Calcium-dependent proteolytic activity of a cysteine protease caldonopain is detected during Leishmania infection

A calcium-activated protease caldonopain in the cytosolic fraction of Leishmania donovani has been found to digest different endogenous proteins when subjected to SDS-PAGE. Gelatin-embedded gel electrophoresis confirms presence of calcium-dependent protease activity. Ca²⁺ affects proteolytic activity after 10 h. When host–parasite interaction was conducted in vitro, caldonopain was found to be active after 10 h of incubation with calcium. A 67-kDa protein is specifically digested during this time and two new proteins of 45 and 36 kDa appeared in SDS-PAGE electrophoregram. This belated action of calcium towards protease activity may be pre-requisite to facilitate invasion of host tissues and thereby mediate protein metabolism during survival of this pathogen both independently and intracellularly. It is likely that calcium metabolism in promastigotes and amastigotes does not propagate in the same manner. Involvement of calcium to initiate caldonopain activity may be critically associated with signal transduction pathways which may be responsible for the pathobiological action of this parasite. We propose that caldonopain could be a potential target to develop new chemotherapeutic approach against leishmaniasis.     

Relationships between plant roots,proteolytic organisms and activity of protease

Proteolytic bacteria represented 18–58% of the bacterial population isolated from the rhizoplane of different crops. The activity of protease was considerably higher on roots of wheat growing in the soil than in the rhizosphere or free soil. However, only a slightly positive rhizosphere effect in the relative occurrence of casein-hydrolyzing bacteria could be observed. An indirect relationship between numbers of bacteria hydrolyzing casein and the activity of the enzyme could be found. The activity of protease related to a unit of culturable proteolytic bacteria was considerably higher on the root than in the rhizosphere and in the soil. A relationship between characteristics of the production of the enzyme by proteolytic bacteria and the protease activity on the surface of roots was demonstrated. The resulting enzyme activity on the surface of roots depended apparently on growth conditions of the plant and nature of root exudates and was influenced both by inactivation and protection due to adsorption of the enzyme by roots.     

Maturation of giardiavirus capsid protein involves posttranslational proteolytic processing by a cysteine protease.

The double-stranded RNA genome of giardiavirus (GLV) has only two large open reading frame (ORFs). The 100-kDa capsid polypeptide (p100) is encoded by ORF1, whereas the only other viral polypeptide, the 190-kDa GLV RNA-dependent RNA polymerase (p190), is synthesized as an ORF1-ORF2 fusion protein by a (-1) ribosomal frameshifting. Edman degradation revealed that p100 was N-terminally blocked except for 2 to 5% of it that showed free N terminus starting from amino acid residue 33 of ORF1. Studies using antiserum targeted against amino acid residues 6 to 27 indicated that this region (NT) is absent from viral p100 and p190, while pulse-labelling experiments showed that NT is present in nascent p100 synthesized in GLV-infected Giardia lamblia but removed subsequently. In contrast, this region was retained in the two viral proteins synthesized in vitro, and it was not removed upon prolonged incubation or inclusion of microsomal fraction in the in vitro translation reaction mixtures. These results suggest that endoplasmic reticulum is not involved in the protein processing and that the precursors of p100 and p190 are incapable of cleaving themselves or each other. This specific cleavage was reproduced when lysates from GLV-infected G. lamblia were added, but not those from uninfected cells. The cleavage activity was relatively insensitive to phenylmethylsulfonyl fluoride, but it was inhibitable by leupeptin or E-64, two known specific inhibitors of cysteine protease. The possible origin of this processing activity is discussed.     

Antitrypanosomal and cysteine protease inhibitory activities of alkyldiamine cryptolepine derivatives

Cryptolepine derivatives containing alkyldiamine side-chains, 2, with potent inhibitory activity against Trypanosoma brucei brucei are reported. Compounds 2 showed improved activity and selectivity to T. b. brucei when compared to the lead compound. The most selective compound, 2k, presents a selectivity index value of 6200 and an IC(50) of 10nM against the parasite. These derivatives are also potent inhibitors of the trypanosome papain-like cysteine proteases cruzain, which could, at least in part, explain their antitrypanosomal activity. Overall, these compounds with good antitrypanosomal activity and selectivity provide an encouraging starting point for the rational design of new and effective antitrypanosomal agents.     

Stage-specific antimalarial activity of cysteine protease inhibitors

Cysteine proteases of the malaria parasite Plasmodium falciparum, known as falcipains, are promising targets for antimalarial chemotherapy. We evaluated cultured parasites for the stage-specific expression of cysteine proteases and sensitivity to cysteine protease inhibitors. Protease activity and inhibitor sensitivity varied markedly over time. Cysteine protease activity was greatest in early trophozoites, while sensitivity to cysteine protease inhibitors was greatest in mature trophozoites. Our results indicate the importance of considering the stage-specific effects of antimalarials and are consistent with the conclusion that the principal antimalarial activity of cysteine protease inhibitors is due to a block in hemoglobin hydrolysis.     

Homocysteine modulates the proteolytic potential of human arterial smooth muscle cells through a reactive oxygen species dependant mechanism

Suberoyl bishydroxamic acid (SBHA) as a histone deacetylase (HDAC) inhibitor has various cellular effects such as cell growth and apoptosis. In the present study, we evaluated the effects of SBHA on the growth and death of A549 lung cancer cells. SBHA inhibited the growth of A549 cells with an IC₅₀ of approximately 50 μM at 72 h in a dose-dependent manner. DNA flow cytometric analysis indicated that SBHA induced a G2/M phase arrest of the cell cycle. This agent also induced apoptosis, as evidenced by sub-G1 cells and annexin V-FITC staining cells. SBHA-induced apoptosis was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨ_m), Bcl-2 decrease, Bax increase, and the activation of caspase-3. All of the tested caspase inhibitors significantly rescued some cells from SBHA-induced A549 cell death. However, none of the caspase inhibitors prevented the loss of MMP (ΔΨ_m) induced by SBHA. Intracellular reactive oxygen species (ROS) levels including O ₂ ^?? were increased in 50 μM SBHA-treated A549 cells. None of the caspase inhibitors attenuated ROS levels in these cells. SBHA also elevated the number of glutathione (GSH)-depleted cells in A549 cells, which was reduced by treatment with caspase inhibitors. In conclusion, this is the first report that SBHA inhibited the growth of A549 lung cancer cells via caspase-dependent apoptosis, which was related to GSH depletion rather than changes in ROS level.     

Purification and partial characterisation of a novel trypsin-like cysteine protease from Metarhizium anisopliae

Abstract Trypsin-like enzymes from the entomopathogenic fungus Metarhizium anisopliae have been characterised. Two proteases with tryptic activity were purified by narrow range isoelectric focussing and affinity chromatography. One of these proteases, with an isoelectric point of 5.4 and a molecular mass of 28.8 kDa is a 'classical' trypsin belonging to the serine protease class. The other protease, with an isoelectric point of 4.6 and a molecular mass of 26.7 kDa, demonstrates trypsin-like specificity but, on the basis of inhibition and activation studies, belongs to the cysteine protease family and as such is the first fungal protease to be found of this type. The amino acid composition, kinetic constants and activity against proteinaceous substrates, including locust cuticle have been determined.     

Hen egg white ovomacroglobulin has a protease inhibitory activity

Hen egg white ovomacroglobulin purified by Miller and Feeney without reference to its activity was shown to have a protease inhibitory activity towards trypsin, papain, and thermolysin. It has four subunits of equal molecular weight (175,000 by SDS-PAGE) and each two of which are disulfide bonded. Upon incubation with trypsin it yields a fragment of Mr = 80,000 plus smaller ones. The subunit composition, amino acid composition and a newly found protease inhibitory activity place ovomacroglobulin as a closely related protein to human serum alpha 2-macroglobulin.     

Ornithine decarboxylase-antizyme is rapidly degraded through a mechanism that requires functional ubiquitin-dependent proteolytic activity.

Antizyme is a polyamine-induced cellular protein that binds to ornithine decarboxylase (ODC), and targets it to rapid ubiquitin-independent degradation by the 26S proteasome. However, the metabolic fate of antizyme is not clear. We have tested the stability of antizyme in mammalian cells. In contrast with previous studies demonstrating stability in vitro in a reticulocyte lysate-based degradation system, in cells antizyme is rapidly degraded and this degradation is inhibited by specific proteasome inhibitors. While the degradation of ODC is stimulated by the presence of cotransfected antizyme, degradation of antizyme seems to be independent of ODC, suggesting that antizyme degradation does not occur while presenting ODC to the 26S proteasome. Interestingly, both species of antizyme, which represent initiation at two in-frame initiation codons, are rapidly degraded. The degradation of both antizyme proteins is inhibited in ts20 cells containing a thermosensitive ubiquitin-activating enzyme, E1. Therefore we conclude that in contrast with ubiquitin-independent degradation of ODC, degradation of antizyme requires a functional ubiquitin system.     

Dissecting processing and apoptotic activity of a cysteine protease by mutant analysis

We have compared the behavior of wild-type mouse NEDD-2, a neural precursor cell-expressed, developmentally down-regulated cysteine protease gene, to various mutant forms of the gene in both apoptotic activity in neuronal cells and proteolytic cleavage in the Semliki Forest virus and rabbit reticulocyte protein expression systems. Our results confirm that NEDD-2 processing and apoptotic activity are linked phenomena. They identify aspartate residues as likely targets for autocatalytic cleavage. They establish that cleavage events only occur at specific sites. Finally, they pinpoint differential effects of individual mutations on the overall proteolytic cleavage patterns, raising interesting questions related to the mechanisms of subunit assembly.     

Tissue-type plasminogen activator rescues neurones from serum deprivation-induced apoptosis through a mechanism independent of its proteolytic activity

Although the mechanism of action of tissue-type plasminogen activator (tPA) in excitotoxic necrosis is well documented, whether this serine protease can influence the apoptotic cascade remains a subject of debate. Here, we report that tPA protects cultured cortical neurones against apoptotic cell death induced by serum deprivation, an effect associated with a reduction of caspase-3 activation. Interestingly, blocking tPA proteolytic activity by either tPA stop or neuroserpin did not prevent this neuroprotection. Similarly, prevention of the interaction between tPA and its receptor low-density lipoprotein receptor-related protein (LRP) could not alter tPA anti-apoptotic activity. Interestingly, the survival-promoting effect of tPA was blocked by the phosphatidylinositol-3 (PI-3) kinase inhibitor, LY294002, but not by the mitogen-activated protein (MAP) kinase inhibitor, U0126. In conclusion, the present demonstration of an anti-apoptotic effect of tPA, independent of its enzymatic activity, reveals an additional level of complexity in our understanding of this critical mediator of brain physiology and pathology.     

Antiprotozoal and cysteine proteases inhibitory activity of dipeptidyl enoates

A family of dipeptidyl enoates has been prepared and tested against the parasitic cysteine proteases rhodesain, cruzain and falcipain-2 related to sleeping sickness, Chagas disease and malaria, respectively. They have also been tested against human cathepsins B and L1 for selectivity. Dipeptidyl enoates resulted to be irreversible inhibitors of these enzymes. Some of the members of the family are very potent inhibitors of parasitic cysteine proteases displaying k_2nd (M^?1s^?1) values of seven orders of magnitude. In vivo antiprotozoal testing was also performed. Inhibitors exhibited IC₅₀ values in the micromolar range against Plasmodium falciparum, Trypanosoma brucei, Trypanosoma cruzi and even more promising lower values against Leishmania donovanii.     

肾素-血管紧张素系统通过氧化应激机制参与血管反应性的调节

肾素-血管紧张素系统过度激活导致血管氧化应激损伤,进而影响血管功能.xanthine氧化酶、NAD(P)H氧化酶和脱耦联的NO合酶是血管组织中活性氧的主要来源.超氧化物阴离子和活性氧簇中的其他成分通过多种机制失活NO在心血管疾病的发生和发展中具有重要作用.随着对氧化应激损伤参与血管反应性调节机制的认识逐渐加深,有希望通过抑制氧化应激损伤改善血管内皮功能.     

Determination of protease activity of plant roots

The colorimetric method described by Charney and Tomarelli (1974) for the assay of the proteolytic activity in the duodenal juice was adapted to measuring protease activity of roots. Diazotized casein served as substrate and the amount of degraded azocasein was measured colorimetrically. A linear relationship between the incubation time or concentration of the enzyme (roots) and the amount of the hydrolyzed substrate was found. The rate increase of the enzyme reaction was proportional to enzyme concentration up to 1 g roots and incubation time of 3 h, and up to 0.5 g of roots and incubation time of 4 h. The optimum of the protease activity was at pH 8.2–8.6.     

Fusion of a soybean cysteine protease inhibitor and a legume lectin enhances anti-insect activity synergistically

Abstract 1 The soybean cysteine protease inhibitor soyacystatin N (scN) and Griffonia simplicifolia lectin II (rGSII) have defense functions against the coleopteran cowpea bruchid beetle Callosobruchus maculatus. However, the ability of the insect to activate scN‐insensitive digestive proteases and the relatively low potency of rGSII have hindered their practical application in plant protection. 2 Recent research suggests that defense proteins may achieve increased toxicity and durability when used in combination. Based on the structures of several natural toxin molecules, we hypothesized that covalently linked scN and rGSII could exhibit greater anti‐insect efficacy than the mixture containing individual proteins. 3 To test this hypothesis, a recombinant scN‐rGSII fusion protein that retained both protease inhibitor and lectin functions was constructed. 4 When fed to cowpea bruchid, this new protein showed a synergistic delay in insect development, whereas a mixture of the separate proteins only showed an additive effect. 5 Our results suggest that tethering digestive protease inhibitors to gut epithelium‐interacting lectins could give plant protection superior to strategies based on single genes or mixtures of single gene products.     

Novel bifunctional alkaline protease inhibitor: protease inhibitory activity as the biochemical basis of antifungal activity

An alkaline protease inhibitor (API) from a Streptomyces sp. NCIM 5127 was shown to possess antifungal activity against several phytopathogenic fungi besides its antiproteolytic (anti-feedent) activity [J. V. Vernekar et al. (1999) Biochem. Biophys. Res. Commun. 262, 702-707]. Based on the correlation between antiproteolytic and antifungal activities in several tests such as copurification, heat inactivation, chemical modification, and its binding interaction with the fungal protease, we demonstrate, for the first time, that the dual function of API is a consequence of its ability to inhibit the essential alkaline protease. The parallel enrichment of both the functions during purification together with the heat inactivation of API leading to the concomitant loss of the two activities suggested their presence on a single molecule. Chemical modification of API with NBS resulted in the complete loss of antiproteolytic and antifungal activities, with no gross change in conformation implying the involvement of a Trp residue in the active site of the inhibitor and the presence of a single active site for the two activities. Treatment of API with DTT abolished both the activities although the native structure of API remained virtually unaffected, indicating the catalytic role of the disulfide bonds. Inactivation of API either by active site modification or by conformational changes leads to the concurrent loss of both the antiproteolytic and antifungal activities. Experimental evidences presented here serve to implicate that the antifungal activity of API is a consequence of its protease inhibitory activity.     

Hemoglobinase activity of a cysteine protease from the ixodid tick Haemaphysalis longicornis

We report here the molecular characterization and possible function of a cysteine protease (termed HlCPL-A) identified in the midgut of the hard tick Haemaphysalis longicornis. HlCPL-A is a 333 amino acid protein belonging to the papain family of the cysteine protease. A construct encoding proHlCPL-A was expressed in Escherichia coli and purified as both procathepsin L and active processed cathepsin L forms. The HlCPL-A gene expression was up-regulated by blood-feeding process. HlCPL-A exhibited substrate specificity against synthetic peptidyl substrates (Z-Phe-Arg-MCA and Z-Arg-Arg-MCA; k_cat / K_m = 0.19 and 0.0023 M^− 1 S^− 1, respectively). The proteolytic activity of HlCPL-A was inhibited by leupeptin, antipain and E-64 but was unaffected by pepstatin. HlCPL-A was capable of degrading bovine hemoglobin at pH 3.2 to 5.6. These results suggest that HlCPL-A may play important roles in the digestion of host hemoglobin in ticks.