A cathepsin B-like protease is required for host protein degradation in Trypanosoma brucei

Identification and analysis of Clan CA (papain) cysteine proteases in primitive protozoa and metazoa have suggested that this enzyme family is more diverse and biologically important than originally thought. The protozoan parasite Trypanosoma brucei is the etiological agent of African sleeping sickness. The cysteine protease activity of this organism is a validated drug target as first recognized by the killing of the parasite with the diazomethane inhibitor Z-Phe-Ala-CHN(2) (where Z is benzyloxycarbonyl). Whereas the presumed target of this inhibitor was rhodesain (also brucipain, trypanopain), the major cathepsin L-like cysteine protease of T. brucei, genomic analysis has now identified tbcatB, a cathepsin B-like cysteine protease as a possible inhibitor target. The mRNA of tbcatB is more abundantly expressed in the bloodstream versus the procyclic form of the parasite. Induction of RNA interference against rhodesain did not result in an abnormal phenotype in cultured T. brucei. However, induction of RNA interference against tbcatB led to enlargement of the endosome, accumulation of fluorescein isothiocyanate-transferrin, defective cytokinesis after completion of mitosis, and ultimately the death of cultured parasites. Therefore, tbcatB, but not rhodesain, is essential for T. brucei survival in culture and is the most likely target of the diazomethane protease inhibitor Z-Phe-Ala-CHN(2) in T. brucei.     

A cathepsin B-like enzyme from mackerel white muscle is a precursor of cathepsin B

A cathepsin B-like enzyme from the white muscle of common mackerel Scomber japonicus was a cysteine protease that hydrolyzed Z-Arg-Arg-MCA, the substrate for cathepsin B. In a partial purified cathepsin B-like enzyme preparation at 4 degrees C left over time, a converted enzyme that hydrolyzes Z-Arg-Arg-MCA and Z-Phe-Arg-MCA appeared in the preparation. The converted enzyme was purified from the cathepsin B-like enzyme, characterized and was identified as mackerel cathepsin B. These results suggested that the mackerel cathepsin B-like enzyme was a precursor of cathepsin B. Mackerel cathepsin B formed in the purified cathepsin B-like enzyme preparation by adding of a small amount of the purified cathepsin B to the preparation. Therefore, mackerel cathepsin B-like enzyme was converted to the mature form of cathepsin B by autoactivation. The conversion of the cathepsin B-like enzyme (molecular mass 60 kDa) to cathepsin B (molecular mass 23 kDa) was detected by immunoblotting by using human anti-(cathepsin B) antibody. The intermediate forms of 40 kDa and 38 kDa were also detected during the conversion.     

Identification and characterization of a cathepsin B-like protease in Physarum sclerotium

In response to dry stress the plasmodium of a true slime mold, Physarum polycephalum, undergoes formation of sclerotium, which is a dormant body resistant to desiccation. The sclerotium can germinate within several hours after addition of water, followed by generation of the plasmodium. In the early phase of the germination many enzymes and other proteins of the sclerotium are required for formation of the plasmodium. As dehydration of proteins often leads to destruction of their structure or reduction in their activity, it is important to elucidate whether the dehydrated enzymes are present as the intact in the sclerotium. In this study three peaks of protease activity were detected with anion exchange column chromatography of the extract from the sclerotia. From among them, an acid protease was purified to homogeneity by gel filtration column chromatography, hydroxyapatite column chromatography, acid treatment, and cation-exchange column chromatography. Treatment of the protease fractions with pH 4.0 resulted in approximately 20-fold activation of the activity. The purified protease was a monomer with a molecular mass of 35 kDa. The optimum pH and temperature were 6.3 and 40 degrees C, respectively. Beta-casein, histone H1, and H2B were degraded by the 35 kDa protease, but human hemoglobin and human serum albumin were very poor substrates. In addition, the enzyme was sensitive to the cysteine protease inhibitors chymostatin, E-64, and leupeptin. These results indicate that, in the sclerotium, a premature form of a cathepsin B-like protease remains non-denatured under dehydrated conditions.     

Drosophila cathepsin B-like proteinase: a suggested role in yolk degradation

A cysteine, cathepsin B-like proteinase activity has been found in Drosophila embryos. It appears associated with yolk granules and its activity during embryogenesis correlates well with the degradation of these organelles. In mature oocytes, the enzyme is found in an inactive form which may be activated by limited proteolysis by a serine proteinase also present in oocytes. In early embryos, when solubilized in vitro, the cathepsin B-like proteinase is found in a form of high molecular mass (approx 1000 kDa). This decreases with development down to about 39 kDa, likely the mass of the free proteinase. The heavy form apparently results from the tight association with a yolk protein complex. The proteinase has been found in vitro to degrade readily the yolk polypeptides. The proteinase activity increases during early embryogenesis in parallel with the decrease in molecular weight of the heavy form, and decreases to low values in late embryos. We have also found that ammonium chloride can inhibit in vivo the degradation of yolk and, in parallel, the developmental inactivation of the proteinase. The results altogether suggest that the cathepsin B-like proteinase is implicated in yolk degradation in Drosophila.     

Yeast KEX1 gene encodes a putative protease with a carboxypeptidase B-like function involved in killer toxin and alpha-factor precursor processing

The yeast KEX1 gene product has homology to yeast carboxypeptidase Y. A mutant replacing serine at the putative active site of the KEX1 protein abolished activity in vivo. A probable site of processing by the KEX1 product is the C-terminus of the alpha-subunit of killer toxin, where toxin is followed in the precursor by 2 basic residues. Processing involves endoproteolysis following these basic residues and trimming of their C-terminal by a carboxypeptidase. Consistent with the KEX1 product being this carboxypeptidase is its role in alpha-factor pheromone production. In wild-type yeast, KEX1 is not essential for alpha-factor production, as the final pheromone repeat needs no C-terminal processing. However, in a mutant in which alpha-factor production requires a carboxypeptidase, pheromone production is KEX1-dependent.     

Molecular and biochemical characterization of a cathepsin B-like protease family unique to Trypanosoma congolense

Cysteine proteases have been shown to be essential virulence factors and drug targets in trypanosomatids and an attractive antidisease vaccine candidate for Trypanosoma congolense. Here, we describe an important amplification of genes encoding cathepsin B-like proteases unique to T. congolense. More than 13 different genes were identified, whereas only one or two highly homologous genes have been identified in other trypanosomatids. These proteases grouped into three evolutionary clusters: TcoCBc1 to TcoCBc5 and TcoCBc6, which possess the classical catalytic triad (Cys, His, and Asn), and TcoCBs7 to TcoCBs13, which contains an unusual catalytic site (Ser, Xaa, and Asn). Expression profiles showed that members of the TcoCBc1 to TcoCBc5 and the TcoCBs7 to TcoCBs13 groups are expressed mainly in bloodstream forms and localize in the lysosomal compartment. The expression of recombinant representatives of each group (TcoCB1, TcoCB6, and TcoCB12) as proenzymes showed that TcoCBc1 and TcoCBc6 are able to autocatalyze their maturation 21 and 31 residues, respectively, upstream of the predicted start of the catalytic domain. Both displayed a carboxydipeptidase function, while only TcoCBc1 behaved as an endopeptidase. TcoCBc1 exhibited biochemical differences regarding inhibitor sensitivity compared to that of other cathepsin B-like proteases. Recombinant pro-TcoCBs12 did not automature in vitro, and the pepsin-matured enzyme was inactive in tests with cathepsin B fluorogenic substrates. In vivo inhibition studies using CA074Me (a cell-permeable cathepsin B-specific inhibitor) demonstrated that TcoCB are involved in lysosomal protein degradation essential for survival in bloodstream form. Furthermore, TcoCBc1 elicited an important immune response in experimentally infected cattle. We propose this family of proteins as a potential therapeutic target and as a plausible antigen for T. congolense diagnosis.     

CylA is a sequence-specific protease involved in toxin biosynthesis

Journal of Industrial Microbiology & Biotechnology - CylA is a subtilisin-like protein belonging to a recently expanded serine protease family related to class II lanthipeptide biosynthesis. As...     

Lysosomal cysteine protease cathepsin S is involved in cancer cell motility by regulating store-operated Ca2+ entry

Cathepsin S (CTSS), a lysosomal cysteine protease, has been reported to be associated with extracellular matrix (ECM) degradation, thus promoting cell migration and invasion, but whether CTSS regulates other intracellular mechanisms during metastasis remains unknown. The expression of CTSS was knocked down using siRNA transfection, and enzymatic activity was inhibited by the highly-selective CTSS inhibitor RJW-58. The results of in vitro functional assays, western blot analysis, and an in vivo colonization model demonstrated that CTSS was positively related to cellular adhesive ability. Moreover, both CTSS knockdown and inhibition significantly decreased Ca²⁺ influx via store-operated Ca²⁺ entry (SOCE) without changing STIM1 and Orai1 expression levels, while RJW-58 dose-dependently reduced the activation of the Ca²⁺-dependent downstream effectors, NFAT1 and Rac1. The results of immunoprecipitation assays demonstrated that CTSS could bind to STIM1, which was reversed by CTSS inhibition. In addition, confocal microscopy and super-resolution imaging showed that CTSS inhibition led to STIM1 puncta accumulation in the endoplasmic reticulum and reduced the interaction between active STIM1 and EB1. In conclusion, we have demonstrated for the first time that the lysosomal cysteine protease, CTSS, plays an important role in mediating Ca²⁺ homeostasis by regulating STIM1 trafficking, which leads to the suppression of cell migration and invasion.     

Purification and characterization of a novel thiol protease involved in processing the enkephalin precursor

Proteolytic processing enzymes are required to convert the enkephalin precursor to active opioid peptides. In this study, a novel 33-kDa thiol protease that cleaves complete precursor in the form of [35S]methionine preproenkephalin was purified from bovine adrenal medullary chromaffin granules. Chromatography on concanavalin A-Sepharose and Sephacryl S-200, chromatofocusing, and chromatography on thiopropyl-Sepharose resulted in an 88,000-fold purification with a recovery of 35% of enzyme activity. The thiol protease is a glycoprotein with a pI of 6.0. It cleaves [35S]methionine preproenkephalin with a pH optimum of 5.5, indicating that it is functional at the intragranular pH of 5.5-6.0. Interestingly, production of trichloroacetic acid-soluble products was optimal at pH 4.0, suggesting that processing of initial precursor and intermediates may require slightly different pH conditions. The protease requires dithiothreitol for activity and is inhibited by the thiol protease inhibitors iodoacetate, p-hydroxymercuribenzoate, mercuric chloride, and cystatin. These properties distinguish it from other thiol proteases (cathepsins B, H, L, N, and S), indicating that a unique thiol protease has been identified. The enzyme converted [35S]cysteine preproenkephalin (possessing [35S]cysteine residues specifically within the precursor's NH2-terminal segment) to 22.1-, 21.6-, 17.7-, 17.3-, and 15.0-kDa intermediates that contain the precursor's NH2-terminal segment; proenkephalin in vivo is converted to similar intermediates. The enzyme cleaves peptide F at Lys-Arg and Lys-Lys dibasic amino acid sites to generate methionine enkephalin and intermediates. The appropriate vesicular localization, pH optimum, proteolytic products, and cleavage site specificity suggest that this thiol protease may be involved in enkephalin precursor processing. Most interestingly, [35S]methionine beta-preprotachykinin, a precursor of substance P, is minimally cleaved, suggesting that the thiol protease may possess some selectivity for the enkephalin precursor.     

The noncanonical role of the protease cathepsin D as a cofilin phosphatase

Cathepsin D (cathD) is traditionally regarded as a lysosomal protease that degrades substrates in acidic compartments. Here we report cathD plays an unconventional role as a cofilin phosphatase orchestrating actin remodeling. In neutral pH environments, the cathD precursor directly dephosphorylates and activates the actin-severing protein cofilin independent of its proteolytic activity, whereas mature cathD degrades cofilin in acidic pH conditions. During development, cathD complements the canonical cofilin phosphatase slingshot and regulates the morphogenesis of actin-based structures. Moreover, suppression of cathD phosphatase activity leads to defective actin organization and cytokinesis failure. Our findings identify cathD as a dual-function molecule, whose functional switch is regulated by environmental pH and its maturation state, and reveal a novel regulatory role of cathD in actin-based cellular processes.Subject terms: Actin, Cytokinesis     

The processing of a cathepsin L precursor in vitro

Subcultured rat fibroblasts secreted a cathepsin L precursor when maintained for 24 h in serum-free medium containing 20 mM ammonium ions. The precursor was identified by immunoblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis using polyclonal antibodies to cathepsin L. The molecular mass of the precursor was found to be approximately 39 kDa, which confirms the result originally reported by Y. Nishimura et al. (1988, Arch. Biochem. Biophys. 263, 107-116). Treatment of the precursor containing medium with cathepsin D at pH values ranging from 3.5 to 5.5 caused a limited cleavage of the precursor molecule. The resultant polypeptides are an unstable intermediate form with Mr 35,000 and a stable single chain form of cathepsin L showing a Mr about 32,500. The cathepsin D-mediated conversion was strongly accelerated by Hg2+ ions. A further proteolytic cleavage of the 32.5-kDa polypeptide has not been observed. The enzymatic activity toward Z-Phe-Arg-NHMec at pH 5.5 increased during the conversion, indicating that active cathepsin L was formed from an inactive precursor molecule.     

Demonstration of a cell-associated, inactive precursor of an exocellular protease produced by Pseudomonas aeruginosa

The enzymatically active form of protease 1, the major exocellular protein produced by Pseudomonas aeruginosa strain 34362, has been shown to exist exclusively exocellularly with no significant cell-associated activity. However, the presence of a cell-associated, enzymatically inactive protein which is serologically cross-reactive with, and convertible to, active enzyme has been demonstrated. One method of conversion of "precursor" to active enzyme is via limited proteolysis. Two assay systems for precursor were developed, one a radioimmune assay, and the other a proteolytic activation procedure. Localization studies suggest that the association while more tenacious than classical periplasmic enzymes is still an ionic rather than a covalent one. Kinetics of production studies showed to precursor to be synthesized early in the growth cycle and to accumulate prior to the rapid release of the active enzyme. Molecular weight studies showed only slight changes produced upon activation.     

A recruited protease is involved in catabolism of pyrimidines

In nature, the same biochemical reaction can be catalyzed by enzymes having fundamentally different folds, reaction mechanisms and origins. For example, the third step of the reductive catabolism of pyrimidines, the conversion of N-carbamyl-β-alanine to β-alanine, is catalyzed by two β-alanine synthase (βASase, EC 3.5.1.6) subfamilies. We show that the “prototype” eukaryote βASases, such as those from Drosophila melanogaster and Arabidopsis thaliana, are relatively efficient in the conversion of N-carbamyl-βA compared with a representative of fungal βASases, the yeast Saccharomyces kluyveri βASase, which has a high K_m value (71 mM). S. kluyveri βASase is specifically inhibited by dipeptides and tripeptides, and the apparent K_i value of glycyl-glycine is in the same range as the substrate K_m. We show that this inhibitor binds to the enzyme active center in a similar way as the substrate. The observed structural similarities and inhibition behavior, as well as the phylogenetic relationship, suggest that the ancestor of the fungal βASase was a protease that had modified its profession and become involved in the metabolism of nucleic acid precursors.     

A precursor form of latent collagenase produced in a cell-free system with mRNA from rabbit synovial cells

mRNA extracted from rabbit synovial fibroblasts which had been induced to produce large amounts of collagenase (EC 3.4.23.7) by urate crystals was translated in a cell-free wheat germ system. Collagenase was identified by immunoprecipitation using mono-specific antibody to rabbit synovial collagenase. In the absence of microsomal membranes, a single precursor with Mr = 59,000 was synthesized. This polypeptide was susceptible to proteolytic degradation. In the presence of canine pancreatic microsomes, the nascent protein was processed to a polypeptide with Mr = 57,000 (identical in mobility on sodium dodecyl sulfate-gel electrophoresis to the major latent collagenase secreted from cells) and was protected from tryptic digestion unless a detergent was used to disrupt the membranes. In addition to Mr = 57,000 material, cells secreted immunologically reactive latent collagenase with Mr = 61,000. High molecular weight collagenase was separated from Mr = 57,000 species by binding to concanavalin a-Sepharose, suggesting that this enzyme was a product of post-translational glycosylation. Both latent enzymes were activated by trypsin and human plasma kallikrein to Mr = 45,000 and 49,000. The evidence indicates that rabbit synovial fibroblast collagenase is synthesized and secreted as a single polypeptide zymogen, not as an enzyme-inhibitor complex.