TcZC3HTTP, a regulatory element that contributes to Trypanosoma cruzi cell proliferation.

Post-transcriptional regulation of gene expression is a critical process for adapting to and surviving Trypanosoma cruzi, a parasite with a complex life cycle. RNA-binding proteins (RBPs) are key players in this regulation, forming ribonucleoprotein complexes (messenger ribonucleoproteins) and RNA granules that control transcript stability, localization, degradation, and translation modulation. Understanding the specific roles of individual RBPs is crucial for unraveling the details of this regulatory network. In this study, we generated null mutants of the TcZC3HTTP gene, a specific RBP in the Trypanosoma family characterized by a C3H zinc finger and a DNAJ domain associated with RNA and protein binding, respectively. Through cell growth assays, we demonstrated that the absence of TcZC3HTTP or the expression of an additional tagged version impacted epimastigote growth, indicating its contribution to cell proliferation. TcZC3HTTP was found to associate with mRNAs involved in cell cycle and division in epimastigotes, while in nutritionally stressed parasites it exhibited associations with mRNAs coding for other RBPs and rRNA. Furthermore, our analysis identified that TcZC3HTTP protein partners were different during normal growth conditions compared to starvation conditions, with the latter showing enrichment of ribosomal proteins and other RBPs. Therefore, this study provides insights into TcZC3HTTP's role in the post-transcriptional regulation of gene expression during normal growth and nutritional stress in T. cruzi, uncovering its versatile functions in different cellular contexts.IMPORTANCEUnderstanding how Trypanosoma cruzi, the causative agent of Chagas disease, regulates gene expression is crucial for developing targeted interventions. In this study, we investigated the role of TcZC3HTTP, an RNA-binding protein, in post-transcriptional regulation. Our findings demonstrate that TcZC3HTTP is relevant for the growth and proliferation of epimastigotes, a stage of the parasite's life cycle. We identified its associations with specific mRNAs involved in cell cycle and division and its interactions with enzymes and other RNA-binding proteins (RBPs) under normal and starvation conditions. These insights shed light on the regulatory network underlying gene expression in T. cruzi and reveal the multifaceted functions of RBPs in this parasite. Such knowledge enhances our understanding of the parasite's biology and opens avenues for developing novel therapeutic strategies targeting post-transcriptional gene regulation in T. cruzi.

Modulation of Klebsiella pneumoniae Outer Membrane Vesicle Protein Cargo under Antibiotic Treatment.

Klebsiella pneumoniae is a nosocomial pathogen and an important propagator of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains. Like other Gram-negative bacteria, they secrete outer membrane vesicles (OMVs) that distribute virulence and resistance factors. Here, we subjected a K. pneumoniae-XDR to subinhibitory concentrations of meropenem, amikacin, polymyxin B, and a combination of these agents to evaluate changes in the protein cargo of OMVs through liquid chromatography-tandem mass spectrometry (LC-MS/MS). Genome sequencing of the clinical isolate K. pneumoniae strain HCD1 (KpHCD1) revealed the presence of 41 resistance genes and 159 virulence factors. We identified 64 proteins in KpHCD1-OMVs modulated with different antibiotic treatments involved in processing genetic information, environmental information, cell envelope formation, energy metabolism, and drug resistance. The OMV proteome expression profile suggests that OMVs may be associated with pathogenicity, survival, stress response, and resistance dissemination.

Non-histone protein methylation in Trypanosoma cruzi epimastigotes.

Post-translational methylation of proteins, which occurs in arginines and lysines, modulates several biological processes at different levels of cell signaling. Recently, methylation has been demonstrated in the regulation beyond histones, for example, in the dynamics of protein-protein and protein-nucleic acid interactions. However, the presence and role of non-histone methylation in Trypanosoma cruzi, the etiologic agent of Chagas disease, has not yet been elucidated. Here, we applied mass spectrometry-based-proteomics (LC-MS/MS) to profile the methylproteome of T. cruzi epimastigotes, describing a total of 1252 methyl sites in 824 proteins. Functional enrichment and protein-protein interaction analysis show that protein methylation impacts important biological processes of the parasite, such as translation, RNA and DNA binding, amino acid, and carbohydrate metabolism. In addition, 171 of the methylated proteins were previously reported to bear phosphorylation sites in T. cruzi, including flagellar proteins and RNA binding proteins, indicating that there may be an interplay between these different modifications in non-histone proteins. Our results show that a broad spectrum of functions is affected by methylation in T. cruzi, indicating its potential to impact important processes in the biology of the parasite and other trypanosomes.

Integrated analysis of label-free quantitative proteomics and bioinformatics reveal insights into signaling pathways in male breast cancer.

Male breast cancer (MBC) is a rare malignancy that accounts for about 1.8% of all breast cancer cases. In contrast to the high number of the "omics" studies in breast cancer in women, only recently molecular approaches have been performed in MBC research. High-throughput proteomics based methodologies are promisor strategies to characterize the MBC proteomic signatures and their association with clinico-pathological parameters. In this study, the label-free quantification-mass spectrometry and bioinformatics approaches were applied to analyze the proteomic profiling of a MBC case using the primary breast tumor and the corresponding axillary metastatic lymph nodes and adjacent non-tumor breast tissues. The differentially expressed proteins were identified in the signaling pathways of granzyme B, sirtuins, eIF2, actin cytoskeleton, eNOS, acute phase response and calcium and were connected to the upstream regulators MYC, PI3K SMARCA4 and cancer-related chemical drugs. An additional proteomic comparative analysis was performed with a primary breast tumor of a female patient and revealed an interesting set of proteins, which were mainly involved in cancer biology. Together, our data provide a relevant data source for the MBC research that can help the therapeutic strategies for its management.

Cross-Kingdom Extracellular Vesicles EV-RNA Communication as a Mechanism for Host-Pathogen Interaction.

The extracellular vesicle (EVs) traffic has been highlighted as a very important pathway of cellular communication. EVs are produced by prokaryotes and eukaryotes organisms and can carry molecules to help maintain homeostasis, responding to general disbalance, infections, and allowing rapid modulation of the immune system. In the context of infection, EVs from both the host and the pathogen have been identified as playing roles in the recruitment of immunological molecules that can lead to the resolution of the infection or the host's defeat. Bacterial vesicles RNA cargo play roles in the host cell by regulating gene expression and modulating immune response. In fungi the RNA molecules present in EVs are diverse and participate in communication between the host and pathogenic fungi. Little is known about how cross-kingdom sRNA trafficking occurs, although in recent years, there has been an increase in studies that relate EV participation in sRNA delivery. This review aims to elucidate and update the reader concerning the role of extracellular vesicles, with emphasis in the RNA content. We describe the EVs during infection from the host point-of-view, as well as the bacteria and fungi pathogens producing EVs that help the establishment of the disease.

Genome-Wide Proteomics and Phosphoproteomics Analysis of Trypanosoma cruzi During Differentiation.

Trypanosoma cruzi is a pathogenic protozoan that still has an impact on public health, despite the decrease in the number of infection cases along the years. T. cruzi possesses an heteroxenic life cycle in which it differentiates in at least four forms. Among the differentiation processes, metacyclogenesis has been exploited in different views by researchers. An intriguing question that rises is how metacyclogenesis is triggered and controlled by cell signaling and which are the differentially expressed proteins and posttranslational modifications involved in this process. An important cell signaling pathway is the protein phosphorylation, and it is reinforced in T. cruzi in which the gene expression control occurs almost exclusively posttranscriptionally. Additionally, the number of protein kinases in T. cruzi is relatively high compared to other organisms. A way to approach these questions is evaluating the cells through phosphoproteomics and proteomics. In this chapter, we will describe the steps from the cell protein extraction, digestion and fractionation, phosphopeptide enrichment, to LC-MS/MS analysis as well as a brief overview on peptide identification. In addition, a published method for in vitro metacyclogenesis will be detailed.

Quantitative phosphoproteome and proteome analyses emphasize the influence of phosphorylation events during the nutritional stress of Trypanosoma cruzi: the initial moments of in vitro metacyclogenesis.

Phosphorylation is an important event in cell signaling that is modulated by kinases and phosphatases. In Trypanosoma cruzi, the etiological agent of Chagas disease, approximately 2% of the protein-coding genes encode for protein kinases. This parasite has a heteroxenic life cycle with four different development stages. In the midgut of invertebrate vector, epimastigotes differentiate into metacyclic trypomastigotes in a process known as metacyclogenesis. This process can be reproduced in vitro by submitting parasites to nutritional stress (NS). Aiming to contribute to the elucidation of mechanisms that trigger metacyclogenesis, we applied super-SILAC (super-stable isotope labeling by amino acids in cell culture) and LC-MS/MS to analyze different points during NS. This analysis resulted in the identification of 4205 protein groups and 3643 phosphopeptides with the location of 4846 phosphorylation sites. Several phosphosites were considered modulated along NS and are present in proteins associated with various functions, such as fatty acid synthesis and the regulation of protein expression, reinforcing the importance of phosphorylation and signaling events to the parasite. These modulated sites may be triggers of metacyclogenesis.

High-throughput mass spectrometry and bioinformatics analysis of breast cancer proteomic data.

Data present here describe a comparative proteomic analysis among the malignant [primary breast tumor (PT) and axillary metastatic lymph nodes (LN)], and the non-tumor [contralateral (NCT) and adjacent (ANT)] breast tissues. Protein identification and quantification were performed through label-free mass spectrometry using a nano-liquid chromatography coupled to an electrospray ionization-mass spectrometry (nLC-ESI-MS/MS). The mass spectrometry proteomic data have been deposited to the ProteomeXchange Consortium via PRIDE partner repository with the dataset identifier PXD012431. A total of 462 differentially expressed proteins was identified among these tissues and was analyzed in six groups' comparisons (named NCTxANT, PTxNCT, PTxANT, LNxNCT, LNxANT and PTxLN). Proteins at 1.5 log2 fold change were submitted to the Ingenuity® Pathway Analysis (IPA) software version 2.3 (QIAGEN Inc.) to identify biological pathways, disease and function annotation, and interaction networks related to cancer biology. The detailed data present here provides information about the proteome alterations and their role on breast tumorigenesis. This information can lead to novel biological insights on cancer research. For further interpretation of these data, please see our research article 'Quantitative label-free mass spectrometry using contralateral and adjacent breast tissues reveal differentially expressed proteins and their predicted impacts on pathways and cellular functions in breast cancer' [2].

Quantitative label-free mass spectrometry using contralateral and adjacent breast tissues reveal differentially expressed proteins and their predicted impacts on pathways and cellular functions in breast cancer.

Proteins play an essential role in the biological processes associated with cancer. Their altered expression levels can deregulate critical cellular pathways and interactive networks. In this study, the mass spectrometry-based label-free quantification followed by functional annotation was performed to investigate the most significant deregulated proteins among tissues of primary breast tumor (PT) and axillary metastatic lymph node (LN) and corresponding non-tumor tissues contralateral (NCT) and adjacent (ANT) from patients diagnosed with invasive ductal carcinoma. A total of 462 proteins was observed as differentially expressed (DEPs) among the groups analyzed. A high level of similarity was observed in the proteome profile of both non-tumor breast tissues and DEPs (n = 12) were mainly predicted in the RNA metabolism. The DEPs among the malignant and non-tumor breast tissues [n = 396 (PTxNCT) and n = 410 (LNxNCT)] were related to pathways of the LXR/RXR, NO, eNOS, eIF2 and sirtuins, tumor-related functions, fatty acid metabolism and oxidative stress. Remarkable similarity was observed between both malignant tissues, which the DEPs were related to metastatic capabilities. Altogether, our findings revealed differential proteomic profiles that affected cancer associated and interconnected signaling processes. Validation studies are recommended to demonstrate the potential of individual proteins and/or pathways as biological markers in breast cancer. SIGNIFICANCE: The proteomic analysis of this study revealed high similarity in the proteomic profile of the contralateral and adjacent non-tumor breast tissues. Significant differences were identified among the proteome of the malignant and non-tumor tissue groups of the same patients, providing relevant insights into the hallmarks, signaling pathways, biological functions, and interactive protein networks that act during tumorigenesis and breast cancer progression. These proteins are suggested as targets of relevant interest to be explored as potential biological markers related to tumor development and metastatic progression in the breast cancer disease.