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1.
J of Cell Sci, v. 136, n. 22. nov. 2023
Artigo em Inglês | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP | ID: bud-5163

RESUMO

The nucleolus is sensitive to stress and can orchestrate a chain of cellular events in response to stress signals. Despite being a growth factor, FGF2 has antiproliferative and tumor-suppressive functions in some cellular contexts. In this work, we investigated how the antiproliferative effect of FGF2 modulates chromatin-, nucleolus-, and rDNA-associated proteins. The chromatin and nucleolar proteome indicated that FGF2 stimulation modulates proteins related to transcription, rRNA expression, and chromatin remodeling proteins. The global transcriptional rate and nucleolus area increased along with nucleolar disorganization upon 24 h of FGF2 stimulation. FGF2 stimulation induced immature rRNA accumulation by increasing rRNA transcription. The rDNA-associated protein analysis reinforced that FGF2 stimulus interferes with transcription and rRNA processing. RNA Pol I inhibition partially reversed the growth arrest induced by FGF2, indicating that changes in rRNA expression may be crucial for triggering the antiproliferative effect. Taken together, we demonstrate that the antiproliferative FGF2 stimulus triggers significant transcriptional changes and modulates the main cell transcription site, the nucleolus.

2.
Microorganisms, v. 11, n. 6, 1558, jun. 2023
Artigo em Inglês | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP | ID: bud-4955

RESUMO

Toxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa and causes toxoplasmosis infections, a disease that affects a quarter of the world’s population and has no effective cure. Epigenetic regulation is one of the mechanisms controlling gene expression and plays an essential role in all organisms. Lysine deacetylases (KDACs) act as epigenetic regulators affecting gene silencing in many eukaryotes. Here, we focus on TgKDAC4, an enzyme unique to apicomplexan parasites, and a class IV KDAC, the least-studied class of deacetylases so far. This enzyme shares only a portion of the specific KDAC domain with other organisms. Phylogenetic analysis from the TgKDAC4 domain shows a putative prokaryotic origin. Surprisingly, TgKDAC4 is located in the apicoplast, making it the only KDAC found in this organelle to date. Transmission electron microscopy assays confirmed the presence of TgKDAC4 in the periphery of the apicoplast. We identified possible targets or/and partners of TgKDAC4 by immunoprecipitation assays followed by mass spectrometry analysis, including TgCPN60 and TgGAPDH2, both located at the apicoplast and containing acetylation sites. Understanding how the protein works could provide new insights into the metabolism of the apicoplast, an essential organelle for parasite survival.

3.
mSphere, v. 7, n. 6, e00403-22, nov-dez. 2022
Artigo em Inglês | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP | ID: bud-4713

RESUMO

Chromatin dynamics can regulate all DNA-dependent processes. Access to DNA within chromatin is orchestrated mainly by histones and their posttranslational modifications (PTMs). Like other eukaryotes, the apicomplexan parasite Toxoplasma gondii encodes four canonical histones and five histone variants. In contrast, the linker histone (H1) has never been identified in apicomplexan parasites. In other eukaryotes, histone H1 compacts the chromatin by linking the nucleosome and increasing the DNA compaction. H1 is a multifunctional protein and can be involved in different steps of DNA metabolism or associated with protein complexes related to distinct biological processes. We have identified a novel protein in T. gondii (“TgH1-like”) that, although lacking the globular domain of mammalian H1, is remarkably like the H1-like proteins of bacteria and trypanosomatids. Our results demonstrate that TgH1-like is a nuclear protein associated with chromatin and other histones. Curiously, TgH1-like is also in the nucleolus and associated with ribosomal proteins, indicating a versatile function in this parasite. Although knockout of the tgh1-like gene does not affect the cell cycle, it causes endopolygeny and asynchronous division. Interestingly, mutation of posttranslationally modified amino acids results in defects in cell division like those in the Δtgh1-like mutant, showing that these sites are important for protein function. Furthermore, in the bradyzoite stage, this protein is expressed only in dividing parasites, reinforcing its importance in cell division. Indeed, the absence of TgH1-like decreases compaction of peripheral chromatin, confirming its role in the chromatin modulation in T. gondii. Histone H1, or linker histone, is an important protein that binds to the nucleosome, aiding chromatin compaction. Here, we characterize for the first time a linker histone in T. gondii, named TgH1-like. It is a small and basic protein that corresponds only to the C-terminal portion of the human H1 but is similar to histone H1 from trypanosomatids and bacteria. TgH1-like is located in the nucleus, interacts with nucleosome histones, and acts in chromatin structure and cell division. Our findings show for the first time the presence of a histone H1 protein in an apicomplexan parasite and will provide new insights into cell division and chromatin dynamics in T. gondii and related parasites.

4.
Mol Omics, v. 17, n. 5, p. 725-739, maio. 2021
Artigo em Inglês | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP | ID: bud-3872

RESUMO

Paradoxically, oncogenes that drive cell cycle progression may also trigger pathways leading to senescence, thereby inhibiting the growth of tumorigenic cells. Knowledge of how these pathways operate, and how tumor cells may evade these pathways, is important for understanding tumorigenesis. The Y1 cell line, which harbors an amplification of the proto-oncogene Ras, rapidly senesces in response to the mitogen fibroblast growth factor-2 (FGF-2). To gain a more complete picture of how FGF-2 promotes senescence, we employed a multi-omics approach to analyze histone modifications, mRNA and protein expression, and protein phosphorylation in Y1 cells treated with FGF-2. Compared to control cells treated with serum alone, FGF-2 caused a delayed accumulation of acetylation on histone H4 and higher levels of H3K27me3. Sequencing analysis revealed decreased expression of cell cycle-related genes with concomitant loss of H3K27ac. At the same time, FGF-2 promoted the expression of p21, various cytokines, and MAPK-related genes. Nuclear envelope proteins, particularly lamin B1, displayed increased phosphorylation in response to FGF-2. Proteome analysis suggested alterations in cellular metabolism, as evident by modulated expression of enzymes involved in purine biosynthesis, tRNA aminoacylation, and the TCA cycle. We propose that Y1 cells senesce due to an inability to progress through the cell cycle, which may stem from DNA damage or TGFb signaling. Altogether, the phenotype of Y1 cells is consistent with rapidly established oncogene-induced senescence, demonstrating the synergy between growth factors and oncogenes in driving senescence and bringing additional insight into this tumor suppressor mechanism.

5.
Nucleic Acids Res, v. 49, n. 12, p. 7053–7074, jun. 2021
Artigo em Inglês | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP | ID: bud-3861

RESUMO

Eukaryotic ribosome biogenesis is an elaborate process during which ribosomal proteins assemble with the pre-rRNA while it is being processed and folded. Hundreds of assembly factors (AF) are required and transiently recruited to assist the sequential remodeling events. One of the most intricate ones is the stepwise removal of the internal transcribed spacer 2 (ITS2), between the 5.8S and 25S rRNAs, that constitutes together with five AFs the pre-60S ‘foot’. In the transition from nucleolus to nucleoplasm, Nop53 replaces Erb1 at the basis of the foot and recruits the RNA exosome for the ITS2 cleavage and foot disassembly. Here we comprehensively analyze the impact of Nop53 recruitment on the pre-60S compositional changes. We show that depletion of Nop53, different from nop53 mutants lacking the exosome-interacting motif, not only causes retention of the unprocessed foot in late pre-60S intermediates but also affects the transition from nucleolar state E particle to subsequent nuclear stages. Additionally, we reveal that Nop53 depletion causes the impairment of late maturation events such as Yvh1 recruitment. In light of recently described pre-60S cryo-EM structures, our results provide biochemical evidence for the structural role of Nop53 rearranging and stabilizing the foot interface to assist the Nog2 particle formation

6.
PLoS Pathog, v. 17, n. 4, e1009502, abr. 2021
Artigo em Inglês | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP | ID: bud-3655

RESUMO

Trypanosoma cruzi is the etiologic agent of Chagas’ disease. Infected cells with T. cruzi activate several responses that promote unbalance of reactive oxygen species (ROS) that may cause DNA damage that activate cellular responses including DNA repair processes. In this work, HeLa cells and AC16 human cardiomyocyte cell line were infected with T. cruzi to investigate host cell responses at genome level during parasites intracellular life cycle. In fact, alkaline sensitive sites and oxidized DNA bases were detected in the host cell genetic material particularly in early stages of infection. These DNA lesions were accompanied by phosphorylation of the histone H2Ax, inducing γH2Ax, a marker of genotoxic stress. Moreover, Poly [ADP-ribose] polymerase) and 8-oxoguanine glycosylase (OGG1) are recruited to host cell nuclei, indicating activation of the DNA repair process. In infected cells, chromatin-associated proteins are carbonylated, as a possible consequence of oxidative stress and the nuclear factor erythroid 2–related factor 2 (NRF2) is induced early after infection, suggesting that the host cell antioxidant defenses are activated. However, at late stages of infection, NRF2 is downregulated. Interestingly, host cells pretreated with glutathione precursor, N-acetyl cysteine, NRF2 activator (Sulforaphane), and also Benznidonazol (BNZ) reduce parasite burst significantly, and DNA damage. These data indicate that the balance of oxidative stress and DNA damage induction in host cells may play a role during the process of infection itself, and interference in these processes may hamper T. cruzi infection, revealing potential target pathways for the therapy support.

7.
J Proteomics ; 225: 103847, 2020 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-32480077

RESUMO

Trypanosome histone N-terminal sequences are very divergent from the other eukaryotes, although they are still decorated by post-translational modifications (PTMs). Here, we used a highly robust workflow to analyze histone PTMs in the parasite Trypanosoma cruzi using mass spectrometry-based (MS-based) data-independent acquisition (DIA). We adapted the workflow for the analysis of the parasite's histone sequences by modifying the software EpiProfile 2.0, improving peptide and PTM quantification accuracy. This workflow could now be applied to the study of 141 T. cruzi modified histone peptides, which we used to investigate the dynamics of histone PTMs along the metacyclogenesis and the life cycle of T. cruzi. Global levels of histone acetylation and methylation fluctuates along metacyclogenesis, however most critical differences were observed between parasite life forms. More than 66 histone PTM changes were detected. Strikingly, the histone PTM pattern of metacyclic trypomastigotes is more similar to epimastigotes than to cellular trypomastigotes. Finally, we highlighted changes at the H4 N-terminus and at H3K76 discussing their impact on the trypanosome biology. Altogether, we have optimized a workflow easily applicable to the analysis of histone PTMs in T. cruzi and generated a dataset that may shed lights on the role of chromatin modifications in this parasite. SIGNIFICANCE: Trypanosomes are unicellular parasites that have divergent histone sequences, no chromosome condensation and a peculiar genome/gene regulation. Genes are transcribed from divergent polycistronic regions and post-transcriptional gene regulation play major role on the establishment of transcripts and protein levels. In this regard, the fact that their histones are decorated with multiple PTMs raises interesting questions about their role. Besides, this digenetic organism must adapt to different environments changing its metabolism accordingly. As metabolism and epigenetics are closely related, the study of histone PTMs in trypanosomes may enlighten this strikingly, and not yet fully understood, interplay. From a biomedical perspective, the comprehensive study of molecular mechanisms associated to the metacyclogenesis process is essential to create better strategies for controlling Chagas disease.


Assuntos
Parasitos , Trypanosoma cruzi , Animais , Epigênese Genética , Histonas/metabolismo , Estágios do Ciclo de Vida , Parasitos/metabolismo , Processamento de Proteína Pós-Traducional , Trypanosoma cruzi/metabolismo
8.
Clin Epigenetics, v. 12, 127, ago. 2020
Artigo em Inglês | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP | ID: bud-3143

RESUMO

Background We have previously developed a murine cellular system that models the transformation from melanocytes to metastatic melanoma cells. This model was established by cycles of anchorage impediment of melanocytes and consists of four cell lines: differentiated melanocytes (melan-a), pre-malignant melanocytes (4C), malignant (4C11−), and metastasis-prone (4C11+) melanoma cells. Here, we searched for transcriptional and epigenetic signatures associated with melanoma progression and metastasis by performing a gene co-expression analysis of transcriptome data and a mass-spectrometry-based profiling of histone modifications in this model. Results Eighteen modules of co-expressed genes were identified, and some of them were associated with melanoma progression, epithelial-to-mesenchymal transition (EMT), and metastasis. The genes in these modules participate in biological processes like focal adhesion, cell migration, extracellular matrix organization, endocytosis, cell cycle, DNA repair, protein ubiquitination, and autophagy. Modules and hub signatures related to EMT and metastasis (turquoise, green yellow, and yellow) were significantly enriched in genes associated to patient survival in two independent melanoma cohorts (TCGA and Leeds), suggesting they could be sources of novel prognostic biomarkers. Clusters of histone modifications were also linked to melanoma progression, EMT, and metastasis. Reduced levels of H4K5ac and H4K8ac marks were seen in the pre-malignant and tumorigenic cell lines, whereas the methylation patterns of H3K4, H3K56, and H4K20 were related to EMT. Moreover, the metastatic 4C11+ cell line showed higher H3K9me2 and H3K36me3 methylation, lower H3K18me1, H3K23me1, H3K79me2, and H3K36me2 marks and, in agreement, downregulation of the H3K36me2 methyltransferase Nsd1. Conclusions We uncovered transcriptional and histone modification signatures that may be molecular events driving melanoma progression and metastasis, which can aid in the identification of novel prognostic genes and drug targets for treating the disease.

9.
J Proteomics, v. 225, 103847, ago. 2020
Artigo em Inglês | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP | ID: bud-3126

RESUMO

Trypanosome histone N-terminal sequences are very divergent from the other eukaryotes, although they are still decorated by post-translational modifications (PTMs). Here, we used a highly robust workflow to analyze histone PTMs in the parasite Trypanosoma cruzi using mass spectrometry-based (MS-based) data-independent acquisition (DIA). We adapted the workflow for the analysis of the parasite's histone sequences by modifying the software EpiProfile 2.0, improving peptide and PTM quantification accuracy. This workflow could now be applied to the study of 141 T. cruzi modified histone peptides, which we used to investigate the dynamics of histone PTMs along the metacyclogenesis and the life cycle of T. cruzi. Global levels of histone acetylation and methylation fluctuates along metacyclogenesis, however most critical differences were observed between parasite life forms. More than 66 histone PTM changes were detected. Strikingly, the histone PTM pattern of metacyclic trypomastigotes is more similar to epimastigotes than to cellular trypomastigotes. Finally, we highlighted changes at the H4 N-terminus and at H3K76 discussing their impact on the trypanosome biology. Altogether, we have optimized a workflow easily applicable to the analysis of histone PTMs in T. cruzi and generated a dataset that may shed lights on the role of chromatin modifications in this parasite.

10.
Parasitology, v. 146, n. 4, p. 543-552, abr. 2019
Artigo em Inglês | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP | ID: bud-2708

RESUMO

Trypanosoma cruzi, the causative agent of Chagas disease, is a public health concern in Latin America. Epigenetic events, such as histone acetylation, affect DNA topology, replication and gene expression. Histone deacetylases (HDACs) are involved in chromatin compaction and post-translational modifications of cytoplasmic proteins, such as tubulin. HDAC inhibitors, like trichostatin A (TSA), inhibit tumour cell proliferation and promotes ultrastructural modifications. In the present study, TSA effects on cell proliferation, viability, cell cycle and ultrastructure were evaluated, as well as on histone acetylation and tubulin expression of the T. cruzi epimastigote form. Protozoa proliferation and viability were reduced after treatment with TSA. Quantitative proteomic analyses revealed an increase in histone acetylation after 72 h of TSA treatment. Surprisingly, results obtained by different microscopy methodologies indicate that TSA does not affect chromatin compaction, but alters microtubule cytoskeleton dynamics and impair kDNA segregation, generating polynucleated cells with atypical morphology. Confocal fluorescence microscopy and flow cytometry assays indicated that treated cell microtubules were more intensely acetylated. Increases in tubulin acetylation may be directly related to the higher number of parasites in the G2/M phase after TSA treatment. Taken together, these results suggest that deacetylase inhibitors represent excellent tools for understanding trypanosomatid cell biology.

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