Showing papers on "Cysteine protease published in 2001"
TL;DR: This chapter focuses on the most well characterized inhibitors—cystatin C—and provides some information on its structure, biochemical properties, its role in normal and abnormal physiological processes, as well as on its use as a diagnostic marker.
Abstract: Publisher Summary This chapter focuses on the most well characterized inhibitors—cystatin C—and provide some information on its structure, biochemical properties, its role in normal and abnormal physiological processes, as well as on its use as a diagnostic marker. A major part of the cysteine proteases are evolutionary related to the structurally well–defined cysteine protease papain and are called papain–like cysteine proteases. The biological roles and the cystatin superfamily inhibitors of papain–like cystein proteases are also discussed. The aminoacid sequence and schematic structure of human cystatin C is also presented. The evolutionary relationships among all known inhibitory active human cystatins and kininogen cystatin domains are diagrammatically represented. The distribution of cystatins in body fluids and additional functions attributed to cystatin C are described. The serum or plasma cystatin C is used as a marker for glomerular filtration rate (GFR). The urine cystatin C is used as a marker for proximal tubular damage. The two types of brain hemorrhage associated with Cystatin C amyloid deposits are also demonstrated. The conditions connected with deposition of amyloid β–protein in cystatin C and cerebral hemorrhage is also provided.
402 citations
TL;DR: The majority of mitochondrial, but not cytoplasmic, caspase-3 zymogens contain this inhibitory modification S-nitrosylated and these studies suggest that S-Nitrosylation plays an important role in regulating mitochondrial caspases function and that the S- nitrogenous state of a given protein depends on its subcellular localization.
Abstract: Caspase-3 is a cysteine protease located in both the cytoplasm and mitochondrial intermembrane space that is a central effector of many apoptotic pathways. In resting cells, a subset of caspase-3 zymogens is S-nitrosylated at the active site cysteine, inhibiting enzyme activity. During Fas-induced apoptosis, caspases are denitrosylated, allowing the catalytic site to function. In the current studies, we sought to identify the subpopulation of caspases that is regulated by S-nitrosylation. We report that the majority of mitochondrial, but not cytoplasmic, caspase-3 zymogens contain this inhibitory modification. In addition, the majority of mitochondrial caspase-9 is S-nitrosylated. These studies suggest that S-nitrosylation plays an important role in regulating mitochondrial caspase function and that the S-nitrosylation state of a given protein depends on its subcellular localization.
362 citations
TL;DR: It is suggested that translocation of lysosomal proteases is an early event in NZ‐induced apoptosis and that the release and increased activity of cathepsin D allow this protease to exert an apoptosis‐mediating effect upstream of the caspase cascade.
Abstract: SPECIFIC AIMSThe overall objective of the present study was to examine lysosomal stability during apoptosis and the relationship between caspase activation and the lysosomal proteases cathepsins D and B with regard to their effects on apoptosis.PRINCIPAL FINDINGS1. Naphthazarin-induced apoptosis is dependent on the lysosomal protease cathepsin DThe caspase-3-like activity increased after 12 h of treatment with the redox cycling quinone naphthazarine (NZ). By comparison, the activity of cathepsin D was augmented after 4 h of NZ treatment and peaked at 16 h (Fig. 1A⤻ , B⤻ ). Moreover, increased levels of p53, a transcription factor for cathepsin D, were detected after exposure to NZ for 4 h (Fig. 1D⤻ ). The total protein level did not change in response to p53 induction and was estimated to 8.2 ± 3.5, 8.0 ± 2.1, and 7.1 ± 1.4 μg protein/10 000 cells after 0, 12, and 20 h respectively. We also found that NZ caused a rapid decrease in the activity of the lysosomal cysteine protease cathepsin B (Fig. 1C⤻ ). Fi...
284 citations
TL;DR: It is suggested that the exclusion domain originates from a metallo‐protease inhibitor, characterized in people suffering from Haim–Munk and Papillon–Lefevre syndromes, suggests how they disrupt the fold and function of the enzyme.
Abstract: Dipeptidyl peptidase I (DPPI) or cathepsin C is the physiological activator of groups of serine proteases from immune and inflammatory cells vital for defense of an organism. The structure presented shows how an additional domain transforms the framework of a papain-like endopeptidase into a robust oligomeric protease-processing enzyme. The tetrahedral arrangement of the active sites exposed to solvent allows approach of proteins in their native state; the massive body of the exclusion domain fastened within the tetrahedral framework excludes approach of a polypeptide chain apart from its termini; and the carboxylic group of Asp1 positions the N-terminal amino group of the substrate. Based on a structural comparison and interactions within the active site cleft, it is suggested that the exclusion domain originates from a metallo-protease inhibitor. The location of missense mutations, characterized in people suffering from Haim-Munk and Papillon-Lefevre syndromes, suggests how they disrupt the fold and function of the enzyme.
238 citations
TL;DR: These studies provide the first example of a product from a eukaryotic parasite that can directly interfere with antigen presentation, which, in turn, may suggest how filarial parasites might inactivate the host immune response to a helminth invader.
220 citations
TL;DR: Falcipain-3 is a second P. falciparum haemoglobinase that is particularly suited for the hydrolysis of native haemochemistry in the acidic food vacuole, and may offer optimized hydroolysis of both native ha Hemoglobin and globin peptides.
Abstract: In the malaria parasite Plasmodium falciparum, erythrocytic trophozoites hydrolyse haemoglobin to provide amino acids for parasite protein synthesis. Cysteine protease inhibitors block parasite haemoglobin hydrolysis and development, indicating that cysteine proteases are required for these processes. Three papain-family cysteine protease sequences have been identified in the P. falciparum genome, but the specific roles of their gene products and other plasmodial proteases in haemoglobin hydrolysis are uncertain. Falcipain-2 was recently identified as a principal trophozoite cysteine protease and potential drug target. The present study characterizes the related P. falciparum cysteine protease falcipain-3. As is the case with falcipain-2, falcipain-3 is expressed by trophozoites and appears to be located within the food vacuole, the site of haemoglobin hydrolysis. Both proteases require a reducing environment and acidic pH for optimal activity, and both prefer peptide substrates with leucine at the P(2) position. The proteases differ, however, in that falcipain-3 undergoes efficient processing to an active form only at acidic pH, is more active and stable at acidic pH, and has much lower specific activity against typical papain-family peptide substrates, but has greater activity against native haemoglobin. Thus falcipain-3 is a second P. falciparum haemoglobinase that is particularly suited for the hydrolysis of native haemoglobin in the acidic food vacuole. The redundancy of cysteine proteases may offer optimized hydrolysis of both native haemoglobin and globin peptides. Consideration of both proteases will be necessary to evaluate cysteine protease inhibitors as antimalarial drugs.
213 citations
TL;DR: This work expressed and purified rhodesain, the target protease of Trypanosoma brucei rhodesiense (MVAT4 strain), in reagent quantities from Pichia pastoris, and identified an invariable ER(A/V)FNAA motif in the pro-peptide sequence of r Rhodesain that was identified as being unique to the genus Trypano-Brucei.
169 citations
TL;DR: The first generation of 'immune evasion genes' from parasites such as the filarial nematode Brugia malayi are identified, and the major transcript present in mosquito-borne infective larvae, Bm-ALT, is a credible vaccine candidate for use against lymphatic filariasis, while a second abundantly-expressed gene is similar to a likely vaccine antigen being developed against hookworm parasites.
169 citations
TL;DR: Results indicate that the intermediate of RD21 was accumulated in the vacuoles as an aggregate, and then slowly matured to make a soluble protease by removing the granulin domain during leaf senescence.
Abstract: Arabidopsis RD21 is a cysteine protease of the papain family. Unlike other members of the papain family, RD21 has a C-terminal extension sequence composed of two domains, a 2-kD proline-rich domain and a 10-kD domain homologous to animal epithelin/granulin family proteins. The RD21 protein was accumulated as 38- and 33-kD proteins in Arabidopsis leaves. An immunoblot showed that the 38-kD protein had the granulin domain, whereas the 33-kD protein did not. A pulse-chase experiment with Bright-Yellow 2 transformant cells expressing RD21 showed that RD21 was synthesized as a 57-kD precursor and was then slowly processed to make the 33-kD mature protein via the 38-kD intermediate. After a 12-h chase, the 38-kD intermediate was still detected in the cells. These results indicate that the N-terminal propeptide was first removed from the 57-kD precursor, and the C-terminal granulin domain was then slowly removed to yield the 33-kD mature protein. Subcellular fractionation of the Bright-Yellow 2 transformant showed that the intermediate and mature forms of RD21 were localized in the vacuoles. Under the acidic conditions of the vacuolar interior, the intermediate was found to be easily aggregated. The intermediate and the mature protein were accumulated in association with leaf senescence. Taken together, these results indicate that the intermediate of RD21 was accumulated in the vacuoles as an aggregate, and then slowly matured to make a soluble protease by removing the granulin domain during leaf senescence.
163 citations
TL;DR: Recombinant onchocystatin has the potential to contribute to a state of cellular hyporesponsiveness and is a possible pathogenicity factor essential for the persistence of O. volvulus within its human host.
Abstract: Immune responses of individuals infected with filarial nematodes are characterized by a marked cellular hyporesponsiveness and a shift of the cytokine balance toward a Th2/Th3 response. This modulation of cellular immune responses is considered as an important mechanism to avoid inflammatory immune responses that could eliminate the parasites. We investigated the immunomodulatory potential of a secreted cysteine protease inhibitor (onchocystatin) of the human pathogenic filaria Onchocerca volvulus. Recombinant onchocystatin (rOv17), a biologically active cysteine protease inhibitor that inhibited among others the human cysteine proteases cathepsins L and S, suppressed the polyclonally stimulated and the Ag-driven proliferation of human PBMC. Stimulated as well as unstimulated PBMC in the presence of rOv17 produced significantly more IL-10, which was paralleled in some situations by a decrease of IL-12p40 and preceded by an increase of TNF-alpha. At the same time, rOv17 reduced the expression of HLA-DR proteins and of the costimulatory molecule CD86 on human monocytes. Neutralization of IL-10 by specific Abs restored the expression of HLA-DR and CD86, whereas the proliferative block remained unaffected. Depletion of monocytes from the PBMC reversed the rOv17-induced cellular hyporeactivity, indicating monocytes to be the target cells of immunomodulation. Therefore, onchocystatin has the potential to contribute to a state of cellular hyporesponsiveness and is a possible pathogenicity factor essential for the persistence of O. volvulus within its human host.
163 citations
TL;DR: It can be concluded that the serine protease is the most important protease in the release of cell-bound FnBPs and protein A, which requires aureolysin to be activated.
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 in sarA 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 of sarA mutant cells in the presence of the global protease inhibitor alpha(2)-macroglobulin resulted in a 16-fold increase in cell-bound FnBPs, indicating that extracellular proteases were responsible for the decreased amounts of FnBPs in sarA mutant 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.
TL;DR: It is concluded that SspA is required for maturation of SspB and plays a role in controlling autolytic activity but does not by itself exert a significant contribution to the development of tissue abscess infections.
Abstract: Signature tagged mutagenesis has recently revealed that the Ssp serine protease (V8 protease) contributes to in vivo growth and survival of Staphylococcus aureus in different infection models, and our previous work indicated that Ssp could play a role in controlling microbial adhesion. In this study, we describe an operon structure within the ssp locus of S. aureus RN6390. The ssp gene encoding V8 protease is designated as sspA, and is followed by sspB, which encodes a 40.6-kDa cysteine protease, and sspC, which encodes a 12.9-kDa protein of unknown function. S. aureus SP6391 is an isogenic derivative of RN6390, in which specific loss of SspA function was achieved through a nonpolar allelic replacement mutation. In addition to losing SspA, the culture supernatant of SP6391 showed a loss of 22- to 23-kDa proteins and the appearance of a 40-kDa protein corresponding to SspB. Although the 40-kDa SspB protein could degrade denatured collagen, our data establish that this is a precursor form which is normally processed by SspA to form a mature cysteine protease. Culture supernatant of SP6391 also showed a new 42-kDa glucosaminidase and enhanced glucosaminidase activity in the 29 to 32 kDa range. Although nonpolar inactivation of sspA exerted a pleiotropic effect, S. aureus SP6391 exhibited enhanced virulence in a tissue abscess infection model relative to RN6390. Therefore, we conclude that SspA is required for maturation of SspB and plays a role in controlling autolytic activity but does not by itself exert a significant contribution to the development of tissue abscess infections.
TL;DR: A strong variation within the latter activities was shown within one family and between protease inhibitor families, and a broad spectrum of enzyme inhibition was shown.
Abstract: Protease inhibitors from potato juice of cv. Elkana were purified and quantified. The protease inhibitors represent ca. 50% of the total soluble proteins in potato juice. The protease inhibitors were classified into seven different families: potato inhibitor I (PI-1), potato inhibitor II (PI-2), potato cysteine protease inhibitor (PCPI), potato aspartate protease inhibitor (PAPI), potato Kunitz-type protease inhibitor (PKPI), potato carboxypeptidase inhibitor (PCI), and "other serine protease inhibitors". The most abundant families were the PI-2 and PCPI families, representing 22 and 12% of all proteins in potato juice, respectively. Potato protease inhibitors show a broad spectrum of enzyme inhibition. All the families (except PCI) inhibited trypsin and/or chymotrypsin. PI-2 isoforms exhibit 82 and 50% of the total trypsin and chymotrypsin inhibiting activity, respectively. A strong variation within the latter activities was shown within one family and between protease inhibitor families.
TL;DR: The isolation, biochemical characterization, developmental stage distribution and subcellular localization of chagasin, an endogenous cysteine protease inhibitor in T. cruzi is reported, indicating that the expression of chgasin is developmentally regulated and inversely correlated with that of cruzipain.
Abstract: 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.
TL;DR: N. brasiliensis appears to skillfully evade host immune systems by secreting nippocystatin, which modulates antigen processing in antigen-presenting cells of hosts.
Abstract: During infection, parasites evade the host immune system by modulating or exploiting the immune system; eg, they suppress expression of major histocompatibility complex class II molecules or secrete cytokine-like molecules However, it is not clear whether helminths disturb the immune responses of their hosts by controlling the antigen-processing pathways of the hosts In this study, we identified a new cysteine protease inhibitor, nippocystatin, derived from excretory-secretory (ES) products of an intestinal nematode, Nippostrongylus brasiliensis Nippocystatin, which belongs to cystatin family 2, consists of 144 amino acids and is secreted as a 14-kDa mature form In vivo treatment of ovalbumin (OVA)-immunized mice with recombinant nippocystatin (rNbCys) profoundly suppressed OVA-specific proliferation of splenocytes but not non-antigen-specific proliferation of splenocytes OVA-specific cytokine production was also greatly suppressed in rNbCys-treated mice Although the serum levels of both OVA-specific immunoglobulin G1 (IgG1) and IgG2a were not affected by rNbCys treatment, OVA-specific IgE was preferentially downregulated in rNbCys-treated mice In vitro rNbCys inhibited processing of OVA by lysosomal cysteine proteases from the spleens of mice Mice with anti-nippocystatin antibodies became partially resistant to infection with N brasiliensis Based on these findings, N brasiliensis appears to skillfully evade host immune systems by secreting nippocystatin, which modulates antigen processing in antigen-presenting cells of hosts
TL;DR: This review presents the state of the art in the design of such enzyme inhibitors with potential therapeutic applications, as well as recent advances in the use of some of these proteases in therapy.
Abstract: Proteases of the serine-, cysteine- and metallo- type are widely spread in many pathogenic bacteria, where they play critical functions related to colonisation and evasion of host immune defences, acquisition of nutrients for growth and proliferation, facilitation of dissemination, or tissue damage during infection. Since all the antibiotics currently used clinically share a common mechanism of action, i.e., inhibition of bacterial cell wall biosynthesis, resistance to these pharmacological agents represents a serious medical problem, which might be resolved by using a new generation of antibiotics with a different mechanism of action. Bacterial protease inhibitors constitute an interesting possibility, due to the fact that many specific and ubiquitous proteases have recently been characterised in some detail in both Gram-positive and Gram-negative pathogens. Unfortunately, at this moment few potent, specific inhibitors for such bacterial proteases have been reported, except for signal peptidase, clostrip...
TL;DR: These studies provide a promising alternative for protection against leishmaniasis with a switch of CD4+ differentiation from Th2 to Th1, indicative of long-term resistance.
Abstract: The virulence of Leishmania donovani in mammals depends at least in part on cysteine proteases because they play a key role in CD4 + T cell differentiation. A 6-fold increase in NO production was observed with 0.5 μM chicken cystatin, a natural cysteine protease inhibitor, in IFN-γ-activated macrophages. In a 45-day BALB/c mouse model of visceral leishmaniasis, complete elimination of spleen parasite burden was achieved by cystatin in synergistic activation with a suboptimal dose of IFN-γ. In contrast to the case with promastigotes, cystatin and IFN-γ inhibited the growth of amastigotes in macrophages. Although in vitro cystatin treatment of macrophages did not induce any NO generation, significantly enhanced amounts of NO were generated by macrophages of cystatin-treated animals. Their splenocytes secreted soluble factors required for the induction of NO biosynthesis, and the increased NO production was paralleled by a concomitant increase in antileishmanial activity. Moreover, splenocyte supernatants treated with anti-IFN-γ or anti-TNF-α Abs suppressed inducible NO generation, whereas i.v. administration of these anticytokine Abs along with combined therapy reversed protection against infection. mRNA expression and flow cytometric analysis of infected spleen cells suggested that cystatin and IFN-γ treatment, in addition to greatly reducing parasite numbers, resulted in reduced levels of IL-4 but increased levels of IL-12 and inducible NO synthase. Not only was this treatment curative when administered 15 days postinfection, but it also imparted resistance to reinfection. These studies provide a promising alternative for protection against leishmaniasis with a switch of CD4 + differentiation from Th2 to Th1, indicative of long-term resistance.
TL;DR: A role for α3 gap junctions in maintaining calcium homeostasis is established that in turn is required to control activity of the calcium-dependent cysteine protease Lp82, shown here to be a key initiator of the process of cataractogenesis.
TL;DR: The crystal structure of the cysteine protease caspase-8 in complex with the inhibitor p35 has revealed a new family of suicide protease inhibitors, and helped elucidate many aspects of the mechanism of action of serpins.
TL;DR: The identification of the ASFV protease will allow a better understanding of the role of polyprotein processing in virus assembly and may contribute to the knowledge of the emerging family of SUMO-1-specific proteases.
TL;DR: It is demonstrated that ER stress in cortical neurons leads to activation of several cysteine proteases within diverse neuronal compartments and indicates that Alzheimer's disease-linked PS-1 mutations do not invariably alter the proteolytic, chaperone induction, translational suppression, and apoptotic responses to ER stress.
TL;DR: When cultured in feather-containing broth with a growth optimum of pH 7.0 and 47 degrees C, a Bacillus licheniformis strain exhibited a high chicken feather-degrading activity and a trypsin-like protease was isolated from its ferment broth and was partially characterized.
Abstract: When cultured in feather-containing broth with a growth optimum of pH 7.0 and 47 degrees C, a Bacillus licheniformis strain exhibited a high chicken feather-degrading activity. A trypsin-like protease was isolated from its ferment broth and was partially characterized. The enzyme was constitutively secreted and was highly active towards N-benzoyl-Phe-Val-Arg-p-nitroanilide as chromogenic substrate. Its pH optimum was 8.5 and it exhibited the highest activity at 52 degrees C. Fractionation on Sephadex G-100 column revealed that its molecular mass was about 42 kDa. The enzyme, which is new for the genus Bacillus, is a thiol protease, as tosyl-L-phenylalanine chloromethyl ketone, tosyl-L-lysine chloromethyl ketone, phenylmethylsulfonyl fluoride and ethylenediamine tetraacetate did not inhibit it, while HgCl2 and para-chloromercuribenzoate lowered its activity.
TL;DR: Cross-resistance between cysteine protease inhibitors and other antimalarial agents is not expected in parasites that are now circulating and falcipain-2 remains a promising chemotherapeutic target.
Abstract: Falcipain-2, a cysteine protease and essential hemoglobinase of Plasmodium falciparum, is a potential antimalarial drug target. We compared the falcipain-2 sequences and sensitivities to cysteine protease inhibitors of five parasite strains that differ markedly in sensitivity to established antimalarial drugs. The sequence of falcipain-2 was highly conserved, and the sensitivities of all of the strains to falcipain-2 inhibitors were very similar. Thus, cross-resistance between cysteine protease inhibitors and other antimalarial agents is not expected in parasites that are now circulating and falcipain-2 remains a promising chemotherapeutic target.
TL;DR: The proteolytic activity of this enzyme was rapidly and efficiently inhibited by human α-2-macroglobulin; however, human kininogen as well as cystatins were not inhibitory and the protease was capable of inactivating, by limited proteolysis, both α-1-antitrypsin and HMWkininogen.
Abstract: We report the complete coding sequence and the partial amino acid sequence (determined by chemical sequencing) of Staphylococcus epidermidis extracellular cysteine (Ecp) and serine (Esp) proteases. The first enzyme shows an extended sequence similarity to Staphylococcus aureus cysteine protease (staphopain) and the second one resembles the serine protease produced by that species. The region directly upstream of the sequence coding for the mature protein in both enzymes displays significant homology to the profragments encoded by sspB and sspA, respectively, thus suggesting that the characterised enzymes may also be produced as proproteins. Furthermore, we report some biological properties of the cysteine protease, contributing to a better understanding of its role as a possible virulence factor. The proteolytic activity of this enzyme was rapidly and efficiently inhibited by human alpha-2-macroglobulin; however, human kininogen as well as cystatins (A, C and D) were not inhibitory. Moreover, the protease was capable of inactivating, by limited proteolysis, both alpha-1-antitrypsin and HMW-kininogen, but neither alpha-1-antichymotrypsin nor antithrombin III.
TL;DR: Human cysteine protease cathepsin L was inactivated at acid pH by a first-order process, showing that the inactivation is accompanied by large structural changes, a decrease in alpha-helix content being especially pronounced.
Abstract: Human cysteine protease cathepsin L was inactivated at acid pH by a first-order process. The inactivation rate decreased with increasing concentrations of a small synthetic substrate, suggesting that substrates stabilize the active conformation. The substrate-independent inactivation rate constant increased with organic solvent content of the buffer, consistent with internal hydrophobic interactions, disrupted by the organic solvent, also stabilizing the enzyme. Circular dichroism showed that the inactivation is accompanied by large structural changes, a decrease in α-helix content being especially pronounced. The high activation energy of the reaction at pH 3.0 (200 kJ·mol−1) supported such a major conformational change occurring. The acid inactivation of cathepsin L was irreversible, consistent with the propeptide being needed for proper folding of the enzyme. Aspartic protease cathepsin D was shown to cleave denatured, but not active cathepsin L, suggesting a potential mechanism for in-vivo regulation and turnover of cathepsin L inside lysosomes.
TL;DR: Heterologous expression of the catalytic domain of cruzipain 2 in Saccharomyces cerevisae yielded an enzyme that differs markedly from cruzain with respect to pH stability, substrate specificity and sensitivity to inhibition by natural and synthetic inhibitors of cysteine proteases.
TL;DR: It is demonstrated that the levels of active CatL are strongly reduced in bone marrow‐derived antigen‐presenting cells that lack p41, which suggests that p41 is not merely an inhibitor of CatL enzymatic activity, but serves as a chaperone to help maintain a pool of mature enzyme in late‐endocytic compartments of antigen‐ presenting cells.
Abstract: The p41 splice variant of major histocompatibility complex (MHC) class II-associated invariant chain (Ii) contains a 65 aa segment that binds to the active site of cathepsin L (CatL), a lysosomal cysteine protease involved in MHC class II-restricted antigen presentation. This segment is absent from the predominant form of Ii, p31. Here we document the in vivo significance of the p41–CatL interaction. By biochemical means and electron microscopy, we demonstrate that the levels of active CatL are strongly reduced in bone marrow-derived antigen-presenting cells that lack p41. This defect mainly concerns the mature two-chain forms of CatL, which depend on p41 to be expressed at wild-type levels. Indeed, pulse–chase analysis suggests that these mature forms of CatL are degraded by endocytic proteases when p41 is absent. We conclude that p41 is required for activity of CatL by stabilizing the mature forms of the enzyme. This suggests that p41 is not merely an inhibitor of CatL enzymatic activity, but serves as a chaperone to help maintain a pool of mature enzyme in late-endocytic compartments of antigen-presenting cells.
TL;DR: Serratia marcescens ATCC 25419 was chosen for production, purification and characterization of secreted proteases when growing on reconstituted whey and was strongly inhibited by ethylenediaminetetraacetic acid and 1,10-phenanthroline, whereas the serine protease was inhibited by phenylmethylsulfonyl fluoride.
TL;DR: The results suggest that the biosynthesis, processing, and turnover of cat K in human osteoclasts is constitutive and occurs in a manner similar to that of other known cysteine proteases.
TL;DR: Although vaccination did not confer any protection against infection with H. contortus, cystatin will be a valuable tool in the analysis of the function of the cysteine proteases which are the subject of on-going study as potential vaccine components.
Abstract: A cDNA encoding a cysteine protease inhibitor (cystatin) was identified by immunoscreening a Haemonchus contortus cDNA library with antisera from lambs vaccinated with a protective membrane protein complex (H-gal-GP) derived from the gut of the parasite. The cDNA sequence, designated Cys-1, showed significant levels of similarity with cystatins from several species of nematode as well as with human cystatinNucleotide and amino acid sequence data reported in this paper are available in the GenBankTM database under the accession number AF035945.
. Recombinant H. contortus cystatin was expressed in Escherichia coli in a soluble and functionally active form, which proved to be a potent inhibitor of both mammalian cathepsin B and native H. contortus cysteine proteases. Immunolocalization studies using antisera raised against recombinant H. contortus cystatin showed that the inhibitor was predominantly expressed in the cytoplasm of intestinal cells. To determine whether H. contortus had any protective capacity against infection, lambs were vaccinated with the recombinant molecule and subsequently given a single challenge infection. Although vaccination did not confer any protection against infection with H. contortus, as judged by faecal egg output or worm counts, cystatin will be a valuable tool in the analysis of the function of the cysteine proteases which are the subject of on-going study as potential vaccine components.