RESUMO
The mammalian target of rapamycin (mTOR) pathway plays a key role in determining immune cells function through modulation of their metabolic status. By specific deletion of Rictor in CD11c+ myeloid cells (referred to here as CD11cRicΔ/Δ), we investigated the role of mTOR complex 2 (mTORC2) signaling in dendritic cells (DCs) function in mice. We showed that upon dextran sulfate sodium-induced colitis, the lack of mTORC2 signaling CD11c+ cells diminishes the colitis score and abrogates DC migration to the mesenteric lymph nodes, thereby diminishing the infiltration of T helper 17 cells in the lamina propria and subsequent inflammation. These findings corroborate with the abrogation of cytoskeleton organization and the decreased activation of Rac1 and Cdc42 GTPases observed in CD11c+-mTORC2-deficient cells. Meta-analysis on colonic samples from ulcerative colitis patients revealed increased gene expression of proinflammatory cytokines, which coincided with augmented expression of the mTOR pathway, a positive correlation between the DC marker ITGAX and interleukin-6, the expression of RICTOR, and CDC42. Together, this work proposes that targeting mTORC2 on DCs offers a key to hamper inflammatory responses, and this way, ameliorates the progression and severity of intestinal inflammatory diseases.
Assuntos
Movimento Celular , Colite , Células Dendríticas , Sulfato de Dextrana , Alvo Mecanístico do Complexo 2 de Rapamicina , Células Mieloides , Transdução de Sinais , Animais , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Camundongos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Colite/patologia , Colite/induzido quimicamente , Colite/imunologia , Células Mieloides/metabolismo , Células Mieloides/imunologia , Sulfato de Dextrana/toxicidade , Proteína Companheira de mTOR Insensível à Rapamicina/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina/genética , Antígeno CD11c/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Humanos , Proteínas rac1 de Ligação ao GTP/metabolismo , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , Camundongos Knockout , Neuropeptídeos , Antígenos CD11RESUMO
Resistance to radio and chemotherapy in Glioblastoma (GBM) is correlated with its malignancy, invasiveness, and aggressiveness. The Rho GTPase pathway plays important roles in these processes, but its involvement in the GBM response to genotoxic treatments remains unsolved. Inhibition of this signaling pathway has emerged as a promising approach for the treatment of CNS injuries and diseases, proving to be a strong candidate for therapeutic approaches. To this end, Rho-associated kinases (ROCK), classic downstream effectors of small Rho GTPases, were targeted for pharmacological inhibition using Y-27632 in GBM cells, expressing the wild-type or mutated p53 gene, and exposed to genotoxic stress by gamma ionizing radiation (IR) or cisplatin (PT). The use of the ROCK inhibitor (ROCKi) had opposite effects in these cells: in cells expressing wild-type p53, ROCKi reduced survival and DNA repair capacity (reduction of γH2AX foci and accumulation of strand breaks) after stress promoted by IR or PT; in cells expressing the mutant p53 protein, both treatments promoted longer survival and more efficient DNA repair, responses further enhanced by ROCKi. The target DNA repair mechanisms of ROCK inhibition were, respectively, an attenuation of NHEJ and NER pathways in wild-type p53 cells, and a stimulation of HR and NER pathways in mutant p53 cells. These effects were accompanied by the formation of reactive oxygen species (ROS) induced by genotoxic stress only in mutant p53 cells but potentiated by ROCKi and reversed by p53 knockdown. N-acetyl-L-cysteine (NAC) treatment or Rac1 knockdown completely eliminated ROCKi's p53-dependent actions, since ROCK inhibition specifically elevated Rac-GTP levels only in mutant p53 cells. Combining IR or PT and ROCKi treatments broadens our understanding of the sensitivity and resistance of, respectively, GBM expressing wild-type or mutant p53 to genotoxic agents. Our proposal may be a determining factor in improving the efficiency and assertiveness of CNS antitumor therapies based on ROCK inhibitors. SIGNIFICANCE: The use of ROCK inhibitors in association with radio or chemotherapy modulates GBM resistance and sensitivity depending on the p53 activity, suggesting the potential value of this protein as therapeutic target for tumor pre-sensitization strategies.
Assuntos
Glioblastoma , Humanos , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismo , Dano ao DNA , Linhagem Celular TumoralRESUMO
Glioblastoma (GBM), the most common aggressive brain tumor, is characterized by rapid cellular infiltration and is routinely treated with ionizing radiation (IR), but therapeutic resistance inevitably recurs. The actin cytoskeleton of glioblastoma cells provides their high invasiveness, but it remains unclear whether Rho GTPases modulate DNA damage repair and therapeutic sensitivity. Here, we irradiated glioblastoma cells with different p53 status and explored the effects of Rho pathway inhibition to elucidate how actin cytoskeleton disruption affects the DNA damage response and repair pathways. p53-wild-type and p53-mutant cells were subjected to Rho GTPase pathway modulation by treatment with C3 toxin; knockdown of mDia-1, PFN1 and MYPT1; or treatment with F-actin polymerization inhibitors. Rho inhibition increased the sensitivity of glioma cells to IR by increasing the number of DNA double-strand breaks and delaying DNA repair by nonhomologous end-joining in p53-wild-type cells. p53 knockdown reversed this phenotype by reducing p21 expression and Rho signaling activity, whereas reactivation of p53 in p53-mutant cells by treatment with PRIMA-1 reversed these effects. The interdependence between p53 and Rho is based on nuclear p53 translocation facilitated by G-actin and enhanced by IR. Isolated IR-resistant p53-wild-type cells showed an altered morphology and increased stress fiber formation: inhibition of Rho or actin polymerization decreased cell viability in a p53-dependent manner and reversed the resistance phenotype. p53 silencing reversed the Rho inhibition-induced sensitization of IR-resistant cells. Rho inhibition also impaired the repair of IR-damaged DNA in 3D spheroid models. Rho GTPase activity and actin cytoskeleton dynamics are sensitive targets for the reversal of acquired resistance in GBM tumors with wild-type p53.
Assuntos
Reparo do DNA , Glioblastoma , Proteínas rho de Ligação ao GTP , Humanos , DNA , Reparo do DNA/genética , Reparo do DNA/efeitos da radiação , Regulação para Baixo , Glioblastoma/genética , Glioblastoma/radioterapia , Glioblastoma/metabolismo , Recidiva Local de Neoplasia , Profilinas/genética , Radiação Ionizante , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/efeitos da radiação , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
PURPOSE: Transcriptome analysis of pancreatic ductal adenocarcinoma (PDAC) has been useful to identify gene expression changes that sustain malignant phenotypes. Yet, most studies examined only tumor tissues and focused on protein-coding genes, leaving long non-coding RNAs (lncRNAs) largely underexplored. METHODS: We generated total RNA-Seq data from patient-matched tumor and nonmalignant pancreatic tissues and implemented a computational pipeline to survey known and novel lncRNAs. siRNA-mediated knockdown in tumor cell lines was performed to assess the contribution of PDAC-associated lncRNAs to malignant phenotypes. Gene co-expression network and functional enrichment analyses were used to assign deregulated lncRNAs to biological processes and molecular pathways. RESULTS: We detected 9,032 GENCODE lncRNAs as well as 523 unannotated lncRNAs, including transcripts significantly associated with patient outcome. Aberrant expression of a subset of novel and known lncRNAs was confirmed in patient samples and cell lines. siRNA-mediated knockdown of a subset of these lncRNAs (LINC01559, LINC01133, CCAT1, LINC00920 and UCA1) reduced cell proliferation, migration and invasion. Gene co-expression network analysis associated PDAC-deregulated lncRNAs with diverse biological processes, such as cell adhesion, protein glycosylation and DNA repair. Furthermore, UCA1 knockdown was shown to specifically deregulate co-expressed genes involved in DNA repair and to negatively impact DNA repair following damage induced by ionizing radiation. CONCLUSIONS: Our study expands the repertoire of lncRNAs deregulated in PDAC, thereby revealing novel candidate biomarkers for patient risk stratification. It also provides a roadmap for functional assays aimed to characterize novel mechanisms of action of lncRNAs in pancreatic cancer, which could be explored for therapeutic development.
Assuntos
Adenocarcinoma , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , RNA Longo não Codificante , Adenocarcinoma/genética , Adenocarcinoma/patologia , Carcinoma Ductal Pancreático/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Neoplasias Pancreáticas/patologia , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Interferente Pequeno , Neoplasias PancreáticasRESUMO
Stearoyl-CoA desaturase (SCD) is a central lipogenic enzyme for the synthesis of monounsaturated fatty acids (MUFA). SCD1 overexpression is associated with a genetic predisposition to hepatocarcinogenesis in mice and rats. This work hypothesized possible roles of SCD1 to genomic stability, lipogenesis, cell proliferation, and survival that contribute to the malignant transformation of non-tumorigenic liver cells. Therefore, HepG2 tumor cells were treated with the SCD1 inhibitor (CAY10566) to ensure a decrease in proliferation/survival, as confirmed by a lipidomic analysis that detected an efficient decrease in the concentration of MUFA. According to that, we switched to a model of normal hepatocytes, the HepaRG cell line, where we: (i) overexpressed SCD1 (HepaRG-SCD1 clones), (ii) inhibited the endogenous SCD1 activity with CAY10566, or (iii) treated with two monounsaturated (oleic OA and/or palmitoleic PA) fatty acids. SCD1 overexpression or MUFA stimulation increased cell proliferation, survival, and the levels of AKT, phospho-AKT(Ser473), and proliferating cell nuclear antigen (PCNA) proteins. By contrast, opposite molecular and cellular responses were observed in HepaRG cells treated with CAY10566. To assess genomic stability, HepaRG-SCD1 clones were treated with ionizing radiation (IR) and presented reduced levels of DNA damage and higher survival at doses of 5 Gy and 10 Gy compared to parental cells. In sum, this work suggests that modulation of SCD1 activity not only plays a role in cell proliferation and survival, but also in maintaining genomic stability, and therefore, contributes to a better understanding of this enzyme in molecular mechanisms of hepatocarcinogenesis projecting SCD1 as a potential translational target.
Assuntos
Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Neoplasias Hepáticas/tratamento farmacológico , Ácidos Oleicos/farmacologia , Oxidiazóis/farmacologia , Piridazinas/farmacologia , Estearoil-CoA Dessaturase/metabolismo , Apoptose , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Ciclo Celular , Proliferação de Células , Instabilidade Genômica , Hepatócitos/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Estearoil-CoA Dessaturase/genética , Células Tumorais CultivadasRESUMO
DUSP3 is a phosphatase expressed and active in several tissues that dephosphorylates tyrosine residues in many regulatory proteins of cellular activities such as proliferation, survival, and cell death. Recently, two new independent functions were assigned to this enzyme: dephosphorylation of focal adhesion kinase (FAK) and regulation of nucleotide-excision repair (NER) pathway. Genotoxic stress by UV radiation is known to affect cell morphology, adhesion, and migration for affecting, for example, the Rho GTPases that regulate actin cytoskeleton. This work investigated the intersection of DUSP3 function, XPA protein activity, and UV toxicity by examining cell migration, FAK, and SRC kinase phosphorylation status, in addition to cell morphology, in fibroblast cells proficient (MRC-5) or deficient (XPA) of the NER pathway. DUSP3 loss reduced cell migration of normal cells, which was stimulated by the genotoxic stress, effects evidenced in presence of serum mitogenic stimulus. However, NER-deficient cells migration response was the opposite since DUSP3 loss increased migration, especially after cells being exposed to UV stress. The levels of pFAK(Y397) peaked 15 min and 1 h after UV radiation in normal cells, but only slightly increased in repair-deficient cells. However, the DUSP3 knockdown strongly raised pFAK(Y397) levels in both cells, but especially in XPA cells as supported by the higher SRC activity. These effects impacted on the dynamics of actin-based structures formation, such as stress fibres, apparently dependent on DUSP3 and DNA-repair (NER) proficiency of the cells. Altogether our findings suggest this dual-phosphatase is bridging gaps between the complex regulation of cell morphology, motility, and genomic stability.
Assuntos
Movimento Celular/efeitos da radiação , Fosfatase 3 de Especificidade Dupla/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Raios Ultravioleta , Adesão Celular/efeitos da radiação , Linhagem Celular , Reparo do DNA/efeitos da radiação , Fosfatase 3 de Especificidade Dupla/antagonistas & inibidores , Fosfatase 3 de Especificidade Dupla/genética , Proteína-Tirosina Quinases de Adesão Focal/genética , Humanos , Fosforilação/efeitos da radiação , Interferência de RNA , RNA Interferente Pequeno/metabolismoRESUMO
The small GTPase Cdc42, a member of the Rho family, regulates essential biological processes such as cytoskeleton remodeling, migration, vesicular trafficking and cell cycle. It was demonstrated that Cdc42 overactivation through different molecular strategies increases cell sensitivity to genotoxic stress and affects the phosphorylation status of DNA damage response proteins by unknown mechanisms. By using a combination of approaches including affinity purification/mass spectrometry (AP/MS) and colocalization microscopy analysis we were able to identify Cdc42EP3/Borg2 as a putative molecular effector of these molecular and cellular events that seem to be independent of cell line or DNA damage stimuli. We then investigated the influence of Cdc42EP3/Borg2 and other potential protein partners, such as the NCK and Septin2 proteins, which could mediate cellular responses to genotoxic stress under different backgrounds of Cdc42 activity. Clonogenic assays showed a reduced cell survival when ectopically expressing the Cdc42EP3/Borg2, NCK2 or Septin2 in an overactivated Cdc42-dependent background. Moreover, endogenous NCK appears to relocate into the nucleus upon Cdc42 overactivation, especially under genotoxic stress, and promotes the suppression of Chk1 phosphorylation. In sum, our findings reinforce Cdc42 as an important player involved in the DNA damage response acting through Cdc42EP3/Borg2 and NCK proteins following genomic instability conditions.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , DNA/metabolismo , Reguladores de Proteínas de Ligação ao GTP/metabolismo , Proteínas Oncogênicas/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Movimento Celular/fisiologia , Sobrevivência Celular/fisiologia , Citoesqueleto/metabolismo , Instabilidade Genômica/genética , Transdução de Sinais/fisiologiaRESUMO
The DUSP3 phosphatase regulates cell cycle, proliferation, apoptosis and senescence of different cell types, lately shown as a mediator of DNA repair processes. This work evaluated the impact of DUSP3 loss of function (lof) on DNA repair-proficient fibroblasts (MRC-5), NER-deficient cell lines (XPA and XPC) and translesion DNA synthesis (TLS)-deficient cells (XPV), after UV-radiation stress. The levels of DNA strand breaks, CPDs and 6-4-PPs have accumulated over time in all cells under DUSP3 lof, with a significant increase in NER-deficient lines. The inefficient repair of these lesions increased sub-G1 population of XPA and XPC cells 24 hours after UV treatment, notably marked by DUSP3 lof, which is associated with a reduced cell population in G1, S and G2/M phases. It was also detected an increase in S and G2/M populations of XPV and MRC-5 cells after UV-radiation exposure, which was slightly attenuated by DUSP3 lof due to a discrete increase in sub-G1 cells. The cell cycle progression was accompanied by changes in the levels of the main Cyclins (A1, B1, D1 or E1), CDKs (1, 2, 4 or 6), and the p21 Cip1 inhibitor, in a DUSP3-dependent manner. DUSP3 lof affected the proliferation of MRC-5 and XPA cells, with marked worsening of the XP phenotype after UV radiation. This work highlights the roles of DUSP3 in DNA repair fitness and in the fine control of regulatory proteins of cell cycle, essential mechanisms to maintenance of genomic stability.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Reparo do DNA/genética , Fosfatase 3 de Especificidade Dupla/metabolismo , Instabilidade Genômica , Ciclo Celular/efeitos da radiação , Proliferação de Células/genética , Proliferação de Células/efeitos da radiação , Dano ao DNA , Reparo do DNA/efeitos da radiação , Inativação Gênica/efeitos da radiação , Instabilidade Genômica/efeitos da radiação , Humanos , Dímeros de Pirimidina/metabolismo , Estresse Fisiológico/efeitos da radiação , Raios UltravioletaRESUMO
Antibiotic-induced dysbiosis is a key factor predisposing intestinal infection by Clostridium difficile. Here, we show that interventions that restore butyrate intestinal levels mitigate clinical and pathological features of C. difficile-induced colitis. Butyrate has no effect on C. difficile colonization or toxin production. However, it attenuates intestinal inflammation and improves intestinal barrier function in infected mice, as shown by reduced intestinal epithelial permeability and bacterial translocation, effects associated with the increased expression of components of intestinal epithelial cell tight junctions. Activation of the transcription factor HIF-1 in intestinal epithelial cells exerts a protective effect in C. difficile-induced colitis, and it is required for butyrate effects. We conclude that butyrate protects intestinal epithelial cells from damage caused by C. difficile toxins via the stabilization of HIF-1, mitigating local inflammatory response and systemic consequences of the infection.
Assuntos
Butiratos/administração & dosagem , Clostridioides difficile/patogenicidade , Colite/prevenção & controle , Fator 1 Induzível por Hipóxia/metabolismo , Administração Oral , Animais , Antibacterianos/farmacologia , Butiratos/farmacologia , Clostridioides difficile/metabolismo , Colite/etiologia , Colite/microbiologia , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Ácidos Graxos Voláteis/metabolismo , Humanos , Insulina/administração & dosagem , Mucosa Intestinal/citologia , Mucosa Intestinal/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microbiota/efeitos dos fármacos , Permeabilidade/efeitos dos fármacos , Junções Íntimas/metabolismo , Toxinas Biológicas/toxicidade , Triglicerídeos/administração & dosagemRESUMO
Dual Specificity Phosphatase 12 is a member of the Atypical DUSP Protein Tyrosine Phosphatase family, meaning that it does not contain typical MAP kinase targeting motifs, while being able to dephosphorylate tyrosine and serine/threonine residues. DUSP12 contains, apart from its catalytic domain, a zinc finger domain, making it one of the largest DUSPs, which displays strong nuclear expression in several tissues. In this work we identified nuclear targets of DUSP12 in two different cancer cell lines (A549 and MCF-7), challenging them with genotoxic stimuli to observe the effect on the networks and to link existing information about DUSP12 functions to the data obtained though mass spectrometry. We found network connections to the cytoskeleton (e.g. IQGAP1), to the chromatin (e.g. HP1BP3), to the splicing machinery and to the previously known pathway of ribosome maturation (e.g. TCOF1), which draw insight into many of the functions of this phosphatase, much likely connecting it to distinct, previously unknown genomic stability mechanisms.
Assuntos
Núcleo Celular/metabolismo , Dano ao DNA , Fosfatases de Especificidade Dupla/metabolismo , Proteínas Nucleares/metabolismo , Mapas de Interação de Proteínas , Células A549 , Núcleo Celular/patologia , Humanos , Células MCF-7RESUMO
Protein tyrosine kinases (PTK), discovered in the 1970s, have been considered master regulators of biological processes with high clinical significance as targets for human diseases. Their actions are countered by protein tyrosine phosphatases (PTP), enzymes yet underrepresented as drug targets because of the high homology of their catalytic domains and high charge of their catalytic pocket. This scenario is still worse for some PTP subclasses, for example, for the atypical dual-specificity phosphatases (ADUSPs), whose biological functions are not even completely known. In this sense, the present work focuses on the dual-specificity phosphatase 3 (DUSP3), also known as VH1-related phosphatase (VHR), an uncommon regulator of mitogen-activated protein kinase (MAPK) phosphorylation. DUSP3 expression and activities are suggestive of a tumor suppressor or tumor-promoting enzyme in different types of human cancers. Furthermore, DUSP3 has other biological functions involving immune response mediation, thrombosis, hemostasis, angiogenesis, and genomic stability that occur through either MAPK-dependent or MAPK-independent mechanisms. This broad spectrum of actions is likely due to the large substrate diversity and molecular mechanisms that are still under scrutiny. The growing advances in characterizing new DUSP3 substrates will allow the development of pharmacological inhibitors relevant for possible future clinical trials. This review covers all aspects of DUSP3, since its gene cloning and crystallographic structure resolution, in addition to its classical and novel substrates and the biological processes involved, followed by an update of what is currently known about the DUSP3/VHR-inhibiting compounds that might be considered potential drugs to treat human diseases.
Assuntos
Fosfatase 3 de Especificidade Dupla/genética , Fosfatase 3 de Especificidade Dupla/fisiologia , Fosfatase 3 de Especificidade Dupla/antagonistas & inibidores , Humanos , Proteínas Quinases Ativadas por Mitógeno , Neoplasias/enzimologia , Neovascularização Patológica , Fosforilação , Proteínas Tirosina Fosfatases , Proteínas Tirosina QuinasesRESUMO
Protein tyrosine phosphatases have long been considered key regulators of biological processes and are therefore implicated in the origins of various human diseases. Heterozygosity, mutations, deletions, and the complete loss of some of these enzymes have been reported to cause neurodegenerative diseases, autoimmune syndromes, genetic disorders, metabolic diseases, cancers, and many other physiological imbalances. Vaccinia H1-related phosphatase, also known as dual-specificity phosphatase 3, is a protein tyrosine phosphatase enzyme that regulates the phosphorylation of the mitogen-activated protein kinase signaling pathway, a central mediator of a diversity of biological responses. It has been suggested that vaccinia H1-related phosphatase can act as a tumor suppressor or tumor-promoting phosphatase in different cancers. Furthermore, emerging evidence suggests that this enzyme has many other biological functions, such as roles in immune responses, thrombosis, hemostasis, angiogenesis, and genomic stability, and this broad spectrum of vaccinia H1-related phosphatase activity is likely the result of its diversity of substrates. Hence, fully identifying and characterizing these substrate-phosphatase interactions will facilitate the identification of pharmacological inhibitors of vaccinia H1-related phosphatase that can be evaluated in clinical trials. In this review, we describe the biological processes mediated by vaccinia H1-related phosphatase, especially those related to genomic stability. We also focus on validated substrates and signaling circuitry with clinical relevance in human diseases, particularly oncogenesis.
Assuntos
Fosfatase 3 de Especificidade Dupla/fisiologia , Neoplasias/enzimologia , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neoplasias/mortalidade , Transdução de Sinais , Análise de SobrevidaRESUMO
Several lines of indirect evidence, such as mutations or dysregulated expression of genes related to cytoskeleton, have suggested that cytoskeletal dynamics, a process essential for axons and dendrites development, is compromised in autism spectrum disorders (ASD). However, no study has yet examined whether cytoskeleton dynamics is functionally altered in cells from ASD patients. Here we investigated the regulation of actin cytoskeleton dynamics in stem cells from human exfoliated deciduous teeth (SHEDs) of 13 ASD patients and 8 control individuals by inducing actin filament depolymerization and then measuing their reconstruction upon activation of the RhoGTPases Rac, Cdc42 or RhoA. We observed that stem cells from seven ASD individuals (53%) presented altered dymanics of filament reconstruction, including a patient recently studied by our group whose iPSC-derived neuronal cells show shorten and less arborized neurites. We also report potentially pathogenic genetic variants that might be related to the alterations in actin repolymerization dynamics observed in some patient-derived cells. Our results suggest that, at least for a subgroup of ASD patients, the dynamics of actin polymerization is impaired, which might be ultimately leading to neuronal abnormalities.
Assuntos
Citoesqueleto de Actina/química , Actinas/química , Transtorno do Espectro Autista/genética , Neurônios/química , Citoesqueleto de Actina/genética , Actinas/genética , Animais , Transtorno do Espectro Autista/metabolismo , Transtorno do Espectro Autista/patologia , Regulação da Expressão Gênica/genética , Humanos , Células-Tronco Pluripotentes Induzidas/química , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Esfoliação de Dente , Proteína cdc42 de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Protein tyrosine phosphatases have long been considered key regulators of biological processes and are therefore implicated in the origins of various human diseases. Heterozygosity, mutations, deletions, and the complete loss of some of these enzymes have been reported to cause neurodegenerative diseases, autoimmune syndromes, genetic disorders, metabolic diseases, cancers, and many other physiological imbalances. Vaccinia H1-related phosphatase, also known as dual-specificity phosphatase 3, is a protein tyrosine phosphatase enzyme that regulates the phosphorylation of the mitogen-activated protein kinase signaling pathway, a central mediator of a diversity of biological responses. It has been suggested that vaccinia H1-related phosphatase can act as a tumor suppressor or tumor-promoting phosphatase in different cancers. Furthermore, emerging evidence suggests that this enzyme has many other biological functions, such as roles in immune responses, thrombosis, hemostasis, angiogenesis, and genomic stability, and this broad spectrum of vaccinia H1-related phosphatase activity is likely the result of its diversity of substrates. Hence, fully identifying and characterizing these substrate-phosphatase interactions will facilitate the identification of pharmacological inhibitors of vaccinia H1-related phosphatase that can be evaluated in clinical trials. In this review, we describe the biological processes mediated by vaccinia H1-related phosphatase, especially those related to genomic stability. We also focus on validated substrates and signaling circuitry with clinical relevance in human diseases, particularly oncogenesis.
Assuntos
Humanos , Fosfatase 3 de Especificidade Dupla/fisiologia , Neoplasias/enzimologia , Transdução de Sinais , Análise de Sobrevida , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neoplasias/mortalidadeRESUMO
Ultraviolet radiation is the main cause of DNA damage to melanocytes and development of melanoma, one of the most lethal human cancers, which leads to metastasis due to uncontrolled cell proliferation and migration. These phenotypes are mediated by RhoA, a GTPase overexpressed or overactivated in highly aggressive metastatic tumors that plays regulatory roles in cell cycle progression and cytoskeleton remodeling. This work explores whether the effects of UV on DNA damage, motility, proliferation, and survival of human metastatic melanoma cells are mediated by the RhoA pathway. Mutant cells expressing dominant-negative (MeWo-RhoA-N19) or constitutively active RhoA (MeWo-RhoA-V14) were generated and subjected to UV radiation. A slight reduction in migration and invasion was observed in MeWo and MeWo-RhoA-V14 cells but not in MeWo-RhoA-N19 cells, which presented inefficient motility and invasiveness associated with stress fibers fragmentation. Proliferation and survival of RhoA-deficient cells were drastically reduced by UV compared to cells displaying normal or high RhoA activity, suggesting increased sensitivity to UV. Loss of RhoA activity also caused less efficient DNA repair, with elevated levels of DNA lesions such as strand breaks and cyclobutane pyrimidine dimers (CPDs). Thus, RhoA mediates genomic stability and represents a potential target for sensitizing metastatic tumors to genotoxic agents.
Assuntos
Melanoma/patologia , Raios Ultravioleta , Proteína rhoA de Ligação ao GTP/metabolismo , Proteínas Reguladoras de Apoptose , Linhagem Celular Tumoral/efeitos da radiação , Movimento Celular , Proliferação de Células , Ensaio Cometa , Dano ao DNA , Reparo do DNA , Proteínas de Ligação ao GTP , Glutationa Transferase/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Melanoma/metabolismo , Microscopia de Fluorescência , Invasividade Neoplásica , Metástase Neoplásica , Dímeros de Pirimidina/química , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologiaRESUMO
Rac1 GTPase controls essential cellular functions related to the cytoskeleton, such as motility and adhesion. Rac1 is overexpressed in many tumor cells, including breast cancers, where it is also involved in the proliferation and checkpoint control necessary for the cell's recovery after exposure to ionizing radiation. However, its role in DNA damage and repair remains obscure in other tumor cells and under different genotoxic conditions. Here, we compare HeLa cells with mutants exogenously expressing a dominant-negative Rac1 (HeLa-Rac1-N17) by their responses to DNA damage induced by gamma or UV radiation. In HeLa cells, these treatments led to increased levels of active Rac1 (Rac1-GTP) and of stress fibers, with a diminished ability to migrate compared to untreated cells. However, the reduction of Rac1-GTP in Rac1-N17-deficient clones resulted in much higher levels of polymerized stress fibers accompanied by a strong impairment of cell migration, even after both radiation treatments. With regard to proliferation and genomic stability, dominant-negative Rac1 cells were more sensitive to gamma and UV radiation, exhibiting reduced proliferation and survival consistent with increased DNA damage and delayed or reduced DNA repair observed in this Rac1-deficient clone. The DNA damage response, as indicated by pH2AX and pChk1 levels, was increased in HeLa cells but was not effectively triggered in the Rac1-N17 clone after radiation treatment, which is likely the main cause of DNA damage accumulation. These data suggest that Rac1 GTPase plays an important role in signaling and contributes to the sensitivity of cervical cancer cells under UV or gamma radiation treatments.
Assuntos
Proliferação de Células/genética , Reparo do DNA/genética , Transdução de Sinais/genética , Proteínas rac1 de Ligação ao GTP/genética , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/genética , Sobrevivência Celular/efeitos da radiação , Raios gama , Células HeLa , Humanos , Transdução de Sinais/efeitos da radiação , Raios Ultravioleta , Proteínas rac1 de Ligação ao GTP/deficiênciaRESUMO
The atypical dual-specificity phosphatases (aDUSPs) are a group of protein tyrosine phosphatases (PTPs) that have been increasingly studied recently, but little is known about their substrates or their roles and regulation. aDUSPs are typically low-molecular-weight enzymes that are distinct from the mitogen-activated protein kinase phosphatases (MKPs) but that still function in the regulation of the MAPK signalling cascade. aDUSPs may also have non-MAPK substrates, based on homologies observed in the sequences flanking potential phosphotyrosine target sites of other proteins and the cell type-specific characteristics of certain aDUSPs. Here, we combined experimental and computational tools to identify new substrates and protein partners of VHR (DUSP3) phosphatase in HeLa cells exposed to genotoxic stress. Experimental approaches confirmed the good stability of VHR and its nuclear co-localisation with classical MAPK substrates. The bioinformatics analysis of 4539 human nuclear proteins to identify a subset with functions related to DNA damage response and repair or to checkpoints and cell cycle control, that contain the phosphorylatable Thr-X-Tyr motif of MAPK with a high probability of dual phosphorylation, and that have structural homology to the MAPK activation loop resulted in a list of 57 putative VHR substrates. Fluorescence confocal microscopy and pull-down experiments followed by immunoblots revealed that VHR co-localised and interacted with components of the MRN complex and pH2AX, a DNA double-strand break sensor. Our platform, which combines experimental data from structure-function and bioinformatics analyses based on MAPK substrate similarities, provides a low-cost and rapid approach for the identification of novel aDUSP-interacting proteins with unknown roles in genotoxic stress response and genome stability maintenance.
Assuntos
Núcleo Celular/metabolismo , Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Fosfatase 3 de Especificidade Dupla/metabolismo , Proteínas Nucleares/metabolismo , Sítios de Ligação , Biologia Computacional/métodos , Fosfatase 3 de Especificidade Dupla/química , Células HeLa , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Nucleares/química , Ligação Proteica , Mapeamento de Interação de Proteínas/métodos , Análise de Sequência de Proteína/métodosRESUMO
The protein kinase C (PKC) family of serine/threonine kinases participate in embryonic stem cell (ESC) proliferation/self-renewal. A few stimuli that induce ESC proliferation activate several PKC isoenzymes including δPKC, however, the role of this isoenzyme under basal conditions that maintain undifferentiated ESCs remains to be determined. Herewith, we aimed to characterize signaling events that occur in undifferentiated ESCs upon δPKC activation. Using phosphoproteomics and a δPKC specific activator peptide, ψδRACK, it was seen that the majority of proteins whose phosphorylation increased upon δPKC activation participate in cell proliferation. Network analysis of these proteins directly connected δPKC to Raf1 and 14-3-3. Experimental validation studies showed that activation of δPKC increased its binding to 14-3-3, transiently activated ERK1/2 and increased ESC proliferation. Independently inhibiting MEK or PI3 kinase both led to a decrease in proliferation of approximately 50%, but δPKC activation only recovered the effect of PI3 kinase inhibition suggesting that ERK1/2 activation via δPKC is probably a parallel pathway to PI3 kinase and that both pathways are necessary for undifferentiated ESC proliferation. BIOLOGICAL SIGNIFICANCE: The use of embryonic stem cells and induced pluripotent stem cells for regenerative therapies is still a challenge. Understanding the underlying mechanisms that keep these cells proliferating with the ability to differentiate in more than 200 cell types (self-renewal) will aid in the future use of these cells therapeutically. Using a targeted phosphoproteomics study, insights into signaling pathways involved in ESC proliferation can be obtained. Modulating these pathways will aid the obtention of a larger number of self-renewing stem cells and induced pluripotent stem cells that can be used therapeutically.
Assuntos
Proliferação de Células/efeitos dos fármacos , Células-Tronco Embrionárias/enzimologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Peptídeos/farmacologia , Proteína Quinase C-delta/metabolismo , Proteínas 14-3-3/metabolismo , Linhagem Celular , Células-Tronco Embrionárias/citologia , Ativação Enzimática/efeitos dos fármacos , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismoRESUMO
DUSP3 (or Vaccinia virus phosphatase VH1-related; VHR) is a small dual-specificity phosphatase known to dephosphorylate c-Jun N-terminal kinases and extracellular signal-regulated kinases. In human cervical cancer cells, DUSP3 is overexpressed, localizes preferentially to the nucleus, and plays a key role in cellular proliferation and senescence triggering. Other DUSP3 functions are still unknown, as illustrated by recent and unpublished results from our group showing that this enzyme mediates DNA damage response or repair processes. In this study, we sought to identify new interactions between DUSP3 and proteins directly or indirectly involved in or correlated with its biological roles in HeLa cells exposed to gamma or UV radiation. By using GST-DUSP as bait, we pulled down interacting proteins and identified them by LC-MS/MS. Of the 46 proteins obtained, six hits were extensively validated by immune techniques; the proteins Nucleophosmin, HnRNP C1/C2, and Nucleolin were the most promising targets found to directly interact with DUSP3. We then analyzed the DUSP3 interactomes using physical protein-protein interaction networks using our hits as the seed list. The validated hits as well as unvalidated hits fluctuated on the DUSP3 interactomes of HeLa cells, independent of the time post radiation, which confirmed our proteomic and experimental data and clearly showed the proximity of DUSP3 to proteins involved in processes intimately related to DNA repair and senescence, such as Ku70 and Tert, via interactions with nucleolar proteins, which were identified in this study, that regulate DNA/RNA structure and functions.
Assuntos
Reparo do DNA , DNA/genética , Fosfatase 3 de Especificidade Dupla/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo C/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Cromatografia Líquida , DNA/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fosfatase 3 de Especificidade Dupla/genética , Raios gama , Regulação da Expressão Gênica , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas Grupo C/genética , Humanos , Imunoprecipitação , Autoantígeno Ku , Proteínas Nucleares/genética , Nucleofosmina , Fosfoproteínas/genética , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Espectrometria de Massas em Tandem , Telomerase/genética , Telomerase/metabolismo , Raios Ultravioleta , NucleolinaRESUMO
Manganese (Mn) is an essential trace element and trivalent Mn complexes have been used as oxidation catalysts and enzyme mimetics. We studied the cytotoxicity of Mn(III) derivatives of citrate, pyrophosphate and salicylene diamine (respectively, MnCit, MnPPi and EUK8) toward HeLa cells stressed by ultraviolet irradiation and the effect of the co-administration of ascorbate and para-amino salicylate (PAS) on cell viability. Metal complexes enhanced the lethality of irradiated cells, and this effect was even more pronounced when ascorbate was co-administered with Mn(III) species. The active role of Mn(III) compounds in the antitumor activity was demonstrated by the treatment of the cells with the chelator PAS, which restored the viability of both non-irradiated and UV-irradiated cells. The association of the Mn(III) metallodrugs with radiation and an antioxidant proved to be a very effective approach to chemotherapy.