Refolding of metacaspase 5 from Trypanosoma cruzi, structural characterization and the influence of c-terminal in protein recombinant production.

Metacaspases are known to have a fundamental role in apoptosis-like, a programmed cellular death (PCD) in plants, fungi, and protozoans. The last includes several parasites that cause diseases of great interest to public health, mostly without adequate treatment and included in the neglected tropical diseases category. One of them is Trypanosoma cruzi which causes Chagas disease and has two metacaspases involved in its PCD: TcMCA3 and TcMCA5. Their roles seemed different in PCD, TcMCA5 appears as a proapoptotic protein negatively regulated by its C-terminal sequence, while TcMCA3 is described as a cell cycle regulator. Despite this, the precise role of TcMCA3 and TcMCA5 and their atomic structures remain elusive. Therefore, developing methodologies to allow investigations of those metacaspases is relevant. Herein, we produced full-length and truncated versions of TcMCA5 and applied different strategies for their folded recombinant production from E. coli inclusion bodies. Biophysical assays probed the efficacy of the production method in providing a high yield of folded recombinant TcMCA5. Moreover, we modeled the TcMCA5 protein structure using experimental restraints obtained by XLMS. The experimental design for novel methods and the final protocol provided here can guide studies with other metacaspases. The production of TcMCA5 allows further investigations as protein crystallography, HTS drug discovery to create potential therapeutic in the treatment of Chagas' disease and in the way to clarify how the PCD works in the parasite.

Procaspase-activating compound-1 induces apoptosis in Trypanosoma cruzi.

Some therapeutics for parasitic, cardiac and neurological diseases activate apoptosis. Therefore, the study of apoptotic proteins in pathogenic organisms is relevant. However, the molecular mechanism of apoptosis in unicellular organisms remain elusive, despite morphological evidence of its occurrence. In Trypanosoma cruzi, the causative agent of Chagas disease, metacaspase 3 (TcMCA3), seems to have a key role in parasite apoptosis. Accordingly, this work provides data concerning TcMCA3 regulation through its interaction with procaspase-activating compound 1 (PAC-1), a procaspase 3 activator. Indeed, PAC-1 reduced T. cruzi epimastigote viability with an IC50 of 14.12 µM and induced loss of mitochondrial potential and exposure of phosphatidylserine, features of the apoptotic process. Notwithstanding, those PAC-1-inducible effects were not conserved in metacyclic trypomastigotes. Moreover, PAC-1 reduced the viability of mammalian cells with a greater IC50 (25.70 µM) compared to T. cruzi epimastigotes, indicating distinct modes of binding between caspases and metacaspases. To shed light on the selectivity of metacaspases and caspases, we determined the structural features related to the PAC-1 binding sites in both types of proteins. These data are important for improving the understanding of the apoptosis pathway in T. cruzi so that TcMCA3 could be better targeted with future pharmaceuticals.

Processing of metacaspase 2 from Trypanosoma brucei (TbMCA2) broadens its substrate specificity.

Metacaspases are members of the cysteine peptidase family and may be implicated in programmed cell death in plants and lower eukaryotes. These proteases exhibit calcium-dependent activity and specificity for arginine residues at P1. In contrast to caspases, they do not require processing or dimerization for activity. Indeed, unprocessed metacaspase-2 of Trypanosoma brucei (TbMCA2) is active; however, it has been shown that cleavages at Lys55 and Lys268 increase TbMCA2 hydrolytic activity on synthetic substrates. The processed TbMCA2 comprises 3 polypeptide chains that remain attached by non-covalent bonds. Replacement of Lys55 and Lys268 with Gly via site-directed mutagenesis results in non-processed but enzymatically active mutant, TbMCA2 K55/268G. To investigate the importance of this processing for the activity and specificity of TbMCA2, we performed activity assays comparing the non-processed mutant (TbMCA2 K55/268G) with the processed TbMCA2 form. Significant differences between TbMCA2 WT (processed form) and TbMCA2 K55/268G (non-processed form) were observed. Specifically, we verified that although non-processed TbMCA2 is active when assayed with small synthetic substrates, the TbMCA2 form does not exhibit hydrolytic activity on large substrates such as azocasein, while processed TbMCA2 is able to readily digest this protein. Such differences can be relevant for understanding the physiological regulation and function of TbMCA2.

Genome-wide identification and expression analysis of the metacaspase gene family in Hevea brasiliensis.

Metacaspases, a family of cysteine proteases, have been suggested to play important roles in programmed cell death (PCD) during plant development and stress responses. To date, no systematic characterization of this gene family has been reported in rubber tree (Hevea brasiliensis). In the present study, nine metacaspase genes, designated as HbMC1 to HbMC9, were identified from whole-genome sequence of rubber tree. Multiple sequence alignment and phylogenetic analyses suggested that these genes were divided into two types: type I (HbMC1-HBMC7) and type II (HbMC8 and HbMC9). Gene structure analysis demonstrated that type I and type II HbMCs separately contained four and two introns, indicating the conserved exon-intron organization of HbMCs. Quantitative real-time PCR analysis revealed that HbMCs showed distinct expression patterns in different tissues, suggesting the functional diversity of HbMCs in various tissues during development. Most of the HbMCs were regulated by drought, cold, and salt stress, implying their possible functions in regulating abiotic stress-induced cell death. Of the nine HbMCs, HbMC1, HbMC2, HbMC5, and HbMC8 displayed a significantly higher relative transcript accumulation in barks of tapping panel dryness (TPD) trees compared with healthy trees. In addition, the four genes were up-regulated by ethephon (ET) and methyl jasmonate (MeJA), indicating their potential involvement in TPD resulting from ET- or JA-induced PCD. In summary, this work provides valuable information for further functional characterization of HbMC genes in rubber tree.

Two death pathways induced by sorafenib in myeloma cells: Puma-mediated apoptosis and necroptosis.

PURPOSE: Sorafenib is a multikinase inhibitor that targets the MAPK pathway and is currently used for the treatment of hepatocellular and renal carcinoma. Recently, it has been shown that sorafenib is also cytotoxic to multiple myeloma (MM) cells. Here, we have further analyzed the mechanism of sorafenib-induced death in MM cells. METHODS: Cell death induced by sorafenib in MM cell lines and in plasma cells from MM patients was evaluated by analysis of gene expression by RT-MLPA and quantitative PCR, protein levels and functionality by Western blot and flow cytometry and gene silencing with siRNA. RESULTS: Cell death was characterized by phosphatidylserine exposure, ΔΨm loss, cytochrome c release and caspase activation, hallmarks of apoptosis. DL50 at 24 h ranged from 6 to 10 µM. Ex vivo treatment with 20 µM sorafenib induced apoptosis in around 80 % myeloma cells from six multiple myeloma patients. Sorafenib induced caspase-dependent degradation of Bcl-xL and Mcl-1 proteins, destabilizing the mitochondria and speeding up the development of apoptosis. Sorafenib treatment increased levels of Puma at mRNA and protein level and gene silencing with siRNA confirmed a relevant role for Puma in the induction of apoptosis. Co-treatment with the pan-caspase inhibitor Z-VAD-fmk prevented cell death to a variable degree depending on the cell line. In RPMI 8226 cells, Z-VAD-fmk prevented most of sorafenib-induced death. However, death in MM.1S was only prevented by co-incubation with both Z-VAD-fmk and the RIP1K inhibitor necrostatin-1, indicating that under conditions of inefficient caspase activation, sorafenib induces death by necroptosis. CONCLUSION: Our results demonstrate a key role for Puma in the triggering of sorafenib-induced apoptosis and that this drug can also induce death by necroptosis in multiple myeloma cells.

Identification and structural-functional analysis of cyclin-dependent kinases of the cattle tick Rhipicephalus (Boophilus) microplus.

Cyclin-dependent kinases (CDKs) are a family of serine/threonine kinases essential for cell cycle progression. Herein, we describe the participation of CDKs in the physiology of Rhipicephalus microplus, the southern cattle tick and an important disease vector. Firstly, amino acid sequences homologous with CDKs of other organisms were identified from a R. microplus transcriptome database in silico. The analysis of the deduced amino acid sequences of CDK1 and CDK10 from R. microplus showed that both have caspase-3/7 cleavage motifs despite their differences in motif position and length of encoded proteins. CDK1 has two motifs (DKRGD and SAKDA) located opposite to the ATP binding site while CDK10 has only one motif (SLLDN) for caspase 3-7 near the ATP binding site. Roscovitine (Rosco), a purine derivative that inhibits CDK/cyclin complexes by binding to the catalytic domain of the CDK molecule at the ATP binding site, which prevents the transfer of ATP's γphosphoryl group to the substrate. To determine the effect of Rosco on tick CDKs, BME26 cells derived from R. microplus embryo cells were utilized in vitro inhibition assays. Cell viability decreased in the Rosco-treated groups after 24 hours of incubation in a concentration-dependent manner and this was observed up to 48 hours following incubation. To our knowledge, this is the first report on characterization of a cell cycle protein in arachnids, and the sensitivity of BME26 tick cell line to Rosco treatment suggests that CDKs are potential targets for novel drug design to control tick infestation.

Metacaspases, autophagins and metallocarboxypeptidases: potential new targets for chemotherapy of the trypanosomiases.

During the last decade, de novo drug discovery approaches have come into focus due to the increased number of parasite pathogen genomes sequenced and the subsequent availability of genome-scale functional datasets. In order to prioritize target proteins, these approaches consider traits commonly thought to be desirable in a drug target, including essentiality, druggability (whether drug-like molecules are likely to interact with the target), assayability, importance in lifecycle stages of the pathogen relevant to human health, and specificity (i.e. the target is absent from, or substantially different in, the host). Proteases from protozoan parasites have become popular drug targets since these enzymes accomplish both housekeeping tasks common to many eukaryotes as well as functions highly specific to the parasite life style. Trypanosoma cruzi, the parasitic flagellate, agent of Chagas Disease, contains several cysteine, serine, threonine and metallo proteinases. This review will deal with peculiar families described in this parasite. Among them, two eukaryote homologues of the carboxypeptidases Taq are promising targets due to their particular phylogenetic distribution. Also absent in metazoans, metacaspases are essential peptidases playing important roles in cell growth, death and differentiation of trypanosomatids. Finally, autophagins are involved in the regulation of a conserved degradative pathway, the autophagy pathway, and result important for parasite survival under nutritional stress conditions and differentiation. Although so far there are no specific inhibitors for these families, the increasing knowledge of their biochemical properties, including substrate specificity, crystal structure, and biological functions, is an essential step towards the development of inhibitors.

Nuclear morphometric analysis (NMA): screening of senescence, apoptosis and nuclear irregularities.

Several cellular mechanisms affect nuclear morphology which can therefore be used to assess certain processes. Here, we present an analytic tool to quantify the number of cells in a population that present characteristics of senescence, apoptosis or nuclear irregularities through nuclear morphometric analysis. The tool presented here is based on nuclear image analysis and evaluation of size and regularity of adhered cells in culture. From 46 measurements of nuclear morphometry, principal component analysis filtered four measurements that best separated regular from irregular nuclei. These measurements, namely aspect, area box, radius ratio and roundness were combined into a single nuclear irregularity index (NII). Normal nuclei are used to set the parameters for a given cell type, and different nuclear phenotypes are separated in an area versus NII plot. The tool was validated with ß-gal staining for senescence and annexin or caspases inhibitor for apoptosis as well as several treatments that induce different cellular phenotypes. This method provides a direct and objective way of screening normal, senescent, apoptotic and nuclear irregularities which may occur during failed mitosis or mitotic catastrophe, which may be very useful in basic and clinical research.

Antagonic activities of Trypanosoma cruzi metacaspases affect the balance between cell proliferation, death and differentiation.

Metacaspases are distant relatives of animal caspases present in plants, fungi and protozoa. At variance with caspases, metacaspases exhibit stringent specificity for basic amino-acid residues and are absolutely dependent on millimolar concentrations of calcium. In the protozoan parasite Trypanosoma cruzi, metacaspases have been suggested to be involved in an apoptosis-like phenomenon upon exposure of the parasite to fresh human serum (FHS). Nuclear relocalization of metacaspases was observed after FHS treatment and overexpression of metacaspase-5 led to enhanced sensitivity to this stimulus. Here we report some biochemical properties of T. cruzi metacaspases. Performing fluorescent-activated cell sorting (FACS) analysis of epimastigotes inducibly overexpressing metacaspase-3, we demonstrate a role for this metacaspase in cell cycle progression, protection of epimastigotes from naturally occurring cell death and differentiation to infective metacyclic trypomastigotes. We also show that regulation of metacaspase-3 activity is important for cell cycle completion inside the mammalian host. On the other hand, inducible overexpression of metacaspase-5 lacking its C-terminal domain caused an apoptotic-like response. These results suggest that the two T. cruzi metacaspases could play an important role in the life cycle and bring to light the close relationship between cell division, death and differentiation in this ancient unicellular eukaryote.

CaSPredictor: a new computer-based tool for caspase substrate prediction.

MOTIVATION: In vitro studies have shown that the most remarkable catalytic features of caspases, a family of cysteineproteases, are their stringent specificity to Asp (D) in the S1 subsite and at least four amino acids to the left of scissile bound. However, there is little information about the substrate recognition patterns in vivo. The prediction and characterization of proteolytic cleavage sites in natural substrates could be useful for uncovering these structural relationships. RESULTS: PEST-like sequences rich in the amino acids Ser (S), Thr (T), Pro (P), Glu or Asp (E/D), including Asn (N) and Gln (Q) are adjacent structural/sequential elements in the majority of cleavage site regions of the natural caspase substrates described in the literature, supporting its possible implication in the substrate selection by caspases. We developed CaSPredictor, a software which incorporated a PEST-like index and the position-dependent amino acid matrices for prediction of caspase cleavage sites in individual proteins and protein datasets. The program predicted successfully 81% (111/137) of the cleavage sites in experimentally verified caspase substrates not annotated in its internal data file. Its accuracy and confidence was estimated as 80% using ROC methodology. The program was much more efficient in predicting caspase substrates when compared with PeptideCutter and PEPS software. Finally, the program detected potential cleavage sites in the primary sequences of 1644 proteins in a dataset containing 9986 protein entries. AVAILABILITY: Requests for software should be made to Dr José E. Belizário SUPPLEMENTARY INFORMATION: Supplementary information is available for academic users at site http://icb.usp.br/~farmaco/Jose/CaSpredictorfiles.