Journal articles on the topic 'Insect, protease, peptides'
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1
COPLEY, S. Kathrin, M. Sheri ALM, A. David SCHOOLEY, and E. William COURCHESNE. "Expression, processing and secretion of a proteolytically-sensitive insect diuretic hormone by Saccharomyces cerevisiae requires the use of a yeast strain lacking genes encoding the Yap3 and Mkc7 endoproteases found in the secretory pathway." Biochemical Journal 330, no. 3 (March 15, 1998): 1333–40. http://dx.doi.org/10.1042/bj3301333.
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A system is described for the heterologous expression of peptides in Saccharomyces cerevisiae. A synthetic gene encoding a precursor of the 41 amino acid Manduca sexta diuretic hormone (Mas-DH) was expressed at 0.8 mg/l purified peptide. A precursor of a mutant peptide of Mas-DH, Mas-DH[K22Q] was also expressed. The peptides were purified, then treated with peptidylglycine α-amidating enzyme to generate the α-amidated, mature, form of Mas-DH or Mas-DH[K22Q], which were biologically active. Successful expression of full-length Mas-DH+Gly depended upon the use of a protease-deficient yeast strain. In wild-type strains, Mas-DH+Gly was recovered only as proteolytic fragments, even in the presence of various protease inhibitors. Expression of Mas-DH+Gly in strains deficient in either the Mkc7 or the Yap3 protease reduced proteolysis, while no proteolysis of Mas-DH+Gly was detectable in a strain lacking both proteases. This protease-deficient strain may prove of general utility for expression of peptides. Analysis of recovered proteolytic fragments revealed a complex pattern of cleavage sites. Both the Yap3 and Mkc7 proteases preferred to cleave at a single Glu-Lys↓-Glu-Arg site. Analysis of secondary cleavage sites showed that Yap3 preferred to cleave after either Lys or Arg and Mkc7 after Lys. This paper is the first report on the in vivo activity and specificity of Yap3 and Mkc7 expressed at physiological levels.
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Deng, Yuping, James Gibbs, Igor Bačík, Angel Porgador, James Copeman, Paul Lehner, Bodo Ortmann, Peter Cresswell, Jack R. Bennink, and Jonathan W. Yewdell. "Assembly of MHC Class I Molecules with Biosynthesized Endoplasmic Reticulum-Targeted Peptides Is Inefficient in Insect Cells and Can Be Enhanced by Protease Inhibitors." Journal of Immunology 161, no. 4 (August 15, 1998): 1677–85. http://dx.doi.org/10.4049/jimmunol.161.4.1677.
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Abstract To study the requirements for assembly of MHC class I molecules with antigenic peptides in the endoplasmic reticulum (ER), we studied Ag processing in insect cells. Insects lack a class I recognition system, and their cells therefore provide a “blank slate” for identifying the proteins that have evolved to facilitate assembly of class I molecules in vertebrate cells. H-2Kb heavy chain, mouse β2-microglobulin, and an ER-targeted version of a peptide corresponding to Ova257–264 were expressed in insect cells using recombinant vaccinia viruses. Cell surface expression of Kb-OVA257–264 complexes was quantitated using a recently described complex-specific mAb (25-D1.16). Relative to TAP-deficient human cells, insect cells expressed comparable levels of native, peptide-receptive cell surface Kb molecules, but generated cell surface Kb-OVA257–264 complexes at least 20-fold less efficiently from ER-targeted peptides. The inefficient assembly of Kb-OVA257–264 complexes in the ER of insect cells cannot be attributed solely to a requirement for human tapasin, since first, human cells lacking tapasin expressed endogenously synthesized Kb-OVA257–264 complexes at levels comparable to tapasin-expressing cells, and second, vaccinia virus-mediated expression of human tapasin in insect cells did not detectably enhance the expression of Kb-OVA257–264 complexes. The assembly of Kb-OVA257–264 complexes could be greatly enhanced in insect but not human cells by a nonproteasomal protease inhibitor. These findings indicate that insect cells lack one or more factors required for the efficient assembly of class I-peptide complexes in vertebrate cells and are consistent with the idea that the missing component acts to protect antigenic peptides or their immediate precursors from degradation.
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Caldas, C., A. Cherqui, A. Pereira, and N. Simões. "Purification and Characterization of an Extracellular Protease from Xenorhabdus nematophila Involved in Insect Immunosuppression." Applied and Environmental Microbiology 68, no. 3 (March 2002): 1297–304. http://dx.doi.org/10.1128/aem.68.3.1297-1304.2002.
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ABSTRACT Xenorhabdus nematophila, a bacterium pathogenic for insects associated with the nematode Steinernema carpocapsae, releases high quantities of proteases, which may participate in the virulence against insects. Zymogram assays and cross-reactions of antibodies suggested that two distinct proteases were present. The major one, protease II, was purified and shown to have a molecular mass of 60 kDa and an estimated isoelectric point of 8.5. Protease II digested the chromogenic substrate N-tosyl-Gly-Pro-Arg-paranitroanilide (pNA) with V max and Km values of 0.0551 μM/min and 234 μM, respectively, and the substrate dl-Val-Leu-Arg-pNA with V max and Km values of 0.3830 μM/min and 429 μM, respectively. Protease II activity was inhibited 93% by Pefabloc SC and 45% by chymostatin. The optimum pH for protease II was 7, and the optimum temperature was 23°C. Proteolytic activity was reduced by 90% at 60°C for 10 min. Sequence analysis was performed on four internal peptides that resulted from the digestion of protease II. Fragments 29 and 45 are 75 and 68% identical to alkaline metalloproteinase produced by Pseudomonas aeruginosa. Fragment 29 is 79% identical to a metalloprotease of Erwinia amylovora and 75% identical to the protease C precursor of Erwinia chrysanthemi. Protease II showed no toxicity to hemocytes but destroyed antibacterial activity on the hemolymph of inoculated insects' larvae and reduced 97% of the cecropin A bacteriolytic activity.
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Pfrepper, Klaus-Ingmar, Hans-Richard Rackwitz, Martina Schnölzer, Hans Heid, Martin Löchelt, and Rolf M. Flügel. "Molecular Characterization of Proteolytic Processing of the Pol Proteins of Human Foamy Virus Reveals Novel Features of the Viral Protease." Journal of Virology 72, no. 9 (September 1, 1998): 7648–52. http://dx.doi.org/10.1128/jvi.72.9.7648-7652.1998.
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ABSTRACT Spumaviruses, or foamy viruses, express a pol-specific transcript that codes for a Pol polyprotein that consists of the protease, reverse transcriptase, ribonuclease H, and the integrase domains. To delineate the proteolytic cleavage sites between the Pol subdomains, recombinant human foamy virus (HFV) Pol proteins were expressed, purified by affinity chromatography, and subjected to either HFV protease assays or autocatalytic processing. In control experiments, HFV protease-deficient mutant proteins in which the active site Asp was replaced by an Ala residue were used to rule out unspecific processing by nonviral proteases. Specific proteolytic cleavage products were isolated, and the cleavage sites were analyzed by amino acid sequencing. Peptides spanning the resulting cleavage sites were chemically synthesized and assayed with HFV protease, and the cleaved peptides were subjected to mass spectrometry. The cleavage site sequences obtained were in complete agreement with the amino-terminal sequences from amino acid sequencing of authentic cleavage products of the HFV Pol proteins. Analysis by fast-protein liquid chromatography of a short version of the active HFV protease revealed that the enzyme predominantly formed dimeric molecules.
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Lin, Ying-Chuan, Zachary Beck, Taekyu Lee, Van-Duc Le, Garrett M. Morris, Arthur J. Olson, Chi-Huey Wong, and John H. Elder. "Alteration of Substrate and Inhibitor Specificity of Feline Immunodeficiency Virus Protease." Journal of Virology 74, no. 10 (May 15, 2000): 4710–20. http://dx.doi.org/10.1128/jvi.74.10.4710-4720.2000.
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ABSTRACT Feline immunodeficiency virus (FIV) protease is structurally very similar to human immunodeficiency virus (HIV) protease but exhibits distinct substrate and inhibitor specificities. We performed mutagenesis of subsite residues of FIV protease in order to define interactions that dictate this specificity. The I37V, N55M, M56I, V59I, and Q99V mutants yielded full activity. The I37V, N55M, V59I, and Q99V mutants showed a significant increase in activity against the HIV-1 reverse transcriptase/integrase and P2/nucleocapsid junction peptides compared with wild-type (wt) FIV protease. The I37V, V59I, and Q99V mutants also showed an increase in activity against two rapidly cleaved peptides selected by cleavage of a phage display library with HIV-1 protease. Mutations at Q54K, I98P, and L101I dramatically reduced activity. Mutants containing a I35D or I57G substitution showed no activity against either FIV or HIV substrates. FIV proteases all failed to cut HIV-1 matrix/capsid, P1/P6, P6/protease, and protease/reverse transcriptase junctions, indicating that none of the substitutions were sufficient to change the specificity completely. The I37V, N55M, M56I, V59I, and Q99V mutants, compared with wt FIV protease, all showed inhibitor specificity more similar to that of HIV-1 protease. The data also suggest that FIV protease prefers a hydrophobic P2/P2′ residue like Val over Asn or Glu, which are utilized by HIV-1 protease, and that S2/S2′ might play a critical role in distinguishing FIV and HIV-1 protease by specificity. The findings extend our observations regarding the interactions involved in substrate binding and aid in the development of broad-based inhibitors.
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Pfrepper, Klaus-Ingmar, Martin Löchelt, Hans-Richard Rackwitz, Martina Schnölzer, Hans Heid, and Rolf M. Flügel. "Molecular Characterization of Proteolytic Processing of the Gag Proteins of Human Spumavirus." Journal of Virology 73, no. 9 (September 1, 1999): 7907–11. http://dx.doi.org/10.1128/jvi.73.9.7907-7911.1999.
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ABSTRACT Spumaviruses, or foamy viruses, express Gag proteins that are incompletely processed by the viral protease in cell cultures. To delineate the proteolytic cleavage sites between potential Gag subdomains, recombinant human spumaretrovirus (HSRV) Gag proteins of different lengths were expressed, purified by affinity chromatography, and subjected to HSRV protease assays. HSRV-specific proteolytic cleavage products were isolated and characterized by Western blotting. Peptides spanning potential cleavage sites, as deduced from the sizes of the proteolytic cleavage products, were chemically synthesized and assayed with HSRV protease. The cleaved peptides were then subjected to mass spectrometry. In control experiments, HSRV protease-deficient mutant proteins were used to rule out unspecific processing by nonviral proteases. The cleavage site junctions identified and the calculated sizes of the cleavage products were in agreement with those of the authentic cleavage products of the HSRV Gag proteins detectable in viral proteins from purified HSRV particles and in virus-infected cells. The biological significance of the data was confirmed by mutational analysis of the cleavage sites in a recombinant Gag protein and in the context of the infectious HSRV DNA provirus.
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SIMONET, Gert, Bert BREUGELMANS, Paul PROOST, Ilse CLAEYS, Jozef VAN DAMME, Arnold DE LOOF, and Jozef VANDEN BROECK. "Characterization of two novel pacifastin-like peptide precursor isoforms in the desert locust (Schistocerca gregaria): cDNA cloning, functional analysis and real-time RT-PCR gene expression studies." Biochemical Journal 388, no. 1 (May 10, 2005): 281–89. http://dx.doi.org/10.1042/bj20041414.
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In the last decade, a new serine protease inhibitor family has been described in arthropods. Eight members of the family were purified from locusts and share a conserved cysteine array (Cys-Xaa9–12-Cys-Asn-Xaa-Cys-Xaa-Cys-Xaa2–3-Gly-Xaa3–6-Cys-Thr-Xaa3-Cys) with nine inhibitory domains of the light chain of the crayfish protease inhibitor, pacifastin (PLDs; pacifastin light chain domains). Using cDNA cloning, several pacifastin-related precursors have been identified, encoding additional PLD-related peptides in different insect species. In the present study, two isoforms of a novel pacifastin-related precursor (SGPP-4) have been identified in the desert locust, predicting the previously identified SGPI-5 (Schistocerca gregaria PLD-related inhibitor-5) peptide and two novel PLD-related peptide sequences. One novel peptide (SGPI-5A) was synthesized chemically, and its inhibitory activity was assessed in vitro. Although proteases from a locust midgut extract were very sensitive to SGPI-5A, the same peptide proved to be a relatively poor inhibitor of bovine trypsin. By an in silico datamining approach, a novel pacifastin-related precursor with seven PLD-related domains was identified in the mosquito, Aedes aegypti. As in other insect pacifastin-related precursors, the Aedes precursor showed a particular domain architecture that is not encountered in other serine protease inhibitor families. Finally, a comparative real-time RT-PCR analysis of SGPP-4 transcripts in different tissues of isolated- (solitarious) and crowded-reared (gregarious) locusts was performed. This showed that SGPP-4 mRNA levels are higher in the brain, testes and fat body of gregarious males than of solitarious males. These results have been compared with data from a similar study on SGPP-1–3 transcripts and discussed with respect to a differential regulation of serine-protease-dependent pathways as a possible mechanism underlying locust phase polymorphism.
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Beck, Zachary Q., Ying-Chuan Lin, and John H. Elder. "Molecular Basis for the Relative Substrate Specificity of Human Immunodeficiency Virus Type 1 and Feline Immunodeficiency Virus Proteases." Journal of Virology 75, no. 19 (October 1, 2001): 9458–69. http://dx.doi.org/10.1128/jvi.75.19.9458-9469.2001.
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ABSTRACT We have used a random hexamer phage library to delineate similarities and differences between the substrate specificities of human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus (FIV) proteases (PRs). Peptide sequences were identified that were specifically cleaved by each protease, as well as sequences cleaved equally well by both enzymes. Based on amino acid distinctions within the P3-P3′ region of substrates that appeared to correlate with these cleavage specificities, we prepared a series of synthetic peptides within the framework of a peptide sequence cleaved with essentially the same efficiency by both HIV-1 and FIV PRs, Ac-KSGVF↓VVNGLVK-NH2 (arrow denotes cleavage site). We used the resultant peptide set to assess the influence of specific amino acid substitutions on the cleavage characteristics of the two proteases. The findings show that when Asn is substituted for Val at the P2 position, HIV-1 PR cleaves the substrate at a much greater rate than does FIV PR. Likewise, Glu or Gln substituted for Val at the P2′ position also yields peptides specifically susceptible to HIV-1 PR. In contrast, when Ser is substituted for Val at P1′, FIV PR cleaves the substrate at a much higher rate than does HIV-1 PR. In addition, Asn or Gln at the P1 position, in combination with an appropriate P3 amino acid, Arg, also strongly favors cleavage by FIV PR over HIV PR. Structural analysis identified several protease residues likely to dictate the observed specificity differences. Interestingly, HIV PR Asp30 (Ile-35 in FIV PR), which influences specificity at the S2 and S2′ subsites, and HIV-1 PR Pro-81 and Val-82 (Ile-98 and Gln-99 in FIV PR), which influence specificity at the S1 and S1′ subsites, are residues which are often involved in development of drug resistance in HIV-1 protease. The peptide substrate KSGVF↓VVNGK, cleaved by both PRs, was used as a template for the design of a reduced amide inhibitor, Ac-GSGVFΨ(CH2NH)VVNGL-NH2. This compound inhibited both FIV and HIV-1 PRs with approximately equal efficiency. These findings establish a molecular basis for distinctions in substrate specificity between human and feline lentivirus PRs and offer a framework for development of efficient broad-based inhibitors.
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Zhang, T. T., G. C. Zhang, F. F. Zeng, C. Y. Liu, and J. J. Mao. "Insulin-like peptides regulate vitellogenesis and oviposition in the green lacewing, Chrysopa septempunctata." Bulletin of Entomological Research 107, no. 2 (August 30, 2016): 148–54. http://dx.doi.org/10.1017/s0007485316000742.
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AbstractInsulin-like peptides (ILPs) act through a conserved insulin signaling pathway and play crucial roles in insect metabolism, growth, reproduction, and aging. Application of bovine insulin is able to increase vitellogenin (Vg) mRNA and protein levels in female insects. Here, we first show that injection of bovine insulin into previtellogenic Chrysopa septempunctata female adults promoted ovarian growth, increased Vg protein abundance, elevated reproductive performance, and enhanced protease activity. These data suggested that ILPs play crucial roles in reproductive regulation of the green lacewing, C. septempunctata.
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You, Liwen, Daniel Garwicz, and Thorsteinn Rögnvaldsson. "Comprehensive Bioinformatic Analysis of the Specificity of Human Immunodeficiency Virus Type 1 Protease." Journal of Virology 79, no. 19 (October 1, 2005): 12477–86. http://dx.doi.org/10.1128/jvi.79.19.12477-12486.2005.
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ABSTRACT Rapidly developing viral resistance to licensed human immunodeficiency virus type 1 (HIV-1) protease inhibitors is an increasing problem in the treatment of HIV-infected individuals and AIDS patients. A rational design of more effective protease inhibitors and discovery of potential biological substrates for the HIV-1 protease require accurate models for protease cleavage specificity. In this study, several popular bioinformatic machine learning methods, including support vector machines and artificial neural networks, were used to analyze the specificity of the HIV-1 protease. A new, extensive data set (746 peptides that have been experimentally tested for cleavage by the HIV-1 protease) was compiled, and the data were used to construct different classifiers that predicted whether the protease would cleave a given peptide substrate or not. The best predictor was a nonlinear predictor using two physicochemical parameters (hydrophobicity, or alternatively polarity, and size) for the amino acids, indicating that these properties are the key features recognized by the HIV-1 protease. The present in silico study provides new and important insights into the workings of the HIV-1 protease at the molecular level, supporting the recent hypothesis that the protease primarily recognizes a conformation rather than a specific amino acid sequence. Furthermore, we demonstrate that the presence of 1 to 2 lysine residues near the cleavage site of octameric peptide substrates seems to prevent cleavage efficiently, suggesting that this positively charged amino acid plays an important role in hindering the activity of the HIV-1 protease.
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Otvos, Laszlo, Krisztina Bokonyi, Istvan Varga, Hildegund C. J. Ertl, Ralf Hoffmann, Philippe Bulet, Balint I. Otvos, John D. Wade, Ailsa M. Mcmanus, and David J. Craik. "Insect peptides with improved protease-resistance protect mice against bacterial infection." Protein Science 9, no. 4 (December 31, 2008): 742–49. http://dx.doi.org/10.1110/ps.9.4.742.
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Ueno, Takamasa, Satoru Misawa, Yoichi Ohba, Mitsuhiro Matsumoto, Makiko Mizunuma, Nobuhiro Kasai, Kouhei Tsumoto, Izumi Kumagai, and Hideya Hayashi. "Isolation and Characterization of Monoclonal Antibodies That Inhibit Hepatitis C Virus NS3 Protease." Journal of Virology 74, no. 14 (July 15, 2000): 6300–6308. http://dx.doi.org/10.1128/jvi.74.14.6300-6308.2000.
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ABSTRACT A series of mouse monoclonal antibodies (MAbs) to the nonstructural protein 3 (NS3) of hepatitis C virus was prepared. One of these MAbs, designated 8D4, was found to inhibit NS3 protease activity. This inhibition was competitive with respect to the substrate peptide (Ki = 39 nM) but was significantly decreased by the addition of the NS4A peptide, a coactivator of the NS3 protease. 8D4 also showed marked inhibition of the NS3-dependentcis processing of the NS3/4A polyprotein but had virtually no effect on the succeeding NS3/4A-dependent transprocessing of the NS5A/5B polyprotein in vitro. Epitope mapping of 8D4 with a random peptide library revealed a consensus sequence, DxDLV, that matched residues 79 to 83 (DQDLV) of NS3, a region containing the catalytic residue Asp-81. Furthermore, synthetic peptides including this sequence were shown to block the ability of 8D4 to bind to NS3, indicating that 8D4 interacts with the catalytic region of NS3. The data showing decreased inhibition potency of 8D4 against the NS3/4A complex suggest that 8D4 recognizes the conformational state of the protease active site caused by the association of NS4A with the protease.
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Shaikh, Faiyaz Khudaboddin, Sarwan W. Hamad, Saber W. Hamad, and Ashok A. Shinde. "In Vitro Screening of Seed Extracts of Medicinal Plants for Protease Inhibitory Activity." Cihan University-Erbil Scientific Journal 3, no. 1 (June 30, 2019): 61–65. http://dx.doi.org/10.24086/cuesj.v3n1y2019.pp61-65.
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Protease inhibitors (PIs) are deployed in the plant kingdom as storage proteins or peptides, regulators of endogenous proteases, and plant protection agents against insect pests and pathogen attack. In humans, they are identified as chemopreventive agents against a range of cancers and have potential as drug to treat an array of disease associated with aberrant activity of proteases. The present investigation reports PIs activity data from 30 medicinal plants. The screening for PIs activity was done by dot blot assay using X-ray film coated with gelatin. Among screened seed extracts, Albizia lebbeck, Raphanus sativus, Mucuna pruriens, Achyranthes aspera, and Coffea arabica showed high inhibitory activities with trypsin protease. Most of seed extracts exhibited moderate activity, whereas Ocimum sanctum showed moderate to low activity against trypsin. The presence of varied protein content is reported from all seed extracts with highest in A. lebbeck (50.0 ± 3.4 mg/ml). The data produced in the present investigation could be helpful for further exploration of PIs as therapeutic agent.
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Oparin, Peter B., Konstantin S. Mineev, Yakov E. Dunaevsky, Alexander S. Arseniev, Mikhail A. Belozersky, Eugene V. Grishin, Tsezi A. Egorov, and Alexander A. Vassilevski. "Buckwheat trypsin inhibitor with helical hairpin structure belongs to a new family of plant defence peptides." Biochemical Journal 446, no. 1 (July 27, 2012): 69–77. http://dx.doi.org/10.1042/bj20120548.
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A new peptide trypsin inhibitor named BWI-2c was obtained from buckwheat (Fagopyrum esculentum) seeds by sequential affinity, ion exchange and reversed-phase chromatography. The peptide was sequenced and found to contain 41 amino acid residues, with four cysteine residues involved in two intramolecular disulfide bonds. Recombinant BWI-2c identical to the natural peptide was produced in Escherichia coli in a form of a cleavable fusion with thioredoxin. The 3D (three-dimensional) structure of the peptide in solution was determined by NMR spectroscopy, revealing two antiparallel α-helices stapled by disulfide bonds. Together with VhTI, a trypsin inhibitor from veronica (Veronica hederifolia), BWI-2c represents a new family of protease inhibitors with an unusual α-helical hairpin fold. The linker sequence between the helices represents the so-called trypsin inhibitory loop responsible for direct binding to the active site of the enzyme that cleaves BWI-2c at the functionally important residue Arg19. The inhibition constant was determined for BWI-2c against trypsin (1.7×10−10 M), and the peptide was tested on other enzymes, including those from various insect digestive systems, revealing high selectivity to trypsin-like proteases. Structural similarity shared by BWI-2c, VhTI and several other plant defence peptides leads to the acknowledgement of a new widespread family of plant peptides termed α-hairpinins.
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Robel, Ivonne, Julia Gebhardt, Jeroen R. Mesters, Alexander Gorbalenya, Bruno Coutard, Bruno Canard, Rolf Hilgenfeld, and Jacques Rohayem. "Functional Characterization of the Cleavage Specificity of the Sapovirus Chymotrypsin-Like Protease." Journal of Virology 82, no. 16 (June 11, 2008): 8085–93. http://dx.doi.org/10.1128/jvi.00693-08.
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ABSTRACT Sapovirus is a positive-stranded RNA virus with a translational strategy based on processing of a polyprotein precursor by a chymotrypsin-like protease. So far, the molecular mechanisms regulating cleavage specificity of the viral protease are poorly understood. In this study, the catalytic activities and substrate specificities of the predicted forms of the viral protease, the 3C-like protease (NS6) and the 3CD-like protease-polymerase (NS6-7), were examined in vitro. The purified NS6 and NS6-7 were able to cleave synthetic peptides (15 to 17 residues) displaying the cleavage sites of the sapovirus polyprotein, both NS6 and NS6-7 proteins being active forms of the viral protease. High-performance liquid chromatography and subsequent mass spectrometry analysis of digested products showed a specific trans cleavage of peptides bearing Gln-Gly, Gln-Ala, Glu-Gly, Glu-Pro, or Glu-Lys at the scissile bond. In contrast, peptides bearing Glu-Ala or Gln-Asp at the scissile bond (NS4-NS5 and NS5-NS6, or NS6-NS7 junctions, respectively) were resistant to trans cleavage by NS6 or NS6-7 proteins, whereas cis cleavage of the Glu-Ala scissile bond of the NS5-NS6 junction was evidenced. Interestingly, the presence of a Phe at position P4 overruled the resistance to trans cleavage of the Glu-Ala junction (NS5-NS6), whereas substitutions at the P1 and P2′ positions altered the cleavage efficiency. The differential cleavage observed is supported by a model of the substrate-binding site of the sapovirus protease, indicating that the P4, P1, and P2′ positions in the substrate modulate the cleavage specificity and efficiency of the sapovirus chymotrypsin-like protease.
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Gallinari, Paola, Debra Brennan, Chiara Nardi, Mirko Brunetti, Licia Tomei, Christian Steinkühler, and Raffaele De Francesco. "Multiple Enzymatic Activities Associated with Recombinant NS3 Protein of Hepatitis C Virus." Journal of Virology 72, no. 8 (August 1, 1998): 6758–69. http://dx.doi.org/10.1128/jvi.72.8.6758-6769.1998.
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ABSTRACT The hepatitis C virus (HCV) nonstructural 3 protein (NS3) contains at least two domains associated with multiple enzymatic activities; a serine protease activity resides in the N-terminal one-third of the protein, whereas RNA helicase activity and RNA-stimulated nucleoside triphosphatase activity are associated with the C-terminal portion. To study the possible mutual influence of these enzymatic activities, a full-length NS3 polypeptide of 67 kDa was expressed as a nonfusion protein in Escherichia coli, purified to homogeneity, and shown to retain all three enzymatic activities. The protease activity of the full-length NS3 was strongly dependent on the activation by a synthetic peptide spanning the central hydrophobic core of the NS4A cofactor. Once complexed with the NS4A-derived peptide, the full-length NS3 protein and the isolated N-terminal protease domain cleaved synthetic peptide substrates with comparable efficiency. We show that, as in the case of the isolated protease domain, the protease activity of full-length NS3 undergoes inhibition by the N-terminal cleavage products of substrate peptides corresponding to the NS4A-NS4B and NS5A-NS5B. We have also characterized and quantified the NS3 ATPase, RNA helicase, and RNA-binding activities under optimized reaction conditions. Compared with the isolated N-terminal and C-terminal domains, recombinant full-length NS3 did not show significant differences in the three enzymatic activities analyzed in independent in vitro assays. We have further explored the possible interdependence of the NS3 N-terminal and C-terminal domains by analyzing the effect of polynucleotides on the modulation of all NS3 enzymatic functions. Our results demonstrated that the observed inhibition of the NS3 proteolytic activity by single-stranded RNA is mediated by direct interaction with the protease domain rather than with the helicase RNA-binding domain.
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Young, John K., Donghui Li, Matthew C. Abramowitz, and Trudy G. Morrison. "Interaction of Peptides with Sequences from the Newcastle Disease Virus Fusion Protein Heptad Repeat Regions." Journal of Virology 73, no. 7 (July 1, 1999): 5945–56. http://dx.doi.org/10.1128/jvi.73.7.5945-5956.1999.
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ABSTRACT Typical of many viral fusion proteins, the sequence of the Newcastle disease virus (NDV) fusion protein has several heptad repeat regions. One, HR1, is located just carboxyl terminal to the fusion peptide, while the other, HR2, is located adjacent to the transmembrane domain. The structure and function of a synthetic peptide with a sequence from the region of the NDV HR1 region (amino acids 150 to 173) were characterized. The peptide inhibited fusion with a half-maximal concentration of approximately 2 μM; however, inhibition was observed only if the peptide was added prior to protease activation of the fusion protein. This inhibition was virus specific since the peptide had minimal effect on fusion directed by the Sendai virus glycoproteins. To explore the mechanism of action, the potential HR1 peptide interaction with a previously characterized fusion inhibitory peptide with a sequence from the HR2 domain (J. K. Young, R. P. Hicks, G. E. Wright, and T. G. Morrison, Virology 238:291–304, 1997) was characterized. The results demonstrated an interaction between the two peptides both functionally and directly. First, while the individual peptides each inhibit fusion, equimolar mixtures of the two peptides had minimal effect on fusion, suggesting that the two peptides form a complex preventing their interaction with a target protein. Second, an HR2 peptide covalently linked with biotin was found to bind specifically to HR1 peptide in a Western blot. The structure of the HR1 peptide was analyzed by nuclear magnetic resonance spectroscopy and found to be an α helix.
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Khan, Selina, Albert Zimmermann, Michael Basler, Marcus Groettrup, and Hartmut Hengel. "A Cytomegalovirus Inhibitor of Gamma Interferon Signaling Controls Immunoproteasome Induction." Journal of Virology 78, no. 4 (February 15, 2004): 1831–42. http://dx.doi.org/10.1128/jvi.78.4.1831-1842.2004.
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ABSTRACT Both human and mouse cytomegaloviruses (HCMV and MCMV) avoid peptide presentation through the major histocompatibility complex (MHC) class I pathway to CD8+ T cells. Within the MHC class I pathway, the vast majority of antigenic peptides are generated by the proteasome system, a multicatalytic protease complex consisting of constitutive subunits, three of which can be replaced by enzymatically active gamma interferon (IFN-γ)-inducible subunits, i.e., LMP2, LMP7, and MECL1, to form the so-called immunoproteasomes. Here, we show that steady-state levels of immunoproteasomes are readily formed in response to MCMV infection in the liver. In contrast, the incorporation of immunoproteasome subunits was prevented in MCMV-infected, as well as HCMV-infected, fibroblasts in vitro. Likewise, the expression of the IFN-γ-inducible proteasome regulator PA28αβ was also impaired in MCMV-infected cells. Both MCMV and HCMV did not alter the constitutive-subunit composition of proteasomes in infected cells. Quantitative assessment of LMP2, MECL1, and LMP7 transcripts revealed that the inhibition of immunoproteasome formation occurred at a pretranscriptional level. Remarkably, a targeted deletion of the MCMV gene M27, encoding an inhibitor of STAT2 that disrupts IFN-γ receptor signaling, largely restored transcription and protein expression of immunoproteasome subunits in infected cells. While CMV block peptide transport and MHC class I assembly by posttranslational strategies, immunoproteasome assembly, and thus the repertoire of proteasomal peptides, is controlled by pretranscriptional mechanisms. We hypothesize that the blockade of immunoproteasome formation has considerable consequences for shaping the CD8+-T-cell repertoire during the effector phase of the immune response.
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Mitpuangchon, Natrada, Kwan Nualcharoen, Singtoe Boonrotpong, and Patamarerk Engsontia. "Identification of Novel Toxin Genes from the Stinging Nettle Caterpillar Parasa lepida (Cramer, 1799): Insights into the Evolution of Lepidoptera Toxins." Insects 12, no. 5 (April 29, 2021): 396. http://dx.doi.org/10.3390/insects12050396.
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Many animal species can produce venom for defense, predation, and competition. The venom usually contains diverse peptide and protein toxins, including neurotoxins, proteolytic enzymes, protease inhibitors, and allergens. Some drugs for cancer, neurological disorders, and analgesics were developed based on animal toxin structures and functions. Several caterpillar species possess venoms that cause varying effects on humans both locally and systemically. However, toxins from only a few species have been investigated, limiting the full understanding of the Lepidoptera toxin diversity and evolution. We used the RNA-seq technique to identify toxin genes from the stinging nettle caterpillar, Parasa lepida (Cramer, 1799). We constructed a transcriptome from caterpillar urticating hairs and reported 34,968 unique transcripts. Using our toxin gene annotation pipeline, we identified 168 candidate toxin genes, including protease inhibitors, proteolytic enzymes, and allergens. The 21 P. lepida novel Knottin-like peptides, which do not show sequence similarity to any known peptide, have predicted 3D structures similar to tarantula, scorpion, and cone snail neurotoxins. We highlighted the importance of convergent evolution in the Lepidoptera toxin evolution and the possible mechanisms. This study opens a new path to understanding the hidden diversity of Lepidoptera toxins, which could be a fruitful source for developing new drugs.
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Welch, Brett D., J. Nicholas Francis, Joseph S. Redman, Suparna Paul, Matthew T. Weinstock, Jacqueline D. Reeves, Yolanda S. Lie, et al. "Design of a Potent d-Peptide HIV-1 Entry Inhibitor with a Strong Barrier to Resistance." Journal of Virology 84, no. 21 (August 18, 2010): 11235–44. http://dx.doi.org/10.1128/jvi.01339-10.
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ABSTRACT The HIV gp41 N-trimer pocket region is an ideal viral target because it is extracellular, highly conserved, and essential for viral entry. Here, we report on the design of a pocket-specific d-peptide, PIE12-trimer, that is extraordinarily elusive to resistance and characterize its inhibitory and structural properties. d-Peptides (peptides composed of d-amino acids) are promising therapeutic agents due to their insensitivity to protease degradation. PIE12-trimer was designed using structure-guided mirror-image phage display and linker optimization and is the first d-peptide HIV entry inhibitor with the breadth and potency required for clinical use. PIE12-trimer has an ultrahigh affinity for the gp41 pocket, providing it with a reserve of binding energy (resistance capacitor) that yields a dramatically improved resistance profile compared to those of other fusion inhibitors. These results demonstrate that the gp41 pocket is an ideal drug target and establish PIE12-trimer as a leading anti-HIV antiviral candidate.
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Krishnan, Manigandan, Joonhyeok Choi, Ahjin Jang, and Yangmee Kim. "A Novel Peptide Antibiotic, Pro10-1D, Designed from Insect Defensin Shows Antibacterial and Anti-Inflammatory Activities in Sepsis Models." International Journal of Molecular Sciences 21, no. 17 (August 27, 2020): 6216. http://dx.doi.org/10.3390/ijms21176216.
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Owing to the challenges faced by conventional therapeutics, novel peptide antibiotics against multidrug-resistant (MDR) gram-negative bacteria need to be urgently developed. We had previously designed Pro9-3 and Pro9-3D from the defensin of beetle Protaetia brevitarsis; they showed high antimicrobial activity with cytotoxicity. Here, we aimed to develop peptide antibiotics with bacterial cell selectivity and potent antibacterial activity against gram-negative bacteria. We designed 10-meric peptides with increased cationicity by adding Arg to the N-terminus of Pro9-3 (Pro10-1) and its D-enantiomeric alteration (Pro10-1D). Among all tested peptides, the newly designed Pro10-1D showed the strongest antibacterial activity against Escherichia coli, Acinetobacter baumannii, and MDR strains with resistance against protease digestion. Pro10-1D can act as a novel potent peptide antibiotic owing to its outstanding inhibitory activities against bacterial film formation with high bacterial cell selectivity. Dye leakage and scanning electron microscopy revealed that Pro10-1D targets the bacterial membrane. Pro10-1D inhibited inflammation via Toll Like Receptor 4 (TLR4)/Nuclear factor-κB (NF-κB) signaling pathways in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Furthermore, Pro10-1D ameliorated multiple-organ damage and attenuated systemic infection-associated inflammation in an E. coli K1-induced sepsis mouse model. Overall, our results suggest that Pro10-1D can potentially serve as a novel peptide antibiotic for the treatment of gram-negative sepsis.
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Cabral, C. M., A. Cherqui, A. Pereira, and N. Simões. "Purification and Characterization of Two Distinct Metalloproteases Secreted by the Entomopathogenic Bacterium Photorhabdus sp. Strain Az29." Applied and Environmental Microbiology 70, no. 7 (July 2004): 3831–38. http://dx.doi.org/10.1128/aem.70.7.3831-3838.2004.
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ABSTRACT Photorhabdus sp. strain Az29 is symbiotic with an Azorean nematode of the genus Heterorhabditis in a complex that is highly virulent to insects even at low temperatures. The virulence of the bacteria is mainly attributed to toxins and bacterial enzymes secreted during parasitism. The bacteria secrete proteases during growth, with a peak at the end of the exponential growth phase. Protease secretion was higher in cultures growing at lower temperatures. At 10°C the activity was highest and remained constant for over 7 days, whereas at 23 and 28°C it showed a steady decrease. Two proteases, PrtA and PrtS, that are produced in the growth medium were purified by liquid chromatography. PrtA was inhibited by 1,10-phenantroline and by EDTA and had a molecular mass of 56 kDa and an optimal activity at pH 9 and 50°C. Sequences of three peptides of PrtA showed strong homologies with alkaline metalloproteases from Photorhabdus temperata K122 and Photorhabdus luminescens W14. Peptide PrtA-36 contained the residues characteristic of metzincins, known to be involved in bacterial virulence. In vitro, PrtA inhibited antibacterial factors of inoculated Lepidoptera and of cecropins A and B. PrtS had a molecular mass of 38 kDa and was inhibited by 1,10-phenanthroline but not by EDTA. Its activity ranged between 10 and 80°C and was optimal at pH 7 and 50°C. PrtS also destroyed insect antibacterial factors. Three fragments of PrtS showed homology with a putative metalloprotease of P. luminescens TTO1. Polyclonal antibody raised against PrtA did not recognize PrtS, showing they are distinct molecules.
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Leni, G., L. Soetemans, J. Jacobs, S. Depraetere, N. Gianotten, L. Bastiaens, A. Caligiani, and S. Sforza. "Protein hydrolysates from Alphitobius diaperinus and Hermetia illucens larvae treated with commercial proteases." Journal of Insects as Food and Feed 6, no. 4 (August 11, 2020): 393–404. http://dx.doi.org/10.3920/jiff2019.0037.
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Insect proteins have been proposed as a promising alternative for feed and food formulations. In the present work protease-assisted extraction was studied as a way to separate and extract proteins from two different insect species: Alphitobius diaperinus (AD) and Hermetia illucens (HI). The proteolytic activity of seven enzymes (papain, pancreatin, dispase I, pepsin, protease from Bacillus licheniformis, bromelain and trypsin) was evaluated determining the protein extraction yield, the degree of hydrolysis (DH) and the released free amino acids (FAA). Both insects represent an interesting source of proteins, not only for their amount (more than 40% on dry matter) but also for the nutritional value, with essential amino acid profile exceeding the requirements proposed for human nutrition. Enzyme-assisted protein extraction, performed at laboratory scale, gave for HI an average yield of extraction of 71±8% and for AD 67±6%. Hydrolysates produced from HI gave a DH% ranging between 3 to 18%, whereas hydrolysates produced from AD yielded a DH% between 7 to 23%. The protein hydrolysates were composed by peptides and FAA (which accounted for more than 30% of the extracted protein fraction), which were released according to their abundance in initial protein. A moderate correlation between the DH% and the total amount of FAA was found, except for AD hydrolysed with trypsin and HI with papain. Based on these results, the production of hydrolysates was preliminary scaled up in a proof-of-concept experiment, focusing on the most promising insect-enzyme combination. The final product resulted to be rich in protein (60% on dry matter). This work support enzymatic hydrolysis as an effective method to extract and isolate proteins from insects, with minimal sample preparation, tailoring their composition, preserving the nutritional quality, decreasing the risk of allergic reactions and making them more accessible for their future use as feed/food supplements.
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Chabry, Joëlle, Suzette A. Priola, Kathy Wehrly, Jane Nishio, James Hope, and Bruce Chesebro. "Species-Independent Inhibition of Abnormal Prion Protein (PrP) Formation by a Peptide Containing a Conserved PrP Sequence." Journal of Virology 73, no. 8 (August 1, 1999): 6245–50. http://dx.doi.org/10.1128/jvi.73.8.6245-6250.1999.
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ABSTRACT Conversion of the normal protease-sensitive prion protein (PrP) to its abnormal protease-resistant isoform (PrP-res) is a major feature of the pathogenesis associated with transmissible spongiform encephalopathy (TSE) diseases. In previous experiments, PrP conversion was inhibited by a peptide composed of hamster PrP residues 109 to 141, suggesting that this region of the PrP molecule plays a crucial role in the conversion process. In this study, we used PrP-res derived from animals infected with two different mouse scrapie strains and one hamster scrapie strain to investigate the species specificity of these conversion reactions. Conversion of PrP was found to be completely species specific; however, despite having three amino acid differences, peptides corresponding to the hamster and mouse PrP sequences from residues 109 to 141 inhibited both the mouse and hamster PrP conversion systems equally. Furthermore, a peptide corresponding to hamster PrP residues 119 to 136, which was identical in both mouse and hamster PrP, was able to inhibit PrP-res formation in both the mouse and hamster cell-free systems as well as in scrapie-infected mouse neuroblastoma cell cultures. Because the PrP region from 119 to 136 is very conserved in most species, this peptide may have inhibitory effects on PrP conversion in a wide variety of TSE diseases.
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Freimoser, Florian M., Steven Screen, Gang Hu, and Raymond St. Leger. "EST analysis of genes expressed by the zygomycete pathogen Conidiobolus coronatus during growth on insect cuticle." Microbiology 149, no. 7 (July 1, 2003): 1893–900. http://dx.doi.org/10.1099/mic.0.26252-0.
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Conidiobolus coronatus (Zygomycota) is a facultative saprobe that is a pathogen of many insect species. Almost 2000 expressed sequence tag (EST) cDNA clones were sequenced to analyse gene expression during growth on insect cuticle. Sixty percent of the ESTs that could be clustered into functional groups (E⩽10−5) had their best blast hits among fungal sequences. These included chitinases and multiple subtilisins, trypsin, metalloprotease and aspartyl protease activities with the potential to degrade host tissues and disable anti-microbial peptides. Otherwise, compared to the ascomycete entomopathogen Metarhizium anisopliae, Con. coronatus produced many fewer types of hydrolases (e.g. no phospholipases), antimicrobial agents, toxic secondary metabolites and no ESTs with putative roles in the generation of antibiotics. Instead, Con. coronatus produced a much higher proportion of ESTs encoding ribosomal proteins and enzymes of intermediate metabolism that facilitate its rapid growth. These results are consistent with Con. coronatus having adapted a modification of the saprophytic ruderal-selected strategy, using rapid growth to overwhelm the host and exploit the cadaver before competitors overrun it. This strategy does not preclude specialization to pathogenicity, as Con. coronatus produces the greatest complexity of proteases on insect cuticle, indicating an ability to respond to conditions in the cuticle.
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Stratov, Ivan, C. Jane Dale, Socheata Chea, James McCluskey, and Stephen J. Kent. "Induction of T-Cell Immunity to Antiretroviral Drug-Resistant Human Immunodeficiency Virus Type 1." Journal of Virology 79, no. 12 (June 15, 2005): 7728–37. http://dx.doi.org/10.1128/jvi.79.12.7728-7737.2005.
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ABSTRACT Antiretroviral drug-resistant human immunodeficiency virus type 1 (HIV-1) is a major, growing, public health problem. Immune responses targeting epitopes spanning drug resistance sites could ameliorate development of drug resistance. We studied 25 individuals harboring multidrug-resistant HIV-1 for T-cell immunity to HIV-1 proteins and peptides spanning all common drug resistance mutations. CD8 T cells targeting epitopes spanning drug-induced mutations were detected but only in the 3 individuals with robust HIV-specific T-cell activity. Novel CD8 T-cell responses were detected against the common L63P and L10I protease inhibitor fitness mutations. Induction of T-cell immunity to drug-resistant variants was demonstrated in simian human immunodeficiency virus-infected macaques, where both CD8 and CD4 T-cell immune responses to reverse transcriptase and protease antiretroviral mutations were elicited using a novel peptide-based immunotherapy. T-cell responses to antiretroviral resistance mutations were strongest in the most immunocompetent animals. This study suggests feasible strategies to further evaluate the potential of limiting antiretroviral drug resistance through induction of T-cell immunity.
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Vincent, Martin J., Angela J. Sanchez, Bobbie R. Erickson, Ajoy Basak, Michel Chretien, Nabil G. Seidah, and Stuart T. Nichol. "Crimean-Congo Hemorrhagic Fever Virus Glycoprotein Proteolytic Processing by Subtilase SKI-1." Journal of Virology 77, no. 16 (August 15, 2003): 8640–49. http://dx.doi.org/10.1128/jvi.77.16.8640-8649.2003.
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ABSTRACT Crimean-Congo hemorrhagic fever (CCHF) virus is a tick-borne member of the genus Nairovirus, family Bunyaviridae. The mature virus glycoproteins, Gn and Gc (previously referred to as G2 and G1), are generated by proteolytic cleavage from precursor proteins. The amino termini of Gn and Gc are immediately preceded by tetrapeptides RRLL and RKPL, respectively, leading to the hypothesis that SKI-1 or related proteases may be involved (A. J. Sanchez, M. J. Vincent, and S. T. Nichol, J. Virol. 76:7263-7275, 2002). In vitro peptide cleavage data show that an RRLL peptide representing the Gn processing site is efficiently cleaved by SKI-1 protease, whereas an RKPL peptide representing the Gc processing site is cleaved at negligible levels. The efficient cleavage of RRLL peptide is consistent with the known recognition sequences of SKI-1, including the sequence determinants involved in the cleavage of the Lassa virus (family Arenaviridae) glycoprotein precursor. These in vitro findings were confirmed by expression of wild-type or mutant CCHF virus glycoproteins in CHO cells engineered to express functional or nonfunctional SKI-1. Gn processing was found to be dependent on functional SKI-1, whereas Gc processing was not. Gn processing occurred in the endoplasmic reticulum-cis Golgi compartments and was dependent on an R at the −4 position within the RRLL recognition motif, consistent with the known cleavage properties of SKI-1. Comparison of SKI-1 cleavage efficiency between peptides representing Lassa virus GP2 and CCHF virus Gn cleavage sites suggests that amino acids flanking the RRLL may modulate the efficiency. The apparent lack of SKI-1 cleavage at the CCHF virus Gc RKPL site indicates that related proteases, other than SKI-1, are likely to be involved in the processing at this site and identical or similar sites utilized in several New World arenaviruses.
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Da Costa, Bruno, Stéphanie Soignier, Christophe Chevalier, Celine Henry, Corinne Thory, Jean-Claude Huet, and Bernard Delmas. "Blotched Snakehead Virus Is a New Aquatic Birnavirus That Is Slightly More Related to Avibirnavirus Than to Aquabirnavirus." Journal of Virology 77, no. 1 (January 1, 2003): 719–25. http://dx.doi.org/10.1128/jvi.77.1.719-725.2003.
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ABSTRACT By different approaches, we characterized the birnavirus blotched snakehead virus (BSNV). The sequence of genomic segment A revealed the presence of two open reading frames (ORFs): a large ORF with a 3,207-bp-long nucleotide sequence and a 417-nucleotide-long small ORF located within the N-terminal half of the large ORF, but in a different reading frame. The large ORF was found to encode a polyprotein cotranslationally processed by the viral protease VP4 to generate pVP2 (the VP2 precursor), a 71-amino-acid-long peptide ([X]), VP4, and VP3. The two cleavage sites at the [X]-VP4 and VP4-VP3 junctions were identified by N-terminal sequencing. We showed that the processing of pVP2 generated VP2 and several small peptides (amino acids [aa] 418 to 460, 461 to 467, 468 to 474, and 475 to 486). Two of these peptides (aa 418 to 460 and 475 to 486) were positively identified in the viral particles with 10 additional peptides derived from further processing of the peptide aa 418 to 460. The results suggest that VP4 cleaves multiple Pro-X-Ala↓Ala motifs, with the notable exception of the VP4-VP3 junction. Replacement of the members of the predicted VP4 catalytic dyad (Ser-692 and Lys-729) confirmed their indispensability in the polyprotein processing. The genomic segment B sequence revealed a single large ORF encoding a putative polymerase, VP1. Our results demonstrate that BSNV should be considered a new aquatic birnavirus species, slightly more related to IBDV than to IPNV.
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St. Leger, Raymond J. "The role of cuticle-degrading proteases in fungal pathogenesis of insects." Canadian Journal of Botany 73, S1 (December 31, 1995): 1119–25. http://dx.doi.org/10.1139/b95-367.
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The proteinaceous outer integument of insects forms an effective barrier against most microbes. Only the 700 known species of entomopathogenic fungi effect entry into their hosts by breaching the cuticle. There is accumulating evidence that the ability of fungi to degrade protein may aid their invasion of and growth in this orderly complex structure. Evidence for the particular importance of proteinases derives largely from studies of their production in infected cuticles associated with cuticle degradation, the effects of proteinase inhibitors on pathogen behavior, and by the analysis of protease-deficient mutants. More recently, studies have included the cloning, identification, and manipulation of specific protease genes of Metarhizium anisopliae, particularly those of the subtilisin (chymoelastase) type (designated Pr1) also produced by many other entomopathogenic fungi. Following solubilization of cuticle proteins by Pr1-type endoproteases, complete degradation of the cuticle involves a number of interacting enzyme species including a family of trypsin-like proteinases, metalloproteinases, several aminopeptidases, and a carboxypeptidase. Testing genetically engineered M. anisopliae null mutants of Pr1 indicated that the other endopeptidases can partially substitute for Pr1. The exopeptidases further degrade peptides released by the endopeptidases producing free amino acids for uptake and metabolism. Utilization of these enzymes has assisted investigators in understanding cuticle structure and how the cuticle is degraded naturally, and could lead to improved strain selection of entomopathogenic fungi or the introduction of their genes into other microbes and plants for the purpose of insect control. Key words: proteinaceous insect cuticle, pathogen endopeptidases, exopeptidases, multiple isozymes, enzyme regulation.
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Ujike, Makoto, Hiroki Nishikawa, Akira Otaka, Naoki Yamamoto, Norio Yamamoto, Masao Matsuoka, Eiichi Kodama, Nobutaka Fujii, and Fumihiro Taguchi. "Heptad Repeat-Derived Peptides Block Protease-Mediated Direct Entry from the Cell Surface of Severe Acute Respiratory Syndrome Coronavirus but Not Entry via the Endosomal Pathway." Journal of Virology 82, no. 1 (October 17, 2007): 588–92. http://dx.doi.org/10.1128/jvi.01697-07.
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ABSTRACT The peptides derived from the heptad repeat (HRP) of severe acute respiratory syndrome coronavirus (SCoV) spike protein (sHRPs) are known to inhibit SCoV infection, yet their efficacies are fairly low. Recently our research showed that some proteases facilitated SCoV's direct entry from the cell surface, resulting in a more efficient infection than the previously known infection via endosomal entry. To compare the inhibitory effect of the sHRP in each pathway, we selected two sHRPs, which showed a strong inhibitory effect on the interaction of two heptad repeats in a rapid and virus-free in vitro assay system. We found that they efficiently inhibited SCoV infection of the protease-mediated cell surface pathway but had little effect on the endosomal pathway. This finding suggests that sHRPs may effectively prevent infection in the lungs, where SCoV infection could be enhanced by proteases produced in this organ. This is the first observation that HRP exhibits different effects on virus that takes the endosomal pathway and virus that enters directly from the cell surface.
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Gordon, M. M., T. Howard, M. J. Becich, and D. H. Alpers. "Cathepsin L mediates intracellular ileal digestion of gastric intrinsic factor." American Journal of Physiology-Gastrointestinal and Liver Physiology 268, no. 1 (January 1, 1995): G33—G40. http://dx.doi.org/10.1152/ajpgi.1995.268.1.g33.
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Although acidic proteases of lysosomal origin are implicated in the degradation of intrinsic factor (IF) during cobalamin (cbl) transport across enterocytes and proximal renal tubule cell lines, the enzyme(s) involved in this process is not known. Recombinant (baculovirus-produced) rat 125I-labeled IF (125I-rIF), 43 kDa, added in vivo to the lumen of rat ileum was converted intracellularly to peptides of 33 and 26 kDa. In vitro rat 125I-rIF was degraded to peptides of 33 and 31 kDa by addition of cathepsin L; this conversion was fully inhibited by leupeptin. Western blot analysis using antiserum against denatured native rat IF identified additional cathepsin L degradation products in the 17- to 23-kDa range. In vitro the binding of cobalamin partially inhibited cathepsin L degradation of IF. Rat rIF produced from either insect (Sf9) or mammalian (CHO) cells and native rat IF were all degraded by cathepsin L, although the prominence of the various products differed in the recombinant preparations, being 33 and 36 kDa, respectively. Native rat IF was most sensitive to proteolysis, and no degradation products were identified. Rat 125I-rIF was taken up by LLC-PK1 cells, and 125I from degraded IF appeared abundantly on the basolateral side of cell monolayers by 1 h. The intracellular products of rat rIF in LLC-PK1 cells were the same size as those produced in vitro by the action of cathepsin L. Antiserum against a human kidney cDNA cathepsin L fusion protein easily demonstrated the protease in rat intestinal mucosa, as well as in all other tissues tested. These data suggest that cathepsin L is the protease responsible for the leupeptin-sensitive intracellular degradation of IF.
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Mason, R. D., M. I. Bowmer, C. M. Howley, and M. D. Grant. "Cross-Reactive Cytotoxic T Lymphocytes against Human Immunodeficiency Virus Type 1 Protease and Gamma Interferon-Inducible Protein 30." Journal of Virology 79, no. 9 (May 1, 2005): 5529–36. http://dx.doi.org/10.1128/jvi.79.9.5529-5536.2005.
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ABSTRACT The gamma interferon (IFN-γ)-inducible protein 30 (IP-30) signal peptide −11 to −3 (LLDVPTAAV) is a prominent self peptide expressed with the class I human histocompatibility leukocyte antigen A2 (HLA-A2). Stimulation of peripheral blood mononuclear cells (PBMC) from HLA-A2 human immunodeficiency virus type 1 (HIV-1)-infected individuals with an HLA-A2-restricted HIV protease (PR) peptide 76-84 (LVGPTPVNI) activated cytotoxic T lymphocytes (CTL) against the IP-30 signal peptide. Since HIV-1 PR 76-84 stimulated CD8+ T cells from these individuals to secrete IFN-γ, we tested whether the activation of IP-30-specific CTL in vitro resulted from T-cell cross-reactivity or from up-regulation of IP-30 by IFN-γ. Neither high levels of exogenous IFN-γ nor incubation of PBMC with other HIV peptides triggering substantial IFN-γ release activated IP-30-specific CTL. Although the IP-30 signal peptide did not stimulate IFN-γ release from freshly isolated PBMC, it activated CTL in vitro against itself and HIV PR 76-84. Peptide-stimulated IFN-γ release, cold target inhibition, and HLA-A2/immunoglobulin dimer-mediated binding and depletion of effector cells all indicated that in vitro stimulation with HIV PR 76-84 or the IP-30 signal peptide activated a comparable population of cross-reactive effector cells. Neither IP-30 nor HIV PR 76-84 activated CTL against themselves following in vitro stimulation of PBMC from non-HIV-infected HLA-A2 individuals. Peptide titrations indicated higher-avidity T-cell interactions with HIV PR 76-84 than with the IP-30 signal peptide. These data indicate that HIV PR 76-84 is a heteroclitic variant of the IP-30 signal peptide −11 to −3, which has implications for immune memory and autoimmunity.
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Nelson, Christian D. S., Eveliina Minkkinen, Magnus Bergkvist, Karin Hoelzer, Mathew Fisher, Brian Bothner, and Colin R. Parrish. "Detecting Small Changes and Additional Peptides in the Canine Parvovirus Capsid Structure." Journal of Virology 82, no. 21 (August 13, 2008): 10397–407. http://dx.doi.org/10.1128/jvi.00972-08.
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ABSTRACT Parvovirus capsids are assembled from multiple forms of a single protein and are quite stable structurally. However, in order to infect cells, conformational plasticity of the capsid is required and this likely involves the exposure of structures that are buried within the structural models. The presence of functional asymmetry in the otherwise icosahedral capsid has also been proposed. Here we examined the protein composition of canine parvovirus capsids and evaluated their structural variation and permeability by protease sensitivity, spectrofluorometry, and negative staining electron microscopy. Additional protein forms identified included an apparent smaller variant of the virus protein 1 (VP1) and a small proportion of a cleaved form of VP2. Only a small percentage of the proteins in intact capsids were cleaved by any of the proteases tested. The capsid susceptibility to proteolysis varied with temperature but new cleavages were not revealed. No global change in the capsid structure was observed by analysis of Trp fluorescence when capsids were heated between 40°C and 60°C. However, increased polarity of empty capsids was indicated by bis-ANS binding, something not seen for DNA-containing capsids. Removal of calcium with EGTA or exposure to pHs as low as 5.0 had little effect on the structure, but at pH 4.0 changes were revealed by proteinase K digestion. Exposure of viral DNA to the external environment started above 50°C. Some negative stains showed increased permeability of empty capsids at higher temperatures, but no effects were seen after EGTA treatment.
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Kellenberger, Christine, Christian Boudier, Isabel Bermudez, Joseph G. Bieth, Bang Luu, and Hélène Hietter. "Serine Protease Inhibition by Insect Peptides Containing a Cysteine Knot and a Triple-stranded β-Sheet." Journal of Biological Chemistry 270, no. 43 (October 1995): 25514–19. http://dx.doi.org/10.1074/jbc.270.43.25514.
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SACRAMENTO, R. S., R. M. MARTINS, A. MIRANDA, A. S. S. DOBROFF, S. DAFFRE, A. S. FORONDA, D. DE FREITAS, and S. SCHENKMAN. "Differential effects of α-helical and β-hairpin antimicrobial peptides against Acanthamoeba castellanii." Parasitology 136, no. 8 (June 2, 2009): 813–21. http://dx.doi.org/10.1017/s0031182009006283.
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SUMMARYIn this work we evaluated the ability of different types of antimicrobial peptides to promote permeabilization and growth inhibition of Acanthamoeba castellanii trophozoites, which cause eye keratitis. We used cationic α-helical peptides P5 and P6, corresponding to the N-terminus of the pore-forming protein from Triatoma infestans, a blood-sucking insect, and a β-hairpin amphipathic molecule (gomesin), of the spider Acanthoscurria gomesiana haemocytes. A. castellanii permeabilization was obtained after 1 h incubation with micromolar concentrations of both types of peptides. While permeabilization induced by gomesin increased with longer incubations, P5 permeabilization did not increase with time and occurred at doses that are more toxic for SIRC cells. P5, however, at doses below the critical dose used to kill rabbit corneal cells was quite effective in promoting growth inhibition. Similarly, P5 was more effective when serine protease inhibitor was added simultaneously to the permeabilization assay. High performance chromatography followed by mass spectrometry analysis confirmed that, in contrast to gomesin, P5 is hydrolysed by A. castellanii culture supernatants. We conclude that the use of antimicrobial peptides to treat A. castellanii infections requires the search of more specific peptides that are resistant to proteolysis.
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Crim, Roberta L., Susette A. Audet, Steven A. Feldman, Howard S. Mostowski, and Judy A. Beeler. "Identification of Linear Heparin-Binding Peptides Derived from Human Respiratory Syncytial Virus Fusion Glycoprotein That Inhibit Infectivity." Journal of Virology 81, no. 1 (October 18, 2006): 261–71. http://dx.doi.org/10.1128/jvi.01226-06.
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ABSTRACT It has been shown previously that the fusion glycoprotein of human respiratory syncytial virus (RSV-F) interacts with cellular heparan sulfate. Synthetic overlapping peptides derived from the F-protein sequence of RSV subtype A (strain A2) were tested for their ability to bind heparin using heparin-agarose affinity chromatography (HAAC). This evaluation identified 15 peptides representing eight linear heparin-binding domains (HBDs) located within F1 and F2 and spanning the protease cleavage activation site. All peptides bound to Vero and A549 cells, and binding was inhibited by soluble heparins and diminished by either enzymatic treatment to remove cell surface glycosaminoglycans or by treatment with sodium chlorate to decrease cellular sulfation. RSV-F HBD peptides were less likely to bind to glycosaminoglycan-deficient CHO-745 cells than parental CHO-K1 cells that express these molecules. Three RSV-F HBD peptides (F16, F26, and F55) inhibited virus infectivity; two of these peptides (F16 and F55) inhibited binding of virus to Vero cells, while the third (F26) did not. These studies provided evidence that two of the linear HBDs mapped by peptides F16 and F55 may mediate one of the first steps in the attachment of virus to cells while the third, F26, inhibited infectivity at a postattachment step, suggesting that interactions with cell surface glycosaminoglycans may play a role in infectivity of some RSV strains.
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Prabu-Jeyabalan, Moses, Ellen A. Nalivaika, Nancy M. King, and Celia A. Schiffer. "Viability of a Drug-Resistant Human Immunodeficiency Virus Type 1 Protease Variant: Structural Insights for Better Antiviral Therapy." Journal of Virology 77, no. 2 (January 15, 2003): 1306–15. http://dx.doi.org/10.1128/jvi.77.2.1306-1315.2003.
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ABSTRACT Under the selective pressure of protease inhibitor therapy, patients infected with human immunodeficiency virus (HIV) often develop drug-resistant HIV strains. One of the first drug-resistant mutations to arise in the protease, particularly in patients receiving indinavir or ritonavir treatment, is V82A, which compromises the binding of these and other inhibitors but allows the virus to remain viable. To probe this drug resistance, we solved the crystal structures of three natural substrates and two commercial drugs in complex with an inactive drug-resistant mutant (D25N/V82A) HIV-1 protease. Through structural analysis and comparison of the protein-ligand interactions, we found that Val82 interacts more closely with the drugs than with the natural substrate peptides. The V82A mutation compromises these interactions with the drugs while not greatly affecting the substrate interactions, which is consistent with previously published kinetic data. Coupled with our earlier observations, these findings suggest that future inhibitor design may reduce the probability of the appearance of drug-resistant mutations by targeting residues that are essential for substrate recognition.
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Darsouei, Reyhaneh, Javad Karimi, and Gary B. Dunphy. "Functional Characterization of Outer Membrane Proteins (OMPs) in Xenorhabdus nematophila and Photorhabdus luminescens through Insect Immune Defense Reactions." Insects 10, no. 10 (October 17, 2019): 352. http://dx.doi.org/10.3390/insects10100352.
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Xenorhabdus nematophila and Photorhabdus luminescens are entomopathogenic bacterial symbionts that produce toxic proteins that can interfere with the immune system of insects. Herein, we show that outer membrane proteins (OMPs) could be involved as bacterial virulence factors. Purified totals OMPs of both bacterial species were injected into fifth instar larvae of Spodoptera exigua Hübner. Larvae were surveyed for cellular defenses fluctuations in total haemocyte counts (THC) and granulocyte percentage and for the humoral defenses protease, phospholipase A2 (PLA2), and phenoloxidase (PO) activities at specific time intervals. Changes in the expression of the three inducible antimicrobial peptides (AMPs), cecropin, attacin, and spodoptericin, were also measured. Larvae treated with OMPs of both bacterial species had more haemocytes than did the negative controls. OMPs of X. nematophila caused more haemocyte destruction than did the OMPs of P. luminescens. The OMPs of both bacterial species initially activated insect defensive enzymes post-injection, the degree of activation varying with enzyme type. The AMPs, attacin, cecropin, and spodoptericin were up-regulated by OMP injections compared with the normal larvae. The expression of these three AMPs was maximal at four hours post injection (hpi) with P. luminescens OMPs treatment. Expression of the three AMPs in X. nematophila treated insects was irregular and lower than in the P. luminescens OMPs treatment. These findings provide insights into the role of OMPs of entomopathogenic nematode bacterial symbionts in countering the physiological defenses of insects.
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Rahman, Khalidur, Mohd Amir F. Abdullah, Suresh Ambati, Milton D. Taylor, and Michael J. Adang. "Differential Protection of Cry1Fa Toxin against Spodoptera frugiperda Larval Gut Proteases by Cadherin Orthologs Correlates with Increased Synergism." Applied and Environmental Microbiology 78, no. 2 (November 11, 2011): 354–62. http://dx.doi.org/10.1128/aem.06212-11.
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ABSTRACTThe Cry proteins produced byBacillus thuringiensis(Bt) are the most widely used biopesticides effective against a range of crop pests and disease vectors. Like chemical pesticides, development of resistance is the primary threat to the long-term efficacy of Bt toxins. Recently discovered cadherin-based Bt Cry synergists showed the potential to augment resistance management by improving efficacy of Cry toxins. However, the mode of action of Bt Cry synergists is thus far unclear. Here we elucidate the mechanism of cadherin-based Cry toxin synergism utilizing two cadherin peptides,Spodoptera frugiperdaCad (SfCad) andManduca sextaCad (MsCad), which differentially enhance Cry1Fa toxicity toSpodoptera frugiperdaneonates. We show that differential SfCad- and MsCad-mediated protection of Cry1Fa toxin in theSpodoptera frugiperdamidgut correlates with differential Cry1Fa toxicity enhancement. Both peptides exhibited high affinity for Cry1Fa toxin and an increased rate of Cry1Fa-induced pore formation inS. frugiperda. However, only SfCad bound theS. frugiperdabrush border membrane vesicle and more effectively prolonged the stability of Cry1Fa toxin in the gut, explaining higher Cry1Fa enhancement by this peptide. This study shows that cadherin fragments may enhanceB. thuringiensistoxicity by at least two different mechanisms or a combination thereof: (i) protection of Cry toxin from protease degradation in the insect midgut and (ii) enhancement of pore-forming ability of Cry toxin.
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Maroto, Beatriz, Juan C. Ramı́rez, and José M. Almendral. "Phosphorylation Status of the Parvovirus Minute Virus of Mice Particle: Mapping and Biological Relevance of the Major Phosphorylation Sites." Journal of Virology 74, no. 23 (December 1, 2000): 10892–902. http://dx.doi.org/10.1128/jvi.74.23.10892-10902.2000.
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ABSTRACT The core of the VP-1 and VP-2 proteins forming the T=1 icosahedral capsid of the prototype strain of the parvovirus minute virus of mice (MVMp) share amino acids sequence and a common three-dimensional structure; however, the roles of these polypeptides in the virus infection cycle differ. To gain insights into this paradox, the nature, distribution, and biological significance of MVMp particle phosphorylation was investigated. The VP-1 and VP-2 proteins isolated from purified empty capsids and from virions containing DNA harbored phosphoserine and phosphothreonine amino acids, which in two-dimensional tryptic analysis resulted in complex patterns reproducibly composed by more than 15 unevenly phosphorylated peptides. Whereas secondary protease digestions and comigration of most weak peptides in the fingerprints revealed common phosphorylation sites in the VP-1 and VP-2 subunits assembled in capsids, the major tryptic phosphopeptides were remarkably characteristic of either polypeptide. The VP-2-specific peptide named B, containing the bulk of the32P label of the MVMp particle in the form of phosphoserine, was mapped to the structurally unordered N-terminal domain of this polypeptide. Mutations in any or all four serine residues present in peptide B showed that the VP-2 N-terminal domain is phosphorylated at multiple sites, even though none of them was essential for capsid assembly or virus formation. Chromatographic analysis of purified wild-type (wt) and mutant peptide B digested with a panel of specific proteases allowed us to identify the VP-2 residues Ser-2, Ser-6, and Ser-10 as the main phosphate acceptors for MVMp capsid during the natural viral infection. Phosphorylation at VP-2 N-terminal serines was not necessary for the externalization of this domain outside of the capsid shell in particles containing DNA. However, the plaque-forming capacity and plaque size of VP-2 N-terminal phosphorylation mutants were severely reduced, with the evolutionarily conserved Ser-2 determining most of the phenotypic effect. In addition, the phosphorylated amino acids were not required for infection initiation or for nuclear translocation of the expressed structural proteins, and thus a role at a late stage of MVMp life cycle is proposed. This study illustrates the complexity of posttranslational modification of icosahedral viral capsids and underscores phosphorylation as a versatile mechanism to modulate the biological functions of their protein subunits.
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Fodor, Sharon K., and Volker M. Vogt. "Characterization of the Protease of a Fish Retrovirus, Walleye Dermal Sarcoma Virus." Journal of Virology 76, no. 9 (May 1, 2002): 4341–49. http://dx.doi.org/10.1128/jvi.76.9.4341-4349.2002.
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ABSTRACT Three fish retroviruses infecting walleyes constitute the recently recognized genus called epsilonretrovirus. The founding member of this group, walleye dermal sarcoma virus (WDSV), induces benign skin tumors in the infected fish and replicates near 4°C. While the viral genomic sequence is known, biochemical characterization of the virus has been limited to the identification of the mature structural and envelope proteins present in virions. We undertook this study to determine the cleavage sites in the WDSV Pro and Pol proteins and to characterize the viral protease (PR) in vitro. A recombinant PR was expressed in and purified from Escherichia coli as a larger fusion with additional nucleocapsid and reverse transcriptase residues flanking the PR domain. Autocleavage produced a functional, mature PR. Autocleavage as well as cleavage of peptides and of Gag protein by the mature PR occurred at a pH optimum of 7.0, higher than that of other retroviral proteases. Analysis of the cleavage sites identified a glutamine residue in the P2 position of all WDSV sites, both in Gag and in Pol. Amino acid sequence alignments of Gag-Pro-Pol from WDSV, walleye epidermal hyperplasia virus type 1, and walleye epidermal hyperplasia virus type 2 showed the P2 glutamine to be conserved in all cleavage sites in these three viruses. Such conservation is unprecedented in other retroviruses.
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Merkulov, Gennady V., Joseph F. Lawler, Yolanda Eby, and Jef D. Boeke. "Ty1 Proteolytic Cleavage Sites Are Required for Transposition: All Sites Are Not Created Equal." Journal of Virology 75, no. 2 (January 15, 2001): 638–44. http://dx.doi.org/10.1128/jvi.75.2.638-644.2001.
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ABSTRACT The retroviral protease is a key enzyme in a viral multienzyme complex that initiates an ordered sequence of events leading to virus assembly and propagation. Viral peptides are initially synthesized as polyprotein precursors; these precursors undergo a number of proteolytic cleavages executed by the protease in a specific and presumably ordered manner. To determine the role of individual protease cleavage sites in Ty1, a retrotransposon from Saccharomyces cerevisiae, the cleavage sites were systematically mutagenized. Altering the cleavage sites of the yeast Ty1 retrotransposon produces mutants with distinct retrotransposition phenotypes. Blocking the Gag/PR site also blocks cleavage at the other two cleavage sites, PR/IN and IN/RT. In contrast, mutational block of the PR/IN or IN/RT sites does not prevent cleavage at the other two sites. Retrotransposons with mutations in each of these sites have transposition defects. Mutations in the PR/IN and IN/RT sites, but not in the Gag/PR site, can be complemented in trans by endogenous Ty1 copies. Hence, the digestion of the Gag/PR site and release of the protease N terminus is a prerequisite for processing at the remaining sites; cleavage of PR/IN is not required for the cleavage of IN/RT, and vice versa. Of the three cleavage sites in the Gag-Pol precursor, the Gag/PR site is processed first. Thus, Ty1 Gag-Pol processing proceeds by an ordered pathway.
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Zhang, R., J. Durkin, W. T. Windsor, C. McNemar, L. Ramanathan, and H. V. Le. "Probing the substrate specificity of hepatitis C virus NS3 serine protease by using synthetic peptides." Journal of virology 71, no. 8 (1997): 6208–13. http://dx.doi.org/10.1128/jvi.71.8.6208-6213.1997.
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Cordingley, M. G., R. B. Register, P. L. Callahan, V. M. Garsky, and R. J. Colonno. "Cleavage of small peptides in vitro by human rhinovirus 14 3C protease expressed in Escherichia coli." Journal of Virology 63, no. 12 (1989): 5037–45. http://dx.doi.org/10.1128/jvi.63.12.5037-5045.1989.
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Wang, Lei, Liu Yang, Xiao-San Zhou, Tao-Hong Li, and Chao-Liang Liu. "A clip domain serine protease stimulates melanization activation and expression of antimicrobial peptides in the Chinese oak silkworm, Antheraea pernyi." Journal of Asia-Pacific Entomology 21, no. 3 (September 2018): 864–71. http://dx.doi.org/10.1016/j.aspen.2018.06.008.
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Lindemann, Dirk, Thomas Pietschmann, Marcus Picard-Maureau, Angelika Berg, Martin Heinkelein, Jana Thurow, Petra Knaus, Hanswalter Zentgraf, and Axel Rethwilm. "A Particle-Associated Glycoprotein Signal Peptide Essential for Virus Maturation and Infectivity." Journal of Virology 75, no. 13 (July 1, 2001): 5762–71. http://dx.doi.org/10.1128/jvi.75.13.5762-5771.2001.
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ABSTRACT Signal peptides (SP) are key determinants for targeting glycoproteins to the secretory pathway. Here we describe the involvement in particle maturation as an additional function of a viral glycoprotein SP. The SP of foamy virus (FV) envelope glycoprotein is predicted to be unusually long. Using an SP-specific antiserum, we demonstrate that its proteolytic removal occurs posttranslationally by a cellular protease and that the major N-terminal cleavage product, gp18, is found in purified viral particles. Analysis of mutants in proposed signal peptidase cleavage positions and N-glycosylation sites revealed an SP about 148 amino acids (aa) in length. FV particle release from infected cells requires the presence of cognate envelope protein and cleavage of its SP sequence. An N-terminal 15-aa SP domain with two conserved tryptophan residues was found to be essential for the egress of FV particles. While the SP N terminus was found to mediate the specificity of FV Env to interact with FV capsids, it was dispensable for Env targeting to the secretory pathway and FV envelope-mediated infectivity of murine leukemia virus pseudotypes.
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Stączek, Sylwia, Agnieszka Zdybicka-Barabas, Iwona Wojda, Adrian Wiater, Paweł Mak, Piotr Suder, Krzysztof Skrzypiec, and Małgorzata Cytryńska. "Fungal α-1,3-Glucan as a New Pathogen-Associated Molecular Pattern in the Insect Model Host Galleria mellonella." Molecules 26, no. 16 (August 23, 2021): 5097. http://dx.doi.org/10.3390/molecules26165097.
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Recognition of pathogen-associated molecular patterns (PAMPs) by appropriate pattern recognition receptors (PRRs) is a key step in activating the host immune response. The role of a fungal PAMP is attributed to β-1,3-glucan. The role of α-1,3-glucan, another fungal cell wall polysaccharide, in modulating the host immune response is not clear. This work investigates the potential of α-1,3-glucan as a fungal PAMP by analyzing the humoral immune response of the greater wax moth Galleria mellonella to Aspergillus niger α-1,3-glucan. We demonstrated that 57-kDa and 61-kDa hemolymph proteins, identified as β-1,3-glucan recognition proteins, bound to A. niger α-1,3-glucan. Other hemolymph proteins, i.e., apolipophorin I, apolipophorin II, prophenoloxidase, phenoloxidase activating factor, arylphorin, and serine protease, were also identified among α-1,3-glucan-interacting proteins. In response to α-1,3-glucan, a 4.5-fold and 3-fold increase in the gene expression of antifungal peptides galiomicin and gallerimycin was demonstrated, respectively. The significant increase in the level of five defense peptides, including galiomicin, corresponded well with the highest antifungal activity in hemolymph. Our results indicate that A. niger α-1,3-glucan is recognized by the insect immune system, and immune response is triggered by this cell wall component. Thus, the role of a fungal PAMP for α-1,3-glucan can be postulated.
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Ray, L. Bryan. "How Flies Find Fungal Foes." Science's STKE 2007, no. 369 (January 16, 2007): tw21. http://dx.doi.org/10.1126/stke.3692007tw21.
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Responses of the immune systems of plants and animals show what appears to be evidence of ancient attacks and counterattacks by pathogens and their hosts in the battle for survival. Drosophila have developed receptors that recognize constituents of bacterial cell walls and mount an immune response that causes proteolytic cleavage of the cytokine Spätzle. The Spätzle fragment then activates Toll receptors and leads to production of antimicrobial peptides. Gottar et al. explored the response of Drosophila to fungal infections and found a similar defense mechanism but also unveiled a second signaling pathway that detects a virulence factor produced by the fungus. The authors infected flies by pricking them with a needle dipped in fungus-containing solution and monitored survival or Toll-dependent expression of the gene encoding an antifungal peptide. They found that the receptor GNBP3 (Gram-negative binding protein 3) was required for detection of cell wall components of the fungi and consequent activation of Toll receptors. However, cells with a mutated GNB3 protein could still respond to fungi and activate Toll, but in this case cell wall-derived components were not the trigger. This response depended on the presence of live fungi and, presumably, the production of virulence factors. One such factor is the protease PR1, and the authors showed that expression of PR1 alone led to activation of the Toll pathway. Knowing that a fly protease PSH (Persephone), which is thought to participate in a cascade of proteases that lead to Spätzle cleavage and activation of the Toll pathway in response to fungi, itself requires proteolytic removal of a prodomain for activity, the authors tested whether PR1 might activate PSH. Indeed, studies in vitro and in vivo indicated that PSH appears to be a direct substrate of PR1. The fungi use the PR1 protease to break down the protective cuticle of the insect and allow infection. The authors propose that PSH acts like a sensor to monitor the status of the cuticle. If the presence of PR1 shows that the defense barrier is being broken, PSH is cleaved and the antimicrobial signaling is initiated. Whether humans have such a sensor system to recognize fungal virulence factors remains unknown.M. Gottar, V. Gobert, A. A. Matskevich, J.-M. Reichhart, C. Wang, T. M. Butt, M. Belvin, J. A. Hoffmann, D. Ferrandon, Dual detection of fungal infections in Drosophila via recognition of glucans and sensing of virulence factors. Cell127, 1425-1437 (2006). [Online Journal]
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Li, Beibei, Hongmei Li, Ye Tian, Nazir Ahmed Abro, Xiangqun Nong, Zehua Zhang, and Guangjun Wang. "Molecular Identification and Immunity Functional Characterization of Lmserpin1 in Locusta migratoria manilensis." Insects 12, no. 2 (February 18, 2021): 178. http://dx.doi.org/10.3390/insects12020178.
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Serine protease inhibitors (Serpins) are a broadly distributed superfamily of proteins that exist in organisms with the role of immune responses. Lmserpin1 gene was cloned firstly from Locusta migratoria manilensis and then was detected in all tested stages from eggs to adults and six different tissues through qRT-PCR analysis. The expression was significantly higher in the 3rd instars and within integument. After RNAi treatment, the expression of Lmserpin1 was significantly down-regulated at four different time points. Moreover, it dropped significantly in the fat body and hemolymph at 24 h after treatment. The bioassay results indicated that the mortality of L. migratoria manilensis treated with dsSerpin1 + Metarhizium was significantly higher than the other three treatments. Furthermore, the immune-related genes (PPAE, PPO, and defensin) treated by dsSerpin1 + Metarhizium were significantly down-regulated compared with the Metarhizium treatment, but the activities of phenoloxidase (PO), peroxidase (POD), superoxide dismutase (SOD), glutathione S-transferase (GST), and multifunctional oxidase (MFO) were fluctuating. Our results suggest that Lmserpin1 plays a crucial role in the innate immunity of L. migratoria manilensis. Lmserpin1 probably took part in regulation of melanization and promoted the synthesis of antimicrobial peptides (AMPs).
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Saikhedkar, Nidhi S., Rakesh S. Joshi, Amit K. Yadav, Shubhendu Seal, Moneesha Fernandes, and Ashok P. Giri. "Phyto-inspired cyclic peptides derived from plant Pin-II type protease inhibitor reactive center loops for crop protection from insect pests." Biochimica et Biophysica Acta (BBA) - General Subjects 1863, no. 8 (August 2019): 1254–62. http://dx.doi.org/10.1016/j.bbagen.2019.05.003.
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