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1
Mitchell, Angela M., and R. Jude Samulski. "Mechanistic Insights into the Enhancement of Adeno-Associated Virus Transduction by Proteasome Inhibitors." Journal of Virology 87, no. 23 (September 11, 2013): 13035–41. http://dx.doi.org/10.1128/jvi.01826-13.
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Proteasome inhibitors (e.g., bortezomib, MG132) are known to enhance adeno-associated virus (AAV) transduction; however, whether this results from pleotropic proteasome inhibition or off-target serine and/or cysteine protease inhibition remains unresolved. Here, we examined recombinant AAV (rAAV) effects of a new proteasome inhibitor, carfilzomib, which specifically inhibits chymotrypsin-like proteasome activity and no other proteases. We determined that proteasome inhibitors act on rAAV through proteasome inhibition and not serine or cysteine protease inhibition, likely through positive changes late in transduction.
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Dubin, G., J. Stec-Niemczyk, T. Dylag, J. Silberring, A. Dubin, and J. Potempa. "Characterisation of a highly specific, endogenous inhibitor of cysteine protease from Staphylococcus epidermidis, a new member of the staphostatin family." Biological Chemistry 385, no. 6 (June 7, 2004): 543–46. http://dx.doi.org/10.1515/bc.2004.064.
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AbstractStaphostatins, a novel family of cysteine protease inhibitors with a unique mechanism of action and distinct protein fold has recently been discovered. In this report we describe the properties ofStaphylococcus epidermidisstaphostatin A (EcpB), a new member of the family. As for other staphostatins, the recombinantS. epidermidisstaphostatin A exerted very narrow inhibitory specificity, limited to cysteine protease from the same species. The closely related proteases fromS. aureuscleaved the inhibitor at the reactive site peptide bond and inactivated it. The EcpB homologue,S. aureusstaphostatin A (ScpB), was also susceptible to proteolytic cleavage at the same site by nontarget cysteine proteases. Conversely,S. aureusstaphostatin B (SspC) was resistant to such proteolysis. The difference in the susceptibility of individual inhibitors to proteolytic cleavage at the reactive site suggests subtle variations in the mechanism of interaction with cysteine proteases.
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Tušar, Livija, Aleksandra Usenik, Boris Turk, and Dušan Turk. "Mechanisms Applied by Protein Inhibitors to Inhibit Cysteine Proteases." International Journal of Molecular Sciences 22, no. 3 (January 20, 2021): 997. http://dx.doi.org/10.3390/ijms22030997.
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Protein inhibitors of proteases are an important tool of nature to regulate and control proteolysis in living organisms under physiological and pathological conditions. In this review, we analyzed the mechanisms of inhibition of cysteine proteases on the basis of structural information and compiled kinetic data. The gathered structural data indicate that the protein fold is not a major obstacle for the evolution of a protease inhibitor. It appears that nature can convert almost any starting fold into an inhibitor of a protease. In addition, there appears to be no general rule governing the inhibitory mechanism. The structural data make it clear that the “lock and key” mechanism is a historical concept with limited validity. However, the analysis suggests that the shape of the active site cleft of proteases imposes some restraints. When the S1 binding site is shaped as a pocket buried in the structure of protease, inhibitors can apply substrate-like binding mechanisms. In contrast, when the S1 binding site is in part exposed to solvent, the substrate-like inhibition cannot be employed. It appears that all proteases, with the exception of papain-like proteases, belong to the first group of proteases. Finally, we show a number of examples and provide hints on how to engineer protein inhibitors.
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Sallai, Roberto Carlos, Bruno Ramos Salu, Rosemeire Aparecida Silva-Lucca, Flávio Lopes Alves, Thiago Henrique Napoleão, Patrícia Maria Guedes Paiva, Rodrigo da Silva Ferreira, Misako Uemura Sampaio, and Maria Luiza Vilela Oliva. "Biotechnological Potential of Araucaria angustifolia Pine Nuts Extract and the Cysteine Protease Inhibitor AaCI-2S." Plants 9, no. 12 (November 30, 2020): 1676. http://dx.doi.org/10.3390/plants9121676.
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Protease inhibitors are involved in the regulation of endogenous cysteine proteases during seed development and play a defensive role because of their ability to inhibit exogenous proteases such as those present in the digestive tracts of insects. Araucaria angustifolia seeds, which can be used in human and animal feed, were investigated for their potential for the development of agricultural biotechnology and in the field of human health. In the pine nuts extract, which blocked the activities of cysteine proteases, it was detected potent insecticidal activity against termites (Nasutitermes corniger) belonging to the most abundant termite genus in tropical regions. The cysteine inhibitor (AaCI-2S) was purified by ion-exchange, size exclusion, and reversed-phase chromatography. Its functional and structural stability was confirmed by spectroscopic and circular dichroism studies, and by detection of inhibitory activity at different temperatures and pH values. Besides having activity on cysteine proteases from C. maculatus digestive tract, AaCI-2S inhibited papain, bromelain, ficin, and cathepsin L and impaired cell proliferation in gastric and prostate cancer cell lines. These properties qualify A. angustifolia seeds as a protein source with value properties of natural insecticide and to contain a protease inhibitor with the potential to be a bioactive molecule on different cancer cells.
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Cornwall, Gail A., Angus Cameron, Iris Lindberg, Daniel M. Hardy, Nathaly Cormier, and Nelson Hsia. "The Cystatin-Related Epididymal Spermatogenic Protein Inhibits the Serine Protease Prohormone Convertase 2." Endocrinology 144, no. 3 (March 1, 2003): 901–8. http://dx.doi.org/10.1210/en.2002-220997.
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The cystatin-related epididymal spermatogenic (CRES) protein is related to the family 2 cystatins of the cystatin superfamily of cysteine protease inhibitors. However, CRES lacks sequences important for cysteine protease inhibitory activity and is specifically expressed in reproductive and neuroendocrine tissues. Thus, CRES is distinct from cystatins and may perform unique tissue-specific functions. The purpose of the present study was to determine whether CRES functions as a protease inhibitor in in vitro assays. In contrast to mouse recombinant cystatin C, recombinant CRES did not inhibit the cysteine proteases papain and cathepsin B, suggesting that it probably does not function as a typical cystatin. CRES, however, inhibited the serine protease prohormone convertase 2 (PC2), a protease involved in prohormone processing in the neuroendocrine system, whereas cystatin C showed no inhibition. CRES did not inhibit subtilisin, trypsin, or the convertase family members, PC1 and furin, indicating that it selectively inhibits PC2. Kinetic analysis showed that CRES is a competitive inhibitor of PC2 with a Ki of 25 nm. The removal of N-terminal sequences from CRES decreased its affinity for PC2, suggesting that the N terminus may be important for CRES to function as an inhibitor. These studies suggest that CRES is a cross-class inhibitor that may regulate proprotein processing within the reproductive and neuroendocrine systems.
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Van Wyk, S. G., K. J. Kunert, B. J. Vorster, and U. Schluter. "Interaction of cysteine protease inhibitor mutants with cysteine proteases." South African Journal of Botany 76, no. 2 (April 2010): 406. http://dx.doi.org/10.1016/j.sajb.2010.02.053.
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Parikh, Sunil, Jun Liu, Puran Sijwali, Jiri Gut, Daniel E. Goldberg, and Philip J. Rosenthal. "Antimalarial Effects of Human Immunodeficiency Virus Type 1 Protease Inhibitors Differ from Those of the Aspartic Protease Inhibitor Pepstatin." Antimicrobial Agents and Chemotherapy 50, no. 6 (June 2006): 2207–9. http://dx.doi.org/10.1128/aac.00022-06.
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ABSTRACT Human immunodeficiency virus type 1 protease inhibitors (HIVPIs) and pepstatin are aspartic protease inhibitors with antimalarial activity. In contrast to pepstatin, HIVPIs were not synergistic with a cysteine protease inhibitor or more active against parasites with the cysteine protease falcipain-2 knocked out than against wild-type parasites. As with pepstatin, HIVPIs were equally active against wild-type parasites and against parasites with the food vacuole plasmepsin aspartic proteases knocked out. The antimalarial mechanism of HIVPIs differs from that of pepstatin.
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Bevec, T., V. Stoka, G. Pungercic, I. Dolenc, and V. Turk. "Major histocompatibility complex class II-associated p41 invariant chain fragment is a strong inhibitor of lysosomal cathepsin L." Journal of Experimental Medicine 183, no. 4 (April 1, 1996): 1331–38. http://dx.doi.org/10.1084/jem.183.4.1331.
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The invariant chain (Ii) is associated with major histocompatibility complex class II molecules during early stages of their intracellular transport. In an acidic endosomal/lysosomal compartment, it is proteolytically cleaved and removed from class II heterodimers. Participation of aspartic and cysteine proteases has been observed in in vitro degradation of Ii, but the specific enzymes responsible for its in vivo processing are as yet undefined. We have previously isolated a noncovalent complex of the lysosomal cysteine protease cathepsin L with a peptide fragment derived from the p41 form of Ii from human kidney. Here we show that this Ii fragment, which is identical to the alternatively spliced segment of p41, is a very potent competitive inhibitor of cathepsin L (equilibrium inhibition constant Ki = 1.7 X 10(-12) M). It inhibits two other cysteine proteases, cathepsin H and papain, but to much lesser extent. Cysteine proteases cathepsins B, C, and S, as well as representatives of serine, aspartic, and metalloproteases, are not inhibited at all. These findings suggest a novel role for p41 in the regulation of various proteolytic activities during antigen processing and presentation. The Ii inhibitory fragment shows no sequence homology with the known cysteine protease inhibitors, and may, therefore, represent a new class.
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Monteiro, Ana C. S., Magnus Abrahamson, Ana P. C. A. Lima, Marcos A. Vannier-Santos, and Julio Scharfstein. "Identification, characterization and localization of chagasin, a tight-binding cysteine protease inhibitor inTrypanosoma cruzi." Journal of Cell Science 114, no. 21 (November 1, 2001): 3933–42. http://dx.doi.org/10.1242/jcs.114.21.3933.
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Lysosomal cysteine proteases from mammalian cells and plants are regulated by endogenous tight-binding inhibitors from the cystatin superfamily. The presence of cystatin-like inhibitors in lower eukaryotes such as protozoan parasites has not yet been demonstrated, although these cells express large quantities of cysteine proteases and may also count on endogenous inhibitors to regulate cellular proteolysis. Trypanosoma cruzi, the causative agent of Chagas’ heart disease, is a relevant model to explore this possibility because these intracellular parasites rely on their major lysosomal cysteine protease (cruzipain) to invade and multiply in mammalian host cells. Here we report the isolation, biochemical characterization, developmental stage distribution and subcellular localization of chagasin, an endogenous cysteine protease inhibitor in T. cruzi. We used high temperature induced denaturation to isolate a heat-stable cruzipain-binding protein (apparent molecular mass, 12 kDa) from epimastigote lysates. This protein was subsequently characterized as a tight-binding and reversible inhibitor of papain-like cysteine proteases. Immunoblotting indicated that the expression of chagasin is developmentally regulated and inversely correlated with that of cruzipain. Gold-labeled antibodies localized chagasin to the flagellar pocket and cytoplasmic vesicles of trypomastigotes and to the cell surface of amastigotes. Binding assays performed by probing living parasites with fluorescein (FITC)-cruzipain or FITC-chagasin revealed the presence of both inhibitor and protease at the cell surface of amastigotes. The intersection of chagasin and cruzipain trafficking pathways may represent a checkpoint for downstream regulation of proteolysis in trypanosomatid protozoa.
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Lee, Jung-Yub, Su-Min Song, Eun-Kyung Moon, Yu-Ran Lee, Bijay Kumar Jha, Dinzouna-Boutamba Sylvatrie Danne, Hee-Jae Cha, et al. "Cysteine Protease Inhibitor (AcStefin) Is Required for Complete Cyst Formation of Acanthamoeba." Eukaryotic Cell 12, no. 4 (February 8, 2013): 567–74. http://dx.doi.org/10.1128/ec.00308-12.
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ABSTRACTThe encystation ofAcanthamoebaleads to the formation of resilient cysts from vegetative trophozoites. This process is essential for parasite survival under unfavorable conditions, such as those associated with starvation, low temperatures, and biocides. Furthermore, cysteine proteases have been implicated in the massive turnover of intracellular components required for encystation. Thus, strict modulation of the activities of cysteine proteases is required to protectAcanthamoebafrom intracellular damage. However, mechanisms underlying the control of protease activity during encystation have not been established inAcanthamoeba. In the present study, we identified and characterizedAcanthamoebacysteine protease inhibitor (AcStefin), which was found to be highly expressed during encystation and to be associated with lysosomes by fluorescence microscopy. Recombinant AcStefin inhibited various cysteine proteases, including human cathepsin B, human cathepsin L, and papain. Transfection with small interfering RNA against AcStefin increased cysteine protease activity during encystation and resulted in incomplete cyst formation, reduced excystation efficiency, and a significant reduction in cytoplasmic area. Taken together, these results indicate that AcStefin is involved in the modulation of cysteine proteases and that it plays an essential role during the encystation ofAcanthamoeba.
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Davis, David A., Keisuke Yusa, Laura A. Gillim, Fonda M. Newcomb, Hiroaki Mitsuya, and Robert Yarchoan. "Conserved Cysteines of the Human Immunodeficiency Virus Type 1 Protease Are Involved in Regulation of Polyprotein Processing and Viral Maturation of Immature Virions." Journal of Virology 73, no. 2 (February 1, 1999): 1156–64. http://dx.doi.org/10.1128/jvi.73.2.1156-1164.1999.
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ABSTRACT We investigated the role of the two highly conserved cysteine residues, cysteines 67 and 95, of the human immunodeficiency virus type 1 (HIV-1) protease in regulating the activity of that protease during viral maturation. To this end, we generated four HIV-1 molecular clones: the wild type, containing both cysteine residues; a protease mutant in which the cysteine at position 67 was replaced by an alanine (C67A); a C95A protease mutant; and a double mutant (C67A C95A). When immature virions were produced in the presence of an HIV-1 protease inhibitor, KNI-272, and the inhibitor was later removed, limited polyprotein processing was observed for wild-type virion preparations over a 20-h period. Treatment of immature wild-type virions with the reducing agent dithiothreitol considerably improved the rate and extent of Gag processing, suggesting that the protease is, in part, reversibly inactivated by oxidation of the cysteine residues. In support of this, C67A C95A virions processed Gag up to fivefold faster than wild-type virions in the absence of a reducing agent. Furthermore, oxidizing agents, such as H2O2 and diamide, inhibited Gag processing of wild-type virions, and this effect was dependent on the presence of cysteine 95. Electron microscopy revealed that a greater percentage of double-mutant virions than wild-type virions developed a mature-like morphology on removal of the inhibitor. These studies provide evidence that under normal culture conditions the cysteines of the HIV-1 protease are susceptible to oxidation during viral maturation, thus preventing immature virions from undergoing complete processing following their release. This is consistent with the cysteines being involved in the regulation of viral maturation in cells under oxidative stress.
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Mihelič, Marko, and Dušan Turk. "Two decades of thyroglobulin type-1 domain research." Biological Chemistry 388, no. 11 (November 1, 2007): 1123–30. http://dx.doi.org/10.1515/bc.2007.155.
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Abstract Thyroglobulin type-1 repeats are primarily found in thyroglobulin and several other functionally unrelated proteins. Because a few of them exhibit inhibitory activity against cysteine proteases they were named thyropins (thyroglobulin type-1 domain protease inhibitors). In contrast to cystatins, the best-characterized group of papain-like protease inhibitors, they exhibit greater selectivity in their interactions with target proteases. Interestingly, a few members inhibit aspartic protease cathepsin D and metalloproteases. In contrast to the inhibitory fragment of the major histocompatibility complex class II-associated p41 form of invariant chain, whose structural integrity appears mandatory for its inhibitory properties, short polypeptides derived from insulin-like growth factor-binding proteins exhibit the same activity as the structure of the whole fragment. Taken together, the results indicate that the thyroglobulin type-1 repeat is a structural motif occasionally employed as an inhibitor of proteases.
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Draper, Deborah, William Donohoe, Leo Mortimer, and R. Phillip Heine. "Cysteine Proteases ofTrichomonas vaginalisDegrade Secretory Leukocyte Protease Inhibitor." Journal of Infectious Diseases 178, no. 3 (September 1998): 815–19. http://dx.doi.org/10.1086/515366.
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OFOSU, Frederick A., John FREEDMAN, Lori DEWAR, Yinqi SONG, and John W. FENTON. "A trypsin-like platelet protease propagates protease-activated receptor-1 cleavage and platelet activation." Biochemical Journal 336, no. 2 (December 1, 1998): 283–85. http://dx.doi.org/10.1042/bj3360283.
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Protease-activated receptor-1 (PAR-1) is a G-protein-linked receptor on platelets and perivascular cells activated by α-thrombin and the PAR-1-activating peptide, SFLLRN. α-Thrombin activates PAR-1 by cleaving it at R41–S42 to release the 41-residue peptide TR(1–41). Unexpectedly, platelet activation with SFLLRN was also associated with PAR-1 cleavage and the release of TR(1–41). Both PAR-1 cleavage and platelet activation resulting from SFLLRN addition to platelets were markedly inhibited by the serine protease inhibitor 4,2-(aminoethyl)-benzene sulphonylfluoride·HCl (pefabloc SC) and soybean trypsin inhibitor, but not by inhibitors of calpain, cysteine proteases or metalloproteases. Thus, a trypsin-like platelet protease propagates SFLLRN-dependent PAR-1 cleavage and platelet activation.
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Brüning, Mareke, Martina Lummer, Caterina Bentele, Marcel M. W. Smolenaars, Kees W. Rodenburg, and Hermann Ragg. "The Spn4 gene from Drosophila melanogaster is a multipurpose defence tool directed against proteases from three different peptidase families." Biochemical Journal 401, no. 1 (December 11, 2006): 325–31. http://dx.doi.org/10.1042/bj20060648.
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By alternative use of four RSL (reactive site loop) coding exon cassettes, the serpin (serine protease inhibitor) gene Spn4 from Drosophila melanogaster was proposed to enable the synthesis of multiple protease inhibitor isoforms, one of which has been shown to be a potent inhibitor of human furin. Here, we have investigated the inhibitory spectrum of all Spn4 RSL variants. The analyses indicate that the Spn4 gene encodes inhibitors that may inhibit serine proteases of the subtilase family (S8), the chymotrypsin family (S1), and the papain-like cysteine protease family (C1), most of them at high rates. Thus a cohort of different protease inhibitors is generated simply by grafting enzyme-adapted RSL sequences on to a single serpin scaffold, even though the target proteases contain different types and/or a varying order of catalytic residues and are descendents of different phylogenetic lineages. Since all of the Spn4 RSL isoforms are produced as intracellular residents and additionally as variants destined for export or associated with the secretory pathway, the Spn4 gene represents a versatile defence tool kit that may provide multiple antiproteolytic functions.
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PARAMÁ, A., R. IGLESIAS, M. F. ÁLVAREZ, J. LEIRO, F. M. UBEIRA, and M. L. SANMARTÍN. "Cysteine proteinase activities in the fish pathogenPhilasterides dicentrarchi(Ciliophora: Scuticociliatida)." Parasitology 128, no. 5 (May 2004): 541–48. http://dx.doi.org/10.1017/s0031182004004883.
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This study investigated protease activities in a crude extract andin vitroexcretion/secretion (E/S) products ofPhilasterides dicentrarchi, a ciliate fish parasite causing economically significant losses in aquaculture. Gelatin/SDS–PAGE analysis (pH 4, reducing conditions) detected 7 bands with gelatinolytic activity (approximate molecular weights 30–63 kDa) in the crude extract. The banding pattern observed in analysis of E/S products was practically identical, except for 1 low-molecular-weight band detected in the crude extract but not in the E/S products. In assays with synthetic peptidep-nitroanilide substrates, the crude extract hydrolysed substrates characteristic of cysteine proteases, namely Z-Arg-Arg pNA, Bz-Phe-Val-Arg pNA and Z-Phe-Arg pNA. These activities were strongly inhibited by the cysteine protease inhibitor E-64 and by Ac-Leu-Val-Lys aldehyde, a potent inhibitor of cysteine proteases of the cathepsin B protease subfamily. The proteases present in the crude extract degraded both type-I collagen and haemoglobinin vitro, consistent with roles in tissue invasion and nutrition respectively. Again, E-64 completely (collagen) or markedly (haemoglobin) inhibited this degradation. Finally, the histolytic activity of the ciliate in turbot fibroblast monolayers was strongly reduced in the presence of E-64, confirming the importance of secreted cysteine proteinases in the biology ofPhilasterides dicentrarchi.
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Masler, Edward P. "Characterisation of the effects on proteases of Heterodera glycines and Meloidogyne incognita second-stage juveniles by inhibitors obtained from cysts of H. glycines." Nematology 20, no. 5 (2018): 461–70. http://dx.doi.org/10.1163/15685411-00003151.
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Summary The protease inhibitor component of Heterodera glycines cyst contents was explored using a battery of peptide substrates and H. glycines and Meloidogyne incognita second-stage juveniles as enzyme sources. Protease inhibitors were prepared by heat-denaturing H. glycines cyst-egg extract (hHglCE), which was used in all inhibition exploration. Eight substrates targeting four endoprotease groups (aspartic, cysteine, metallo- and serine proteases) revealed that protease inhibition by hHglCE varied significantly between H. glycines and M. incognita with seven of the eight substrates. Only cysteine protease activity was inhibited equally between H. glycines and M. incognita. Aspartic protease activity was inhibited more strongly in H. glycines and serine protease activity was inhibited more strongly in M. incognita. Digestion of five matrix metalloprotease (MMP) substrates was inhibited more strongly in H. glycines (two substrates) and M. incognita (three substrates). These variations were particularly intriguing given the potential association of MMP proteases with developing embryos. Inhibition of digestion of nematode FMRFamide-like peptides (FLPs) showed less variation between nematode species than the targeted substrates, but inhibition did vary significantly across substrates within each species. Digestion of FLP-6 was the least affected by hHglCE but was inhibited significantly more in M. incognita than in H. glycines. Residue differences between two FLP-14 sequences significantly affected inhibition of FLP-14 digestion in both H. glycines and M. incognita. RP-HPLC fractionation of hHglCE clearly demonstrated the presence of high (Fr No.5) and low (Fr No.14) polarity inhibitor components. Potency of inhibition of M. incognita serine protease activity, based upon IC50 values (1.68 and 2.78 hHglCEeq reaction−1 for Fr No.5 and Fr No.14, respectively), was reduced significantly from unfractionated hHglCE (IC50 = 0.61), suggesting inhibitor dilution, loss of component synergy, or both, due to fractionation.
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Saitoh, Eiichi, Shinya Yamamoto, Eishiro Okamoto, Yoshimi Hayakawa, Takashi Hoshino, Ritsuko Sato, Satoko Isemura, Sadami Ohtsubo, and Masayuki Taniguchi. "Identification of Cysteine Proteases and Screening of Cysteine Protease Inhibitors in Biological Samples by a Two-Dimensional Gel System of Zymography and Reverse Zymography." Analytical Chemistry Insights 2 (January 2007): 117739010700200. http://dx.doi.org/10.4137/117739010700200011.
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We have developed a two-dimensional (2D-) gel system of zymography and reverse zymography for the detection and characterization of proteases and protease inhibitors. Isoelectric focusing (IEF) agarose gels with pH gradients were employed for separation in the first-dimension and sodium dodecyl sulfate (SDS)-polyacrylamide gel copolymerized with gelatin used for the second dimension. Proteases and protease inhibitors separated by IEF gel were applied on the second gel without trichloroacetic acid (TCA) fixation. Protease activity in the 2D-gel was visualized as transparent spots where gelatin substrate was digested after commassie brilliant blue (CBB) staining. Some of the transparent spots from the skin mucus extract of rainbow trout were determined to be a cysteine protease through use of E-64 or CA-074. In the reverse zymography technique, the gel was incubated with papain solution at 37°C for 18 h. Cysteine protease inhibitors from broad bean seeds were detected as clear blue spots after CBB staining. The amino (N-) terminal sequences of four papain inhibitor spots thus detected were demonstrated to be identical to that of favin β chain, a broad bean lectin. Taken together, our system can be considered to be an efficient technique for discovering and characterizing new proteases and protease inhibitors in biological samples. This is the first report describing a 2D-gel system of zymography and reverse zymography.
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NA, Byoung-Kuk, Bhaskar R. SHENAI, Puran S. SIJWALI, Youngchool CHOE, Kailash C. PANDEY, Ajay SINGH, Charles S. CRAIK, and Philip J. ROSENTHAL. "Identification and biochemical characterization of vivapains, cysteine proteases of the malaria parasite Plasmodium vivax." Biochemical Journal 378, no. 2 (March 1, 2004): 529–38. http://dx.doi.org/10.1042/bj20031487.
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Cysteine proteases play important roles in the life cycles of malaria parasites. Cysteine protease inhibitors block haemoglobin hydrolysis and development in Plasmodium falciparum, suggesting that the cysteine proteases of this major human pathogen, termed falcipains, are appropriate therapeutic targets. To expand our understanding of plasmodial proteases to Plasmodium vivax, the other prevalent human malaria parasite, we identified and cloned genes encoding the P. vivax cysteine proteases, vivapain-2 and vivapain-3, and functionally expressed the proteases in Escherichia coli. The vivapain-2 and vivapain-3 genes predicted papain-family cysteine proteases, which shared a number of unusual features with falcipain-2 and falcipain-3, including large prodomains and short N-terminal extensions on the catalytic domain. Recombinant vivapain-2 and vivapain-3 shared properties with the falcipains, including acidic pH optima, requirements for reducing conditions for activity and hydrolysis of substrates with positively charged residues at P1 and Leu at P2. Both enzymes hydrolysed native haemoglobin at acidic pH and the erythrocyte cytoskeletal protein 4.1 at neutral pH, suggesting similar biological roles to the falcipains. Considering inhibitor profiles, the vivapains were inhibited by fluoromethylketone and vinyl sulphone inhibitors that also inhibited falcipains and have demonstrated potent antimalarial activity.
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Engel, J. C., P. S. Doyle, J. Palmer, I. Hsieh, D. F. Bainton, and J. H. McKerrow. "Cysteine protease inhibitors alter Golgi complex ultrastructure and function in Trypanosoma cruzi." Journal of Cell Science 111, no. 5 (March 1, 1998): 597–606. http://dx.doi.org/10.1242/jcs.111.5.597.
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Cruzain, the major cysteine protease of the protozoan parasite Trypanosoma cruzi, is a target of rational drug design for chemotherapy of Chagas' disease. The precise biological role of cruzain in the parasite life cycle and the mechanism involved in the trypanocidal effect of cysteine protease inhibitors are still unclear. Here we report biological and ultrastructural alterations caused by cysteine protease inhibitors in T. cruzi epimastigotes. Cruzain, a glycoprotein that transits the Golgi-endosomal pathway, localized to pre-lysosomes/lysosomes in the posterior end of untreated epimastigotes by fluorescent microscopy utilizing either a biotinylated cysteine protease inhibitor to tag the active site, or a specific anti-cruzain antibody. Radiolabeled or biotinylated cysteine protease inhibitors bound exclusively to cruzain in intact epimastigotes confirming that cruzain is accessible to, and is targeted by the inhibitors. Treatment of T. cruzi epimastigotes with specific cysteine protease inhibitors arrested growth, altered the intracellular localization of cruzain, and induced major alterations in the Golgi complex. Following treatment, cruzain accumulated in peripheral dilations of Golgi cisternae. There was a concomitant 70% reduction in gold-labeled cruzain transported to lysosomes. Cisternae abnormalities in the Golgi compartment were followed by distention of ER and nuclear membranes. Brefeldin A increased the number and size of cisternae in epimastigotes. Pre-treatment of epimastigotes with cysteine protease inhibitors followed by exposure to brefeldin A induced a more rapid appearance of the cysteine protease inhibitor-induced Golgi alterations. Our results suggest that cysteine protease inhibitors prevent the normal autocatalytic processing and trafficking of cruzain within the Golgi apparatus. Accumulation of cruzain may decrease mobility of Golgi membranes and result in peripheral distention of cisternae. These major alterations of the Golgi complex parallel the death of T. cruzi epimastigotes.
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Drabik, P., E. Politowska, C. Czaplewski, F. Kasprzykowski, L. Lankiewicz, and J. Ciarkowski. "Theoretical studies of binding modes of two covalent inhibitors of cysteine proteases." Acta Biochimica Polonica 47, no. 4 (December 31, 2000): 1061–66. http://dx.doi.org/10.18388/abp.2000_3959.
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Physiological and pathological roles of cysteine proteases make them important targets for inhibitor development. Although highly potent inhibitors of this group of enzymes are known, their major drawback is a lack of sufficient specificity. Two cysteine protease covalent inhibitors, viz. (i) Z-RL-deoxo-V-peptide-epoxysuccinyl hybrid, and (ii) Z-RLVG-methyl-, have been developed and modeled in the catalytic pocket of papain, an archetypal thiol protease. A number of configurations have been generated and relaxed for each system using the AMBER force field. The catalytic pockets S3 and S4 appear rather elusive in view of the observed inhibitors' flexibility. This suggest rather limited chances for the development of selective structure-based inhibitors of thiol proteases, designed to exploit differences in the structure of catalytic pockets of various members of this family.
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Vorster, Barend, Urte Schlüter, Magdeleen du Plessis, Stefan van Wyk, Matome Makgopa, Ignatious Ncube, Marian Quain, Karl Kunert, and Christine Foyer. "The Cysteine Protease–Cysteine Protease Inhibitor System Explored in Soybean Nodule Development." Agronomy 3, no. 3 (August 20, 2013): 550–70. http://dx.doi.org/10.3390/agronomy3030550.
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Coppi, Alida, Melissa Cabinian, David Mirelman, and Photini Sinnis. "Antimalarial Activity of Allicin, a Biologically Active Compound from Garlic Cloves." Antimicrobial Agents and Chemotherapy 50, no. 5 (May 2006): 1731–37. http://dx.doi.org/10.1128/aac.50.5.1731-1737.2006.
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ABSTRACT The incidence of malaria is increasing, and there is an urgent need to identify new drug targets for both prophylaxis and chemotherapy. Potential new drug targets include Plasmodium proteases that play critical roles in the parasite life cycle. We have previously shown that the major surface protein of Plasmodium sporozoites, the circumsporozoite protein (CSP), is proteolytically processed by a parasite-derived cysteine protease, and this processing event is temporally associated with sporozoite invasion of host cells. E-64, a cysteine protease inhibitor, inhibits CSP processing and prevents invasion of host cells in vitro and in vivo. Here we tested allicin, a cysteine protease inhibitor found in garlic extracts, for its ability to inhibit malaria infection. At low concentrations, allicin was not toxic to either sporozoites or mammalian cells. At these concentrations, allicin inhibited CSP processing and prevented sporozoite invasion of host cells in vitro. In vivo, mice injected with allicin had decreased Plasmodium infections compared to controls. When sporozoites were treated with allicin before injection into mice, malaria infection was completely prevented. We also tested allicin on erythrocytic stages and found that a 4-day regimen of allicin administered either orally or intravenously significantly decreased parasitemias and increased the survival of infected mice by 10 days. Together, these experiments demonstrate that the same cysteine protease inhibitor can target two different life cycle stages in the vertebrate host.
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Lisk, Godfrey, Margaret Pain, Ilya Y. Gluzman, Shivkumar Kambhampati, Tetsuya Furuya, Xin-zhuan Su, Michael P. Fay, Daniel E. Goldberg, and Sanjay A. Desai. "Changes in the Plasmodial Surface Anion Channel Reduce Leupeptin Uptake and Can Confer Drug Resistance in Plasmodium falciparum-Infected Erythrocytes." Antimicrobial Agents and Chemotherapy 52, no. 7 (April 28, 2008): 2346–54. http://dx.doi.org/10.1128/aac.00057-08.
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ABSTRACT Cysteine protease inhibitors kill malaria parasites and are being pursued for development as antimalarial agents. Because they have multiple targets within bloodstream-stage parasites, workers have assumed that resistance to these inhibitors would not be acquired easily. In the present study, we used in vitro selection to generate a parasite resistant to growth inhibition by leupeptin, a broad-profile cysteine and serine protease inhibitor. Resistance was not associated with upregulation of cysteine protease activity, reduced leupeptin sensitivity of this activity, or expression level changes for putative cysteine or serine proteases in the parasite genome. Instead, it was associated with marked changes in the plasmodial surface anion channel (PSAC), an ion channel on infected erythrocytes that functions in nutrient and bulky organic solute uptake. Osmotic fragility measurements, electrophysiological recordings, and leupeptin uptake studies revealed selective reductions in organic solute permeability via PSAC, altered single-channel gating, and reduced inhibitor affinity. These changes yielded significantly reduced leupeptin uptake and could fully account for the acquired resistance. PSAC represents a novel route for the uptake of bulky hydrophilic compounds acting against intraerythrocytic parasite targets. Drug development based on such compounds should proceed cautiously in light of possible resistance development though the selection of PSAC mutants.
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KANG, J. M., H. L. JU, J. R. YU, W. M. SOHN, and B. K. NA. "Cryptostatin, a chagasin-family cysteine protease inhibitor of Cryptosporidium parvum." Parasitology 139, no. 8 (March 23, 2012): 1029–37. http://dx.doi.org/10.1017/s0031182012000297.
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SUMMARYCysteine proteases of pathogenic protozoan parasites play pivotal roles in the life cycle of parasites, but strict regulation of their activities is also essential for maintenance of parasite physiology and interaction with hosts. In this study, we identified and characterized cryptostatin, a novel inhibitor of cysteine protease (ICP) of Cryptosporidium parvum. Cryptostatin showed low sequence identity to other chagasin-family ICPs, but 3 motifs (NPTTG, GXGG, and RPW/F motifs), which are evolutionarily conserved in chagasin-family ICPs, were found in the sequence. The overall structure of cryptostatin consisted of 8 β-strands that progressed in parallel and closely resembled the immunoglobulin fold. Recombinant cryptostatin inhibited various cysteine proteases, including papain, human cathepsin B, human cathepsin L, and cryptopain-1, with Ki's in the picomolar range. Cryptostatin was active over a wide pH range and was highly stable under physiological conditions. The protein was thermostable and retained its inhibitory activity even after incubation at 95°C. Cryptostatin formed tight complexes with cysteine proteases, so the complexes remained intact in the presence of sodium dodecyl sulfate and β-mercaptoethanol, but they were disassembled by boiling. An immunogold electron microscopy analysis demonstrated diffused localization of cryptostatin within oocystes and meronts, but not within trophozoites, which suggests a possible role for cryptostatin in host cell invasion by C. parvum.
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Nga, Bui T. T., Yuki Takeshita, Misa Yamamoto, and Yoshimi Yamamoto. "Studies of Inhibitory Mechanisms of Propeptide-Like Cysteine Protease Inhibitors." Enzyme Research 2014 (June 19, 2014): 1–10. http://dx.doi.org/10.1155/2014/848937.
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Mouse cytotoxic T-lymphocyte antigen-2α (CTLA-2α), Drosophila CTLA-2-like protein (crammer), and Bombyx cysteine protease inhibitor (BCPI) belong to a novel family of cysteine protease inhibitors (I29). Their inhibitory mechanisms were studied comparatively. CTLA-2α contains a cysteine residue (C75), which is essential for its inhibitory potency. The CTLA-2α monomer was converted to a disulfide-bonded dimer in vitro and in vivo. The dimer was fully inhibitory, but the monomer, which possessed a free thiol residue, was not. A disulfide-bonded CTLA-2α/cathepsin L complex was isolated, and a cathepsin L subunit with a molecular weight of 24,000 was identified as the interactive enzyme protein. Crammer also contains a cysteine residue (C72). Both dimeric and monomeric forms of crammer were inhibitory. A crammer mutant with Cys72 to alanine (C72A) was fully inhibitory, while the replacement of Gly73 with alanine (G73A) caused a significant loss in inhibitory potency, which suggests a different inhibition mechanism from CTLA-2α. BCPI does not contain cysteine residue. C-terminal region (L77-R80) of BCPI was essential for its inhibitory potency. CTLA-2α was inhibitory in the acidic pH condition but stabilized cathepsin L under neutral pH conditions. The different inhibition mechanisms and functional considerations of these inhibitors are discussed.
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Yoo Im, Sonia, Camila Ramalho Bonturi, Adriana Miti Nakahata, Clóvis Ryuichi Nakaie, Arnildo Pott, Vali Joana Pott, and Maria Luiza Vilela Oliva. "Differences in the Inhibitory Specificity Distinguish the Efficacy of Plant Protease Inhibitors on Mouse Fibrosarcoma." Plants 10, no. 3 (March 23, 2021): 602. http://dx.doi.org/10.3390/plants10030602.
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Metastasis, the primary cause of death from malignant tumors, is facilitated by multiple protease-mediated processes. Thus, effort has been invested in the development of protease inhibitors to prevent metastasis. Here, we investigated the effects of protease inhibitors including the recombinant inhibitors rBbKI (serine protease inhibitor) and rBbCI (serine and cysteine inhibitor) derived from native inhibitors identified in Bauhinia bauhinioides seeds, and EcTI (serine and metalloprotease inhibitor) isolated from the seeds of Enterolobium contortisiliquum on the mouse fibrosarcoma model (lineage L929). rBbKI inhibited 80% of cell viability of L929 cells after 48 h, while EcTI showed similar efficacy after 72 h. Both inhibitors acted in a dose and time-dependent manner. Conversely, rBbCI did not significantly affect the viability of L929 cells. Confocal microscopy revealed the binding of rBbKI and EcTI to the L929 cell surface. rBbKI inhibited approximately 63% of L929 adhesion to fibronectin, in contrast with EcTI and rBbCI, which did not significantly interfere with adhesion. None of the inhibitors interfered with the L929 cell cycle phases. The synthetic peptide RPGLPVRFESPL-NH2, based on the BbKI reactive site, inhibited 45% of the cellular viability of L929, becoming a promising protease inhibitor due to its ease of synthesis.
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Mootz, Joe M., Cheryl L. Malone, Lindsey N. Shaw, and Alexander R. Horswill. "Staphopains Modulate Staphylococcus aureus Biofilm Integrity." Infection and Immunity 81, no. 9 (June 24, 2013): 3227–38. http://dx.doi.org/10.1128/iai.00377-13.
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ABSTRACTStaphylococcus aureusis a known cause of chronic biofilm infections that can reside on medical implants or host tissue. Recent studies have demonstrated an important role for proteinaceous material in the biofilm structure. TheS. aureusgenome encodes many secreted proteases, and there is growing evidence that these enzymes have self-cleavage properties that alter biofilm integrity. However, the specific contribution of each protease and mechanism of biofilm modulation is not clear. To address this issue, we utilized a sigma factor B (ΔsigB) mutant where protease activity results in a biofilm-negative phenotype, thereby creating a condition where the protease(s) responsible for the phenotype could be identified. Using a plasma-coated microtiter assay, biofilm formation was restored to the ΔsigBmutant through the addition of the cysteine protease inhibitor E-64 or by using Staphostatin inhibitors that specifically target the extracellular cysteine proteases SspB and ScpA (called Staphopains). Through construction of gene deletion mutants, we determined that ansspB scpAdouble mutant restored ΔsigBbiofilm formation, and this recovery could be replicated in plasma-coated flow cell biofilms. Staphopain levels were also found to be decreased under biofilm-forming conditions, possibly allowing biofilm establishment. The treatment ofS. aureusbiofilms with purified SspB or ScpA enzyme inhibited their formation, and ScpA was also able to disperse an established biofilm. The antibiofilm properties of ScpA were conserved acrossS. aureusstrain lineages. These findings suggest an underappreciated role of the SspB and ScpA cysteine proteases in modulatingS. aureusbiofilm architecture.
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Sarin, A., D. H. Adams, and P. A. Henkart. "Protease inhibitors selectively block T cell receptor-triggered programmed cell death in a murine T cell hybridoma and activated peripheral T cells." Journal of Experimental Medicine 178, no. 5 (November 1, 1993): 1693–700. http://dx.doi.org/10.1084/jem.178.5.1693.
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The hypothesis that cytoplasmic proteases play a functional role in programmed cell death was tested by examining the effect of protease inhibitors on the T cell receptor-mediated death of the 2B4 murine T cell hybridoma and activated T cells. The cysteine protease inhibitors trans-epoxysuccininyl-L-leucylamido-(4-guanidino) butane (E-64) and leupeptin, the calpain selective inhibitor acetyl-leucyl-leucyl-normethional, and the serine protease inhibitors diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride, all showed dose-dependent blocking of the 2B4 death response triggered by the T cell receptor complex and by anti-Thy-1. These protease inhibitors enhanced rather than inhibited IL-2 secretion triggered by T cell receptor cross-linking, showing that they did not act by preventing signal transduction. Growth inhibition induced by cross-linking the 2B4 T cell receptor, measured by inhibition of thymidine incorporation, was not generally blocked by these protease inhibitors. All five of these protease inhibitors enhanced rather than blocked 2B4 cell death triggered by dexamethasone, an agent previously shown to have a death pathway antagonistic with that of the TCR. 2B4 cytolysis by the cytotoxic agents staphylococcal alpha-toxin and dodecyl imidazole, and that caused by hypotonic conditions, was not significantly affected by the five protease inhibitors tested. The selected protease inhibitors blocked both the apoptotic nuclear morphology changes and DNA fragmentation induced by T cell receptor cross-linking, and enhanced both these properties induced by dexamethasone in 2B4 cells. The T cell receptor-induced death of activated murine lymph node T cells and human peripheral blood CD4+ T cells was blocked by both cysteine and serine protease inhibitors, showing that the protease-dependent death pathway also operates in these systems.
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Carvalho, Thais Batista de, Teresa Cristina Goulart Oliveira-Sequeira, and Semiramis Guimaraes. "In vitro ANTIGIARDIAL ACTIVITY OF THE CYSTEINE PROTEASE INHIBITOR E-64." Revista do Instituto de Medicina Tropical de São Paulo 56, no. 1 (January 2014): 43–47. http://dx.doi.org/10.1590/s0036-46652014000100006.
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The quest for new antiparasitic alternatives has led researchers to base their studies on insights into biology, host-parasite interactions and pathogenesis. In this context, proteases and their inhibitors are focused, respectively, as druggable targets and new therapy alternatives. Herein, we proposed to evaluate the in vitro effect of the cysteine protease inhibitor E-64 on Giardia trophozoites growth, adherence and viability. Trophozoites (105) were exposed to E-64 at different final concentrations, for 24, 48 and 72 h at 37 °C. In the growth and adherence assays, the number of trophozoites was estimated microscopically in a haemocytometer, whereas cell viability was evaluated by a dye-reduction assay using MTT. The E-64 inhibitor showed effect on growth, adherence and viability of trophozoites, however, its better performance was detected in the 100 µM-treated cultures. Although metronidazole was more effective, the E-64 was shown to be able to inhibit growth, adherence and viability rates by ≥ 50%. These results reveal that E-64 can interfere in some crucial processes to the parasite survival and they open perspectives for future investigations in order to confirm the real antigiardial potential of the protease inhibitors.
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Lê, Hương Giang, A.-Jeong Ham, Jung-Mi Kang, Tuấn Cường Võ, Haung Naw, Hae-Jin Sohn, Ho-Joon Shin, and Byoung-Kuk Na. "A Novel Cysteine Protease Inhibitor of Naegleria fowleri That Is Specifically Expressed during Encystation and at Mature Cysts." Pathogens 10, no. 4 (March 24, 2021): 388. http://dx.doi.org/10.3390/pathogens10040388.
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Naegleria fowleri is a free-living amoeba that is ubiquitous in diverse natural environments. It causes a fatal brain infection in humans known as primary amoebic meningoencephalitis. Despite the medical importance of the parasitic disease, there is a great lack of knowledge about the biology and pathogenicity of N. fowleri. In this study, we identified and characterized a novel cysteine protease inhibitor of N. fowleri (NfCPI). NfCPI is a typical cysteine protease inhibitor belonging to the cystatin family with a Gln-Val-Val-Ala-Gly (QVVAG) motif, a characteristic motif conserved in the cystatin family of proteins. Bacterially expressed recombinant NfCPI has a dimeric structure and exhibits inhibitory activity against several cysteine proteases including cathespin Bs of N. fowleri at a broad range of pH values. Expression profiles of nfcpi revealed that the gene was highly expressed during encystation and cyst of the amoeba. Western blot and immunofluorescence assays also support its high level of expression in cysts. These findings collectively suggest that NfCPI may play a critical role in encystation or cyst formation of N. fowleri by regulating cysteine proteases that may mediate encystation or mature cyst formation of the amoeba. More comprehensive studies to investigate the roles of NfCPI in encystation and its target proteases are necessary to elucidate the regulatory mechanism and the biological significance of NfCPI.
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Gumtow, Rebecca, Dongliang Wu, Janice Uchida, and Miaoying Tian. "A Phytophthora palmivora Extracellular Cystatin-Like Protease Inhibitor Targets Papain to Contribute to Virulence on Papaya." Molecular Plant-Microbe Interactions® 31, no. 3 (March 2018): 363–73. http://dx.doi.org/10.1094/mpmi-06-17-0131-fi.
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Papaya fruits, stems, and leaves are rich in papain, a cysteine protease that has been shown to mediate plant defense against pathogens and insects. Yet the oomycete Phytophthora palmivora is a destructive pathogen that infects all parts of papaya plants, suggesting that it has evolved cysteine protease inhibitors to inhibit papain to enable successful infection. Out of five putative extracellular cystatin-like cysteine protease inhibitors (PpalEPICs) from P. palmivora transcriptomic sequence data, PpalEPIC8 appeared to be unique to P. palmivora and was highly induced during infection of papaya. Purified recombinant PpalEPIC8 strongly inhibited papain enzyme activity, suggesting that it is a functional cysteine protease inhibitor. Homozygous PpalEPIC8 mutants were generated using CRISPR/Cas9-mediated gene editing via Agrobacterium-mediated transformation (AMT). Increased papain sensitivity of in-vitro growth and reduced pathogenicity during infection of papaya fruits were observed for the mutants compared with the wild-type strain, suggesting that PpalEPIC8, indeed, plays a role in P. palmivora virulence by inhibiting papain. This study provided genetic evidence demonstrating that plant-pathogenic oomycetes secrete cystatins as important weapons to invade plants. It also established an effective gene-editing system for P. palmivora by the combined use of CRISPR/Cas9 and AMT, which is expected to be applicable to other oomycetes.
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Strachecka, A. J., M. M. Gryzińska, M. Krauze, and K. Grzywnowicz. "Profile of the body surface proteolytic systém in Apis mellifera quee." Czech Journal of Animal Science 56, No. 1 (January 20, 2011): 15–22. http://dx.doi.org/10.17221/150/2009-cjas.
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The proteolytic system on the body surface of the honey bee has been insufficiently researched. In this study the body surface proteolytic activity was examined in queens at various developmental stages (eggs, larvae, pupae and imagines) in different seasons (spring, summer, autumn, winter). Extracts of the body surface material with water and detergent were used for an in vitro analysis of the proteolytic activity and protease inhibitor level assaying, as well as for an electrophoretic separation of the extracts in polyacrylamide gels. The following methods were used: protein content testing by the Lowry method (modified by Schacterle-Pollack), protease activity testing by the Anson method and protease inhibitor activity testing by the Lee and Lin method. Our studies revealed a high protease activity in an acidic environment (pH = 2.4; the material rinsed with detergent), as well as in neutral (pH = 7) and alkaline (pH = 11.2) environments (the material rinsed with water in both cases). The highest protein concentration values were observed in the imagines from summer. The lowest activities of the proteases and protease inhibitors were determined in the eggs from summer. The highest activities of the acidic, neutral and alkaline proteases were observed in the pupae from spring. The highest number of protease activity bands in PAGE zymography was obtained for the neutral and alkaline activities in the queens for all the seasons. In the queens all the catalytic protease types were present: asparagine and cysteine proteases at pH = 2.4; cysteine proteases and metalloproteases at pH = 7 and serine proteases at pH = 11.2. These results were crucial for the analysis of immunity mechanisms on the body surface of the honey bee.
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ALBECK, Amnon, and Sharon KLIPER. "Mechanism of cysteine protease inactivation by peptidyl epoxides." Biochemical Journal 322, no. 3 (March 15, 1997): 879–84. http://dx.doi.org/10.1042/bj3220879.
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Peptidyl epoxides are time- and concentration-dependent selective cysteine protease inhibitors. The lack of recovery of enzymic activity and the retention of 1 molar equivalent of radioactive inhibitor associated with the enzyme on dialysis, shown in this study, indicate that they form a covalent irreversible equimolar complex with the enzyme. It is also shown that the peptidyl epoxide inhibitors alkylate the active-site thiol. This alkylation only occurs when the enzyme is in its native conformation, as the denatured enzyme does not undergo alkylation by the inhibitor to any appreciable extent. Finally, the inactivation process is compared with a model reaction between a peptidyl epoxide and a protected cysteine in neutral and basic aqueous media. The inactivation of cathepsin B by Cbz-Phe-(O-benzyl)-Thr-epoxide is accelerated by 5.5 orders of magnitude relative to the rate of the model reaction at pH 10.0 and 25 °C, and estimated to be at least 108 times faster than the model reaction at pH 7.0. These results, in conjunction with the selectivity exhibited by peptidyl epoxides at all levels, point to a mechanism-based inhibition, and may have mechanistic implications regarding the catalysis carried out by cysteine proteases.
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Krol, J., C. Kopitz, A. Kirschenhofer, M. Schmitt, U. Magdolen, A. Krüger, and V. Magdolen. "Inhibition of Intraperitoneal Tumor Growth of Human Ovarian Cancer Cells by Bi- and Trifunctional Inhibitors of Tumor-Associated Proteolytic Systems." Biological Chemistry 384, no. 7 (July 15, 2003): 1097–102. http://dx.doi.org/10.1515/bc.2003.122.
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AbstractSeveral proteolytic systems are involved in (anti)adhesive, migratory, and proteolytic processes, necessary for tumor progression and metastasis. We analyzed whether multifunctional inhibitors of different tumor-associated proteolytic systems reduce tumor growth and spread of human ovarian cancer cells in vivo. Bifunctional inhibitors are composed of the N-terminal domain of either the human matrix metalloproteinase inhibitors TIMP-1 or TIMP-3 and the cysteine protease inhibitor chicken cystatin (chCysWT); trifunctional inhibitors are composed of N-TIMP-1 or -3 and a chicken cystatin variant harboring the uPAR binding site of uPA, chCys-uPA19-31, which in addition to its inhibitory activity toward cysteine proteases interferes with the interaction of the serine protease uPA with its receptor. OV-MZ-6#8 cancer cells, stably transfected with plasmids expressing the multifunctional inhibitors, displayed similar proliferative and adhesive features as the vector-transfected control, but showed significant reduction in their invasive behavior in vitro. The cell lines expressing the multifunctional inhibitors were inoculated into the peritoneum of nude mice. Expression of three of the four inhibitor variants (NhTIMP-1-chCysWT, N-hTIMP-1- chCys-uPA19-31, and N-hTIMP-3-chCysWT) resulted in a significant reduction of tumor burden compared to the vector-control cell line. These compact and small inhibitors may represent promising agents for gene therapy of solid malignant tumors.
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Karlsson, Anna, Patricia Saravia-Otten, Karin Tegmark, Eva Morfeldt, and Staffan Arvidson. "Decreased Amounts of Cell Wall-Associated Protein A and Fibronectin-Binding Proteins in Staphylococcus aureus sarA Mutants due to Up-Regulation of Extracellular Proteases." Infection and Immunity 69, no. 8 (August 1, 2001): 4742–48. http://dx.doi.org/10.1128/iai.69.8.4742-4748.2001.
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ABSTRACT Data have been presented indicating that Staphylococcus aureus cell surface protein can be degraded by extracellular proteases produced by the same bacterium. We have found that insarA mutant cells, which produce high amounts of four major extracellular proteases (staphylococcal serine protease [V8 protease] [SspA], cysteine protease [SspB], aureolysin [metalloprotease] [Aur], and staphopain [Scp]), the levels of cell-bound fibronectin-binding proteins (FnBPs) and protein A were very low compared to those of wild-type cells, in spite of unaltered or increased transcription of the corresponding genes. Cultivation ofsarA mutant cells in the presence of the global protease inhibitor α2-macroglobulin resulted in a 16-fold increase in cell-bound FnBPs, indicating that extracellular proteases were responsible for the decreased amounts of FnBPs in sarAmutant cells. The protease inhibitor E64 had no effect on the level of FnBPs, indicating that cysteine proteases were not involved. Inactivation of either ssp or aur in the prototype S. aureus strain 8325-4 resulted in a threefold increase in the amount of cell-bound FnBPs. Inactivation of the same protease genes in a sarA mutant of 8325-4 resulted in a 10- to 20-fold increase in cell-bound protein A. As the serine protease requires aureolysin to be activated, it can thus be concluded that the serine protease is the most important protease in the release of cell-bound FnBPs and protein A.
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Mishra, Manasi, Vigyasa Singh, Meenakshi B. Tellis, Rakesh S. Joshi, and Shailja Singh. "Repurposing the McoTI-II Rigid Molecular Scaffold in to Inhibitor of ‘Papain Superfamily’ Cysteine Proteases." Pharmaceuticals 14, no. 1 (December 23, 2020): 7. http://dx.doi.org/10.3390/ph14010007.
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Clan C1A or ‘papain superfamily’ cysteine proteases are key players in many important physiological processes and diseases in most living systems. Novel approaches towards the development of their inhibitors can open new avenues in translational medicine. Here, we report a novel design of a re-engineered chimera inhibitor Mco-cysteine protease inhibitor (CPI) to inhibit the activity of C1A cysteine proteases. This was accomplished by grafting the cystatin first hairpin loop conserved motif (QVVAG) onto loop 1 of the ultrastable cyclic peptide scaffold McoTI-II. The recombinantly expressed Mco-CPI protein was able to bind with micromolar affinity to papain and showed remarkable thermostability owing to the formation of multi-disulphide bonds. Using an in silico approach based on homology modelling, protein–protein docking, the calculation of the free-energy of binding, the mechanism of inhibition of Mco-CPI against representative C1A cysteine proteases (papain and cathepsin L) was validated. Furthermore, molecular dynamics simulation of the Mco-CPI–papain complex validated the interaction as stable. To conclude, in this McoTI-II analogue, the specificity had been successfully redirected towards C1A cysteine proteases while retaining the moderate affinity. The outcomes of this study pave the way for further modifications of the Mco-CPI design for realizing its full potential in therapeutics. This study also demonstrates the relevance of ultrastable peptide-based scaffolds for the development of novel inhibitors via grafting.
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Hartman, Kinga, Przemyslaw Mielczarek, and Jerzy Silberring. "Synthesis of the Novel Covalent Cysteine Proteases Inhibitor with Iodoacetic Functional Group." Molecules 25, no. 4 (February 13, 2020): 813. http://dx.doi.org/10.3390/molecules25040813.
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This work presents the synthesis of the novel covalent inhibitor of cysteine proteases where epoxide has been replaced by the iodoacetyl functional group. The molecule, similar in action to E-64 and DCG-04, the commonly applied inhibitors, is additionally biotinylated and contains tyrosyl iodination sites. The Fmoc solid phase synthesis has been applied. Conjugation of iodoacetic acid with the peptide was optimized by testing different conjugation agents. The purity of the final product was verified by mass spectrometry and its bioactivity was tested by incubation with a model cysteine protease—staphopain C. Finally, it was shown that the synthesized inhibitor binds to the protein at the ratio of 1:1. More detailed analysis by means of tandem mass spectrometry proved that the inhibitor binds to the cysteine present in the active site of the enzyme.
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Nagahama, Masahiro, Keiko Kobayashi, Sadayuki Ochi, and Masaya Takehara. "Internalization of Clostridium botulinum C2 Toxin Is Regulated by Cathepsin B Released from Lysosomes." Toxins 13, no. 4 (April 9, 2021): 272. http://dx.doi.org/10.3390/toxins13040272.
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Clostridium botulinum C2 toxin is a clostridial binary toxin consisting of actin ADP-ribosyltransferase (C2I) and C2II binding components. Activated C2II (C2IIa) binds to cellular receptors and forms oligomer in membrane rafts. C2IIa oligomer assembles with C2I and contributes to the transport of C2I into the cytoplasm of host cells. C2IIa induces Ca2+-induced lysosomal exocytosis, extracellular release of the acid sphingomyelinase (ASMase), and membrane invagination and endocytosis through generating ceramides in the membrane by ASMase. Here, we reveal that C2 toxin requires the lysosomal enzyme cathepsin B (CTSB) during endocytosis. Lysosomes are a rich source of proteases, containing cysteine protease CTSB and cathepsin L (CTSL), and aspartyl protease cathepsin D (CTSD). Cysteine protease inhibitor E64 blocked C2 toxin-induced cell rounding, but aspartyl protease inhibitor pepstatin-A did not. E64 inhibited the C2IIa-promoted extracellular ASMase activity, indicating that the protease contributes to the activation of ASMase. C2IIa induced the extracellular release of CTSB and CTSL, but not CTSD. CTSB knockdown by siRNA suppressed C2 toxin-caused cytotoxicity, but not siCTSL. These findings demonstrate that CTSB is important for effective cellular entry of C2 toxin into cells through increasing ASMase activity.
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NOURRISSON, C., I. WAWRZYNIAK, A. CIAN, V. LIVRELLI, E. VISCOGLIOSI, F. DELBAC, and P. POIRIER. "OnBlastocystissecreted cysteine proteases: a legumain-activated cathepsin B increases paracellular permeability of intestinal Caco-2 cell monolayers." Parasitology 143, no. 13 (September 9, 2016): 1713–22. http://dx.doi.org/10.1017/s0031182016001396.
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SUMMARYBlastocystisspp. pathogenic potential remains unclear as these anaerobic parasitic protozoa are frequently isolated from stools of both symptomatic and asymptomatic subjects.In silicoanalysis of the whole genome sequence ofBlastocystissubtype 7 revealed the presence of numerous proteolytic enzymes including cysteine proteases predicted to be secreted. To assess the potential impact of proteases on intestinal cells and gut function, we focused our study on two cysteine proteases, a legumain and a cathepsin B, which were previously identified inBlastocystissubtype 7 culture supernatants. Both cysteine proteases were produced as active recombinant proteins. Activation of the recombinant legumain was shown to be autocatalytic and triggered by acidic pH, whereas proteolytic activity of the recombinant cathepsin B was only recorded after co-incubation with the legumain. We then measured the diffusion of 4-kDa FITC-labelled dextran across Caco-2 cell monolayers following exposition to eitherBlastocystisculture supernatants or each recombinant protease. BothBlastocystisculture supernatants and recombinant activated cathepsin B induced an increase of Caco-2 cell monolayer permeability, and this effect was significantly inhibited by E-64, a specific cysteine protease inhibitor. Our results suggest that cathepsin B might play a role in pathogenesis ofBlastocystisby increasing intestinal cell permeability.
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Lindvall, Mika. "Molecular Modeling in Cysteine Protease Inhibitor Design." Current Pharmaceutical Design 8, no. 18 (August 1, 2002): 1673–81. http://dx.doi.org/10.2174/1381612023394142.
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Aly, Ahmed S. I., and Kai Matuschewski. "A malarial cysteine protease is necessary for Plasmodium sporozoite egress from oocysts." Journal of Experimental Medicine 202, no. 2 (July 18, 2005): 225–30. http://dx.doi.org/10.1084/jem.20050545.
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The Plasmodium life cycle is a sequence of alternating invasive and replicative stages within the vertebrate and invertebrate hosts. How malarial parasites exit their host cells after completion of reproduction remains largely unsolved. Inhibitor studies indicated a role of Plasmodium cysteine proteases in merozoite release from host erythrocytes. To validate a vital function of malarial cysteine proteases in active parasite egress, we searched for target genes that can be analyzed functionally by reverse genetics. Herein, we describe a complete arrest of Plasmodium sporozoite egress from Anopheles midgut oocysts by targeted disruption of a stage-specific cysteine protease. Our findings show that sporozoites exit oocysts by parasite-dependent proteolysis rather than by passive oocyst rupture resulting from parasite growth. We provide genetic proof that malarial cysteine proteases are necessary for egress of invasive stages from their intracellular compartment and propose that similar cysteine protease–dependent mechanisms occur during egress from liver-stage and blood-stage schizonts.
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Lonsdale-Eccles, J. D., G. W. N. Mpimbaza, Z. R. M. Nkhungulu, J. Olobo, L. Smith, O. M. Tosomba, and D. J. Grab. "Trypanosomatid cysteine protease activity may be enhanced by a kininogen-like moiety from host serum." Biochemical Journal 305, no. 2 (January 15, 1995): 549–56. http://dx.doi.org/10.1042/bj3050549.
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African trypanosomes contain cysteine proteases (trypanopains) the activity of which can be measured by in vitro digestion of fibrinogen, after electrophoresis in fibrinogen-containing SDS/polyacrylamide gels. When assessed by this procedure, trypanopain from Trypanosoma brucei (trypanopain-Tb) is estimated to have a molecular mass of 28 kDa. However, two additional bands of trypanopain activity (87 kDa and 105 kDa) are observed if serum is added to the trypanopain before electrophoresis. Formation of the 87 and 105 kDa bands is frequently accompanied by a reduction in the intensity of the 28 kDa activity which suggests that the extra bands are complexes of the 28 kDa trypanopain-Tb and a molecule from rat serum called rat trypanopain moledulator (rTM). The rTM-induced activation of cysteine proteases is not restricted to T. brucei as it is also observed with proteases from other protozoan parasites such as bloodstream forms of Trypanosoma congolense and the mammalian-infective in vitro-derived promastigote forms of Leishmania donovani and Leishmania major. The physical properties of rTM resemble those of the kininogen family of cysteine protease inhibitors. rTM is an acidic (pI 4.7) heat-stable 68 kDa glycoprotein with 15 kDa protease-susceptible domains. This resemblance between rTM and kininogens was confirmed by the positive, albeit weak, immunoreactivity between anti-(human low-molecular-mass kininogen) antibody and rTM as well as anti-rTM antibody and human low-molecular-mass kininogen. Furthermore, commercial preparations of human-low-molecular-mass kininogen and chicken egg white cystatin mimicked rTM by forming extra bands of proteolytic activity in the presence of trypanopain-Tb. In some instances, low-molecular-mass kininogen was also observed to increase the rate of hydrolysis of 7-(benzyloxycarbonyl-phenylalanyl-arginyl-amido)-4- methylcoumarin by live T. brucei. Although this effect was rather erratic, in no instance was significant inhibition observed when this putative cysteine protease inhibitor was used under these conditions. The activation of parasite cysteine proteases by commonly accepted cysteine protease inhibitors is unexpected and may have important pathological repercussions.
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SNIPAS, Scott J., Henning R. STENNICKE, Stefan RIEDL, Jan POTEMPA, James TRAVIS, Alan J. BARRETT, and Guy S. SALVESEN. "Inhibition of distant caspase homologues by natural caspase inhibitors." Biochemical Journal 357, no. 2 (July 9, 2001): 575–80. http://dx.doi.org/10.1042/bj3570575.
Full textAbstract:
Caspases play an important role in the ability of animal cells to kill themselves by apoptosis. Caspase activity is regulated in vivo by members of three distinct protease inhibitor families, two of which, baculovirus p35 and members of the inhibitor of apoptosis (IAP) family, are thought to be caspase specific. However, caspases are members of the clan of cysteine proteases designated CD, which also includes animal and plant legumains, and the bacterial proteases clostripain, gingipain-R and gingipain-K. Since these proteases have been proposed to have a common mechanism and evolutionary origin, we hypothesized that the caspase inhibitors may also regulate these other proteases. We tested this hypothesis by examining the effect of the natural caspase inhibitors on other members of protease clan CD. The IAP family proteins were found to have only a slight inhibitory effect on gingipain-R. The cowpox viral cytokine-response modifier A (CrmA) serpin had no effect on any of the proteases tested but a single point mutation of CrmA (Asp → Lys) resulted in strong inhibition of gingipain-K. More substantial, with respect to the hypothesis, was the strong inhibition of gingipain-K by wild-type p35. The site in p35, required for inhibition of gingipain-K, was mapped to Lys94, seven residues C-terminal to the caspase inhibitory site. Our data indicate that the virally encoded caspase inhibitors have adopted a mechanism that allows them to regulate disparate members of clan CD proteases.
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Zhu, Bingkuan, Fang Luo, Yi Shen, Wenbin Yang, Chengsong Sun, Jipeng Wang, Jian Li, et al. "Schistosoma japonicum cathepsin B2 (SjCB2) facilitates parasite invasion through the skin." PLOS Neglected Tropical Diseases 14, no. 10 (October 26, 2020): e0008810. http://dx.doi.org/10.1371/journal.pntd.0008810.
Full textAbstract:
Cercariae invasion of the human skin is the first step in schistosome infection. Proteases play key roles in this process. However, little is known about the related hydrolytic enzymes in Schistosoma japonicum. Here, we investigated the biochemical features, tissue distribution and biological roles of a cathepsin B cysteine protease, SjCB2, in the invasion process of S. japonicum cercariae. Enzyme activity analysis revealed that recombinant SjCB2 is a typical cysteine protease with optimum temperature and pH for activity at 37°C and 4.0, respectively, and can be totally inhibited by the cysteine protease inhibitor E-64. Immunoblotting showed that both the zymogen (50 kDa) and mature enzyme (30.5 kDa) forms of SjCB2 are expressed in the cercariae. It was observed that SjCB2 localized predominantly in the acetabular glands and their ducts of cercariae, suggesting that the protease could be released during the invasion process. The protease degraded collagen, elastin, keratin, fibronectin, immunoglobulin (A, G and M) and complement C3, protein components of the dermis and immune system. In addition, proteomic analysis demonstrated that SjCB2 can degrade the human epidermis. Furthermore, it was showed that anti-rSjCB2 IgG significantly reduced (22.94%) the ability of the cercariae to invade the skin. The cysteine protease, SjCB2, located in the acetabular glands and their ducts of S. japonicum cercariae. We propose that SjCB2 facilitates skin invasion by degrading the major proteins of the epidermis and dermis. However, this cysteine protease may play additional roles in host-parasite interaction by degrading immunoglobins and complement protein.
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Overney, S., V. Q. Le, S. Visal, and S. Yelle. "Potential of Proteinase Inhibitors for Insect Pest Control: The Case of the Colorado Potato Beetle." HortScience 30, no. 4 (July 1995): 767D—767. http://dx.doi.org/10.21273/hortsci.30.4.767d.
Full textAbstract:
Bioengineering economically important plants with proteinase inhibitors (PIs) is a promising method for the control of insect pests. In the case of the Colorado potato beetle (CPB; Leptinotarsa decemlineata Say), the major insect pest of potato fields, 80% of the digestive proteases are of cysteine type. We showed that 60% of these cysteine proteases are inhibited by oryzacystatins (OCs). The use of these cysteine protease inhibitor genes therefore appears of great interest for the production of Coleoptera-resistant transgenic plants of potato. Complementary studies of biochemical in vitro assays showed an apparent absence of direct interference between OCs and potato proteases. The high regeneration efficiency of the genetically transformed plants with OC gene and the “normal” phenotypical growth of the resulting transgenic potato plants suggested that these foreign genes do not interact with important physiological processes in the potato plants. In vivo assays of PIs against CPB at various developmental stages suggest the significant potential of OCs as an effective way to control CPB populations and crop damage.
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Taggart, Clifford, Marcus A. Mall, Gilles Lalmanach, Didier Cataldo, Andreas Ludwig, Sabina Janciauskiene, Nicole Heath, et al. "Protean proteases: at the cutting edge of lung diseases." European Respiratory Journal 49, no. 2 (February 2017): 1501200. http://dx.doi.org/10.1183/13993003.01200-2015.
Full textAbstract:
Proteases were traditionally viewed as mere protein-degrading enzymes with a very restricted spectrum of substrates. A major expansion in protease research has uncovered a variety of novel substrates, and it is now evident that proteases are critical pleiotropic actors orchestrating pathophysiological processes. Recent findings evidenced that the net proteolytic activity also relies upon interconnections between different protease and protease inhibitor families in the protease web.In this review, we provide an overview of these novel concepts with a particular focus on pulmonary pathophysiology. We describe the emerging roles of several protease families including cysteine and serine proteases.The complexity of the protease web is exemplified in the light of multidimensional regulation of serine protease activity by matrix metalloproteases through cognate serine protease inhibitor processing. Finally, we will highlight how deregulated protease activity during pulmonary pathogenesis may be exploited for diagnosis/prognosis purposes, and utilised as a therapeutic tool using nanotechnologies.Considering proteases as part of an integrative biology perspective may pave the way for the development of new therapeutic targets to treat pulmonary diseases related to intrinsic protease deregulation.
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Shah, Falgun, Prasenjit Mukherjee, Jiri Gut, Jennifer Legac, Philip J. Rosenthal, Babu L. Tekwani, and Mitchell A. Avery. "Identification of Novel Malarial Cysteine Protease Inhibitors Using Structure-Based Virtual Screening of a Focused Cysteine Protease Inhibitor Library." Journal of Chemical Information and Modeling 51, no. 4 (March 23, 2011): 852–64. http://dx.doi.org/10.1021/ci200029y.
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Lavergne, Marion, Audrey Guillon-Munos, Woodys Lenga Ma Bonda, Sylvie Attucci, Thomas Kryza, Aurélia Barascu, Thierry Moreau, et al. "Tissue factor pathway inhibitor 2 is a potent kallikrein-related protease 12 inhibitor." Biological Chemistry 402, no. 10 (May 12, 2021): 1257–68. http://dx.doi.org/10.1515/hsz-2020-0389.
Full textAbstract:
Abstract The protease activities are tightly regulated by inhibitors and dysregulation contribute to pathological processes such as cancer and inflammatory disorders. Tissue factor pathway inhibitor 2 (TFPI-2) is a serine proteases inhibitor, that mainly inhibits plasmin. This protease activated matrix metalloproteases (MMPs) and degraded extracellular matrix. Other serine proteases are implicated in these mechanisms like kallikreins (KLKs). In this study, we identified for the first time that TFPI-2 is a potent inhibitor of KLK5 and 12. Computer modeling showed that the first Kunitz domain of TFPI-2 could interact with residues of KLK12 near the catalytic triad. Furthermore, like plasmin, KLK12 was able to activate proMMP-1 and -3, with no effect on proMMP-9. Thus, the inhibition of KLK12 by TFPI-2 greatly reduced the cascade activation of these MMPs and the cleavage of cysteine-rich 61, a matrix signaling protein. Moreover, when TFPI-2 bound to extracellular matrix, its classical localisation, the KLK12 inhibition was retained. Finally, TFPI-2 was downregulated in human non-small-cell lung tumour tissue as compared with non-affected lung tissue. These data suggest that TFPI-2 is a potent inhibitor of KLK12 and could regulate matrix remodeling and cancer progression mediated by KLK12.
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Sinaga, Ayu Oshin Yap, and David Septian Sumanto Marpaung. "Predicting The Cleavage Sites of Multiple Proteases Families on Rice Alpha Amylase Isozyme 3D Sequences." Agro Bali : Agricultural Journal 3, no. 1 (June 22, 2020): 16–20. http://dx.doi.org/10.37637/ab.v3i1.449.
Full textAbstract:
Proteases is a proteolytic enzyme that often determined the crucial process in degradation pathway occurred in all of organisms. Prediction of novel protease is important action to design the protease inhibitor. In the secretion of rice αAmy3 protein in outside cells will be followed by secretion of recombinant protein target and proteolytic enzymes together, which means potentially also degraded the recombinant protein target In this study, the proteases was detected in rice αAMY 3D protein sequences. Our study resulted the 3 major proteases appeared in rice αAMY 3D protein sequences, they were cysteine proteases, serine proteases and metalloproteases. Based on the literature, such proteases also appeared in rice suspension cells. Design the inhibitor for such proteases will be suggested for reduction of proteases level.