Metacaspase of Saccharomyces cerevisiae (ScMCA-Ia) presents different catalytic cysteine in a processed and non-processed form.

Metacaspases are cysteine proteases belonging to the CD clan of the C14 family. They possess important characteristics, such as specificity for cleavage after basic residues (Arg/Lys) and dependence on calcium ions to exert their catalytic activity. They are defined by the presence of a large subunit (p20) and a small subunit (p10) and are classified into types I, II, and III. Type I metacaspases have a characteristic pro-domain at the N-terminal of the enzyme, preceding a region rich in glutamine and asparagine. In the yeast Saccharomyces cerevisiae, a type I metacaspase is found. This organism encodes a single metacaspase that participates in the process of programmed cell death by apoptosis. The study focuses on cloning, expressing, and mutating Saccharomyces cerevisiae metacaspase (ScMCA-Ia). Mutations in Cys155 and Cys276 were introduced to investigate autoprocessing mechanisms. Results revealed that Cys155 plays a crucial role in autoprocessing, initiating a conformational change that activates ScMCA-Ia. Comparative analysis with TbMCA-IIa highlighted the significance of the N-terminal region in substrate access to the active site. The study proposes a two-step processing mechanism for type I metacaspases, where an initial processing step generates the active form, followed by a distinct intermolecular processing step. This provides new insights into ScMCA-Ia's activation and function. The findings hold potential implications for understanding cellular processes regulated by metacaspases. Overall, this research significantly advances knowledge in metacaspase biology.

In Vitro Study of Cytotoxic Mechanisms of Alkylphospholipids and Alkyltriazoles in Acute Lymphoblastic Leukemia Models.

This study investigates the efficacy of miltefosine, alkylphospholipid, and alkyltriazolederivative compounds against leukemia lineages. The cytotoxic effects and cellular and molecular mechanisms of the compounds were investigated. The inhibitory potential and mechanism of inhibition of cathepsins B and L, molecular docking simulation, molecular dynamics and binding free energy evaluation were performed to determine the interaction of cathepsins and compounds. Among the 21 compounds tested, C9 and C21 mainly showed cytotoxic effects in Jurkat and CCRF-CEM cells, two human acute lymphoblastic leukemia (ALL) lineages. Activation of induced cell death by C9 and C21 with apoptotic and necrosis-like characteristics was observed, including an increase in annexin-V+propidium iodide-, annexin-V+propidium iodide+, cleaved caspase 3 and PARP, cytochrome c release, and nuclear alterations. Bax inhibitor, Z-VAD-FMK, pepstatin, and necrostatin partially reduced cell death, suggesting that involvement of the caspase-dependent and -independent mechanisms is related to cell type. Compounds C9 and C21 inhibited cathepsin L by a noncompetitive mechanism, and cathepsin B by a competitive and noncompetitive mechanism, respectively. Complexes cathepsin-C9 and cathepsin-C21 exhibited significant hydrophobic interactions, water bridges, and hydrogen bonds. In conclusion, alkyltriazoles present cytotoxic activity against acute lymphoblastic lineages and represent a promising scaffold for the development of molecules for this application.

4-Deoxyraputindole C induces cell death and cell cycle arrest in tumor cell lines.

Several molecules extracted from natural products exhibit different biological activities, such as ion channel modulation, activation of signaling pathways, and anti-inflammatory or antitumor activity. In this study, we tested the antitumor ability of natural compounds extracted from the Raputia praetermissa plant. Among the compounds tested, an alkaloid, here called compound S4 (4-Deoxyraputindole C), showed antitumor effects against human tumor lineages. Compound S4 was the most active against Raji, a lymphoma lineage, promoting cell death with characteristics that including membrane permeabilization, dissipation of the mitochondrial potential, increased superoxide production, and lysosomal membrane permeabilization. The use of cell death inhibitors such as Z-VAD-FMK (caspase inhibitor), necrostatin-1 (receptor-interacting serine/threonine-protein kinase 1 inhibitor), E-64 (cysteine peptidases inhibitor), and N-acetyl- L-cysteine (antioxidant) did not decrease compound S4-dependent cell death. Additionally, we tested the effect of cellular activity on adherent human tumor cells. The highest reduction of cellular activity was observed in A549 cells, a lung carcinoma lineage. In this lineage, the effect on the reduction of the cellular activity was due to cell cycle arrest, without plasma membrane permeabilization, loss of the mitochondrial potential or lysosomal membrane permeabilization. Compound S4 was able to inhibit cathepsin B and L by a nonlinear competitive (negative co-operativity) and simple-linear competitive inhibitions, respectively. The potency of inhibition was higher against cathepsin L. Compound S4 promoted cell cycle arrest at G 0 and G 2 phase, and increase the expression of p16 and p21 proteins. In conclusion, compound S4 is an interesting molecule against cancer, promoting cell death in the human lymphoma lineage Raji and cell cycle arrest in the human lung carcinoma lineage A549.

Semysinthetic biflavonoid Morelloflavone-7,4',7″,3‴,4‴-penta-O-butanoyl is a more potent inhibitor of Proprotein Convertases Subtilisin/Kexin PC1/3 than Kex2 and Furin.

BACKGROUND: Garcinia brasiliensis is a species native to the Amazon forest. The white mucilaginous pulp is used in folk medicine as a wound healing agent and for peptic ulcer, urinary, and tumor disease treatments. The activity of the proprotein convertases (PCs) Subtilisin/Kex is associated with the development of viral, bacterial and fungal infections, osteoporosis, hyperglycemia, atherosclerosis, cardiovascular, neurodegenerative and neoplastic diseases. METHODS: Morelloflavone (BF1) and semisynthetic biflavonoid (BF2, 3 and 4) from Garcinia brasiliensis were tested as inhibitor of PCs Kex2, PC1/3 and Furin, and determined IC50, Ki, human proinflammatory cytokines secretion in Caco-2 cells, mechanism of inhibition, and performed molecular docking studies. RESULTS: Biflavonoids were more effective in the inhibition of neuroendocrine PC1/3 than mammalian Furin and fungal Kex2. BF1 presented a mixed inhibition mechanism for Kex2 and PC1, and competitive inhibition for Furin. BF4 has no good interaction with Kex2 and Furin since carboxypropyl groups results in steric hindrance to ligand-protein interactions. Carboxypropyl groups of BF4 promote steric hindrance with Kex2 and Furin, but effective in the affinity of PC1/3. BF4 was more efficient at inhibiting PCl/3 (IC50 = 1.13 µM and Ki = 0,59 µM, simple linear competitive mechanism of inhibition) than Kex2, Furin. Also, our results strongly suggested that BF4 also inhibits the endogenous cellular PC1/3 activity in Caco-2 cells, since PC1/3 inhibition by BF4 causes a large increase in IL-8 and IL-1ß secretion in Caco-2 cells. CONCLUSIONS: BF4 is a potent and selective inhibitor of PC1/3. GENERAL SIGNIFICANCE: BF4 is the best candidate for further clinical studies on inhibition of PC1/3.

Thermodynamic analysis of Kex2 activity: The acylation and deacylation steps are potassium- and substrate-dependent.

Kex2 is the prototype of a large family of eukaryotic subtilisin-related proprotein-processing proteases that cleave at sites containing pairs of basic residues. Here, we studied the effects of KCl on the individual rate constants of association, dissociation, acylation and deacylation and determined the thermodynamic parameters at each step of the Kex2 reaction. Potassium bound Kex2 with KD=20.3mM. The order in which potassium entered the reaction system modified the effect of activation or inhibition, which depended on the size of the substrate. A possible allosteric potassium binding site at the S6 subsite was involved in activation, and a distant site located between the catalytic domain and the P-domain was involved in inhibition. Potassium decreased the energetic barriers of almost all steps of catalysis. The acylation of Ac-PMYKR-AMC in the absence of potassium was the rate-limiting step. Therefore, for substrates containing a P1-Arg, the deacylation step is not necessarily the rate-limiting event, and other residues at the P' positions may participate in controlling the acylation and deacylation steps. Thus, it is reasonable to conclude that potassium is involved in the processing of the α-mating factor that promotes Ca2+ mobilization by activating a high-affinity Ca2+-influx system to increase the cytosolic [Ca2+], resulting in the activation of channels that are essential for the survival of Saccharomyces cerevisiae cells.

Specificity characterization of the α-mating factor hormone by Kex2 protease.

Kex2 is a Ca2+-dependent serine protease from S. cerevisiae. Characterization of the substrate specificity of Kex2 is of particular interest because this protease serves as the prototype of a large family of eukaryotic subtilisin-related proprotein-processing proteases that cleave sites consisting of pairs or clusters of basic residues. Our goal was to study the prime region subsite S' of Kex2 because previous studies have only taken into account non-prime sites using AMC substrates but not the specificity of prime sites identified through structural modeling or predicted cleavage sites. Therefore, we used peptides derived from Abz-KR↓EADQ-EDDnp and Abz-YKR↓EADQ-EDDnp based on the pro-α-mating factor sequence. The specificity of Kex2 due to basic residues at P1' is affected by the type of residue in the P3 position. Some residues in P1' with large or bulky side chains yielded poor substrate specificity. The kcat/KM values for peptides with P2' substitutions containing Tyr in P3 were higher than those obtained for the peptides without Tyr. In fact, P' and P modifications mainly promoted changes in kcat and KM, respectively. The pH profile of Kex2 was fit to a double-sigmoidal pH-titration curve. The specificity results suggest that Kex2 might be involved in the processing of the putative cleavage sites in a polypeptide involved in cell elongation, hyphal formation and the processing of a toxin, which result in host cell lysis. In summary, the specificity of Kex2 is dependent on the set of interactions with prime and non-prime subsites, resulting in synergism.

Benzophenone derivatives as cysteine protease inhibitors and biological activity against Leishmania(L.) amazonensis amastigotes.

The leishmanicidal potential of benzophenones has been described, some of them highlighting their potential as cysteine protease inhibitors. Therefore, this work described leishmanicidal activity of nine benzophenone derivatives (1a-c;2a-c;3a-c) against intramacrophage amastigote forms of Leishmania(L.)amazonensis (IC50) and the cytotoxic effect on murine peritoneal macrophages (CC50). The derivative 1c exhibited a selectivity index SI (CC50/IC50) of 6.7, besides cytotoxicity lower than Amphotericin B (p< 0.05). Moreover it showed inhibitory activity against papain (42.8±0.3, p<0.05), and when tested on trypanosomatids cysteine proteases 1c also proved to be a potent inhibitor of rCPB2.8, rCPB3.0 and cruzain, showing non-competitive inhibition mechanism by enzymatic assays in vitro.So, benzophenone 1c is interesting drug candidate prototype, with a multi-target directed mode of action, inhibiting rCPB2.8, rCPB3.0 and cruzain.

In vitro and in vivo activity of a palladacycle complex on Leishmania (Leishmania) amazonensis.

BACKGROUND: Antitumor cyclopalladated complexes with low toxicity to laboratory animals have shown leishmanicidal effect. These findings stimulated us to test the leishmanicidal property of one palladacycle compound called DPPE 1.2 on Leishmania (Leishmania) amazonensis, an agent of simple and diffuse forms of cutaneous leishmaniasis in the Amazon region, Brazil. METHODOLOGY/PRINCIPAL FINDINGS: Promastigotes of L. (L.) amazonensis and infected bone marrow-derived macrophages were treated with different concentrations of DPPE 1.2. In in vivo assays foot lesions of L. (L.) amazonensis-infected BALB/c mice were injected subcutaneously with DPPE 1.2 and control animals received either Glucantime or PBS. The effect of DPPE 1.2 on cathepsin B activity of L. (L.) amazonensis amastigotes was assayed spectrofluorometrically by use of fluorogenic substrates. The main findings were: 1) axenic L. (L.) amazonensis promastigotes were destroyed by nanomolar concentrations of DPPE 1.2 (IC50 = 2.13 nM); 2) intracellular parasites were killed by DPPE 1.2 (IC50 = 128.35 nM), and the drug displayed 10-fold less toxicity to macrophages (CC50 = 1,267 nM); 3) one month after intralesional injection of DPPE 1.2 infected BALB/c mice showed a significant decrease of foot lesion size and a reduction of 97% of parasite burdens when compared to controls that received PBS; 4) DPPE 1.2 inhibited the cysteine protease activity of L. (L.) amazonensis amastigotes and more significantly the cathepsin B activity. CONCLUSIONS/SIGNIFICANCE: The present results demonstrated that DPPE 1.2 can destroy L. (L.) amazonensis in vitro and in vivo at concentrations that are non toxic to the host. We believe these findings support the potential use of DPPE 1.2 as an alternative choice for the chemotherapy of leishmaniasis.

Propriedades hidrolíticas de cisteíno-peptidases recombinantes de Tripanossomatídeos / Hidrolytic properties of recombinant cysteine peptidases from Trypanosomatides

Trypanosoma cruzi cysteine proteinase cruzipain contains a 130-aminoacid C-terminal extension, in addition to the catalytic domain. Natural cruzipain is a complex of isoforms, because of the simultaneous expression of several genes, and the presence of either high mannone-type, hybrid monoantennary-type or complex biantenary-type oligosacharide chains at Asn255 of the C-terminal extension. Cruzipain and its recombinant form without this extension (cruzain) were studied comparatively in this work. S2 to S2' subsite specificities of these enzymes were examined using four series of substrates derived from the internally quenched fluorescent peptide Abz-KLRFSKQ-EDDnp. Large differences in the kinetic parameters were not observed between the enzymes; however, Km, values were consistently lower for the hydrolysis of most of the substrates by cruzain. No difference in the pH-activity profile between the two enzymes was found, but in 1 M NaCl cruzipain presented a kcat values significantly higher than that of cruzain. The activation energy of denaturation for the enzymes did not differ significantly; however, a negative entropy value was observed for cruzipain denaturation whereas the value for cruzain was positive. We determined the individual rate constants (k1, substrate diffusion; k-1, substrate dissociation; k2, acylation; k3, deacylation) and the respective activation energies and entropies for hydrolysis of Abz-KLRFSKQ-EDDnp determining the temperature dependence of the Michaelis-Menten parameters kcat /Km, and kcat as previously described. Differences between the two enzymes were clearly detected in the activation energies E,1 and E-1, which are significantly higher for cruzipain. The corresponding ÃS, and ÃS-1 were positive and significantly higher for cruzipain…(au)