhance cell-killing functions while still retaining protease resistance. By identifying mutations that can restore either complement- or Fcγ receptor-mediated functions on a protease-resistant scaffold, we were able to generate a novel protease-resistant platform with selective cell-killing functionality. PMID:23986451

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

    PubMed

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

    2012-01-01

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

  9. Intervention for hyperlipidemia associated with protease inhibitors.

    PubMed

    Melroe, N H; Kopaczewski, J; Henry, K; Huebsch, J

    1999-01-01

    In the past 3 years, treatment for HIV infection has significantly improved the prognosis for HIV-infected persons. The administration of protease inhibitors for the treatment of HIV infection has had a significant role in the reduction of AIDS-related complications. Recent findings have indicated that protease inhibitors may significantly increase lipids to levels that pose a health risk that may be greater than the illness itself. This article reviews the initial findings of a study that investigated the impact of interventions for the treatment of protease inhibitor-related hyperlipidemia. The purpose of the study was to determine if initiation of interventions based on the National Cholesterol Education Program Guidelines would be effective in lowering protease inhibitor-related hyperlipidemia without disrupting the effectiveness of the HIV therapy. A total of 45 HIV-infected individuals who were taking a protease inhibitor and had abnormally elevated lipids were enrolled into this study. Mean serum cholesterol level prior to initiation of a protease inhibitor regimen was 170 mg/dl as compared to a mean cholesterol at time of enrollment of 289 mg/dl and triglycerides of 879 mg/dl. Interventions included diet and exercise and the prescription of gemfibrozil alone or in combination with atorvatstatin. During the course of the study, overall intervention significantly reduced serum cholesterol level to 201 mg/dl (p. 01) over a study period of ten months. Case studies of five medical events related to hyperlipidemia are included. Currently, 26 participants continue in the study. Sixteen participants discontinued protease inhibitor therapy during the course of the study and thus ended their participation.

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

    PubMed

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

    2003-01-01

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

  11. Phytophthora infestans effector AVRblb2 prevents secretion of a plant immune protease at the haustorial interface

    PubMed Central

    Bozkurt, Tolga O.; Schornack, Sebastian; Win, Joe; Shindo, Takayuki; Ilyas, Muhammad; Oliva, Ricardo; Cano, Liliana M.; Jones, Alexandra M. E.; Huitema, Edgar; van der Hoorn, Renier A. L.; Kamoun, Sophien

    2011-01-01

    In response to pathogen attack, plant cells secrete antimicrobial molecules at the site of infection. However, how plant pathogens interfere with defense-related focal secretion remains poorly known. Here we show that the host-translocated RXLR-type effector protein AVRblb2 of the Irish potato famine pathogen Phytophthora infestans focally accumulates around haustoria, specialized infection structures that form inside plant cells, and promotes virulence by interfering with the execution of host defenses. AVRblb2 significantly enhances susceptibility of host plants to P. infestans by targeting the host papain-like cysteine protease C14 and specifically preventing its secretion into the apoplast. Plants altered in C14 expression were significantly affected in susceptibility to P. infestans in a manner consistent with a positive role of C14 in plant immunity. Our findings point to a unique counterdefense strategy that plant pathogens use to neutralize secreted host defense proteases. Effectors, such as AVRblb2, can be used as molecular probes to dissect focal immune responses at pathogen penetration sites. PMID:22143776

  12. Diversity and transcription of proteases involved in the maturation of hydrogenases in Nostoc punctiforme ATCC 29133 and Nostoc sp. strain PCC 7120

    PubMed Central

    2009-01-01

    Background The last step in the maturation process of the large subunit of [NiFe]-hydrogenases is a proteolytic cleavage of the C-terminal by a hydrogenase specific protease. Contrary to other accessory proteins these hydrogenase proteases are believed to be specific whereby one type of hydrogenases specific protease only cleaves one type of hydrogenase. In cyanobacteria this is achieved by the gene product of either hupW or hoxW, specific for the uptake or the bidirectional hydrogenase respectively. The filamentous cyanobacteria Nostoc punctiforme ATCC 29133 and Nostoc sp strain PCC 7120 may contain a single uptake hydrogenase or both an uptake and a bidirectional hydrogenase respectively. Results In order to examine these proteases in cyanobacteria, transcriptional analyses were performed of hupW in Nostoc punctiforme ATCC 29133 and hupW and hoxW in Nostoc sp. strain PCC 7120. These studies revealed numerous transcriptional start points together with putative binding sites for NtcA (hupW) and LexA (hoxW). In order to investigate the diversity and specificity among hydrogeanse specific proteases we constructed a phylogenetic tree which revealed several subgroups that showed a striking resemblance to the subgroups previously described for [NiFe]-hydrogenases. Additionally the proteases specificity was also addressed by amino acid sequence analysis and protein-protein docking experiments with 3D-models derived from bioinformatic studies. These studies revealed a so called "HOXBOX"; an amino acid sequence specific for protease of Hox-type which might be involved in docking with the large subunit of the hydrogenase. Conclusion Our findings suggest that the hydrogenase specific proteases are under similar regulatory control as the hydrogenases they cleave. The result from the phylogenetic study also indicates that the hydrogenase and the protease have co-evolved since ancient time and suggests that at least one major horizontal gene transfer has occurred. This co

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

    PubMed

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

    2014-05-01

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

  14. 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

  15. 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

  16. 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

  17. Lysosomal protease expression in mature enamel.

    PubMed

    Tye, Coralee E; Lorenz, Rachel L; Bartlett, John D

    2009-01-01

    The enamel matrix proteins (amelogenin, enamelin and ameloblastin) are degraded by matrix metalloproteinase-20 and kallikrein-4 during enamel development and mature enamel is virtually protein free. The precise mechanism of removal and degradation of the enamel protein cleavage products from the matrix, however, remains poorly understood. It has been proposed that receptor-mediated endocytosis allows for the cleaved proteins to be removed from the matrix during enamel formation and then transported to the lysosome for further degradation. This study aims to identify lysosomal proteases that are present in maturation-stage enamel organ. RNA from first molars of 11-day-old mice was collected and expression was initially assessed by RT-PCR and then quantified by qPCR. The pattern of expression of selected proteases was assessed by immunohistochemical staining of demineralized mouse incisors. With the exception of cathepsin G, all lysosomal proteases assessed were expressed in maturation-stage enamel organ. Identified proteases included cathepsins B, D, F, H, K, L, O, S and Z. Tripeptidyl peptidases I and II as well as dipeptidyl peptidases I, II, III and IV were also found to be expressed. Immunohistochemical staining confirmed that the maturation-stage ameloblasts express cathepsins L and S and tripeptidyl peptidase II. Our results suggest that the ameloblasts are enriched by a large number of lysosomal proteases at maturation that are likely involved in the degradation of the organic matrix.

  18. Bacterial proteases, untapped antimicrobial drug targets.

    PubMed

    Culp, Elizabeth; Wright, Gerard D

    2017-04-01

    Bacterial proteases are an extensive collection of enzymes that have vital roles in cell viability, stress response and pathogenicity. Although their perturbation clearly offers the potential for antimicrobial drug development, both as traditional antibiotics and anti-virulence drugs, they are not yet the target of any clinically used therapeutics. Here we describe the potential for and recent progress in the development of compounds targeting bacterial proteases with a focus on AAA+ family proteolytic complexes and signal peptidases (SPs). Caseinolytic protease (ClpP) belongs to the AAA+ family of proteases, a group of multimeric barrel-shaped complexes whose activity is tightly regulated by associated AAA+ ATPases. The opportunity for chemical perturbation of these complexes is demonstrated by compounds targeting ClpP for inhibition, activation or perturbation of its associated ATPase. Meanwhile, SPs are also a proven antibiotic target. Responsible for the cleavage of targeting peptides during protein secretion, both type I and type II SPs have been successfully targeted by chemical inhibitors. As the threat of pan-antibiotic resistance continues to grow, these and other bacterial proteases offer an arsenal of novel antibiotic targets ripe for development.

  19. Diverse Intestinal Bacteria Contain Putative Zwitterionic Capsular Polysaccharides with Anti-inflammatory Properties.

    PubMed

    Neff, C Preston; Rhodes, Matthew E; Arnolds, Kathleen L; Collins, Colm B; Donnelly, Jody; Nusbacher, Nichole; Jedlicka, Paul; Schneider, Jennifer M; McCarter, Martin D; Shaffer, Michael; Mazmanian, Sarkis K; Palmer, Brent E; Lozupone, Catherine A

    2016-10-12

    Zwitterionic capsular polysaccharides (ZPSs) are bacterial products that modulate T cells, including inducing anti-inflammatory IL-10-secreting T regulatory cells (Tregs). However, only a few diverse bacteria are known to modulate the host immune system via ZPS. We present a genomic screen for bacteria encoding ZPS molecules. We identify diverse host-associated bacteria, including commensals and pathogens with known anti-inflammatory properties, with the capacity to produce ZPSs. Human mononuclear cells stimulated with lysates from putative ZPS-producing bacteria induce significantly greater IL-10 production and higher proportions of Tregs than lysates from non-ZPS-encoding relatives or a commensal strain of Bacteroides cellulosilyticus in which a putative ZPS biosynthetic operon was genetically disrupted. Similarly, wild-type B. cellulosilyticus DSM 14838, but not a close relative lacking a putative ZPS, attenuated experimental colitis in mice. Collectively, this screen identifies bacterial strains that may use ZPSs to interact with the host as well as those with potential probiotic properties. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Phylogeny and evolution of the aspartyl protease family from clinically relevant Candida species.

    PubMed

    Parra-Ortega, B; Cruz-Torres, H; Villa-Tanaca, L; Hernández-Rodríguez, C

    2009-05-01

    Aspartyl proteases are a class of enzymes that include the yeast aspartyl proteases and secreted aspartyl protease (Sap) superfamilies. Several Sap superfamily members have been demonstrated or suggested as virulence factors in opportunistic pathogens of the genus Candida. Candida albicans, Candida tropicalis, Candida dubliniensis and Candida parapsilosis harbour 10, four, eight and three SAP genes, respectively. In this work, genome mining and phylogenetic analyses revealed the presence of new members of the Sap superfamily in C. tropicalis (8), Candida guilliermondii (8), C. parapsilosis(11) and Candida lusitaniae (3). A total of 12 Sap families, containing proteins with at least 50% similarity, were discovered in opportunistic, pathogenic Candida spp. In several Sap families, at least two subfamilies or orthologous groups were identified, each defined by > 90% sequence similitude, functional similarity and synteny among its members. No new members of previously described Sap families were found in a Candida spp. clinical strain collection; however, the universality of SAPT gene distribution among C. tropicalis strains was demonstrated. In addition, several features of opportunistic pathogenic Candida species, such as gene duplications and inversions, similitude, synteny, putative transcription factor binding sites and genome traits of SAP gene superfamily were described in a molecular evolutionary context.

  1. Gene cloning, molecular modeling, and phylogenetics of serine protease P32 and serine carboxypeptidase SCP1 from nematophagous fungi Pochonia rubescens and Pochonia chlamydosporia.

    PubMed

    Larriba, Eduardo; Martín-Nieto, José; Lopez-Llorca, Luis Vicente

    2012-07-01

    The fungi Pochonia chlamydosporia and Pochonia rubescens are parasites of nematode eggs and thus are biocontrol agents of nematodes. Proteolytic enzymes such as the S8 proteases VCP1 and P32, secreted during the pathogenesis of nematode eggs, are major virulence factors in these fungi. Recently, expression of these enzymes and of SCP1, a new putative S10 carboxypeptidase, was detected during endophytic colonization of barley roots by these fungi. In our study, we cloned the genomic and mRNA sequences encoding P32 from P. rubescens and SCP1 from P. chlamydosporia. P32 showed a high homology with the serine proteases Pr1A from the entomopathogenic fungus Metarhizium anisopliae and VCP1 from P. chlamydosporia (86% and 76% identity, respectively). However, the catalytic pocket of P32 showed differences in the amino acids of the substrate-recognition sites compared with the catalytic pockets of Pr1A and VCP1 proteases. Phylogenetic analysis of P32 suggests a common ancestor with protease Pr1A. SCP1 displays the characteristic features of a member of the S10 family of serine proteases. Phylogenetic comparisons show that SCP1 and other carboxypeptidases from filamentous fungi have an origin different from that of yeast vacuolar serine carboxypeptidases. Understanding protease genes from nematophagous fungi is crucial for enhancing the biocontrol potential of these organisms.

  2. Cysteine Protease Secreted by Paragonimus westermani Attenuates Effector Functions of Human Eosinophils Stimulated with Immunoglobulin G

    PubMed Central

    Shin, Myeong Heon; Kita, Hirohito; Park, Hae Young; Seoh, Ju Young

    2001-01-01

    An immunoglobulin G (IgG)-coated surface, such as that found on helminth parasites, is one of the most effective physiologic stimuli for eosinophil activation. The cysteine proteases secreted by tissue-invasive helminth larvae play an important role in evasion of the immune response by degrading the host immunoglobulins. In this study, we investigated whether cysteine proteases in the excretory-secretory product (ESP) produced by Paragonimus westermani newly excysted metacercariae (PwNEM), which cause pulmonary or extrapulmonary paragonimiasis in human beings, could modify effector functions of human eosinophils stimulated with IgG. We coated 96-well plates with human IgG in the absence or presence of the ESP produced by PwNEM. When eosinophils were incubated in the wells coated with IgG in the presence of the ESP, eosinophil degranulation and superoxide production were significantly reduced compared with results for cells incubated in wells coated with IgG alone. This inhibitory effect of the ESP on IgG-induced superoxide production was dose dependent and was significantly abolished by pretreatment of the ESP with heat. These findings suggest that the cysteine proteases secreted by PwNEM attenuate both activation and degranulation of eosinophils stimulated with IgG. Thus, the cysteine proteases produced by tissue-invasive helminth larvae play crucial roles in evasion of IgG-dependent eosinophil helminthotoxicity and in reduction of eosinophil-associated tissue inflammation during the migratory period. PMID:11179333

  3. Negative Regulation of Pathogenesis in Pseudomonas syringae pv. tabaci 11528 by ATP-Dependent Lon Protease

    PubMed Central

    Yang, Hyun Ju; Lee, Jun Seung; Cha, Ji Young; Baik, Hyung Suk

    2011-01-01

    Pseudomonas syringae pv. tabaci causes wildfire disease in tobacco plants. The hrp pathogenicity island (hrp PAI) of P. syringae pv. tabaci encodes a type III secretion system (TTSS) and its regulatory system, which are required for pathogenesis in plants. Three important regulatory proteins- HrpR, HrpS, and HrpL-have been identified to activate hrp PAI gene expression. The bacterial Lon protease regulates the expression of various genes. To investigate the regulatory mechanism of the Lon protease in P. syringae pv. tabaci 11528, we cloned the lon gene, and then a Δlon mutant was generated by allelic exchange. lon mutants showed increased UV sensitivity, which is a typical feature of such mutants. The Δlon mutant produced higher levels of tabtoxin than the wild-type. The lacZ gene was fused with hrpA promoter and activity of β-galactosidase was measured in hrp-repressing and hrp-inducing media. The Lon protease functioned as a negative regulator of hrp PAI under hrp-repressing conditions. We found that strains with lon disruption elicited the host defense system more rapidly and strongly than the wild-type strain, suggesting that the Lon protease is essential for systemic pathogenesis. PMID:21904881

  4. Alternative splicing, a new target to block cellular gene expression by poliovirus 2A protease

    SciTech Connect

    Alvarez, Enrique; Castello, Alfredo; Carrasco, Luis; Izquierdo, Jose M.

    2011-10-14

    Highlights: {yields} Novel role for poliovirus 2A protease as splicing modulator. {yields} Poliovirus 2A protease inhibits the alternative splicing of pre-mRNAs. {yields} Poliovirus 2A protease blocks the second catalytic step of splicing. -- Abstract: Viruses have developed multiple strategies to interfere with the gene expression of host cells at different stages to ensure their own survival. Here we report a new role for poliovirus 2A{sup pro} modulating the alternative splicing of pre-mRNAs. Expression of 2A{sup pro} potently inhibits splicing of reporter genes in HeLa cells. Low amounts of 2A{sup pro} abrogate Fas exon 6 skipping, whereas higher levels of protease fully abolish Fas and FGFR2 splicing. In vitro splicing of MINX mRNA using nuclear extracts is also strongly inhibited by 2A{sup pro}, leading to accumulation of the first exon and the lariat product containing the unspliced second exon. These findings reveal that the mechanism of action of 2A{sup pro} on splicing is to selectively block the second catalytic step.

  5. Cleavage entropy as quantitative measure of protease specificity.

    PubMed

    Fuchs, Julian E; von Grafenstein, Susanne; Huber, Roland G; Margreiter, Michael A; Spitzer, Gudrun M; Wallnoefer, Hannes G; Liedl, Klaus R

    2013-04-01

    A purely information theory-guided approach to quantitatively characterize protease specificity is established. We calculate an entropy value for each protease subpocket based on sequences of cleaved substrates extracted from the MEROPS database. We compare our results with known subpocket specificity profiles for individual proteases and protease groups (e.g. serine proteases, metallo proteases) and reflect them quantitatively. Summation of subpocket-wise cleavage entropy contributions yields a measure for overall protease substrate specificity. This total cleavage entropy allows ranking of different proteases with respect to their specificity, separating unspecific digestive enzymes showing high total cleavage entropy from specific proteases involved in signaling cascades. The development of a quantitative cleavage entropy score allows an unbiased comparison of subpocket-wise and overall protease specificity. Thus, it enables assessment of relative importance of physicochemical and structural descriptors in protease recognition. We present an exemplary application of cleavage entropy in tracing substrate specificity in protease evolution. This highlights the wide range of substrate promiscuity within homologue proteases and hence the heavy impact of a limited number of mutations on individual substrate specificity.

  6. Cleavage Entropy as Quantitative Measure of Protease Specificity

    PubMed Central

    Fuchs, Julian E.; von Grafenstein, Susanne; Huber, Roland G.; Margreiter, Michael A.; Spitzer, Gudrun M.; Wallnoefer, Hannes G.; Liedl, Klaus R.

    2013-01-01

    A purely information theory-guided approach to quantitatively characterize protease specificity is established. We calculate an entropy value for each protease subpocket based on sequences of cleaved substrates extracted from the MEROPS database. We compare our results with known subpocket specificity profiles for individual proteases and protease groups (e.g. serine proteases, metallo proteases) and reflect them quantitatively. Summation of subpocket-wise cleavage entropy contributions yields a measure for overall protease substrate specificity. This total cleavage entropy allows ranking of different proteases with respect to their specificity, separating unspecific digestive enzymes showing high total cleavage entropy from specific proteases involved in signaling cascades. The development of a quantitative cleavage entropy score allows an unbiased comparison of subpocket-wise and overall protease specificity. Thus, it enables assessment of relative importance of physicochemical and structural descriptors in protease recognition. We present an exemplary application of cleavage entropy in tracing substrate specificity in protease evolution. This highlights the wide range of substrate promiscuity within homologue proteases and hence the heavy impact of a limited number of mutations on individual substrate specificity. PMID:23637583

  7. A cysteine protease of Dieffenbachia maculata.

    PubMed

    Chitre, A; Padmanabhan, S; Shastri, N V

    1998-12-01

    Plants of the genus Dieffenbachia, very popular as indoor ornamental plants, are known for their toxic as well as therapeutic properties. Their toxic manifestations have been partly attributed to their proteolytic activity. The work described in the present paper shows that stem leaves and petiole of Dieffenbachia maculata Schott, a commonly grown species, contain significant proteolytic activity, different parts showing different types of protease activities. Stem showed the highest enzyme activity and this protease was purified about 55 fold by solvent precipitation, gel filtration and ion exchange chromatography. The enzyme has a relative molecular mass of 61 kDa as determined by SDS-PAGE and has an optimum pH of 8.0 and optimum temperature of 50 degrees C. Effects of various substrates, inhibitors and activators indicate that the enzyme is a cysteine protease with leucylpeptidase activity.

  8. A repertoire of protease inhibitor families in Amblyomma americanum and other tick species: inter-species comparative analyses.

    PubMed

    Porter, Lindsay M; Radulović, Željko M; Mulenga, Albert

    2017-03-22

    Protease inhibitors (PIs) are important regulators of physiology and represent anti-parasitic druggable and vaccine targets. We conducted bioinformatic analyses of genome and transcriptome data to determine the protease inhibitor (PI) repertoire in Amblyomma americanum and in 25 other ixodid tick species. For A. americanum, we compared the PI repertoires in fed and unfed, male and female A. americanum ticks. We also analyzed PI repertoires of female 48, 96 and 120 h-fed midgut (MG) and salivary gland (SG) tissues. We found 1,595 putative non-redundant PI sequences across 26 ixodid tick species. Ticks express PIs from at least 18 different families: I1, I2, I4, I8, I21, I25, I29, I31, I32, I35, I39, I43, I51, I53, I63, I68, I72 and I74 (MEROPS). The largest PI families were I2, I4 and I8 and lowest in I21, I31, I32, I35 and I68. The majority (75%) of tick PIs putatively inhibit serine proteases, with ~11 and 9% putatively regulating cysteine or metalloprotease-mediated pathways, respectively, and ~4% putatively regulating multiple/mixed protease types. In A. americanum, we found 370 PIs in female and 354 in male ticks. In A. americanum we found 231 and 442 in unfed and fed ticks, respectively. In females, we found 206 and 164 PIs in SG and MG, respectively. The majority of highly cross-tick species conserved PIs were in families I1, I2, I8, I21, I25, I29, I39 and I43. Ticks appear to express large and diverse repertoires of PIs that primarily target serine protease-mediated pathways. We speculate that PI families with the highest repertoires may contain functionally redundant members while those with the lowest repertoires are functionally non-redundant PIs. We found some highly conserved PIs in the latter category, which we propose as potential candidates for broad-spectrum anti-tick vaccine candidates or druggable targets in tick control.

  9. Multicopy suppressors of prc mutant Escherichia coli include two HtrA (DegP) protease homologs (HhoAB), DksA, and a truncated R1pA.

    PubMed Central

    Bass, S; Gu, Q; Christen, A

    1996-01-01

    We have isolated three multicopy suppressors of the conditional lethal phenotype of a prc (tsp) null strain of Escherichia coli. One of these suppressors included two novel putative protease genes in tandem that map to 3400 kb or 72.5 centisomes on the chromosome. We propose the names hhoA and hhoB, for htrA homolog, to denote that these genes encode proteins that are 58 and 35% identical, respectively, to the HtrA (DegP) serine protease and 36% identical to each other. The HhoA and HhoB proteins are predicted to be 455 and 355 amino acids, respectively, in length. The mature HhoA protein is periplasmic in location, and amino-terminal sequencing shows that it arises following cleavage of a 27-amino-acid signal peptide. Searches of the protein and DNA databases reveal a rapidly growing family of homologous genes in a variety of other bacteria, including several which are required for virulence in their host. Deletion of the hhoAB genes shows that they are not required for viability at high temperatures like the homologous htrA but grow more slowly than wild-type strains. A second multicopy prc suppressor is the dksA (dnaK suppressor) gene, which is also a multicopy suppressor of defects in the heat shock genes dnaK, dnaJ, and grpE. The dksA gene was independently isolated as a multicopy suppressor of a mukB mutation, which is required for chromosomal partitioning. A third dosage-dependent prc suppressor includes a truncated rare lipoprotein A (rlpA) gene. PMID:8576052

  10. Seminal and colostral protease inhibitors on leukocytes.

    PubMed

    Veselský, L; Cechová, D; Hruban, V; Klaudy, J

    1982-01-01

    For detection of protease inhibitors from cow colostrum (CTI) and bull seminal plasma (BUSI I and BUSI II) on the surface of leukocytes, immunological methods were used. An agglutination and an immunofluorescence test demonstrated components on the surface of bovine, porcine and ovine granulocytes and lymphocytes which were immunologically identical with the protease inhibitors isolated from cow colostrum and bull seminal plasma. When antisera against (CTI, BUSI and BUSI II were absorbed by bovine and porcine liver, kidney and spleen homogenate or by bovine and porcine granulocytes or lymphocytes, the immunological tests were negative.

  11. Saquinavir, the pioneer antiretroviral protease inhibitor.

    PubMed

    la Porte, Charles J L

    2009-10-01

    The treatment of HIV infection underwent a major change in 1995 when saquinavir was the first protease inhibitor introduced into the market. This drug made the use of combination therapy in the treatment of HIV possible and increased the success rate of treatment. This article will review recent literature on saquinavir to define its current role in HIV treatment, among the newer antiretroviral drugs. Scientific literature and conference presentations were evaluated for relevant information pertaining to saquinavir. Although underused, saquinavir has good efficacy and tolerability when compared to other protease inhibitors. The film-coated tablet formulation improved pill burden. Saquinavir still has potential in the treatment of adults, children and pregnant women.

  12. Current and Novel Inhibitors of HIV Protease

    PubMed Central

    Pokorná, Jana; Machala, Ladislav; Řezáčová, Pavlína; Konvalinka, Jan

    2009-01-01

    The design, development and clinical success of HIV protease inhibitors represent one of the most remarkable achievements of molecular medicine. This review describes all nine currently available FDA-approved protease inhibitors, discusses their pharmacokinetic properties, off-target activities, side-effects, and resistance profiles. The compounds in the various stages of clinical development are also introduced, as well as alternative approaches, aiming at other functional domains of HIV PR. The potential of these novel compounds to open new way to the rational drug design of human viruses is critically assessed. PMID:21994591

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

    DOEpatents

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

    2015-09-29

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

  14. 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.

  15. Amblyomma americanum (L.) (Acari: Ixodidae) tick salivary gland serine protease inhibitor (serpin) 6 is secreted into tick saliva during tick feeding.

    PubMed

    Chalaire, Katelyn Cox; Kim, Tae Kwon; Garcia-Rodriguez, Heidy; Mulenga, Albert

    2011-02-15

    In order to successfully feed and transmit disease agents, ticks are thought to inject serine protease inhibitors (serpins) into the host to modulate host defense responses to tick feeding, such as inflammation, the complement activation pathway and blood coagulation. In this study, we show that Amblyomma americanum (Aam) serpin (S) 6 is putatively injected into the host during tick feeding, in that the antibody to recombinant (r) AamS6 specifically reacted with the expected ∼43/45 kDa AamS6 protein band on western blots of pilocarpine-induced tick saliva. Additionally, antibodies to tick saliva proteins that were generated by repeated 48 h infestations of rabbits with adult A. americanum specifically reacted with rAamS6. We speculate that AamS6 is associated with regulating events at the start of the tick feeding process, as temporal and spatial RT-PCR and western blot analyses revealed that both AamS6 mRNA and protein are strongly expressed during the first 24-72 h of feeding time before starting to fade from 96 h. The AamS6 protein has an apparently slow turnover rate in that, although the injection of AamS6 dsRNA into unfed ticks triggered complete disruption of the AamS6 mRNA by the 48 h feeding time point, western blot analysis of protein extracts of the same animals showed that the AamS6 protein that may have been expressed prior to disruption of the AamS6 mRNA was not depleted. We speculate that the presence of the AamS6 protein in ticks despite the complete disruption of the AamS6 mRNA explains the observation that RNAi-mediated silencing of the AamS6 mRNA did not affect the ability of A. americanum ticks to attach onto host skin, successfully feed and lay eggs. These findings are discussed in regards to advances in the molecular biology of ticks.

  16. A Novel Extracellular Metallopeptidase Domain Shared by Animal Host-Associated Mutualistic and Pathogenic Microbes

    PubMed Central

    Nakjang, Sirintra; Ndeh, Didier A.; Wipat, Anil; Bolam, David N.; Hirt, Robert P.

    2012-01-01

    The mucosal microbiota is recognised as an important factor for our health, with many disease states linked to imbalances in the normal community structure. Hence, there is considerable interest in identifying the molecular basis of human-microbe interactions. In this work we investigated the capacity of microbes to thrive on mucosal surfaces, either as mutualists, commensals or pathogens, using comparative genomics to identify co-occurring molecular traits. We identified a novel domain we named M60-like/PF13402 (new Pfam entry PF13402), which was detected mainly among proteins from animal host mucosa-associated prokaryotic and eukaryotic microbes ranging from mutualists to pathogens. Lateral gene transfers between distantly related microbes explained their shared M60-like/PF13402 domain. The novel domain is characterised by a zinc-metallopeptidase-like motif and is distantly related to known viral enhancin zinc-metallopeptidases. Signal peptides and/or cell surface anchoring features were detected in most microbial M60-like/PF13402 domain-containing proteins, indicating that these proteins target an extracellular substrate. A significant subset of these putative peptidases was further characterised by the presence of associated domains belonging to carbohydrate-binding module family 5/12, 32 and 51 and other glycan-binding domains, suggesting that these novel proteases are targeted to complex glycoproteins such as mucins. An in vitro mucinase assay demonstrated degradation of mammalian mucins by a recombinant form of an M60-like/PF13402-containing protein from the gut mutualist Bacteroides thetaiotaomicron. This study reveals that M60-like domains are peptidases targeting host glycoproteins. These peptidases likely play an important role in successful colonisation of both vertebrate mucosal surfaces and the invertebrate digestive tract by both mutualistic and pathogenic microbes. Moreover, 141 entries across various peptidase families described in the MEROPS

  17. Putative Regulatory Factors Associated with Intramuscular Fat Content

    PubMed Central

    Cesar, Aline S. M.; Regitano, Luciana C. A.; Koltes, James E.; Fritz-Waters, Eric R.; Lanna, Dante P. D.; Gasparin, Gustavo; Mourão, Gerson B.; Oliveira, Priscila S. N.; Reecy, James M.; Coutinho, Luiz L.

    2015-01-01

    Intramuscular fat (IMF) content is related to insulin resistance, which is an important prediction factor for disorders, such as cardiovascular disease, obesity and type 2 diabetes in human. At the same time, it is an economically important trait, which influences the sensorial and nutritional value of meat. The deposition of IMF is influenced by many factors such as sex, age, nutrition, and genetics. In this study Nellore steers (Bos taurus indicus subspecies) were used to better understand the molecular mechanisms involved in IMF content. This was accomplished by identifying differentially expressed genes (DEG), biological pathways and putative regulatory factors. Animals included in this study had extreme genomic estimated breeding value (GEBV) for IMF. RNA-seq analysis, gene set enrichment analysis (GSEA) and co-expression network methods, such as partial correlation coefficient with information theory (PCIT), regulatory impact factor (RIF) and phenotypic impact factor (PIF) were utilized to better understand intramuscular adipogenesis. A total of 16,101 genes were analyzed in both groups (high (H) and low (L) GEBV) and 77 DEG (FDR 10%) were identified between the two groups. Pathway Studio software identified 13 significantly over-represented pathways, functional classes and small molecule signaling pathways within the DEG list. PCIT analyses identified genes with a difference in the number of gene-gene correlations between H and L group and detected putative regulatory factors involved in IMF content. Candidate genes identified by PCIT include: ANKRD26, HOXC5 and PPAPDC2. RIF and PIF analyses identified several candidate genes: GLI2 and IGF2 (RIF1), MPC1 and UBL5 (RIF2) and a host of small RNAs, including miR-1281 (PIF). These findings contribute to a better understanding of the molecular mechanisms that underlie fat content and energy balance in muscle and provide important information for the production of healthier beef for human consumption. PMID:26042666

  18. Collagenolytic activity related to metalloproteases (and serine proteases) in the fish parasite Hysterothylacium aduncum (Nematoda: Anisakidae).

    PubMed

    Malagón, David; Adroher, Francisco Javier; Díaz-López, Manuel; Benítez, Rocío

    2010-06-11

    Proteases play a vital role in both the life cycle of parasites and the parasite-host relationship and are considered important virulence factors. In the present study, the presence of proteases with collagenolytic activity was investigated in the fish nematode Hysterothylacium aduncum during in vitro development. Collagenolytic activity was found in all studied developmental stages of the nematode (third [L3] and fourth [L4] larval stages and adults). In L3, the activity was maximum at pH 6.5 and, in the other stages, at 7.0. Pepsin is known to favour in vitro development of the worm, but, in this study, collagenolytic activity was shown to be significantly greater when no pepsin was added to the culture medium (at pH 6.5, p = 0.011). At pH 7.0, most activity was observed in the immature adult, after the final moult, suggesting that the collagenolytic activity may be involved in remodelling of the cuticle and in sexual maturity. On the other hand, at pH 6.5, activity may be related to tissue migration by L3 within the host. Using specific inhibitors, it was demonstrated that most of the collagenolytic activity detected in all the developmental stages was due to metalloproteases (40 to 100%), although serine proteases were also detected in L4 and adults (10 to 30%).

  19. Outer Membrane Vesicle-Mediated Export of Processed PrtV Protease from Vibrio cholerae

    PubMed Central

    Duperthuy, Marylise; Johnson, Tanya L.; Åhlund, Monika; Lundmark, Richard; Oscarsson, Jan; Sandkvist, Maria; Uhlin, Bernt Eric; Wai, Sun Nyunt

    2015-01-01

    Background Outer membrane vesicles (OMVs) are known to release from almost all Gram-negative bacteria during normal growth. OMVs carry different biologically active toxins and enzymes into the surrounding environment. We suggest that OMVs may therefore be able to transport bacterial proteases into the target host cells. We present here an analysis of the Vibrio cholerae OMV-associated protease PrtV. Methodology/Principal Findings In this study, we demonstrated that PrtV was secreted from the wild type V. cholerae strain C6706 via the type II secretion system in association with OMVs. By immunoblotting and electron microscopic analysis using immunogold labeling, the association of PrtV with OMVs was examined. We demonstrated that OMV-associated PrtV was biologically active by showing altered morphology and detachment of cells when the human ileocecum carcinoma (HCT8) cells were treated with OMVs from the wild type V. cholerae strain C6706 whereas cells treated with OMVs from the prtV isogenic mutant showed no morphological changes. Furthermore, OMV-associated PrtV protease showed a contribution to bacterial resistance towards the antimicrobial peptide LL-37. Conclusion/Significance Our findings suggest that OMVs released from V. cholerae can deliver a processed, biologically active form of PrtV that contributes to bacterial interactions with target host cells. PMID:26222047

  20. msaABCR operon positively regulates biofilm development by repressing proteases and autolysis in Staphylococcus aureus.

    PubMed

    Sahukhal, Gyan S; Batte, Justin L; Elasri, Mohamed O

    2015-02-01

    Staphylococcus aureus is an important human pathogen that causes nosocomial and community-acquired infections. One of the most important aspects of staphylococcal infections is biofilm development within the host, which renders the bacterium resistant to the host's immune response and antimicrobial agents. Biofilm development is very complex and involves several regulators that ensure cell survival on surfaces within the extracellular polymeric matrix. Previously, we identified the msaABCR operon as an additional positive regulator of biofilm formation. In this study, we define the regulatory pathway by which msaABCR controls biofilm formation. We demonstrate that the msaABCR operon is a negative regulator of proteases. The control of protease production mediates the processing of the major autolysin, Atl, and thus regulates the rate of autolysis. In the absence of the msaABCR operon, Atl is processed by proteases at a high rate, leading to increased cell death and a defect in biofilm maturation. We conclude that the msaABCR operon plays a key role in maintaining the balance between autolysis and growth within the staphylococcal biofilm.

  1. Outer Membrane Vesicle-Mediated Export of Processed PrtV Protease from Vibrio cholerae.

    PubMed

    Rompikuntal, Pramod K; Vdovikova, Svitlana; Duperthuy, Marylise; Johnson, Tanya L; Åhlund, Monika; Lundmark, Richard; Oscarsson, Jan; Sandkvist, Maria; Uhlin, Bernt Eric; Wai, Sun Nyunt

    2015-01-01

    Outer membrane vesicles (OMVs) are known to release from almost all Gram-negative bacteria during normal growth. OMVs carry different biologically active toxins and enzymes into the surrounding environment. We suggest that OMVs may therefore be able to transport bacterial proteases into the target host cells. We present here an analysis of the Vibrio cholerae OMV-associated protease PrtV. In this study, we demonstrated that PrtV was secreted from the wild type V. cholerae strain C6706 via the type II secretion system in association with OMVs. By immunoblotting and electron microscopic analysis using immunogold labeling, the association of PrtV with OMVs was examined. We demonstrated that OMV-associated PrtV was biologically active by showing altered morphology and detachment of cells when the human ileocecum carcinoma (HCT8) cells were treated with OMVs from the wild type V. cholerae strain C6706 whereas cells treated with OMVs from the prtV isogenic mutant showed no morphological changes. Furthermore, OMV-associated PrtV protease showed a contribution to bacterial resistance towards the antimicrobial peptide LL-37. Our findings suggest that OMVs released from V. cholerae can deliver a processed, biologically active form of PrtV that contributes to bacterial interactions with target host cells.

  2. TMPRSS12 Is an Activating Protease for Subtype B Avian Metapneumovirus.

    PubMed

    Yun, Bingling; Zhang, Yao; Liu, Yongzhen; Guan, Xiaolu; Wang, Yongqiang; Qi, Xiaole; Cui, Hongyu; Liu, Changjun; Zhang, Yanping; Gao, Honglei; Gao, Li; Li, Kai; Gao, Yulong; Wang, Xiaomei

    2016-12-15

    The entry of avian metapneumovirus (aMPV) into host cells initially requires the fusion of viral and cell membranes, which is exclusively mediated by fusion (F) protein. Proteolysis of aMPV F protein by endogenous proteases of host cells allows F protein to induce membrane fusion; however, these proteases have not been identified. Here, we provide the first evidence that the transmembrane serine protease TMPRSS12 facilitates the cleavage of subtype B aMPV (aMPV/B) F protein. We found that overexpression of TMPRSS12 enhanced aMPV/B F protein cleavage, F protein fusogenicity, and viral replication. Subsequently, knockdown of TMPRSS12 with specific small interfering RNAs (siRNAs) reduced aMPV/B F protein cleavage, F protein fusogenicity, and viral replication. We also found a cleavage motif in the aMPV/B F protein (amino acids 100 and 101) that was recognized by TMPRSS12. The histidine, aspartic acid, and serine residue (HDS) triad of TMPRSS12 was shown to be essential for the proteolysis of aMPV/B F protein via mutation analysis. Notably, we observed TMPRSS12 mRNA expression in target organs of aMPV/B in chickens. Overall, our results indicate that TMPRSS12 is crucial for aMPV/B F protein proteolysis and aMPV/B infectivity and that TMPRSS12 may serve as a target for novel therapeutics and prophylactics for aMPV.

  3. An Accessory Protease Inhibitor to Increase the Yield and Quality of a Tumour-Targeting mAb in Nicotiana benthamiana Leaves

    PubMed Central

    Jutras, Philippe V.; Marusic, Carla; Lonoce, Chiara; Deflers, Carole; Goulet, Marie-Claire; Benvenuto, Eugenio; Donini, Marcello

    2016-01-01

    The overall quality of recombinant IgG antibodies in plants is dramatically compromised by host endogenous proteases. Different approaches have been developed to reduce the impact of endogenous proteolysis on IgGs, notably involving site-directed mutagenesis to eliminate protease-susceptible sites or the in situ mitigation of host protease activities to minimize antibody processing in the cell secretory pathway. We here characterized the degradation profile of H10, a human tumour-targeting monoclonal IgG, in leaves of Nicotiana benthamiana also expressing the human serine protease inhibitor α1-antichymotrypsin or the cysteine protease inhibitor tomato cystatin SlCYS8. Leaf extracts revealed consistent fragmentation patterns for the recombinant antibody regardless of leaf age and a strong protective effect of SlCYS8 in specific regions of the heavy chain domains. As shown using an antigen-binding ELISA and LC-MS/MS analysis of antibody fragments, SlCYS8 had positive effects on both the amount of fully-assembled antibody purified from leaf tissue and the stability of biologically active antibody fragments containing the heavy chain Fc domain. Our data confirm the potential of Cys protease inhibitors as convenient antibody-stabilizing expression partners to increase the quality of therapeutic antibodies in plant protein biofactories. PMID:27893815

  4. An Accessory Protease Inhibitor to Increase the Yield and Quality of a Tumour-Targeting mAb in Nicotiana benthamiana Leaves.

    PubMed

    Jutras, Philippe V; Marusic, Carla; Lonoce, Chiara; Deflers, Carole; Goulet, Marie-Claire; Benvenuto, Eugenio; Michaud, Dominique; Donini, Marcello

    2016-01-01

    The overall quality of recombinant IgG antibodies in plants is dramatically compromised by host endogenous proteases. Different approaches have been developed to reduce the impact of endogenous proteolysis on IgGs, notably involving site-directed mutagenesis to eliminate protease-susceptible sites or the in situ mitigation of host protease activities to minimize antibody processing in the cell secretory pathway. We here characterized the degradation profile of H10, a human tumour-targeting monoclonal IgG, in leaves of Nicotiana benthamiana also expressing the human serine protease inhibitor α1-antichymotrypsin or the cysteine protease inhibitor tomato cystatin SlCYS8. Leaf extracts revealed consistent fragmentation patterns for the recombinant antibody regardless of leaf age and a strong protective effect of SlCYS8 in specific regions of the heavy chain domains. As shown using an antigen-binding ELISA and LC-MS/MS analysis of antibody fragments, SlCYS8 had positive effects on both the amount of fully-assembled antibody purified from leaf tissue and the stability of biologically active antibody fragments containing the heavy chain Fc domain. Our data confirm the potential of Cys protease inhibitors as convenient antibody-stabilizing expression partners to increase the quality of therapeutic antibodies in plant protein biofactories.

  5. Tissue Factor, Protease Activated Receptors and Pathologic Heart Remodeling

    PubMed Central

    Antoniak, Silvio; Sparkenbaugh, Erica; Pawlinski, Rafal

    2015-01-01

    Tissue factor is the primary initiator of coagulation cascade and plays an essential role in hemostasis and thrombosis. In addition, tissue factor and coagulation proteases contribute to the many cellular responses via activation of protease activated receptors. Heart is the organ demonstrating high levels of constitutive tissue factor expression. This review focuses on the role of tissue factor, coagulation proteases and protease activated receptors in heart hemostasis and the pathological heart remodeling associated with myocardial infarction, viral myocarditis and hypertension. PMID:25104210

  6. Porcine Epidemic Diarrhea Virus 3C-Like Protease Regulates Its Interferon Antagonism by Cleaving NEMO

    PubMed Central

    Wang, Dang; Fang, Liurong; Shi, Yanling; Zhang, Huan; Gao, Li; Peng, Guiqing; Chen, Huanchun; Li, Kui

    2015-01-01

    ABSTRACT Porcine epidemic diarrhea virus (PEDV) is an enteropathogenic coronavirus causing lethal watery diarrhea in piglets. Since 2010, a PEDV variant has spread rapidly in China, and it emerged in the United States in 2013, posing significant economic and public health concerns. The ability to circumvent the interferon (IFN) antiviral response, as suggested for PEDV, promotes viral survival and regulates pathogenesis of PEDV infections, but the underlying mechanisms remain obscure. Here, we show that PEDV-encoded 3C-like protease, nsp5, is an IFN antagonist that proteolytically cleaves the nuclear transcription factor kappa B (NF-κB) essential modulator (NEMO), an essential adaptor bridging interferon-regulatory factor and NF-κB activation. NEMO is cleaved at glutamine 231 (Q231) by PEDV, and this cleavage impaired the ability of NEMO to activate downstream IFN production and to act as a signaling adaptor of the RIG-I/MDA5 pathway. Mutations specifically disrupting the cysteine protease activity of PEDV nsp5 abrogated NEMO cleavage and the inhibition of IFN induction. Structural analysis suggests that several key residues outside the catalytic sites of PEDV nsp5 probably impact NEMO cleavage by modulating potential interactions of nsp5 with their substrates. These data show that PEDV nsp5 disrupts type I IFN signaling by cleaving NEMO. Previously, we and others demonstrated that NEMO is also cleaved by 3C or 3C-like proteinases of picornavirus and artertivirus. Thus, NEMO probably represents a prime target for 3C or 3C-like proteinases of different viruses. IMPORTANCE The continued emergence and reemergence of porcine epidemic diarrhea virus (PEDV) underscore the importance of studying how this virus manipulates the immune responses of its hosts. During coevolution with its hosts, PEDV has acquired mechanisms to subvert host innate immune responses for its survival advantage. At least two proteins encoded by PEDV have been identified as interferon (IFN

  7. Actin enables the antimicrobial action of LL-37 peptide in the presence of microbial proteases.

    PubMed

    Sol, Asaf; Skvirsky, Yaniv; Nashef, Rizan; Zelentsova, Katya; Burstyn-Cohen, Tal; Blotnick, Edna; Muhlrad, Andras; Bachrach, Gilad

    2014-08-15

    Host defense peptides play an important host-protective role by their microcidal action, immunomodulatory functions, and tissue repair activities. Proteolysis is a common strategy of pathogens used to neutralize host defense peptides. Here, we show that actin, the most abundant structural protein in eukaryotes, binds the LL-37 host defense peptide, protects it from degradation by the proteases of Pseudomonas aeruginosa and Porphyromonas gingivalis, and enables its antimicrobial activity despite the presence of the proteases. Co-localization of LL-37 with extracellular actin was observed in necrotized regions of samples from oral lesions. Competition assays, cross-linking experiments, limited proteolysis, and mass spectrometry revealed that LL-37 binds by specific hydrophobic interactions to the His-40-Lys-50 segment of actin, located in the DNase I binding loop. The integrity of the binding site of both LL-37 and actin is a prerequisite to the binding. Our results demonstrate that actin, presumably released by dead cells and abundant in infected sites, might be utilized by the immune system to enhance spatio-temporal immunity in an attempt to arrest infection and control inflammation.

  8. Proteases of haematophagous arthropod vectors are involved in blood-feeding, yolk formation and immunity - a review.

    PubMed

    Santiago, Paula Beatriz; de Araújo, Carla Nunes; Motta, Flávia Nader; Praça, Yanna Reis; Charneau, Sébastien; Bastos, Izabela M Dourado; Santana, Jaime M

    2017-02-13

    Ticks, triatomines, mosquitoes and sand flies comprise a large number of haematophagous arthropods considered vectors of human infectious diseases. While consuming blood to obtain the nutrients necessary to carry on life functions, these insects can transmit pathogenic microorganisms to the vertebrate host. Among the molecules related to the blood-feeding habit, proteases play an essential role. In this review, we provide a panorama of proteases from arthropod vectors involved in haematophagy, in digestion, in egg development and in immunity. As these molecules act in central biological processes, proteases from haematophagous vectors of infectious diseases may influence vector competence to transmit pathogens to their prey, and thus could be valuable targets for vectorial control.

  9. Mycobacterium tuberculosis Rv0198c, a putative matrix metalloprotease is involved in pathogenicity.

    PubMed

    Muttucumaru, D G Niranjala; Smith, Debbie A; McMinn, Elizabeth J; Reese, Valerie; Coler, Rhea N; Parish, Tanya

    2011-03-01

    We are interested in the role of proteases in the biology of the global human pathogen, Mycobacterium tuberculosis. We have focused on a putative matrix metalloprotease, Rv0198c. In order to investigate its role we constructed an unmarked chromosomal deletion of the gene and analysed the phenotype of the resulting mutant. No differences in growth in axenic culture were seen and there was no measurable change in overall protease activity in cell-free extracts. Transcriptome analysis revealed a small number of changes in gene expression in aerobic growth, with Rv2488c and Rv1971 being over 40-fold up-regulated and qor (Rv1454c) being 20-fold down-regulated; in addition, changes were seen in members of the heat shock regulon. Virulence assays demonstrated that the mutant was able to replicate in human macrophage-like cells (THP-1 cell line) to a comparable degree with the wild-type. However, the mutant was hyper-virulent in the SCID and C57BL/6 mouse models. Our data suggest that Rv0198c plays a role during infection. Copyright © 2010 Elsevier Ltd. All rights reserved.

  10. Discovery and characterization of a novel plant pathogen protease

    USDA-ARS?s Scientific Manuscript database

    Chitinase modifying proteins are fungal proteases that attack specific plant defense chitinases. At least three unrelated types of proteases have evolved to have this function. They all truncate the targeted chitinases by cleaving near their amino termini, but each protease type targets a different ...

  11. Kiwifruit cysteine protease actinidin compromises the intestinal barrier by disrupting tight junctions.

    PubMed

    Grozdanovic, Milica M; Čavić, Milena; Nešić, Andrijana; Andjelković, Uroš; Akbari, Peyman; Smit, Joost J; Gavrović-Jankulović, Marija

    2016-03-01

    The intestinal epithelium forms a barrier that food allergens must cross in order to induce sensitization. The aim of this study was to evaluate the impact of the plant-derived food cysteine protease--actinidin (Act d1) on the integrity of intestinal epithelium tight junctions (TJs). Effects of Act d1 on the intestinal epithelium were evaluated in Caco-2 monolayers and in a mouse model by measuring transepithelial resistance and in vivo permeability. Integrity of the tight junctions was analyzed by confocal microscopy. Proteolysis of TJ protein occludin was evaluated by mass spectrometry. Actinidin (1 mg/mL) reduced the transepithelial resistance of the cell monolayer by 18.1% (after 1 h) and 25.6% (after 4 h). This loss of barrier function was associated with Act d 1 disruption of the occludin and zonula occludens (ZO)-1 network. The effect on intestinal permeability in vivo was demonstrated by the significantly higher concentration of 40 kDa FITC-dextran (2.33 μg/mL) that passed from the intestine into the serum of Act d1 treated mice in comparison to the control group (0.5 μg/mL). Human occludin was fragmented, and putative Act d1 cleavage sites were identified in extracellular loops of human occludin. Act d1 caused protease-dependent disruption of tight junctions in confluent Caco-2 cells and increased intestinal permeability in mice. In line with the observed effects of food cysteine proteases in occupational allergy, these results suggest that disruption of tight junctions by food cysteine proteases may contribute to the process of sensitization in food allergy. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Molecular characterization of a gene encoding extracellular serine protease isolated from a subtilisin inhibitor-deficient mutant of Streptomyces albogriseolus S-3253.

    PubMed Central

    Taguchi, S; Odaka, A; Watanabe, Y; Momose, H

    1995-01-01

    An extracellular serine protease produced by a mutant, M1, derived from Streptomyces albogriseolus S-3253 that no longer produces a protease inhibitor (Streptomyces subtilisin inhibitor [SSI]) was isolated. A 20-kDa protein was purified by its affinity for SSI and designated SAM-P20. The amino acid sequence of the amino-terminal region of SAM-P20 revealed high homology with the sequences of Streptomyces griseus proteases A and B, and the gene sequence confirmed the relationships. The sequence also revealed a putative amino acid signal sequence for SAM-P20 that apparently functioned to allow secretion of SAM-P20 from Escherichia coli carrying the recombinant gene. SAM-P20 produced by E. coli cells was shown to be sensitive to SSI inhibition. PMID:7887600

  13. A virus essential for insect host-parasite interactions encodes cystatins.

    PubMed

    Espagne, E; Douris, V; Lalmanach, G; Provost, B; Cattolico, L; Lesobre, J; Kurata, S; Iatrou, K; Drezen, J-M; Huguet, E

    2005-08-01

    Cotesia congregata is a parasitoid wasp that injects its eggs in the host caterpillar Manduca sexta. In this host-parasite interaction, successful parasitism is ensured by a third partner: a bracovirus. The relationship between parasitic wasps and bracoviruses constitutes one of the few known mutualisms between viruses and eukaryotes. The C. congregata bracovirus (CcBV) is injected at the same time as the wasp eggs in the host hemolymph. Expression of viral genes alters the caterpillar's immune defense responses and developmental program, resulting in the creation of a favorable environment for the survival and emergence of adult parasitoid wasps. Here, we describe the characterization of a CcBV multigene family which is highly expressed during parasitism and which encodes three proteins with homology to members of the cystatin superfamily. Cystatins are tightly binding, reversible inhibitors of cysteine proteases. Other cysteine protease inhibitors have been described for lepidopteran viruses; however, this is the first description of the presence of cystatins in a viral genome. The expression and purification of a recombinant form of one of the CcBV cystatins, cystatin 1, revealed that this viral cystatin is functional having potent inhibitory activity towards the cysteine proteases papain, human cathepsins L and B and Sarcophaga cathepsin B in assays in vitro. CcBV cystatins are, therefore, likely to play a role in host caterpillar physiological deregulation by inhibiting host target proteases in the course of the host-parasite interaction.

  14. Cloning, expression, and characterization of a milk-clotting aspartic protease gene (Po-Asp) from Pleurotus ostreatus.

    PubMed

    Yin, Chaomin; Zheng, Liesheng; Chen, Liguo; Tan, Qi; Shang, Xiaodong; Ma, Aimin

    2014-02-01

    An aspartic protease gene from Pleurotus ostreatus (Po-Asp) had been cloned based on the 3' portion of cDNA in our previous work. The Po-Asp cDNA contained 1,324 nucleotides with an open reading frame (ORF) of 1,212 bp encoding 403 amino acid residues. The putative amino acid sequence included a signal peptide, an activation peptide, two most possible N-glycosylation sites and two conserved catalytic active site. The mature polypeptide with 327 amino acid residues had a calculated molecular mass of 35.3 kDa and a theoretical isoelectric point of 4.57. Basic Local Alignment Search Tool analysis showed 68-80 % amino acid sequence identical to other basidiomycetous aspartic proteases. Sequence comparison and evolutionary analysis revealed that Po-Asp is a member of fungal aspartic protease family. The DNA sequence of Po-Asp is 1,525 bp in length without untranslated region, consisting of seven exons and six introns. The Po-Asp cDNA without signal sequence was expressed in Pichia pastoris and sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the molecular mass of recombinant Po-Asp was about 43 kDa. The crude recombinant aspartic protease had milk-clotting activity.

  15. Genetic characterization and expression of the novel fungal protease, EPg222 active in dry-cured meat products.

    PubMed

    Benito, María J; Connerton, Ian F; Córdoba, Juan J

    2006-11-01

    EPg222 protease is a novel extracellular enzyme produced by Penicillium chrysogenum (Pg222) isolated from dry-cured hams that has the potential for use over a broad range of applications in industries that produce dry-cured meat products. The gene encoding EPg222 protease has been identified. Peptide sequences of EPg222 were obtained by de novo sequencing of tryptic peptides using mass spectrometry. The corresponding gene was amplified by PCR using degenerated primers based on a combination of conserved serine protease-encoding sequences and reverse translation of the peptide sequences. EPg222 is encoded as a gene of 1,361 bp interrupted by two introns. The deduced amino acid sequence indicated that the enzyme is synthesized as a preproenzyme with a putative signal sequence of 19 amino acids (aa), a prosequence of 96 aa and a mature protein of 283 aa. A cDNA encoding EPg222 has been cloned and expressed as a functionally active enzyme in Pichia pastoris. The recombinant enzyme exhibits similar activities to the native enzyme against a wide range of protein substrates including muscle myofibrillar protein. The mature sequence contains conserved aa residues characteristic of those forming the catalytic triad of serine proteases (Asp42, His76 and Ser228) but notably the food enzyme exhibits specific aa substitutions in the immunoglobulin-E recognition regions that have been identified in protein homologues that are allergenic.

  16. Cathepsin L-like protease can regulate the process of metamorphosis and fat body dissociation in Antheraea pernyi.

    PubMed

    Sun, Yu-Xuan; Tang, Lin; Wang, Pei; Abbas, Muhammad Nadeem; Tian, Ji-Wu; Zhu, Bao-Jian; Liu, Chao-Liang

    2017-09-27

    Cathepsins are a group of protease, located in lysosome and play a vital role in physiological process. Here, we reported cathepsin L-like protease (Ap-cathL), which contained an open reading frame of 1155 bp and encoding 385 amino acid residues protein. The I29 inhibitor domain and peptidase C1A (clan CA of cysteine proteases, papain family C1 subfamily) putative conserved domains were detected in Ap-cathL. Quantitative real-time PCR (qRT-PCR) analysis revealed that Ap-cathL highly expressed in the fat body and midgut. The high expression during the molting stage, pupal stage and following 20E (20-hydroxyecdysone) treatment indicated that it maybe involved in the process of molting and metamorphosis. In addition, depletion of Ap-cathL influenced the expression of apoptosis pathway related genes. The protease inhibitor and RNA interference experiments showed that Ap-cathL was involved in the fat body dissociation of A. pernyi. These results suggest that Ap-cathL may involve in the process of metamorphosis and fat body dissociation of A. pernyi. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Mutations in SERPINB7, encoding a member of the serine protease inhibitor superfamily, cause Nagashima-type palmoplantar keratosis.

    PubMed

    Kubo, Akiharu; Shiohama, Aiko; Sasaki, Takashi; Nakabayashi, Kazuhiko; Kawasaki, Hiroshi; Atsugi, Toru; Sato, Showbu; Shimizu, Atsushi; Mikami, Shuji; Tanizaki, Hideaki; Uchiyama, Masaki; Maeda, Tatsuo; Ito, Taisuke; Sakabe, Jun-ichi; Heike, Toshio; Okuyama, Torayuki; Kosaki, Rika; Kosaki, Kenjiro; Kudoh, Jun; Hata, Kenichiro; Umezawa, Akihiro; Tokura, Yoshiki; Ishiko, Akira; Niizeki, Hironori; Kabashima, Kenji; Mitsuhashi, Yoshihiko; Amagai, Masayuki

    2013-11-07

    "Nagashima-type" palmoplantar keratosis (NPPK) is an autosomal recessive nonsyndromic diffuse palmoplantar keratosis characterized by well-demarcated diffuse hyperkeratosis with redness, expanding on to the dorsal surfaces of the palms and feet and the Achilles tendon area. Hyperkeratosis in NPPK is mild and nonprogressive, differentiating NPPK clinically from Mal de Meleda. We performed whole-exome and/or Sanger sequencing analyses of 13 unrelated NPPK individuals and identified biallelic putative loss-of-function mutations in SERPINB7, which encodes a cytoplasmic member of the serine protease inhibitor superfamily. We identified a major causative mutation of c.796C>T (p.Arg266(∗)) as a founder mutation in Japanese and Chinese populations. SERPINB7 was specifically present in the cytoplasm of the stratum granulosum and the stratum corneum (SC) of the epidermis. All of the identified mutants are predicted to cause premature termination upstream of the reactive site, which inhibits the proteases, suggesting a complete loss of the protease inhibitory activity of SERPINB7 in NPPK skin. On exposure of NPPK lesional skin to water, we observed a whitish spongy change in the SC, suggesting enhanced water permeation into the SC due to overactivation of proteases and a resultant loss of integrity of the SC structure. These findings provide an important framework for developing pathogenesis-based therapies for NPPK.

  18. Molecular markers of serine protease evolution

    PubMed Central

    Krem, Maxwell M.; Di Cera, Enrico

    2001-01-01

    The evolutionary history of serine proteases can be accounted for by highly conserved amino acids that form crucial structural and chemical elements of the catalytic apparatus. These residues display non- random dichotomies in either amino acid choice or serine codon usage and serve as discrete markers for tracking changes in the active site environment and supporting structures. These markers categorize serine proteases of the chymotrypsin-like, subtilisin-like and α/β-hydrolase fold clans according to phylogenetic lineages, and indicate the relative ages and order of appearance of those lineages. A common theme among these three unrelated clans of serine proteases is the development or maintenance of a catalytic tetrad, the fourth member of which is a Ser or Cys whose side chain helps stabilize other residues of the standard catalytic triad. A genetic mechanism for mutation of conserved markers, domain duplication followed by gene splitting, is suggested by analysis of evolutionary markers from newly sequenced genes with multiple protease domains. PMID:11406580

  19. 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

  20. Investigational protease inhibitors as antiretroviral therapies

    PubMed Central

    Midde, Narasimha M.; Patters, Benjamin J.; Rao, PSS; Cory, Theodore J.; Kumar, Santosh

    2017-01-01

    Introduction Highly Active Antiretroviral Therapy (HAART) has tremendously improved the life expectancy of the HIV-infected population over the past three decades. Protease inhibitors have been one of the major classes of drugs in HAART regimens that are effective in treating HIV. However, the emergence of resistance and cross-resistance against protease inhibitors encourages researchers to develop new PIs with broad-spectrum activity, as well as novel means of enhancing the efficacy of existing PIs. Areas covered In this article we discuss recent advances in HIV protease inhibitor (PI) development, focusing on both investigational and experimental agents. We also include a section on pharmacokinetic booster drugs for improved bioavailability of protease inhibitors. Further, we discuss novel drug delivery systems using a variety of nanocarriers for the delivery of PIs across the blood-brain barrier to treat the HIV in the brain. Expert opinion We discuss our opinion on the promises and challenges on the development of novel investigational and experimental PIs that are less toxic and more effective in combating drug-resistance. Further, we discuss the future of novel nanocarriers that have been developed to deliver PIs to the brain cells. Although these are promising findings, many challenges need to be overcome prior to making them a viable option. PMID:27415449

  1. HIV-protease inhibitors block the replication of both vesicular stomatitis and influenza viruses at an early post-entry replication step

    SciTech Connect

    Federico, Maurizio

    2011-08-15

    The inhibitors of HIV-1 protease (PIs) have been designed to block the activity of the viral aspartyl-protease. However, it is now accepted that this family of inhibitors can also affect the activity of cell proteases. Since the replication of many virus species requires the activity of host cell proteases, investigating the effects of PIs on the life cycle of viruses other than HIV would be of interest. Here, the potent inhibition induced by saquinavir and nelfinavir on the replication of both vesicular stomatitis and influenza viruses is described. These are unrelated enveloped RNA viruses infecting target cells upon endocytosis and intracellular fusion. The PI-induced inhibition was apparently a consequence of a block at the level of the fusion between viral envelope and endosomal membranes. These findings would open the way towards the therapeutic use of PIs against enveloped RNA viruses other than HIV.

  2. trans-Protease Activity and Structural Insights into the Active Form of the Alphavirus Capsid Protease

    PubMed Central

    Aggarwal, Megha; Dhindwal, Sonali; Kumar, Pravindra; Kuhn, Richard J.

    2014-01-01

    ABSTRACT The alphavirus capsid protein (CP) is a serine protease that possesses cis-proteolytic activity essential for its release from the nascent structural polyprotein. The released CP further participates in viral genome encapsidation and nucleocapsid core formation, followed by its attachment to glycoproteins and virus budding. Thus, protease activity of the alphavirus capsid is a potential antialphaviral target to arrest capsid release, maturation, and structural polyprotein processing. However, the discovery of capsid protease inhibitors has been hampered due to the lack of a suitable screening assay and of the crystal structure in its active form. Here, we report the development of a trans-proteolytic activity assay for Aura virus capsid protease (AVCP) based on fluorescence resonance energy transfer (FRET) for screening protease inhibitors. Kinetic parameters using fluorogenic peptide substrates were estimated, and the Km value was found to be 2.63 ± 0.62 μM while the kcat/Km value was 4.97 × 104 M−1 min−1. Also, the crystal structure of the trans-active form of AVCP has been determined to 1.81-Å resolution. Structural comparisons of the active form with the crystal structures of available substrate-bound mutant and inactive blocked forms of the capsid protease identify conformational changes in the active site, the oxyanion hole, and the substrate specificity pocket residues, which could be critical for rational drug design. IMPORTANCE The alphavirus capsid protease is an attractive antiviral therapeutic target. In this study, we have described the formerly unappreciated trans-proteolytic activity of the enzyme and for the first time have developed a FRET-based protease assay for screening capsid protease inhibitors. Our structural studies unveil the structural features of the trans-active protease, which has been previously proposed to exist in the natively unfolded form (M. Morillas, H. Eberl, F. H. Allain, R. Glockshuber, and E. Kuennemann, J

  3. Scrapie protein degradation by cysteine proteases in CD11c+ dendritic cells and GT1-1 neuronal cells.

    PubMed

    Luhr, Katarina M; Nordström, Elin K; Löw, Peter; Ljunggren, Hans-Gustaf; Taraboulos, Albert; Kristensson, Krister

    2004-05-01

    Dendritic cells (DC) of the CD11c(+) myeloid phenotype have been implicated in the spread of scrapie in the host. Previously, we have shown that CD11c(+) DC can cause a rapid degradation of proteinase K-resistant prion proteins (PrP(Sc)) in vitro, indicating a possible role of these cells in the clearance of PrP(Sc). To determine the mechanisms of PrP(Sc) degradation, CD11c(+) DC that had been exposed to PrP(Sc) derived from a neuronal cell line (GT1-1) infected with scrapie (ScGT1-1) were treated with a battery of protease inhibitors. Following treatment with the cysteine protease inhibitors (2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane (E-64c), its ethyl ester (E-64d), and leupeptin, the degradation of PrP(Sc) was inhibited, while inhibitors of serine and aspartic and metalloproteases (aprotinin, pepstatin, and phosphoramidon) had no effect. An endogenous degradation of PrP(Sc) in ScGT1-1 cells was revealed by inhibiting the expression of cellular PrP (PrP(C)) by RNA interference, and this degradation could also be inhibited by the cysteine protease inhibitors. Our data show that PrP(Sc) is proteolytically cleaved preferentially by cysteine proteases in both CD11c(+) DC and ScGT1-1 cells and that the degradation of PrP(Sc) by proteases is different from that of PrP(C). Interference by protease inhibitors with DC-induced processing of PrP(Sc) has the potential to modify prion spread, clearance, and immunization in a host.

  4. Expression, purification and identification of a thermolysin-like protease, neutral protease I, from Aspergillus oryzae with the Pichia pastoris expression system.

    PubMed

    Ma, Xiaojian; Liu, Yunyun; Li, Qingqing; Liu, Lu; Yi, Li; Ma, Lixin; Zhai, Chao

    2016-12-01

    Neutral proteases are widely used in the textile, food and medical industries. This study was designed to obtain high expression levels of neutral protease I from Aspergillus oryzae 3.042 by using Pichia pastoris GS115 as the host strain for industrial purposes. The coding sequence of the target gene was modified, synthesized, and then cloned into the expression vector pHBM905BDM, which harbored the d1+2 × 201 AOX1 promoter and the MF4I leader sequence. The recombinant plasmid was transformed into Pichia pastoris GS115. The recombinant strain was used for high-density fermentation in a 4-L fermenter. The yield of the target protein reached 12.87 mg/mL, and the enzyme activity was approximately 49370 U/mL, which indicated that this enzyme was expressed in Pichia pastoris at a high level. The target protein was purified and characterized. Its optimum temperature and pH were 55 °C and 8.0, respectively. This enzyme was extremely sensitive to EDTA, which is consistent with the previous reports that it is a zinc-dependent metalloprotease. Our results indicated that low concentrations of zinc, calcium and magnesium ions stimulated the enzyme activity, whereas high concentrations inhibited its activity. In addition, calcium and magnesium ions increased the thermostability of the enzyme. All of the evidence indicated that this protease is a thermolysin-like peptidase. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. [Peptide hydrolases with catalytic dyad Ser-Lys. Similarity and distinctions of the active centers of ATP-dependent Lon proteases, LexA repressors, signal peptidases and C-terminal processing proteases].

    PubMed

    Rotanova, T V

    2002-01-01

    It is established that ATP-dependent protease Lon family belongs to the serine-lysine peptide hydrolases clan. Significant similarity of amino acid sequences of proteases Lon and repressors LexA in the regions including the catalytic serine and lysine residues is revealed by comparing primary structures of different families of the enzymes with Ser-Lys catalytic dyad. The both Lon and LexA families are shown to be divided into two subfamilies in accordance with the nature of amino acids in the catalytically active serine environment. Putative DNA binding sites are revealed in proteolytic domains of Lon A subfamily. Similarities and distinctions of the all families peptide hydrolases of the clan in the regions of their active centers are discussed.

  6. Species formation by host shifting in avian malaria parasites.

    PubMed

    Ricklefs, Robert E; Outlaw, Diana C; Svensson-Coelho, Maria; Medeiros, Matthew C I; Ellis, Vincenzo A; Latta, Steven

    2014-10-14

    The malaria parasites (Apicomplexa: Haemosporida) of birds are believed to have diversified across the avian host phylogeny well after the origin of most major host lineages. Although many symbionts with direct transmission codiversify with their hosts, mechanisms of species formation in vector-borne parasites, including the role of host shifting, are poorly understood. Here, we examine the hosts of sister lineages in a phylogeny of 181 putative species of malaria parasites of New World terrestrial birds to determine the role of shifts between host taxa in the formation of new parasite species. We find that host shifting, often across host genera and families, is the rule. Sympatric speciation by host shifting would require local reproductive isolation as a prerequisite to divergent selection, but this mechanism is not supported by the generalized host-biting behavior of most vectors of avian malaria parasites. Instead, the geographic distribution of individual parasite lineages in diverse hosts suggests that species formation is predominantly allopatric and involves host expansion followed by local host-pathogen coevolution and secondary sympatry, resulting in local shifting of parasite lineages across hosts.

  7. Calcium-induced Folding and Stabilization of the Pseudomonas aeruginosa Alkaline Protease*

    PubMed Central

    Zhang, Liang; Conway, James F.; Thibodeau, Patrick H.

    2012-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen that contributes to the mortality of immunocompromised individuals and patients with cystic fibrosis. Pseudomonas infection presents clinical challenges due to its ability to form biofilms and modulate host-pathogen interactions through the secretion of virulence factors. The calcium-regulated alkaline protease (AP), a member of the repeats in toxin (RTX) family of proteins, is implicated in multiple modes of infection. A series of full-length and truncation mutants were purified for structural and functional studies to evaluate the role of Ca2+ in AP folding and activation. We find that Ca2+ binding induces RTX folding, which serves to chaperone the folding of the protease domain. Subsequent association of the RTX domain with an N-terminal α-helix stabilizes AP. These results provide a basis for the Ca2+-mediated regulation of AP and suggest mechanisms by which Ca2+ regulates the RTX family of virulence factors. PMID:22170064

  8. Virtual Screening of Indonesian Herbal Database as HIV-1 Protease Inhibitor

    PubMed Central

    Yanuar, Arry; Suhartanto, Heru; Mun׳im, Abdul; Anugraha, Bram Hik; Syahdi, Rezi Riadhi

    2014-01-01

    HIV-1 (Human immunodeficiency virus type 1)׳s infection is considered as one of most harmful disease known by human, the survivability rate of the host reduced significantly when it developed into AIDS. HIV drug resistance is one of the main problems of its treatment and several drug designs have been done to find new leads compound as the cure. In this study, in silico virtual screening approach was used to find lead molecules from the library or database of natural compounds as HIV-1 protease inhibitor. Virtual screening against Indonesian Herbal Database with AutoDock was performed on HIV-1 protease. From the virtual screening, top ten compounds obtained were 8-Hydroxyapigenin 8-(2",4"-disulfatoglucuronide), Isoscutellarein 4'-methyl ether, Amaranthin, Torvanol A, Ursonic acid, 5-Carboxypyranocyanidin 3-O-(6"-O-malonyl-beta-glucopyranoside), Oleoside, Jacoumaric acid, Platanic acid and 5-Carboxypyranocyanidin 3-O-beta-glucopyranoside. PMID:24616554

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

    PubMed Central

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

    2015-01-01

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

  10. 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.

  11. Natural cysteine protease inhibitors in protozoa: Fifteen years of the chagasin family.

    PubMed

    Costa, Tatiana F R; Lima, Ana Paula C A

    2016-03-01

    Chagasin-type inhibitors comprise natural inhibitors of papain-like cysteine proteases that are distributed among Protist, Bacteria and Archaea. Chagasin was identified in the pathogenic protozoa Trypanosoma cruzi as an approximately 11 kDa protein that is a tight-binding and highly thermostable inhibitor of papain, cysteine cathepsins and endogenous parasite cysteine proteases. It displays an Imunoglobulin-like fold with three exposed loops to one side of the molecule, where amino acid residues present in conserved motifs at the tips of each loop contact target proteases. Differently from cystatins, the loop 2 of chagasin enters the active-site cleft, making direct contact with the catalytic residues, while loops 4 and 6 embrace the enzyme from the sides. Orthologues of chagasin are named Inhibitors of Cysteine Peptidases (ICP), and share conserved overall tri-dimensional structure and mode of binding to proteases. ICPs are tentatively distributed in three families: in family I42 are grouped chagasin-type inhibitors that share conserved residues at the exposed loops; family I71 contains Plasmodium ICPs, which are large proteins having a chagasin-like domain at the C-terminus, with lower similarity to chagasin in the conserved motif at loop 2; family I81 contains Toxoplasma ICP. Recombinant ICPs tested so far can inactivate protozoa cathepsin-like proteases and their mammalian counterparts. Studies on their biological roles were carried out in a few species, mainly using transgenic protozoa, and the conclusions vary. However, in all cases, alterations in the levels of expression of chagasin/ICPs led to substantial changes in one or more steps of parasite biology, with higher incidence in influencing their interaction with the hosts. We will cover most of the findings on chagasin/ICP structural and functional properties and overview the current knowledge on their roles in protozoa.

  12. Distinct and stage specific nuclear factors regulate the expression of falcipains, Plasmodium falciparum cysteine proteases

    PubMed Central

    Sunil, Sujatha; Chauhan, Virander S; Malhotra, Pawan

    2008-01-01

    Background Plasmodium falciparum cysteine proteases (falcipains) play indispensable roles in parasite infection and development, especially in the process of host erythrocyte rupture/invasion and hemoglobin degradation. No detailed molecular analysis of transcriptional regulation of parasite proteases especially cysteine proteases has yet been reported. In this study, using a combination of transient transfection assays and electrophoretic mobility shift assays (EMSA), we demonstrate the presence of stage specific nuclear factors that bind to unique sequence elements in the 5'upstream regions of the falcipains and probably modulate the expression of cysteine proteases. Results Falcipains differ in their timing of expression and exhibit ability to compensate each other's functions at asexual blood stages of the parasite. Present study was undertaken to study the transcriptional regulation of falcipains. Transient transfection assay employing firefly luciferase as a reporter revealed that a ~1 kb sequence upstream of translational start site is sufficient for the functional transcriptional activity of falcipain-1 gene, while falcipain-2, -2' and -3 genes that exist within 12 kb stretch on chromosome 11 require ~2 kb upstream sequences for the expression of reporter luciferase activity. EMSA analysis elucidated binding of distinct nuclear factors to specific sequences within the 5'upstream regions of falcipain genes. Analysis of falcipains' 5'upstream regulatory regions did not reveal the presence of sequences known to bind general eukaryotic factors. However, we did find parasite specific sequence elements such as poly(dA) poly(dT) tracts, CCAAT boxes and a single 7 bp-G rich sequence, (A/G)NGGGG(C/A) in the 5' upstream regulatory regions of these genes, thereby suggesting the role(s) of Plasmodium specific transcriptional factors in the regulation of falcipain genes. Conclusion Taken together, these results suggest that expression of Plasmodium cysteine proteases is

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

    PubMed

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

    2013-11-15

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

  14. Mosaic-like organization of IgA protease genes in Neisseria gonorrhoeae generated by horizontal genetic exchange in vivo.

    PubMed Central

    Halter, R; Pohlner, J; Meyer, T F

    1989-01-01

    IgA protease is a putative virulence factor that exists in several allelic forms in Neisseria gonorrhoeae. However, extracellular secretion of these variant IgA proteases occurs by the same pathway involving three steps of autoproteolytic maturation from a large precursor. Two principal precursor types (H1 and H2) can be distinguished with respect to the location of autoproteolytic sites and the sizes of the mature products. By partial DNA sequence analysis, additional variations have been detected which are not unique to one particular gene; rather, otherwise unrelated iga genes often share homology, thus revealing a composite organization. In the context of other gonococcal features, this observation implies that recombination has occurred in vivo between iga genes of different strains, probably via the route of species-specific DNA transformation. This process may be of general significance for the modulation and the natural exchange of virulence properties among pathogenic Neisseriae. Images PMID:2511009

  15. Dipeptidyl peptidase I activates neutrophil-derived serine proteases and regulates the development of acute experimental arthritis

    PubMed Central

    Adkison, April M.; Raptis, Sofia Z.; Kelley, Diane G.; Pham, Christine T.N.

    2002-01-01

    Leukocyte recruitment in inflammation is critical for host defense, but excessive accumulation of inflammatory cells can lead to tissue damage. Neutrophil-derived serine proteases (cathepsin G [CG], neutrophil elastase [NE], and proteinase 3 [PR3]) are expressed specifically in mature neutrophils and are thought to play an important role in inflammation. To investigate the role of these proteases in inflammation, we generated a mouse deficient in dipeptidyl peptidase I (DPPI) and established that DPPI is required for the full activation of CG, NE, and PR3. Although DPPI–/– mice have normal in vitro neutrophil chemotaxis and in vivo neutrophil accumulation during sterile peritonitis, they are protected against acute arthritis induced by passive transfer of monoclonal antibodies against type II collagen. Specifically, there is no accumulation of neutrophils in the joints of DPPI–/– mice. This protective effect correlates with the inactivation of neutrophil-derived serine proteases, since NE–/– × CG–/– mice are equally resistant to arthritis induction by anti-collagen antibodies. In addition, protease-deficient mice have decreased response to zymosan- and immune complex–mediated inflammation in the subcutaneous air pouch. This defect is accompanied by a decrease in local production of TNF-α and IL-1β. These results implicate DPPI and polymorphonuclear neutrophil–derived serine proteases in the regulation of cytokine production at sites of inflammation. PMID:11827996

  16. Effects of NS2B-NS3 protease and furin inhibition on West Nile and Dengue virus replication.

    PubMed

    Kouretova, Jenny; Hammamy, M Zouhir; Epp, Anton; Hardes, Kornelia; Kallis, Stephanie; Zhang, Linlin; Hilgenfeld, Rolf; Bartenschlager, Ralf; Steinmetzer, Torsten

    2017-12-01

    West Nile virus (WNV) and Dengue virus (DENV) replication depends on the viral NS2B-NS3 protease and the host enzyme furin, which emerged as potential drug targets. Modification of our previously described WNV protease inhibitors by basic phenylalanine analogs provided compounds with reduced potency against the WNV and DENV protease. In a second series, their decarboxylated P1-trans-(4-guanidino)cyclohexylamide was replaced by an arginyl-amide moiety. Compound 4-(guanidinomethyl)-phenylacetyl-Lys-Lys-Arg-NH2 inhibits the NS2B-NS3 protease of WNV with an inhibition constant of 0.11 µM. Due to the similarity in substrate specificity, we have also tested the potency of our previously described multibasic furin inhibitors. Their further modification provided chimeric inhibitors with additional potency against the WNV and DENV proteases. A strong inhibition of WNV and DENV replication in cell culture was observed for the specific furin inhibitors, which reduced virus titers up to 10,000-fold. These studies reveal that potent inhibitors of furin can block the replication of DENV and WNV.

  17. 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

  18. Disruption of genes involved in CORVET complex leads to enhanced secretion of heterologous carboxylesterase only in protease deficient Pichia pastoris.

    PubMed

    Marsalek, Lukas; Gruber, Clemens; Altmann, Friedrich; Aleschko, Markus; Mattanovich, Diethard; Gasser, Brigitte; Puxbaum, Verena

    2017-02-23

    The methylotrophic yeast Pichia pastoris (Komagataella spp.) is a popular microbial host for the production of recombinant proteins. Previous studies have shown that mis-sorting to the vacuole can be a bottleneck during production of recombinant secretory proteins in yeast, however, no information was available for P. pastoris. In this work the authors have therefore generated vps (vacuolar protein sorting) mutant strains disrupted in genes involved in the CORVET (class C core vacuole/endosome tethering) complex at the early stages of endosomal sorting. Both Δvps8 and Δvps21 strains contained lower extracellular amounts of heterologous carboxylesterase (CES) compared to the control strain, which could be attributed to a high proteolytic activity present in the supernatants of CORVET engineered strains due to rerouting of vacuolar proteases. Serine proteases were identified to be responsible for this proteolytic degradation by liquid chromatography-mass spectrometry and protease inhibitor assays. Deletion of the major cellular serine protease Prb1 in Δvps8 and Δvps21 strains did not only rescue the extracellular CES levels, but even outperformed the parental CES strain (56 and 80% higher yields, respectively). Further deletion of Ybr139W, another serine protease, did not show a further increase in secretion levels. Higher extracellular CES activity and low proteolytic activity were detected also in fed batch cultivation of Δvps21Δprb1 strains, thus confirming that modifying early steps in the vacuolar pathway has a positive impact on heterologous protein secretion.

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

    PubMed

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

    2017-02-28

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

  20. Inherent dynamics within the Crimean-Congo Hemorrhagic fever virus protease are localized to the same region as substrate interactions

    SciTech Connect

    Eisenmesser, Elan Z.; Capodagli, Glenn; Armstrong, Geoffrey S.; Holliday, Michael; Isern, Nancy G.; Zhang, Fengli; Pegan, Scott D.

    2015-05-01

    Crimean-Congo Hemorrhagic fever virus (CCHFV) is one of several lethal viruses that encodes for a viral ovarian tumor domain (vOTU), which serves to cleave and remove multiple proteins involved in cellular signaling such as ubiquitin (Ub) and interferon stimulated gene produce 15 (ISG15). Such manipulation of the host cell machinery serves to downregulate the host response and, therefore, complete characterization of these proteases is important. While several structures of the CCHFV vOTU protease have been solved, both free and bound to Ub and ISG15, few structural differences have been found and little insight has been gained as to the dynamic plasticity of this protease. Therefore, we have used NMR relaxation experiments to probe the dynamics of CCHV vOTU, both alone and in complex with Ub, thereby discovering a highly dynamic protease that exhibits conformational exchange within the same regions found to engage its Ub substrate. These experiments reveal a structural plasticity around the N-terminal regions of CCHV vOTU, which are unique to vOTUs, and provide a rationale for engaging multiple substrates with the same binding site.

  1. Mechanisms regulating the degradation of dentin matrices by endogenous dentin proteases and their role in dental adhesion. A review.

    PubMed

    Sabatini, Camila; Pashley, David H

    2014-08-01

    This systematic review provides an overview of the different mechanisms proposed to regulate the degradation of dentin matrices by host-derived dentin proteases, particularly as it relates to their role in dental adhesion. Significant developments have taken place over the last few years that have contributed to a better understanding of all the factors affecting the durability of adhesive resin restorations. The complexity of dentin-resin interfaces mandates a thorough understanding of all the mechanical, physical and biochemical aspects that play a role in the formation of hybrid layers. The ionic and hydrophilic nature of current dental adhesives yields permeable, unstable hybrid layers susceptible to water sorption, hydrolytic degradation and resin leaching. The hydrolytic activity of host-derived proteases also contributes to the degradation of the resin-dentin bonds. Preservation of the collagen matrix is critical to the improvement of resin-dentin bond durability. Approaches to regulate collagenolytic activity of dentin proteases have been the subject of extensive research in the last few years. A shift has occurred from the use of proteases inhibitors to the use of collagen cross-linking agents. Data provided by 51 studies published in peer-reviewed journals between January 1999 and December 2013 were compiled in this systematic review. Appraisal of the data provided by the studies included in the present review yielded a summary of the mechanisms which have already proven to be clinically successful and those which need further investigation before new clinical protocols can be adopted.

  2. A novel subtilisin-like serine protease of Batrachochytrium dendrobatidis is induced by thyroid hormone and degrades antimicrobial peptides.

    PubMed

    Thekkiniath, Jose C; Zabet-Moghaddam, Masoud; San Francisco, Susan K; San Francisco, Michael J

    2013-06-01

    Batrachochytrium dendrobatidis (B. dendrobatidis), a chytrid fungus, is one of the major contributors to the global amphibian decline. The fungus infects both tadpoles and adult amphibians. Tadpoles are infected in their keratinized mouthparts, and infected adults exhibit hyperkeratosis and loss of righting reflex. Infections of adults may result in death from cardiac arrest in susceptible species. Thyroid hormone plays a key role in amphibian metamorphosis. The occurrence of B. dendrobatidis in tadpoles during metamorphosis may result in exposure of the fungus to host morphogens including TH. This exposure may induce gene expression in the fungus contributing to invasion and colonization of the host. Here, we demonstrate movement of fungal zoospores toward TH. Additionally, expression of a subtilisin-like serine protease is up-regulated in B. dendrobatidis cells exposed to TH. A gene encoding this protease was cloned from B. dendrobatidis and expressed in Escherichia coli. The protein was partially purified and characterized. The similarity between subtilases of human dermatophytes and the B. dendrobatidis subtilisin-like serine protease suggests the importance of this enzyme in B. dendrobatidis pathogenicity. Cleavage of frog skin antimicrobial peptides (AMPs) by this B. dendrobatidis subtilisin-like serine protease suggests a role for this enzyme in fungal survival and colonization. Copyright © 2013 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  3. [Peculiarities of the expression, structure, and localization of the subtilisin-like protease in the microsporidium Paranosema locustae].

    PubMed

    Timofeev, S A; Sendersky, I V; Pavlova, O A; Dolgikh, V V

    2014-01-01

    Peculiarities of the expression, localization, and structure of the subtilisin-like protease from the microsporidium Paranosema locustae, a parasite of the migratory locust and other orthopteran species, are analyzed. Heterologous expression of the microsporidian ferment in the bacterium Escherichia coli allowed obtaining antibodies to the recombinant protein and to start its examination. In spite of the presence of the N-tail signal peptide in the ferment, potentially able to secret it into the cytoplasm of the infected cell, immunoblotting with obtained antibodies had demonstrated specific accumulation of the protease in the insoluble fraction of spore homogenate. At the same time, the ferment was absent in intracellular stages.of the parasite and also in the cytoplasm of infested host cells. Accumulation of mRNA, coding the studied protein in microsporidian spores was confirmed with the use of RT-PCR method. Heterologous expression of the protease in the methylotrophic yeast Pichiapastoris demonstrated the same result. The ferment of P. locustae was not secreted into a culture medium and was absent in the cytoplasm of yeast cells, accumulating in a dissoluble (membrane) fraction of the homogenate. On the whole, the obtained data testify to the fact that the subtilisin-like protease of P. locustae plays an important role in the physiology of spores rather than participates in host-parasite relations during intra-cellular development.

  4. Mycobacterium tuberculosis evolutionary pathogenesis and its putative impact on drug development.

    PubMed

    Le Chevalier, Fabien; Cascioferro, Alessandro; Majlessi, Laleh; Herrmann, Jean Louis; Brosch, Roland

    2014-01-01

    Mycobacterium tuberculosis, the etiological agent of human TB, is the most important mycobacterial pathogen in terms of global patient numbers and gravity of disease. The molecular mechanisms by which M. tuberculosis causes disease are complex and the result of host-pathogen coevolution that might have started already in the time of its Mycobacterium canettii-like progenitors. Despite research progress, M. tuberculosis still holds many secrets of its successful strategy for circumventing host defences, persisting in the host and developing resistance, which makes anti-TB treatment regimens extremely long and often inefficient. Here, we discuss what we have learned from recent studies on the evolution of the pathogen and its putative new drug targets that are essential for mycobacterial growth under in vitro or in vivo conditions.

  5. SmCL3, a Gastrodermal Cysteine Protease of the Human Blood Fluke Schistosoma mansoni

    PubMed Central

    Dvořák, Jan; Mashiyama, Susan T.; Sajid, Mohammed; Braschi, Simon; Delcroix, Melaine; Schneider, Eric L.; McKerrow, Wilson H.; Bahgat, Mahmoud; Hansell, Elizabeth; Babbitt, Patricia C.; Craik, Charles S.; McKerrow, James H.; Caffrey, Conor R.

    2009-01-01

    Background Blood flukes of the genus Schistosoma are platyhelminth parasites that infect 200 million people worldwide. Digestion of nutrients from the host bloodstream is essential for parasite development and reproduction. A network of proteolytic enzymes (proteases) facilitates hydrolysis of host hemoglobin and serum proteins. Methodology/Principal Findings We identified a new cathepsin L termed SmCL3 using PCR strategies based on S. mansoni EST sequence data. An ortholog is present in Schistosoma japonicum. SmCL3 was heterologously expressed as an active enzyme in the yeast, Pichia pastoris. Recombinant SmCL3 has a broad pH activity range against peptidyl substrates and is inhibited by Clan CA protease inhibitors. Consistent with a function in degrading host proteins, SmCL3 hydrolyzes serum albumin and hemoglobin, is localized to the adult gastrodermis, and is expressed mainly in those life stages infecting the mammalian host. The predominant form of SmCL3 in the parasite exists as a zymogen, which is unusual for proteases. This zymogen includes an unusually long prodomain with alpha helical secondary structure motifs. The striking specificity of SmCL3 for amino acids with large aromatic side chains (Trp and Tyr) at the P2 substrate position, as determined with positional scanning-synthetic combinatorial library, is consistent with a molecular model that shows a large and deep S2 pocket. A sequence similarity network (SSN) view clusters SmCL3 and other cathepsins L in accordance with previous large-scale phylogenetic analyses that identify six super kingdoms. Conclusions/Significance SmCL3 is a gut-associated cathepsin L that may contribute to the network of proteases involved in degrading host blood proteins as nutrients. Furthermore, this enzyme exhibits some unusual sequence and biophysical features that may result in additional functions. The visualization of network inter-relationships among cathepsins L suggests that these enzymes are suitable

  6. Biochemical Properties of a Novel Cysteine Protease of Plasmodium vivax, Vivapain-4

    PubMed Central

    Zo, Young-Gun; Choe, Youngchool; Kim, Seon-Hee; Desai, Prashant V.; Avery, Mitchell A.; Craik, Charles S.; Kim, Tong-Soo; Rosenthal, Philip J.; Kong, Yoon

    2010-01-01

    Background Multiple cysteine proteases of malaria parasites are required for maintenance of parasite metabolic homeostasis and egress from the host erythrocyte. In Plasmodium falciparum these proteases appear to mediate the processing of hemoglobin and aspartic proteases (plasmepsins) in the acidic food vacuole and the hydrolysis of erythrocyte structural proteins at neutral pH. Two cysteine proteases, vivapain (VX)-2 and VX-3 have been characterized in P. vivax, but comprehensive studies of P. vivax cysteine proteases remain elusive. Findings We characterized a novel cysteine protease of P. vivax, VX-4, of which orthologs appears to have evolved differentially in primate plasmodia with strong cladistic affinity toward those of rodent Plasmodium. Recombinant VX-4 demonstrated dual substrate specificity depending on the surrounding micro-environmental pH. Its hydrolyzing activity against benzyloxycarbonyl-Leu-Arg-4-methyl-coumaryl-7-amide (Z-Leu-Arg-MCA) and Z-Phe-Arg-MCA was highest at acidic pH (5.5), whereas that against Z-Arg-Arg-MCA was maximal at neutral pH (6.5–7.5). VX-4 preferred positively charged amino acids and Gln at the P1 position, with less strict specificity at P3 and P4. P2 preferences depended on pH (Leu at pH 5.5 and Arg at pH 7.5). Three amino acids that delineate the S2 pocket were substituted in VX-4 compared to VX-2 and VX-3 (Ala90, Gly157 and Glu180). Replacement of Glu180 abolished activity against Z-Arg-Arg-MCA at neutral pH, indicating the importance of this amino acid in the pH-dependent substrate preference. VX-4 was localized in the food vacuoles and cytoplasm of the erythrocytic stage of P. vivax. VX-4 showed maximal activity against actin at neutral pH, and that against P. vivax plasmepsin 4 and hemoglobin was detected at neutral/acidic and acidic pH, respectively. Conclusion VX-4 demonstrates pH-dependent substrate switching, which might offer an efficient mechanism for the specific cleavage of different substrates in different

  7. Expression of putative virulence factors in the potato pathogen Clavibacter michiganensis subsp. sepedonicus during infection.

    PubMed

    Holtsmark, Ingrid; Takle, Gunnhild W; Brurberg, May Bente

    2008-02-01

    The Gram-positive bacterium Clavibacter michiganensis subsp. sepedonicus is the causal agent of bacterial wilt and ring rot of potato. So far, only two proteins have been shown to be essential for virulence, namely a plasmid-encoded cellulase CelA and a hypersensitive response-inducing protein. We have examined the relative expression of CelA and eight putative virulence factors during infection of potato and in liquid culture, using quantitative real-time PCR. The examined putative virulence genes were celB, a cellulase-encoding gene and genes encoding a pectate lyase, a xylanase and five homologues of the Clavibacter michiganensis subsp. michiganensis pathogenicity factor Pat-1 thought to encode a serine protease. Six of the nine assayed genes were up-regulated during infection of potato, including celA, celB, the xylanase gene, and two of the pat genes. The pectate lyase gene showed only slightly elevated expression, whereas three of the five examined pat genes were down-regulated during infection in potato. Interestingly, the two up-regulated pat genes showed a noticeable sequence difference compared to the three down-regulated pat genes. These results reveal several new proteins that are likely to be involved in Clavibacter michiganensis subsp. sepedonicus pathogenicity.

  8. Ten Putative Contributors to the Obesity Epidemic

    PubMed Central

    McAllister, Emily J.; Dhurandhar, Nikhil V.; Keith, Scott W.; Aronne, Louis J.; Barger, Jamie; Baskin, Monica; Benca, Ruth M.; Biggio, Joseph; Boggiano, Mary M.; Eisenmann, Joe C.; Elobeid, Mai; Fontaine, Kevin R.; Gluckman, Peter; Hanlon, Erin C.; Katzmarzyk, Peter; Pietrobelli, Angelo; Redden, David T.; Ruden, Douglas M.; Wang, Chenxi; Waterland, Robert A.; Wright, Suzanne M.; Allison, David B.

    2010-01-01

    The obesity epidemic is a global issue and shows no signs of abating, while the cause of this epidemic remains unclear. Marketing practices of energy-dense foods and institutionally-driven declines in physical activity are the alleged perpetrators for the epidemic, despite a lack of solid evidence to demonstrate their causal role. While both may contribute to obesity, we call attention to their unquestioned dominance in program funding and public efforts to reduce obesity, and propose several alternative putative contributors that would benefit from equal consideration and attention. Evidence for microorganisms, epigenetics, increasing maternal age, greater fecundity among people with higher adiposity, assortative mating, sleep debt, endocrine disruptors, pharmaceutical iatrogenesis, reduction in variability of ambient temperatures, and intrauterine and intergenerational effects, as contributing factors to the obesity epidemic are reviewed herein. While the evidence is strong for some contributors such as pharmaceutical-induced weight gain, it is still emerging for other reviewed factors. Considering the role of such putative etiological factors of obesity may lead to comprehensive, cause specific, and effective strategies for prevention and treatment of this global epidemic. PMID:19960394

  9. Derivation and evaluation of putative adverse outcome ...

    EPA Pesticide Factsheets

    Cyclooxygenase (COX) inhibition is of concern in fish because COX inhibitors (e.g., ibuprofen) are ubiquitous in aquatic systems/fish tissues, and can disrupt synthesis of prostaglandins that modulate a variety of essential biological functions including reproduction. High content (transcriptomic) empirical data and publicly available high throughput toxicity data (actor.epa.gov) were utilized to develop putative adverse outcome pathways (AOPs) for molecular initiating event (MIE) of COX inhibition. Effects of a waterborne, 96h exposure to indomethacin (IN; 100 µg/L), ibuprofen (IB; 200 µg/L) and celecoxib (CX; 20 µg/L) on liver metabolome and ovarian gene expression (using oligonucleotide microarrays) in sexually mature fathead minnows (n=8) were examined. Metabolomic profiles of IN, IB and CX were not significantly different from control or one another. Exposure to IB and CX resulted in differential expression of comparable numbers of genes (IB = 433, CX= 545). In contrast, 2558 genes were differentially expressed in IN-treated fish. Functional analyses (canonical pathway and gene set enrichment) indicated extensive effects of IN on prostaglandin synthesis pathway, oocyte meiosis and several other processes consistent with physiological roles of prostaglandins. Transcriptomic data was congruent with apical endpoint data - IN reduced plasma prostaglandin F2 alpha concentrations, and ovarian COX activity, whereas IB and CX did not. Putative AOPs pathways for

  10. A putative serine protease, SpSsp1, from Saprolegnia parasitica is recognised by sera of rainbow trout, Oncorhynchus mykiss

    PubMed Central

    Minor, Kirsty L.; Anderson, Victoria L.; Davis, Katie S.; Van Den Berg, Albert H.; Christie, James S.; Löbach, Lars; Faruk, Ali Reza; Wawra, Stephan; Secombes, Chris J.; Van West, Pieter

    2014-01-01

    Saprolegniosis, the disease caused by Saprolegnia sp., results in considerable economic losses in aquaculture. Current control methods are inadequate, as they are either largely ineffective or present environmental and fish health concerns. Vaccination of fish presents an attractive alternative to these control methods. Therefore we set out to identify suitable antigens that could help generate a fish vaccine against Saprolegnia parasitica. Unexpectedly, antibodies against S. parasitica were found in serum from healthy rainbow trout, Oncorhynchus mykiss. The antibodies detected a single band in secreted proteins that were run on a one-dimensional SDS-polyacrylamide gel, which corresponded to two protein spots on a two-dimensional gel. The proteins were analysed by liquid chromatography tandem mass spectrometry. Mascot and bioinformatic analysis resulted in the identification of a single secreted protein, SpSsp1, of 481 amino acid residues, containing a subtilisin domain. Expression analysis demonstrated that SpSsp1 is highly expressed in all tested mycelial stages of S. parasitica. Investigation of other non-infected trout from several fish farms in the United Kingdom showed similar activity in their sera towards SpSsp1. Several fish that had no visible saprolegniosis showed an antibody response towards SpSsp1 suggesting that SpSsp1 might be a useful candidate for future vaccination trial experiments. PMID:25088077

  11. A putative serine protease, SpSsp1, from Saprolegnia parasitica is recognised by sera of rainbow trout, Oncorhynchus mykiss.

    PubMed

    Minor, Kirsty L; Anderson, Victoria L; Davis, Katie S; Van Den Berg, Albert H; Christie, James S; Löbach, Lars; Faruk, Ali Reza; Wawra, Stephan; Secombes, Chris J; Van West, Pieter

    2014-07-01

    Saprolegniosis, the disease caused by Saprolegnia sp., results in considerable economic losses in aquaculture. Current control methods are inadequate, as they are either largely ineffective or present environmental and fish health concerns. Vaccination of fish presents an attractive alternative to these control methods. Therefore we set out to identify suitable antigens that could help generate a fish vaccine against Saprolegnia parasitica. Unexpectedly, antibodies against S. parasitica were found in serum from healthy rainbow trout, Oncorhynchus mykiss. The antibodies detected a single band in secreted proteins that were run on a one-dimensional SDS-polyacrylamide gel, which corresponded to two protein spots on a two-dimensional gel. The proteins were analysed by liquid chromatography tandem mass spectrometry. Mascot and bioinformatic analysis resulted in the identification of a single secreted protein, SpSsp1, of 481 amino acid residues, containing a subtilisin domain. Expression analysis demonstrated that SpSsp1 is highly expressed in all tested mycelial stages of S. parasitica. Investigation of other non-infected trout from several fish farms in the United Kingdom showed similar activity in their sera towards SpSsp1. Several fish that had no visible saprolegniosis showed an antibody response towards SpSsp1 suggesting that SpSsp1 might be a useful candidate for future vaccination trial experiments.

  12. Comparison of innate immune responses to pathogenic and putative non-pathogenic hantaviruses in vitro.

    PubMed

    Shim, So Hee; Park, Man-Seong; Moon, Sungsil; Park, Kwang Sook; Song, Jin-Won; Song, Ki-Joon; Baek, Luck Ju

    2011-09-01

    Hantaviruses are human pathogens that cause hemorrhagic fever with renal syndrome or hantavirus cardiopulmonary syndrome. The mechanisms accounting for the differences in virulence between pathogenic and non-pathogenic hantaviruses are not well known. We have examined the pathogenesis of different hantavirus groups by comparing the innate immune responses induced in the host cell following infection by pathogenic (Sin Nombre, Hantaan, and Seoul virus) and putative non-pathogenic (Prospect Hill, Tula, and Thottapalayam virus) hantaviruses. Pathogenic hantaviruses were found to replicate more efficiently in interferon-competent A549 cells than putative non-pathogenic hantaviruses. The former also suppressed the expression of the interferon-β and myxovirus resistance protein genes, while the transcription level of both genes increased rapidly within 24 h post-infection in the latter. In addition, the induction level of interferon correlated with the activation level of interferon regulatory factor-3. Taken together, these results suggest that the observed differences are correlated with viral pathogenesis and further indicate that pathogenic and putative non-pathogenic hantaviruses differ in terms of early interferon induction via activation of the interferon regulatory factor-3 in infected host cells.

  13. Putative fossil life in a hydrothermal system of the Dellen impact structure, Sweden

    NASA Astrophysics Data System (ADS)

    Lindgren, Paula; Ivarsson, Magnus; Neubeck, Anna; Broman, Curt; Henkel, Herbert; Holm, Nils G.

    2010-07-01

    Impact-generated hydrothermal systems are commonly proposed as good candidates for hosting primitive life on early Earth and Mars. However, evidence of fossil microbial colonization in impact-generated hydrothermal systems is rarely reported in the literature. Here we present the occurrence of putative fossil microorganisms in a hydrothermal system of the 89 Ma Dellen impact structure, Sweden. We found the putative fossilized microorganisms hosted in a fine-grained matrix of hydrothermal alteration minerals set in interlinked fractures of an impact breccia. The putative fossils appear as semi-straight to twirled filaments, with a thickness of 1-2 μm, and a length between 10 and 100 μm. They have an internal structure with segmentation, and branching of filaments occurs frequently. Their composition varies between an outer and an inner layer of a filament, where the inner layer is more iron rich. Our results indicate that hydrothermal systems in impact craters could potentially be capable of supporting microbial life. This could have played an important role for the evolution of life on early Earth and Mars.

  14. 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.

  15. A Multifunctional Protease Inhibitor To Regulate Endolysosomal Function

    PubMed Central

    2011-01-01

    Proteases constitute a major class of drug targets. Endosomal compartments harbor several protease families whose attenuation may be beneficial to a number of biological processes, including inflammation, cancer metastasis, antigen presentation, and parasite clearance. As a step toward the goal of generalized but targeted protease inhibition in the endocytic pathway, we describe here the synthesis, characterization, and cellular application of a novel multifunctional protease inhibitor. We show that pepstatin A, a potent but virtually insoluble inhibitor of cathepsins D and E, can be conjugated to a single site on cystatin C, a potent inhibitor of the papain-like cysteine proteases (PLCP) and of asparagine endopeptidease (AEP), to create a highly soluble compound capable of suppressing the activity of all 3 principal protease families found in endosomes and lysosomes. We demonstrate that this cystatin–pepstatin inhibitor (CPI) can be taken up by cells to modulate protease activity and affect biological responses. PMID:21910425

  16. A multifunctional protease inhibitor to regulate endolysosomal function.

    PubMed

    van Kasteren, Sander I; Berlin, Ilana; Colbert, Jeff D; Keane, Doreen; Ovaa, Huib; Watts, Colin

    2011-11-18

    Proteases constitute a major class of drug targets. Endosomal compartments harbor several protease families whose attenuation may be beneficial to a number of biological processes, including inflammation, cancer metastasis, antigen presentation, and parasite clearance. As a step toward the goal of generalized but targeted protease inhibition in the endocytic pathway, we describe here the synthesis, characterization, and cellular application of a novel multifunctional protease inhibitor. We show that pepstatin A, a potent but virtually insoluble inhibitor of cathepsins D and E, can be conjugated to a single site on cystatin C, a potent inhibitor of the papain-like cysteine proteases (PLCP) and of asparagine endopeptidease (AEP), to create a highly soluble compound capable of suppressing the activity of all 3 principal protease families found in endosomes and lysosomes. We demonstrate that this cystatin-pepstatin inhibitor (CPI) can be taken up by cells to modulate protease activity and affect biological responses.

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

    PubMed

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

    2017-04-15

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

  18. Management of protease inhibitor-associated hyperlipidemia.

    PubMed

    Penzak, Scott R; Chuck, Susan K

    2002-01-01

    Dyslipidemia, characterized by elevated serum levels of triglycerides and reduced levels of total cholesterol, low-density lipoprotein-cholesterol (LDL-C) and high-density lipoprotein-cholesterol, has been recognized in patients with human immunodeficiency virus (HIV) infection. It is thought that elevated levels of circulating cytokines, such as tumor necrosis factor-alpha and interferon-alpha, may alter lipid metabolism in patients with HIV infection. Protease inhibitors, such as saquinavir, indinavir and ritonavir, have been found to decrease mortality and improve quality of life in patients with HIV infection. However, these drugs have been associated with a syndrome of fat redistribution, insulin resistance, and hyperlipidemia. Elevations in serum total cholesterol and triglyceride levels, along with dyslipidemia that typically occurs in patients with HIV infection, may predispose patients to complications such as premature atherosclerosis and pancreatitis. It has been estimated that hypercholesterolemia and hypertriglyceridemia occur in greater than 50% of protease inhibitor recipients after 2 years of therapy, and that the risk of developing hyperlipidemia increases with the duration of treatment with protease inhibitors. In general, treatment of hyperlipidemia should follow National Cholesterol Education Program guidelines; efforts should be made to modify/control coronary heart disease risk factors (i.e. smoking; hypertension; diabetes mellitus) and maximize lifestyle modifications, primarily dietary intervention and exercise, in these patients. Where indicated, treatment usually consists of either pravastatin or atorvastatin for patients with elevated serum levels of LDL-C and/or total cholesterol. Atorvastatin is more potent in lowering serum total cholesterol and triglycerides compared with other hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, but it is also associated with more drug interactions compared with pravastatin. Simvastatin

  19. Species formation by host shifting in avian malaria parasites

    PubMed Central

    Ricklefs, Robert E.; Outlaw, Diana C.; Svensson-Coelho, Maria; Medeiros, Matthew C. I.; Ellis, Vincenzo A.; Latta, Steven

    2014-01-01

    The malaria parasites (Apicomplexa: Haemosporida) of birds are believed to have diversified across the avian host phylogeny well after the origin of most major host lineages. Although many symbionts with direct transmission codiversify with their hosts, mechanisms of species formation in vector-borne parasites, including the role of host shifting, are poorly understood. Here, we examine the hosts of sister lineages in a phylogeny of 181 putative species of malaria parasites of New World terrestrial birds to determine the role of shifts between host taxa in the formation of new parasite species. We find that host shifting, often across host genera and families, is the rule. Sympatric speciation by host shifting would require local reproductive isolation as a prerequisite to divergent selection, but this mechanism is not supported by the generalized host-biting behavior of most vectors of avian malaria parasites. Instead, the geographic distribution of individual parasite lineages in diverse hosts suggests that species formation is predominantly allopatric and involves host expansion followed by local host–pathogen coevolution and secondary sympatry, resulting in local shifting of parasite lineages across hosts. PMID:25271324

  20. Phosphoglycerate dehydrogenase: potential therapeutic target and putative metabolic oncogene.

    PubMed

    Zogg, Cheryl K

    2014-01-01

    Exemplified by cancer cells' preference for glycolysis, for example, the Warburg effect, altered metabolism in tumorigenesis has emerged as an important aspect of cancer in the past 10-20 years. Whether due to changes in regulatory tumor suppressors/oncogenes or by acting as metabolic oncogenes themselves, enzymes involved in the complex network of metabolic pathways are being studied to understand their role and assess their utility as therapeutic targets. Conversion of glycolytic intermediate 3-phosphoglycerate into phosphohydroxypyruvate by the enzyme phosphoglycerate dehydrogenase (PHGDH)-a rate-limiting step in the conversion of 3-phosphoglycerate to serine-represents one such mechanism. Forgotten since classic animal studies in the 1980s, the role of PHGDH as a potential therapeutic target and putative metabolic oncogene has recently reemerged following publication of two prominent papers near-simu