Proteomic changes in Trypanosoma cruzi epimastigotes treated with the proapoptotic compound PAC-1.

Apoptosis is a highly regulated process of cell death in metazoans. Therefore, understanding the biochemical changes associated with apoptosis-like death in Trypanosoma cruzi is key to drug development. PAC-1 was recently shown to induce apoptosis in T. cruzi; with this as motivation, we used quantitative proteomics to unveil alterations of PAC-1-treated versus untreated epimastigotes. The PAC-1 treatment reduced the abundance of putative vesicle-associated membrane protein, putative eukaryotic translation initiation factor 1 eIF1, coatomer subunit beta, putative amastin, and a putative cytoskeleton-associated protein. Apoptosis-like signaling also increases the abundance of proteins associated with actin cytoskeleton remodeling, cell polarization, apoptotic signaling, phosphorylation, methylation, ergosterol biosynthesis, vacuolar proteins associated with autophagy, and flagellum motility. We shortlist seventeen protein targets for possible use in chemotherapy for Chagas disease. Almost all differentially abundant proteins belong to a family of proteins previously associated with apoptosis in metazoans, suggesting that the apoptotic pathway's key functions have been preserved from trypanosomatids and metazoans. SIGNIFICANCE: Approximately 8 million people worldwide are infected with Trypanosoma cruzi. The treatment of Chagas disease comprises drugs with severe side effects, thus limiting their application. Thus, developing new pharmaceutical solutions is relevant, and several molecules targeting apoptosis are therapeutically efficient for parasitic, cardiac, and neurological diseases. Apoptotic processes lead to specific morphological features that have been previously observed in T. cruzi. Here, we investigate changes in epimastigotes' proteomic profile treated with the proapoptotic compound PAC-1, providing data concerning the regulation of both metabolic and cellular processes in nonmetazoan apoptotic cells. We shortlist seventeen protein target candidates for use in chemotherapy for Chagas disease.

Unveiling the partners of the DRBD2-mRNP complex, an RBP in Trypanosoma cruzi and ortholog to the yeast SR-protein Gbp2.

BACKGROUND: RNA-binding proteins (RBPs) are well known as key factors in gene expression regulation in eukaryotes. These proteins associate with mRNAs and other proteins to form mRNP complexes that ultimately determine the fate of target transcripts in the cell. This association is usually mediated by an RNA-recognition motif (RRM). In the case of trypanosomatids, these proteins play a paramount role, as gene expression regulation is mostly posttranscriptional. Despite their relevance in the life cycle of Trypanosoma cruzi, the causative agent of Chagas' disease, to date, few RBPs have been characterized in this parasite. RESULTS: We investigated the role of DRBD2 in T. cruzi, an RBP with two RRM domains that is associated with cytoplasmic translational complexes. We show that DRBD2 is an ortholog of the Gbp2 in yeast, an SR-rich protein involved in mRNA quality control and export. We used an immunoprecipitation assay followed by shotgun proteomics and RNA-seq to assess the interaction partners of the DRBD2-mRNP complex in epimastigotes. The analysis identified mostly proteins involved in RNA metabolism and regulation, such as ALBA1, ALBA3, ALBA4, UBP1, UBP2, DRBD3, and PABP2. The RNA-seq results showed that most of the transcripts regulated by the DRBD2 complex mapped to hypothetical proteins related to multiple processes, such as to biosynthetic process, DNA metabolic process, protein modification, and response to stress. CONCLUSIONS: The identification of regulatory proteins in the DRBD2-mRNP complex corroborates the important role of DRBD2 in gene expression regulation in T. cruzi. We consider these results an important contribution to future studies regarding gene expression regulation in T. cruzi, especially in the field of RNA-binding proteins.

Assessing the partners of the RBP9-mRNP complex in Trypanosoma cruzi using shotgun proteomics and RNA-seq.

Gene expression regulation in trypanosomes differs from other eukaryotes due to absence of transcriptional regulation for most of their genes. RNA-binding proteins (RBPs) associate with mRNAs and other regulatory proteins to form ribonucleoprotein complexes (mRNPs), which play a major role in post-transcriptional regulation. Here, we show that RBP9 is a cytoplasmic RBP in Trypanosoma cruzi with one RNA-recognition motif (RRM). The RBP9 sedimentation profile in a sucrose gradient indicated its presence in cytoplasmic translational complexes, suggesting its involvement in translation regulation. Taking this result as a motivation, we used shotgun proteomics and RNA-seq approaches to assess the core of the RBP9-mRNP complex. In epimastigotes in exponential growth, the complex was composed mostly by RBPs involved in RNA metabolism, such as ZC3H39, UBP1/2, NRBD1, and ALBA3/4. When parasites were subjected to nutritional stress, our analysis identified regulatory RBPs and the translation initiation factors eIF4E5, eIF4G5, eIF4G1, and eIF4G4. The RNA-seq results showed that RBP9-mRNP complex regulates transcripts encoding some RBPs - e.g. RBP5, RBP6, and RBP10 -, and proteins involved in metabolic processes. Therefore, we argue that RBP9 is part of cytoplasmic mRNPs complexes associated with mRNA metabolism and translation regulation in T. cruzi.

Comparing intestinal versus diffuse gastric cancer using a PEFF-oriented proteomic pipeline.

Gastric cancer is the fifth most common malignant neoplasia and the third leading cause of cancer death worldwide. Mac-Cormick et al. recently showed the importance of considering the anatomical region of the tumor in proteomic gastric cancer studies; more differences were found between distinct anatomical regions than when comparing healthy versus diseased tissue. Thus, failing to consider the anatomical region could lead to differential proteins that are not disease specific. With this as motivation, we compared the proteomic profiles of intestinal and diffuse adenocarcinoma from the same anatomical region, the corpus. To achieve this, we used isobaric labeling (iTRAQ) of peptides, a 10-step HILIC fractionation, and reversed-phase nano-chromatography coupled online with a Q-Exactive Plus mass spectrometer. We updated PatternLab to take advantage of the new Comet-PEFF search engine that enables identifying post-translational modifications and mutations included in neXtProt's PSI Extended FASTA Format (PEFF) metadata. Our pipeline then uses a text-mining tool that automatically extracts PubMed IDs from the proteomic result metadata and drills down keywords from manuscripts related with the biological processes at hand. Our results disclose important proteins such as apolipoprotein B-100, S100 and 14-3-3 proteins, among many others, highlighting the different pathways enriched by each cancer type. SIGNIFICANCE: Gastric cancer is a heterogeneous and multifactorial disease responsible for a significant number of deaths every year. Despite the constant improvement of surgical techniques and multimodal treatments, survival rates are low, mostly due to limited diagnostic techniques and late symptoms. Intestinal and diffuse types of gastric cancer have distinct clinical and pathological characteristics; yet little is known about the molecular mechanisms regulating these two types of gastric tumors. Here we compared the proteomic profile of diffuse and intestinal types of gastric cancer from the same anatomical location, the corpus, from four male patients. This methodological design aimed to eliminate proteomic variations resulting from comparison of tumors from distinct anatomical regions. Our PEFF-tailored proteomic pipeline significantly increased the identifications as when compared to previous versions of PatternLab.