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Karl J. Kramer - One of the best experts on this subject based on the ideXlab platform.
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compensatory proteolytic responses to dietary Proteinase inhibitors in the red flour beetle tribolium castaneum coleoptera tenebrionidae
2005Co-Authors: Brenda Oppert, T D Morgan, Kris L Hartzer, Karl J. KramerAbstract:Increasing levels of inhibitors that target cysteine and/or serine Proteinases were fed to Tribolium castaneum larvae, and the properties of digestive Proteinases were compared in vitro. Cysteine Proteinases were the major digestive Proteinase class in control larvae, and serine Proteinase activity was minor. Dietary serine Proteinase inhibitors had minimal effects on either the developmental time or proteolytic activity of T. castaneum larvae. However, when larvae ingested cysteine Proteinase inhibitors, there was a dramatic shift from primarily cysteine Proteinases to serine Proteinases in the Proteinase profile of the midgut. Moreover, a combination of cysteine and serine Proteinase inhibitors in the diet prevented this shift from cysteine Proteinase-based digestion to serine Proteinase-based digestion, and there was a corresponding substantial retardation in growth. These data suggest that the synergistic inhibitory effect of a combination of cysteine and serine Proteinase inhibitors in the diet of T. castaneum larvae on midgut proteolytic activity and beetle developmental time is achieved through the prevention of the adaptive proteolytic response to overcome the activity of either type of inhibitor.
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effects of Proteinase inhibitors on digestive Proteinases and growth of the red flour beetle tribolium castaneum herbst coleoptera tenebrionidae
2003Co-Authors: Brenda Oppert, T D Morgan, Kris L Hartzer, B Lenarcic, K Galesa, J Brzin, V Turk, K Yoza, K Ohtsubo, Karl J. KramerAbstract:The physiology of the gut lumen of the red flour beetle, T. castaneum, was studied to determine the conditions for optimal protein hydrolysis. Although the pH of gut lumen extracts from T. castaneum was 6.5, maximum hydrolysis of casein by gut Proteinases occurred at pH 4.2. The synthetic substrate N-alpha-benzoyl-DL-arginine-rho-nitroanilide was hydrolyzed by T. castaneum gut Proteinases in both acidic and alkaline buffers, whereas hydrolysis of N-succinyl-ala-ala-pro-phe rho-nitroanilide occurred in alkaline buffer. Inhibitors of T. castaneum digestive Proteinases were examined to identify potential biopesticides for incorporation in transgenic seed. Cysteine Proteinase inhibitors from potato, Job's tears, and sea anemone (equistatin) were effective inhibitors of in vitro casein hydrolysis by T. castaneum Proteinases. Other inhibitors of T. castaneum Proteinases included leupeptin, L-trans-epoxysuccinylleucylamido [4-guanidino] butane (E-64), tosyl-L-lysine chloromethyl ketone, and antipain. Casein hydrolysis was inhibited weakly by chymostatin, N-tosyl-L-phenylalanine chloromethyl ketone, and soybean trypsin inhibitor (Kunitz). The soybean trypsin inhibitor had no significant effect on growth when it was bioassayed alone, but it was effective when used in combination with potato cysteine Proteinase inhibitor. In other bioassays with single inhibitors, larval growth was suppressed by the cysteine Proteinase inhibitors from potato, Job's tears, or sea anemone. Levels of inhibition were similar to that observed with E-64, although the moles of proteinaceous inhibitor tested were approximately 1000-fold less. These proteinaceous inhibitors are promising candidates for transgenic seed technology to reduce seed damage by T. castaneum.
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Effects of a potato cysteine Proteinase inhibitor on midgut proteolytic enzyme activity and growth of the southern corn rootworm, Diabrotica undecimpunctata howardi (Coleoptera: Chrysomelidae).
2002Co-Authors: Jeffrey A. Fabrick, Craig A. Behnke, T. H. Czapla, K Bala, A.g Rao, Karl J. Kramer, Gerald R. ReeckAbstract:The major Proteinase activity in extracts of larval midguts from the southern corn rootworm (SCR), Diabrotica undecimpunctata howardi, was identified as a cysteine Proteinase that prefers substrates containing an arginine residue in the P1 position. Gelatinzymogram analysis of the midgut Proteinases indicated that the artificial diet-fed SCR, corn root-fed SCR, and root-fed western corn rootworms (Diabrotica virgifera virgifera) possess a single major Proteinase with an apparent molecular mass of 25 kDa and several minor Proteinases. Similar Proteinase activity pH profiles were exhibited by root-fed and diet-fed rootworms with the optimal activity being slightly acidic. Rootworm larvae reared on corn roots exhibited significantly less caseinolytic activity than those reared on the artificial diet. Midgut proteolytic activity from SCR was most sensitive to inhibition by inhibitors of cysteine Proteinases. Furthermore, rootworm Proteinase activity was particularly sensitive to inhibition by a commercial protein preparation from potato tubers (PIN-II). One of the proteins, potato cysteine Proteinase inhibitor-10, PCPI-10, obtained from PIN-II by ion-exchange chromatography, was the major source of inhibitory activity against rootworm Proteinase activity. PCPI-10 and E-64 were of comparable potency as inhibitors of southern corn rootworm Proteinase activity (IC 50 =31 and 35 nM, respectively) and substantially more effective than chicken egg white cystatin (IC50 =121 nM). Incorporation of PCPI-10 into the diet of SCR larvae in feeding trials resulted in a significant increase in mortality and growth inhibition. We suggest that expression of inhibitors such as PCPI10 by transgenic corn plants in the field is a potentially attractive method of host plant resistance to these Diabrotica species. 2002 Elsevier Science Ltd. All rights reserved.
Brenda Oppert - One of the best experts on this subject based on the ideXlab platform.
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sequence analysis and molecular characterization of larval midgut cdna transcripts encoding peptidases from the yellow mealworm tenebrio molitor l
2007Co-Authors: Sheila Prabhakar, Elena N. Elpidina, K S Vinokurov, Mingshun Chen, C M Smith, Jeremy L Marshall, Brenda OppertAbstract:Peptidase sequences were analysed in randomly picked clones from cDNA libraries of the anterior or posterior midgut or whole larvae of the yellow mealworm, Tenebrio molitor Linnaeus. Of a total of 1528 sequences, 92 encoded potential peptidases, from which 50 full-length cDNA sequences were obtained, including serine and cysteine Proteinases and metallopeptidases. Serine Proteinase transcripts were predominant in the posterior midgut, whereas transcripts encoding cysteine and metallopeptidases were mainly found in the anterior midgut. Alignments with other Proteinases indicated that 40% of the serine Proteinase sequences were serine Proteinase homologues, and the remaining ones were identified as either trypsin, chymotrypsin or other serine Proteinases. Cysteine Proteinase sequences included cathepsin B- and L-like Proteinases, and metallopeptidase transcripts were similar to carboxypeptidase A. Northern blot analysis of representative sequences demonstrated the differential expression profile of selected transcripts across five developmental stages of Te. molitor . These sequences provide insights into peptidases in coleopteran insects as a basis to study the response of coleopteran larvae to external stimuli and to evaluate regulatory features of the response.
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diversity of digestive Proteinases in tenebrio molitor coleoptera tenebrionidae larvae
2006Co-Authors: K S Vinokurov, Yakov E. Dunaevsky, D P Zhuzhikov, Elena N. Elpidina, Brenda Oppert, S Prabhakar, M A BelozerskyAbstract:The spectrum of Tenebrio molitor larval digestive Proteinases was studied in the context of the spatial organization of protein digestion in the midgut. The pH of midgut contents increased from 5.2-5.6 to 7.8-8.2 from the anterior to the posterior. This pH gradient was reflected in the pH optima of the total proteolytic activity, 5.2 in the anterior and 9.0 in the posterior midgut. When measured at the pH and reducing conditions characteristic of each midgut section, 64% of the total proteolytic activity was in the anterior and 36% in the posterior midgut. In the anterior midgut, two-thirds of the total activity was due to cysteine Proteinases, whereas the rest was from serine Proteinases. In contrast, most (76%) of the proteolytic activity in the posterior midgut was from serine Proteinases. Cysteine Proteinases from the anterior were represented by a group of anionic fractions with similar electrophoretic mobility. Trypsin-like activity was predominant in the posterior midgut and was due to one cationic and three anionic Proteinases. Chymotrypsin-like Proteinases also were prominent in the posterior midgut and consisted of one cationic and four anionic Proteinases, four with an extended binding site. Latent Proteinase activity was detected in each midgut section. These data support a complex system of protein digestion, and the correlation of Proteinase activity and pH indicates a physiological mechanism of enzyme regulation in the gut.
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compensatory proteolytic responses to dietary Proteinase inhibitors in the red flour beetle tribolium castaneum coleoptera tenebrionidae
2005Co-Authors: Brenda Oppert, T D Morgan, Kris L Hartzer, Karl J. KramerAbstract:Increasing levels of inhibitors that target cysteine and/or serine Proteinases were fed to Tribolium castaneum larvae, and the properties of digestive Proteinases were compared in vitro. Cysteine Proteinases were the major digestive Proteinase class in control larvae, and serine Proteinase activity was minor. Dietary serine Proteinase inhibitors had minimal effects on either the developmental time or proteolytic activity of T. castaneum larvae. However, when larvae ingested cysteine Proteinase inhibitors, there was a dramatic shift from primarily cysteine Proteinases to serine Proteinases in the Proteinase profile of the midgut. Moreover, a combination of cysteine and serine Proteinase inhibitors in the diet prevented this shift from cysteine Proteinase-based digestion to serine Proteinase-based digestion, and there was a corresponding substantial retardation in growth. These data suggest that the synergistic inhibitory effect of a combination of cysteine and serine Proteinase inhibitors in the diet of T. castaneum larvae on midgut proteolytic activity and beetle developmental time is achieved through the prevention of the adaptive proteolytic response to overcome the activity of either type of inhibitor.
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comparative analysis of Proteinase activities of bacillus thuringiensis resistant and susceptible ostrinia nubilalis lepidoptera crambidae
2004Co-Authors: Brenda Oppert, Randall A Higgins, Fangneng Huang, Kun Yan Zhu, Lawrent L BuschmanAbstract:Proteinase activities were compared in soluble and membrane fractions of guts obtained from larvae of Bacillus thuringiensisresistant and -susceptible Ostrinia nubilalis. Overall, serine Proteinases from soluble fractions of the susceptible strain were more active than those of the resistant strain. The soluble trypsin-like Proteinase activity of the resistant strain was approximately half that of the susceptible strain. The number and relative molecular masses of soluble and membrane serine Proteinases were different. However, there were no significant differences in the activities of serine Proteinases and aminopeptidases extracted from midgut membranes of the two strains. Cry1Ab protoxin hydrolysis by soluble Proteinase extracts of the resistant strain was reduced approximately 20–30% relative to that of the susceptible strain. Reduced protoxin processing due to decreased activities of Bt protoxin activation Proteinases may be associated with resistance to Bt toxin in this resistant strain of O. nubilalis. # 2004 Elsevier Ltd. All rights reserved.
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effects of Proteinase inhibitors on digestive Proteinases and growth of the red flour beetle tribolium castaneum herbst coleoptera tenebrionidae
2003Co-Authors: Brenda Oppert, T D Morgan, Kris L Hartzer, B Lenarcic, K Galesa, J Brzin, V Turk, K Yoza, K Ohtsubo, Karl J. KramerAbstract:The physiology of the gut lumen of the red flour beetle, T. castaneum, was studied to determine the conditions for optimal protein hydrolysis. Although the pH of gut lumen extracts from T. castaneum was 6.5, maximum hydrolysis of casein by gut Proteinases occurred at pH 4.2. The synthetic substrate N-alpha-benzoyl-DL-arginine-rho-nitroanilide was hydrolyzed by T. castaneum gut Proteinases in both acidic and alkaline buffers, whereas hydrolysis of N-succinyl-ala-ala-pro-phe rho-nitroanilide occurred in alkaline buffer. Inhibitors of T. castaneum digestive Proteinases were examined to identify potential biopesticides for incorporation in transgenic seed. Cysteine Proteinase inhibitors from potato, Job's tears, and sea anemone (equistatin) were effective inhibitors of in vitro casein hydrolysis by T. castaneum Proteinases. Other inhibitors of T. castaneum Proteinases included leupeptin, L-trans-epoxysuccinylleucylamido [4-guanidino] butane (E-64), tosyl-L-lysine chloromethyl ketone, and antipain. Casein hydrolysis was inhibited weakly by chymostatin, N-tosyl-L-phenylalanine chloromethyl ketone, and soybean trypsin inhibitor (Kunitz). The soybean trypsin inhibitor had no significant effect on growth when it was bioassayed alone, but it was effective when used in combination with potato cysteine Proteinase inhibitor. In other bioassays with single inhibitors, larval growth was suppressed by the cysteine Proteinase inhibitors from potato, Job's tears, or sea anemone. Levels of inhibition were similar to that observed with E-64, although the moles of proteinaceous inhibitor tested were approximately 1000-fold less. These proteinaceous inhibitors are promising candidates for transgenic seed technology to reduce seed damage by T. castaneum.
Michael R Kanost - One of the best experts on this subject based on the ideXlab platform.
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manduca sexta hemolymph Proteinase 21 activates prophenoloxidase activating Proteinase 3 in an insect innate immune response Proteinase cascade
2007Co-Authors: Maureen J Gorman, Haobo Jiang, Yang Wang, Michael R KanostAbstract:Melanization, an insect immune response, requires a set of hemolymph proteins including pathogen recognition proteins that initiate the response, a cascade of mostly unknown serine Proteinases, and phenoloxidase. Until now, only initial and final Proteinases in the pathways have been conclusively identified. Four such Proteinases have been purified from the larval hemolymph of Manduca sexta: hemolymph Proteinase 14 (HP14), which autoactivates in the presence of microbial surface components, and three prophenoloxidase-activating Proteinases (PAP1-3). In this study, we have used two complementary approaches to identify a serine Proteinase that activates proPAP3. Partial purification from hemolymph of an activator of proPAP3 resulted in an active fraction with two abundant polypeptides of ∼32 and ∼37 kDa. Labeling of these polypeptides with a serine Proteinase inhibitor, diisopropyl fluorophosphate, indicated that they were active serine Proteinases. N-terminal sequencing revealed that both were cleaved forms of the previously identified hemolymph serine Proteinase, HP21. Surprisingly, cleavage of proHP21 had occurred not at the predicted activation site but more N-terminal to it. In vitro reactions carried out with purified HP14 (which activates proHP21), proHP21, proPAP3, and site-directed mutant forms of the latter two Proteinases confirmed that HP21 activates proPAP3 by limited proteolysis. Like the HP21 products purified from hemolymph, HP21 that was activated by HP14 in the in vitro reactions was not cleaved at its predicted activation site.
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molecular identification of a bevy of serine Proteinases in manduca sexta hemolymph
2005Co-Authors: Haobo Jiang, Yang Wang, Xiaoping Guo, Zhen Zou, Frank Scholz, Tina Trenczek, Michael R KanostAbstract:Extracellular serine Proteinase pathways control immune and homeostatic processes in insects. Our current knowledge of their components is limited-prophenoloxidase-activating Proteinases (PAPs) are among the few hemolymph Proteinases (HPs) with known functions. To identify components of Proteinase systems in the hemolymph of Manduca sexta, we amplified cDNAs from larval fat body or hemocytes using degenerate primers coding for two conserved regions in S1 family serine Proteinases. PCR yielded fragments encoding seven known (HP1-HP4, PAP-1, PAP-2 and PAP-3) and 18 unknown (HP5-HP22) serine Proteinases. We screened cDNA libraries and isolated clones for 17 of the newly discovered HPs (HP5-HP22 except for HP11) and prepared antibodies to 14 recombinant proteins (HP6, HP8-HP10, HP12, HP14-HP19, HP21 and HP22). Fourteen of the HPs contain regulatory clip domain(s) at their amino-terminus--HP1, HP2, HP6, HP8, HP13, HP17, HP18, HP21, HP22 and PAP-1 have one, whereas HP12, HP15, PAP-2 and PAP-3 have two clip domains. Multiple sequence alignment of catalytic domains in these and other arthropod serine Proteinases provided useful clues for future functional analysis. Northern blot and reverse transcription PCR (RT-PCR) analyses showed increases in HP2, HP7, HP9, HP10, HP12-HP22 mRNA levels at 24h after a bacterial challenge, and immunoblot analysis confirmed elevated concentrations of HP12, HP14-HP19, HP21 and HP22 proteins in plasma in response to injected bacteria. Hemocytes express HP13 and HP18; fat body produces HP12, HP20-HP22; both tissues synthesize the other HPs. These results collectively indicate the existence of a complex serine Proteinase network in M. sexta hemolymph, predicted to mediate rapid defense responses upon wounding and/or microbial infection.
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Innate immune responses of a lepidopteran insect, Manduca sexta
2004Co-Authors: Michael R Kanost, Haobo Jiang, Xiao Qiang YuAbstract:Many innate immune mechanisms are conserved throughout the animal kingdom. Manduca sexta, a widely used model for insect biochemical research, employs these mechanisms to defend against invading pathogens and parasites. We have isolated from M. sexta hemolymph a group of proteins (hemolin, peptidoglycan recognition proteins, beta-1,3-glucan recognition proteins, and C-type lectins), which serve as a surveillance mechanism by binding to microbial surface molecules (e.g. peptidoglycan, lipopolysaccharide, lipoteichoic acid, and beta-1,3-glucan). The binding triggers diverse responses such as phagocytosis, nodule formation, encapsulation, melanization, and synthesis of anti-microbial peptides/proteins. Some of these responses are mediated and coordinated by serine Proteinase cascades, analogous to the complement system in mammals. Our current research is focused on the proteolytic activation of prophenoloxidase (proPO)--a reaction implicated in melanotic encapsulation, wound healing, and protein cross-linking. We have isolated three proPO-activating Proteinases, each of which requires serine Proteinase homologs as a cofactor for generating active phenoloxidase. The Proteinases and Proteinase-like molecules, containing one to two clip domains at their amino-terminus, are acute-phase proteins induced upon an immune challenge. Inhibitory regulation of the Proteinases by serpins and association of the Proteinase homologs with a bacteria-binding lectin are important for ensuring a localized defense response. Additional serine Proteinases expressed in M. sexta hemocytes and fat body have been discovered. Future research efforts will be aimed at elucidating the Proteinase cascade for proPO activation and investigating the roles of Proteinases in other immune responses such as processing of plasmatocyte-spreading peptide.
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serine Proteinase inhibitors in arthropod immunity
1999Co-Authors: Michael R KanostAbstract:Abstract Arthropod hemolymph contains proteins with serine Proteinase inhibitory activity. These inhibitors may exist in plasma or in hemocyte granules. Serine Proteinase inhibitors from the Kazal, Kunitz, α-macroglobulin, and serpin families have been identified in arthropod hemolymph and have been characterized biochemically. Two new families of low molecular weight serine Proteinase inhibitors have recently been discovered: one in silkworms (the Bombyx family) and another in locusts and a crayfish. The serine Proteinase inhibitors in arthropod hemolymph are likely to function in protecting their hosts from infection by pathogens or parasites. Some may inhibit fungal or bacterial Proteinases. Others probably have roles in regulating endogenous Proteinases involved in coagulation, prophenol oxidase activation, or cytokine activation.
T D Morgan - One of the best experts on this subject based on the ideXlab platform.
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of the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae)
2015Co-Authors: T D Morgan, K. Hartzer, B Lenarcic, K Galesa, J Brzin, V Turk, K Yoza, K Ohtsubo, K. Kramerd J. D AAbstract:The physiology of the gut lumen of the red flour beetle, T. castaneum, was studied to determine the conditions for optimal protein hydrolysis. Although the pH of gut lumen extracts from T. castaneum was 6.5, maximum hydrolysis of casein by gut Proteinases occurred at pH 4.2. The synthetic substrate N-a-benzoyl-DL-arginine-r-nitroanilide was hydrolyzed by T. castaneum gut Proteinases in both acidic and alkaline buffers, whereas hydrolysis of N-succinyl-ala-ala-pro-phe r-nitroanilide occurred in alkaline buffer. Inhibitors of T. castaneum digestive Proteinases were examined to identify potential biopesticides for incorporation in transgenic seed. Cysteine Proteinase inhibitors from potato, Job’s tears, and sea anemone (equistatin) were effective inhibitors of in vitro casein hydrolysis by T. castaneum Proteinases. Other inhibitors of T. castaneum Proteinases included leupeptin, L-trans-epoxysuccinylleucylamido w4-guanidinox butane (E-64), tosyl-L-lysine chloromethyl ketone, and antipain. Casein hydrolysis was inhibited weakly by chymostatin, N-tosyl-L-phenylalanine chloromethyl ketone, and soybean trypsin inhibitor (Kunitz). The soybean trypsin inhibitor had no significant effect on growth when it was bioassayed alone, but it was effective when used in combination with potato cysteine Proteinase inhibitor. In other bioassays with single inhibitors, larval growth was suppressed by the cystein
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compensatory proteolytic responses to dietary Proteinase inhibitors in the red flour beetle tribolium castaneum coleoptera tenebrionidae
2005Co-Authors: Brenda Oppert, T D Morgan, Kris L Hartzer, Karl J. KramerAbstract:Increasing levels of inhibitors that target cysteine and/or serine Proteinases were fed to Tribolium castaneum larvae, and the properties of digestive Proteinases were compared in vitro. Cysteine Proteinases were the major digestive Proteinase class in control larvae, and serine Proteinase activity was minor. Dietary serine Proteinase inhibitors had minimal effects on either the developmental time or proteolytic activity of T. castaneum larvae. However, when larvae ingested cysteine Proteinase inhibitors, there was a dramatic shift from primarily cysteine Proteinases to serine Proteinases in the Proteinase profile of the midgut. Moreover, a combination of cysteine and serine Proteinase inhibitors in the diet prevented this shift from cysteine Proteinase-based digestion to serine Proteinase-based digestion, and there was a corresponding substantial retardation in growth. These data suggest that the synergistic inhibitory effect of a combination of cysteine and serine Proteinase inhibitors in the diet of T. castaneum larvae on midgut proteolytic activity and beetle developmental time is achieved through the prevention of the adaptive proteolytic response to overcome the activity of either type of inhibitor.
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effects of Proteinase inhibitors on digestive Proteinases and growth of the red flour beetle tribolium castaneum herbst coleoptera tenebrionidae
2003Co-Authors: Brenda Oppert, T D Morgan, Kris L Hartzer, B Lenarcic, K Galesa, J Brzin, V Turk, K Yoza, K Ohtsubo, Karl J. KramerAbstract:The physiology of the gut lumen of the red flour beetle, T. castaneum, was studied to determine the conditions for optimal protein hydrolysis. Although the pH of gut lumen extracts from T. castaneum was 6.5, maximum hydrolysis of casein by gut Proteinases occurred at pH 4.2. The synthetic substrate N-alpha-benzoyl-DL-arginine-rho-nitroanilide was hydrolyzed by T. castaneum gut Proteinases in both acidic and alkaline buffers, whereas hydrolysis of N-succinyl-ala-ala-pro-phe rho-nitroanilide occurred in alkaline buffer. Inhibitors of T. castaneum digestive Proteinases were examined to identify potential biopesticides for incorporation in transgenic seed. Cysteine Proteinase inhibitors from potato, Job's tears, and sea anemone (equistatin) were effective inhibitors of in vitro casein hydrolysis by T. castaneum Proteinases. Other inhibitors of T. castaneum Proteinases included leupeptin, L-trans-epoxysuccinylleucylamido [4-guanidino] butane (E-64), tosyl-L-lysine chloromethyl ketone, and antipain. Casein hydrolysis was inhibited weakly by chymostatin, N-tosyl-L-phenylalanine chloromethyl ketone, and soybean trypsin inhibitor (Kunitz). The soybean trypsin inhibitor had no significant effect on growth when it was bioassayed alone, but it was effective when used in combination with potato cysteine Proteinase inhibitor. In other bioassays with single inhibitors, larval growth was suppressed by the cysteine Proteinase inhibitors from potato, Job's tears, or sea anemone. Levels of inhibition were similar to that observed with E-64, although the moles of proteinaceous inhibitor tested were approximately 1000-fold less. These proteinaceous inhibitors are promising candidates for transgenic seed technology to reduce seed damage by T. castaneum.
Guy S Salvesen - One of the best experts on this subject based on the ideXlab platform.
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granzyme b is inhibited by the cowpox virus serpin cytokine response modifier a
1995Co-Authors: Long T Quan, David J. Pickup, Antonio Caputo, Chris R Bleackley, Guy S SalvesenAbstract:Abstract The ability of cytolytic cells to cause apoptosis in target cells is in part due to the action of the serine Proteinase granzyme B. We demonstrate that granzyme B is inhibited, with an association rate constant of 2.9 105 M s, by the cowpox viral serpin cytokine response modifier A (CrmA). Previously we have shown CrmA to be an inhibitor of the cysteine Proteinase interleukin-1β-converting enzyme (ICE). Thus the mechanism of CrmA involves the unusual ability to efficiently inhibit Proteinases from two distinct catalytic classes, in this case serine and cysteine Proteinases. Granzyme B and ICE are both used to combat viral infection, and we propose that cowpox virus uses CrmA to evade the contribution of these two Proteinases. Thus, through CrmA, the virus may influence two of the pathways normally used to kill virus-infected cells: acting on endogenous Proteinases such as ICE and on exogenous Proteinases delivered by cytotoxic lymphocytes to infected cells.
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inhibition of interleukin 1 beta converting enzyme by the cowpox virus serpin crma an example of cross class inhibition
1994Co-Authors: Tomoko Komiyama, David J. Pickup, Caroline A Ray, Andrew D Howard, Nancy A Thornberry, Erin P Peterson, Guy S SalvesenAbstract:Abstract We reported previously that human interleukin-1 beta converting enzyme (ICE) is regulated by the CrmA serpin encoded by cowpox virus. We now report the mechanism and kinetics of this unusual inhibition of a cysteine Proteinase by a member of the serpin superfamily previously thought to inhibit serine Proteinase only. CrmA possesses several characteristics typical of a number of inhibitory serpins. It is conformationally unstable, unfolding around 3 M urea, and stable to denaturation in 8 M urea upon complex formation with ICE. CrmA rapidly inhibits ICE with an association rate constant (kon) of 1.7 x 10(7) M-1 s-1, forming a tight complex with an equilibrium constant for inhibition (Ki) of less than 4 x 10(-12) M. These data indicate that CrmA is a potent inhibitor of ICE, consistent with the dramatic effects of CrmA on modifying host responses to virus infection. The inhibition of ICE by CrmA is an example of a "cross-class" interaction, in which a serpin inhibits a non-serine Proteinase. Since CrmA possesses characteristics shared by inhibitors of serine Proteinases, we presume that ICE, though it is a cysteine Proteinase, has a substrate binding geometry strikingly close to that of serine Proteinases. We reason that it is the substrate binding geometry, not the catalytic mechanism of a Proteinase, that dictates its reactivity with protein inhibitors.
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analysis of the plasma elimination kinetics and conformational stabilities of native Proteinase complexed and reactive site cleaved serpins comparison of alpha 1 Proteinase inhibitor alpha 1 antichymotrypsin antithrombin iii alpha 2 antiplasmin angiotensinogen and ovalbumin
1991Co-Authors: Alan E Mast, Salvatore V Pizzo, Jan J Enghild, Guy S SalvesenAbstract:Abstract Proteinase inhibitors of the serpin superfamily may exist in one of three distinct conformations: the native form, a fully active protein with the reactive site loop intact; the proteolytically modified form in which inhibitory capacity is abolished; and the Proteinase-complexed form, a stable equimolar complex between the inhibitor and a target Proteinase. Here, the specificity and kinetics of the plasma elimination of different serpin conformations are compared. Proteinase-complexed serpins were rapidly cleared from the circulation. However, the native and modified forms were not cleared rapidly, indicating that the receptor-mediated pathways which recognize the complexes fail to recognize the native and modified forms. This result suggests that significant structural differences exist between modified and Proteinase-complexed serpins. The structural differences were probed by using transverse urea gradient gel electrophoresis, a technique that allows comparisons of the conformational stabilities of proteins. With the exception of the noninhibitory serpins ovalbumin and angiotensinogen, the modified and Proteinase-complexed serpins were both stabilized thermodynamically compared to the native forms. In addition, the Proteinase component of the serpin-Proteinase complex was usually thermodynamically stabilized. These data are used to compare the conformations of serpin-Proteinase complexes with those of native and modified serpins; they are discussed in terms of a model whereby serpins inhibit Proteinases in a manner similar to that described for other types of protein inhibitors of serine Proteinases.
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zymogen activation specificity and genomic structures of human neutrophil elastase and cathepsin g reveal a new branch of the chymotrypsinogen superfamily of serine Proteinases
1991Co-Authors: Guy S Salvesen, Jan J EnghildAbstract:On the basis of amino acid sequences inferred from the genes encoding human neutrophil elastase and cathepsin G, it is likely that both are synthesized as precursors containing N- and C-terminal peptide extensions. We show that these extensions are removed about 90 min after onset of synthesis of these proteins in the U937 cell line. Removal of these extensions causes activation of the Proteinases, and it is likely that the N-terminal extension of each enzyme serves as a zymogen activation peptide. Elastase and cathepsin G are, therefore, transiently present as zymogens, presumably to protect the biosynthetic machinery of the cell from adventitious proteolysis. Zymogen activation results from cleavage following a glutamic acid residue, a specificity opposite to most other serine Proteinase zymogens. The specificity is likely to be shared, however, by neutrophil Proteinase 3, rat mast cell Proteinase II, and most members of the granzyme group of Proteinases present in cytotoxic T-lymphocyte granules. The conservation in zymogen activation specificity between these leukocyte Proteinase homologs is mirrored by the preservation of a discrete genomic organization. This suggests that most of the leukocyte serine Proteinases evolved from a common ancestor distinct from the main branches of the chymotrypsinogen superfamily of serine Proteinases.
