Cathepsin S. The cysteine proteinase from bovine lymphoid tissue is distinct from cathepsin L (EC 3.4.22.15).
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ABSTRACT:
SUBMITTER: Kirschke H
PROVIDER: S-EPMC1147438 | biostudies-other | 1986 Dec
REPOSITORIES: biostudies-other
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Project description:The aim of the work was to identify and characterize the cysteine proteinases of bone tissue, as these enzymes appear necessary for bone resorption. Three cysteine-dependent proteolytic activities were separated from a homogenate of mouse calvaria by a fractionation procedure involving (NH4)2SO4 precipitation, gel filtration and ion-exchange chromatography. The first two are typical cathepsins B and L with respect to (1) their reactivity with anti-(cathepsin B) and anti-(cathepsin L) antibodies respectively, (2) their relative rate constants for inhibition by benzyloxycarbonyl-Phe-Phe-CHN2 and L-3-carboxy-trans-2,3-epoxypropionyl-L-leucylamido-(4-guanid ino)butane and (3) their enzymic properties, such as the higher activities of cathepsin L against collagen and gelatin as compared with cathepsin B, and the fact that benzyloxycarbonyl-Arg-Arg 4-methoxy-2-naphthylamide is hydrolysed only by cathepsin B. Cathepsin L was mainly recovered in its precursor form, as indicated by its apparent 40 kDa molecular mass and its relative stability at pH 7.2. The third enzyme is a cathepsin L-like proteinase with an apparent molecular mass of 70 kDa. It is immunoprecipitated by anti-(cathepsin L) antibodies, and appears as the 25 kDa band of mature cathepsin L in Western blots. It further resembles (pro)cathepsin L with regard to its activities against synthetic substrates and proteins such as collagen, and with regard to its response to various inhibitors. However, unlike (pro)cathepsin L, it is eluted as a 70 kDa protein on gel filtration (even in the presence of 1% Brij or 1 M-NaCl), it is stable at pH values as high as 9, and it exhibits stronger affinity for phenyl-Sepharose. It might thus result from a strong complex between mature cathepsin L and another entity that confers stability at alkaline pH and favours hydrophobic interactions. This 70 kDa activity was also detected in mouse muscle and long bones of Ca(2+)-deficient chicks but not in mouse liver, spleen or kidney.
| S-EPMC1151563 | biostudies-other
Project description:Human macrophages mediate the dissolution of elastic lamina by mobilizing tissue-destructive cysteine proteinases. While macrophage-mediated elastin degradation has been linked to the expression of cathepsins L and S, these cells also express cathepsin K, a new member of the cysteine proteinase family whose elastinolytic potential exceeds that of all known elastases. To determine the relative role of cathepsin K in elastinolysis, monocytes were differentiated under conditions in which they recapitulated a gene expression profile similar to that observed at sites of tissue damage in vivo. After a 12-d culture period, monocyte-derived macrophages (MDMs) expressed cathepsin K in tandem with cathepsins L and S. Though cysteine proteinases are acidophilic and normally confined to the lysosomal network, MDMs secreted cathepsin K extracellularly in concert with cathepsins L and S. Simultaneously, MDMs increased the expression of vacuolar-type H(+)-ATPase components, acidified the pericellular milieu, and maintained extracellular cathepsin K in an active form. MDMs from a cathepsin K-deficient individual, however, retained the ability to express, process, and secrete cathepsins L and S, and displayed normal elastin-degrading activity. Thus, matrix-destructive MDMs exteriorize a complex mix of proteolytic cysteine proteinases, but maintain full elastinolytic potential in the absence of cathepsin K by mobilizing cathepsins L and S.
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Project description:We have cloned a cDNA encoding a cysteine proteinase of the Acanthamoeba healyi OC-3A strain isolated from the brain of a granulomatous amoebic encephalitis patient. A DNA probe for an A. healyi cDNA library screening was amplified by PCR using degenerate oligonucleotide primers designed on the basis of conserved amino acids franking the active sites of cysteine and asparagine residues that are conserved in the eukaryotic cysteine proteinases. Cysteine proteinase gene of A. healyi (AhCP1) was composed of 330 amino acids with signal sequence, a proposed pro-domain and a predicted active site made up of the catalytic residues. Cys25, His159, and Asn175. Deduced amino acid sequence analysis indicates that AhCP1 belong to ERFNIN subfamily of C1 peptidases. By Northern blot analysis, no direct correlation was observed between AhCP1 mRNA expression and virulence of Acanthamoeba, but the gene was expressed at higher level in amoebae isolated from soil than amoeba from clinical samples. These findings raise the possibility that Ahcp1 protein may play a role in protein metabolism and digestion of phagocytosed bacteria or host tissue debris rather than in invasion of amoebae into host tissue.
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Project description:Radopholus similis is an important plant parasitic nematode which severely harms many crops. Cathepsin B is present in a wide variety of organisms, and plays an important role in many parasites. Understanding cathepsin B of R. similis would allow us to find new targets and approaches for its control. In this study, we found that Rs-cb-1 mRNA was expressed in esophageal glands, intestines and gonads of females, testes of males, juveniles and eggs in R. similis. Rs-cb-1 expression was the highest in females, followed by juveniles and eggs, and was the lowest in males. The maximal enzyme activity of Rs-CB-1 was detected at pH 6.0 and 40 °C. Silencing of Rs-cb-1 using in vitro RNAi (Soaking with dsRNA in vitro) not only significantly inhibited the development and hatching of R. similis, but also greatly reduced its pathogenicity. Using in planta RNAi, we confirmed that Rs-cb-1 expression in nematodes were significantly suppressed and the resistance to R. similis was significantly improved in T2 generation transgenic tobacco plants expressing Rs-cb-1 dsRNA. The genetic effects of in planta RNAi-induced gene silencing could be maintained in the absence of dsRNA for at least two generations before being lost, which was not the case for the effects induced by in vitro RNAi. Overall, our results first indicate that Rs-cb-1 plays key roles in the development, hatching and pathogenesis of R. similis, and that in planta RNAi is an effective tool in studying gene function and genetic engineering of plant resistance to migratory plant parasitic nematodes.
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Project description:BackgroundThe nematode Radopholus similis is an important migratory endoparasite of plants. Cysteine proteinases such as cathepsin S (CPS) play key roles during embryonic development, invasion, and pathogenesis in nematodes and many other animal parasites. This study was designed to investigate the molecular characterization and functions of a cathepsin S protease in R. similis and to find new targets for its control.ResultsRs-CPS of R. similis, Hg-CPS of Heterodera glycines and Ha-CPS of H. avenae are closely genetically related and share the same branch of the phylogenetic tree. Rs-cps is a multi-copy gene that is expressed in the esophageal glands, ovaries, testes, vas deferens, and eggs of R. similis. Rs-cps mRNA transcripts are expressed at varying levels during all developmental stages of R. similis. Rs-cps expression was highest in females. The neurostimulant octopamine did not significantly enhance the ingestion of the dsRNA soaking solution by R. similis but instead had a detrimental effect on nematode activity. The dsRNA soaking solution diffused into the body of R. similis not only through the esophageal lumen but also through the amphids, excretory duct, vagina, anus and cloacal orifice. We confirmed that RNAi significantly suppressed the expression level of Rs-cps and reproductive capability and pathogenicity of R. similis.ConclusionsOur results demonstrate that Rs-cps plays important roles in the reproduction, parasitism and pathogenesis of R. similis and could be used as a new potential target for controlling plant parasitic nematodes.
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Project description: Not available
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Project description:The pine wood nematode (PWN), Bursaphelenchus xylophilus, is the pathogen of pine wilt disease (PWD), resulting in huge losses in pine forests. However, its pathogenic mechanism remains unclear. The cathepsin L-like cysteine proteinase (CPL) genes are multifunctional genes related to the parasitic abilities of plant-parasitic nematodes, but their functions in PWN remain unclear. We cloned three cpl genes of PWN (Bx-cpls) by rapid amplification of cDNA ends (RACE) and analyzed their characteristics using bioinformatic methods. The tissue specificity of cpl gene of PWN (Bx-cpl) was studied using in situ mRNA hybridization (ISH). The functions of Bx-cpls in development and pathogenicity were investigated using real-time quantitative PCR (qPCR) and RNA interference (RNAi). The results showed that the full-length cDNAs of Bx-cpl-1, Bx-cpl-2, and Bx-cpl-3 were 1163 bp, 1305 bp, and 1302 bp, respectively. Bx-cpls could accumulate specifically in the egg, intestine, and genital system of PWN. During different developmental stages of PWN, the expression of Bx-cpls in the egg stage was highest. After infection, the expression levels of Bx-cpls increased and reached their highest at the initial stage of PWD, then declined gradually. The silencing of Bx-cpl could reduce the feeding, reproduction, and pathogenicity of PWN. These results revealed that Bx-cpls play multiple roles in the development and pathogenic processes of PWN.
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Project description:The inhibition of human liver cathepsin L by two specific proteinase inhibitors present in human serum, namely alpha 2 cysteine-proteinase inhibitor and the low-Mr cysteine-proteinase inhibitor, was studied. Kinetic parameters, including inhibition constants (Ki) and rate constants for association and dissociation (k+1 and K-1), were determined. The values found are consistent with a possible physiological function of these inhibitors to control cathepsin L activity. Furthermore, a transfer of active proteinase from the complex with either cysteine-proteinase inhibitor species to alpha 2-macroglobulin was demonstrated in vitro. Given the rate of dissociation of both cathepsin-L-cysteine-proteinase inhibitor complexes, a function of transitory inhibitor can therefore be hypothesized for these proteins and might then provide an explanation of the clearance of lysosomal proteinases.
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Project description:Tamoxifen is one of the most commonly employed endocrine therapies for patients with estrogen receptor α (ERα)-positive breast cancer. Unfortunately the clinical benefit is limited due to intrinsic and acquired drug resistance. We previously reported a genome-wide association study that identified common SNPs near the CTSO gene and in ZNF423 associated with development of breast cancer during tamoxifen therapy in the NSABP P-1 and P-2 breast cancer prevention trials. Here, we have investigated their roles in ERα-positive breast cancer growth and tamoxifen response, focusing on the mechanism of CTSO. We performed in vitro studies including luciferase assays, cell proliferation, and mass spectrometry-based assays using ERα-positive breast cancer cells and a panel of genomic data-rich lymphoblastoid cell lines. We report that CTSO reduces the protein levels of BRCA1 and ZNF423 through cysteine proteinase-mediated degradation. We also have identified a series of transcription factors of BRCA1 that are regulated by CTSO at the protein level. Importantly, the variant CTSO SNP genotypes are associated with increased CTSO and decreased BRCA1 protein levels that confer resistance to tamoxifen. Characterization of the effect of both CTSO SNPs and ZNF423 SNPs on tamoxifen response revealed that cells with different combinations of CTSO and ZNF423 genotypes respond differently to Tamoxifen, PARP inhibitors or the combination of the two drugs due to SNP dependent differential regulation of BRCA1 levels. Therefore, these genotypes might be biomarkers for selection of individual drug to achieve the best efficacy.
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