RESUMO
Caspases are a family of proteins mostly known for their role in the activation of the apoptotic pathway leading to cell death. In the last decade, caspases have been found to fulfill other tasks regulating the cell phenotype independently to cell death. Microglia are the immune cells of the brain responsible for the maintenance of physiological brain functions but can also be involved in disease progression when overactivated. We have previously described non-apoptotic roles of caspase-3 (CASP3) in the regulation of the inflammatory phenotype of microglial cells or pro-tumoral activation in the context of brain tumors. CASP3 can regulate protein functions by cleavage of their target and therefore could have multiple substrates. So far, identification of CASP3 substrates has been performed mostly in apoptotic conditions where CASP3 activity is highly upregulated and these approaches do not have the capacity to uncover CASP3 substrates at the physiological level. In our study, we aim at discovering novel substrates of CASP3 involved in the normal regulation of the cell. We used an unconventional approach by chemically reducing the basal level CASP3-like activity (by DEVD-fmk treatment) coupled to a Mass Spectrometry screen (PISA) to identify proteins with different soluble amounts, and consequently, non-cleaved proteins in microglia cells. PISA assay identified several proteins with significant change in their solubility after DEVD-fmk treatment, including a few already known CASP3 substrates which validated our approach. Among them, we focused on the Collectin-12 (COLEC12 or CL-P1) transmembrane receptor and uncovered a potential role for CASP3 cleavage of COLEC12 in the regulation of the phagocytic capacity of microglial cells. Taken together, these findings suggest a new way to uncover non-apoptotic substrates of CASP3 important for the modulation of microglia cell physiology.
Assuntos
Microglia , Proteoma , Caspase 3/metabolismo , Microglia/metabolismo , Apoptose/fisiologia , Proteômica , Solubilidade , Caspases/metabolismo , ColectinasRESUMO
Macroautophagy/autophagy is a tightly regulated catabolic process, which contributes at baseline level to cellular homeostasis, and upon its stimulation to the adaptive cellular response to intra- and extracellular stress stimuli. Decrease of autophagy activity is occurring upon aging and thought to contribute to age-related-diseases. Recently, we uncovered, upon autophagy induction, the role of de novo DNMT3A (DNA methyltransferase 3 alpha)-mediated DNA methylation on expression of the MAP1LC3 (microtubule associated protein 1 light chain 3) proteins, core components of the autophagy pathway, which resulted in reduced baseline autophagy activity. Here, we report that serine/threonine kinase ULK3 (unc-51 like kinase 3)-dependent activation of GLI1 (GLI family zinc finger 1) contributes to the transcriptional upregulation of DNMT3A gene expression upon autophagy induction, thereby bringing additional understanding of the long-term effect of autophagy induction and a possible mechanism for its decline upon aging, pathological conditions, or in response to treatment interventions.Abbreviations: CBZ: carbamazepine; ChIP: chromatin immunoprecipitation; Clon: clonidine; DNMT3A: DNA methyltransferase 3 alpha; GLI1: GLI family zinc finger 1; GLI2: GLI family zinc finger 2; MAP1LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; PLA: proximity ligation assay; RT-qPCR: quantitative reverse transcription PCR; shRNA: small hairpin RNA; siRNA: small interfering RNA; Treh: trehalose; ULK3: unc-51 like kinase 3.
Assuntos
Autofagia , Transdução de Sinais , Autofagia/genética , Proteína GLI1 em Dedos de Zinco/genética , Proteína GLI1 em Dedos de Zinco/metabolismo , Proteína GLI1 em Dedos de Zinco/farmacologia , Proteínas Serina-Treonina Quinases , RNA Interferente Pequeno/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismoRESUMO
Macroautophagy/autophagy is an evolutionarily conserved and tightly regulated catabolic process involved in the maintenance of cellular homeostasis whose dysregulation is implicated in several pathological processes. Autophagy begins with the formation of phagophores that engulf cytoplasmic cargo and mature into double-membrane autophagosomes; the latter fuse with lysosomes/vacuoles for cargo degradation and recycling. Here, we report that yeast Set2, a histone lysine methyltransferase, and its mammalian homolog, SETD2, both act as positive transcriptional regulators of autophagy. However, whereas Set2 regulates the expression of several autophagy-related (Atg) genes upon nitrogen starvation, SETD2 effects in mammals were found to be more restricted. In fact, SETD2 appears to primarily regulate the differential expression of protein isoforms encoded by the ATG14 gene. SETD2 promotes the expression of a long ATG14 isoform, ATG14L, that contains an N-terminal cysteine repeats domain, essential for the efficient fusion of the autophagosome with the lysosome, that is absent in the short ATG14 isoform, ATG14S. Accordingly, SETD2 loss of function decreases autophagic flux, as well as the turnover of aggregation-prone proteins such as mutant HTT (huntingtin) leading to increased cellular toxicity. Hence, our findings bring evidence to the emerging concept that the production of autophagy-related protein isoforms can differentially affect core autophagy machinery bringing an additional level of complexity to the regulation of this biological process in more complex organisms.
Assuntos
Autofagossomos , Macroautofagia , Animais , Autofagossomos/metabolismo , Lisossomos/metabolismo , Autofagia/genética , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , MamíferosRESUMO
Macroautophagy/autophagy is a conserved catabolic pathway that targets cytoplasmic components for their degradation and recycling in an autophagosome-dependent lysosomal manner. Under physiological conditions, this process maintains cellular homeostasis. However, autophagy can be stimulated upon different forms of cellular stress, ranging from nutrient starvation to exposure to drugs. Thus, this pathway can be seen as a central component of the integrated and adaptive stress response. Here, we report that even brief induction of autophagy is coupled in vitro to a persistent downregulation of the expression of MAP1LC3 isoforms, which are key components of the autophagy core machinery. In fact, DNA-methylation mediated by de novo DNA methyltransferase DNMT3A of MAP1LC3 loci upon autophagy stimulation leads to the observed long-term decrease of MAP1LC3 isoforms at transcriptional level. Finally, we report that the downregulation of MAP1LC3 expression can be observed in vivo in zebrafish larvae and mice exposed to a transient autophagy stimulus. This epigenetic memory of autophagy provides some understanding of the long-term effect of autophagy induction and offers a possible mechanism for its decline upon aging, pathological conditions, or in response to treatment interventions.Abbreviations: ACTB: actin beta; ATG: autophagy-related; 5-Aza: 5-aza-2'-deoxycytidine; BafA1: bafilomycin A1; CBZ: carbamazepine; CDKN2A: cyclin dependent kinase inhibitor 2A; ChIP: chromatin immunoprecipitation; Clon.: clonidine; CpG: cytosine-guanine dinucleotide: DMSO: dimethyl sulfoxide; DNA: deoxyribonucleic acid; DNMT: DNA methyltransferase; DNMT1: DNA methyltransferase 1; DNMT3A: DNA methyltransferase alpha; DNMT3B: DNA methyltransferase beta; dpf: days post-fertilization; EBSS: Earle's balanced salt solution; EM: Zebrafish embryo medium; GABARAP: GABA type A receptor associated protein; GABARAPL1: GABA type A receptor associated protein like 1; GABARAPL2: GABA type A receptor associated protein like 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GRO-Seq: Global Run-On sequencing; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAP1LC3A: microtubule-associated protein 1 light chain 3 alpha; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MAP1LC3B2: microtubule-associated protein 1 light chain 3 beta 2; MEM: minimum essential medium; MEF: mouse embryonic fibroblasts; mRNA: messenger RNA; MTOR: mechanistic target of rapamycin kinase; PBS: phosphate-buffered saline; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; RB1CC1/FIP200: RB1 inducible coiled-coil 1; RT-qPCR: quantitative reverse transcription polymerase chain reaction; SQSTM1/p62: sequestosome 1; Starv.: starvation; Treh.: trehalose; ULK1: unc-51 like autophagy activating kinase 1.
Assuntos
Autofagia/fisiologia , DNA Metiltransferase 3A/metabolismo , DNA/metabolismo , Memória de Longo Prazo/fisiologia , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Fibroblastos/metabolismo , Humanos , Lisossomos/metabolismo , Metiltransferases/metabolismo , Camundongos , Peixe-Zebra/genéticaRESUMO
Red wine contains antioxidants and is at moderate amounts believed to exert certain positive health effects. Resveratrol is one of the most studied antioxidants in red wine and has been suggested to activate the longevity- and metabolism-associated histone deacetylase SIRT1. Here we show that relatively low concentrations of resveratrol (0.5-3 microM) specifically inhibited neuronal differentiation of neural stem cells in a SIRT1-dependent manner whereas higher concentrations of resveratrol (> or =10 microM) induced a SIRT1-independent cell death. Surprisingly, using a cell based assay, we found that small amounts of red wine (1-5% v/v)--but not white wine--induced a massive and rapid cell death of various cell types, including neural stem cells and several cancer cell lines. This red wine-induced cell death was ethanol-, SIRT1- and resveratrol-independent but associated with increased oxidative stress and inhibition of thioredoxin reductase (TrxR) activity. The TrxR inhibition correlated with the red color (absorbance at 520 nm) of the wines demonstrating that pigment components of red wine can exert profound cellular effects. Our results unveil important roles for SIRT1 and TrxR in resveratrol and red wine-mediated effects on progenitor and cancer cells, and demonstrate that cellular responses to red wine may be more complex than generally appreciated.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Neurônios/efeitos dos fármacos , Sirtuínas/fisiologia , Células-Tronco/efeitos dos fármacos , Estilbenos/farmacologia , Tiorredoxina Dissulfeto Redutase/antagonistas & inibidores , Vinho , Animais , Astrócitos/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Diferenciação Celular , Linhagem Celular Tumoral , Imuno-Histoquímica , Estresse Oxidativo , Ratos , Resveratrol , Sirtuína 1RESUMO
Inactivating mutations in the SETD2 gene, encoding for a nonredundant histone H3 methyltransferase and regulator of transcription, is a frequent molecular feature in clear cell renal cell carcinomas (ccRCC). SETD2 deficiency is associated with recurrence of ccRCC and bears low prognostic values. Targeting autophagy, a conserved catabolic process with critical functions in maintenance of cellular homeostasis and cell conservation under stress condition, is emerging as a potential therapeutic strategy to combat ccRCC. Epigenetics-based pathways are now appreciated as key components in the regulation of autophagy. However, whether loss of function in the SETD2 histone modifying enzyme occurring in ccRCC cells may impact on their ability to undergo autophagy remained to be explored. Here, we report that SETD2 deficiency in RCC cells is associated with the aberrant accumulation of both free ATG12 and of an additional ATG12-containing complex, distinct from the ATG5-ATG12 complex. Rescue of SETD2 functions in the SETD2 deficiency in RCC cells, or reduction of SETD2 expression level in RCC cells wild type for this enzyme, demonstrates that SETD2 deficiency in RCC is directly involved in the acquisition of these alterations in the autophagic process. Furthermore, we revealed that deficiency in SETD2, known regulator of alternative splicing, is associated with increased expression of a short ATG12 spliced isoform at the depend of the canonical long ATG12 isoform in RCC cells. The defect in the ATG12-dependent conjugation system was found to be associated with a decrease autophagic flux, in accord with the role for this ubiquitin-like protein conjugation system in autophagosome formation and expansion. Finally, we report that SETD2 and ATG12 gene expression levels are associated with favorable respective unfavorable prognosis in ccRCC patients. Collectively, our findings bring further argument for considering the SETD2 gene status of ccRCC tumors, when therapeutic interventions, such as targeting the autophagic process, are considered to combat these kidney cancers.
Assuntos
Proteína 12 Relacionada à Autofagia/metabolismo , Autofagia/genética , Carcinoma de Células Renais/genética , Histona-Lisina N-Metiltransferase/genética , Neoplasias Renais/genética , Processamento Alternativo/genética , Proteína 12 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/mortalidade , Linhagem Celular Tumoral , Movimento Celular/genética , Regulação Neoplásica da Expressão Gênica/genética , Histona-Lisina N-Metiltransferase/deficiência , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Humanos , Neoplasias Renais/metabolismo , Neoplasias Renais/mortalidade , Mutação , Prognóstico , RNA Interferente PequenoRESUMO
The pro-inflammatory immune response driven by microglia is a key contributor to the pathogenesis of several neurodegenerative diseases. Though the research of microglia spans over a century, the last two decades have increased our understanding exponentially. Here, we discuss the phenotypic transformation from homeostatic microglia towards reactive microglia, initiated by specific ligand binding to pattern recognition receptors including toll-like receptor-4 (TLR4) or triggering receptors expressed on myeloid cells-2 (TREM2), as well as pro-inflammatory signaling pathways triggered such as the caspase-mediated immune response. Additionally, new research disciplines such as epigenetics and immunometabolism have provided us with a more holistic view of how changes in DNA methylation, microRNAs, and the metabolome may influence the pro-inflammatory response. This review aimed to discuss our current knowledge of pro-inflammatory microglia from different angles, including recent research highlights such as the role of exosomes in spreading neuroinflammation and emerging techniques in microglia research including positron emission tomography (PET) scanning and the use of human microglia generated from induced pluripotent stem cells (iPSCs). Finally, we also discuss current thoughts on the impact of pro-inflammatory microglia in neurodegenerative diseases.
Assuntos
Sistema Nervoso Central/patologia , Inflamação/patologia , Microglia/patologia , Animais , Caspases/metabolismo , Epigênese Genética , Humanos , Microglia/enzimologia , Modelos BiológicosRESUMO
In septic patients, the onset of septic shock occurs due to the over-activation of monocytes. We tested the therapeutic potential of directly targeting innate immune cell activation to limit the cytokine storm and downstream phases. We initially investigated whether caspase-8 could be an appropriate target given it has recently been shown to be involved in microglial activation. We found that LPS caused a mild increase in caspase-8 activity and that the caspase-8 inhibitor IETD-fmk partially decreased monocyte activation. Furthermore, caspase-8 inhibition induced necroptotic cell death of activated monocytes. Despite inducing necroptosis, caspase-8 inhibition reduced LPS-induced expression and release of IL-1ß and IL-10. Thus, blocking monocyte activation has positive effects on both the pro and anti-inflammatory phases of septic shock. We also found that in primary mouse monocytes, caspase-8 inhibition did not reduce LPS-induced activation or induce necroptosis. On the other hand, broad caspase inhibitors, which have already been shown to improve survival in mouse models of sepsis, achieved both. Thus, given that monocyte activation can be regulated in humans via the inhibition of a single caspase, we propose that the therapeutic use of caspase-8 inhibitors could represent a more selective alternative that blocks both phases of septic shock at the source.
Assuntos
Caspase 8/metabolismo , Inibidores de Caspase/farmacologia , Monócitos/enzimologia , Monócitos/imunologia , Choque Séptico/prevenção & controle , Animais , Células Cultivadas , Citocinas/imunologia , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/efeitos dos fármacos , Choque Séptico/enzimologia , Choque Séptico/imunologiaRESUMO
The transcription factor p73, a member of the p53 family of proteins, is involved in the regulation of cell cycle progression and apoptosis. However, the regulatory mechanisms controlling the distinct roles for p73 in these two processes have remained unclear. Here, we report that p73 is able to induce cell cycle arrest independently of its amino-terminal transactivation domain, whereas this domain is crucial for p73 proapoptotic functions. We also characterized a second transactivation domain in the carboxy terminus of p73 within amino acid residues 381 to 399. This carboxy terminus transactivation domain was found to preferentially regulate genes involved in cell cycle progression. Moreover, its activity is regulated throughout the cell cycle and modified by protein kinase C-dependent phosphorylation at serine residue 388. Our results suggest that this novel posttranslational modification within the p73 carboxy terminus transactivation domain is involved in the context-specific guidance of p73 toward the selective induction of cell cycle arrest.
Assuntos
Ciclo Celular , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteína Quinase C/metabolismo , Ativação Transcricional/genética , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/metabolismo , Sequência de Aminoácidos , Animais , Apoptose , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Proteína Vmw65 do Vírus do Herpes Simples/metabolismo , Humanos , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Especificidade de Órgãos , Fosforilação , Regiões Promotoras Genéticas/genética , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Ratos , Serina/metabolismo , Relação Estrutura-AtividadeRESUMO
The p73 gene, a member of the p53 family, encodes several variants through differential splicing and use of alternative promoters. At the NH2 terminus, two different promoters generate the full-length and the DeltaN isoforms, with or without the transactivating domain. At the COOH terminus, seven isoforms generated through alternative splicing have been cloned. Previous studies have demonstrated that DeltaNp73 isoforms exert a dominant-negative effect on p73 by blocking their transactivation activity and hence the ability to induce apoptosis. Considerable efforts are made to identify the functional diversity of the COOH-terminal p73 variants. In this study, we found that p73alpha inhibited drug-induced apoptosis in small cell lung carcinoma cells, whereas p73beta promoted it. p73alpha prevented Bax activation, mitochondrial dysfunction, and caspase activation. In addition, p73alpha was also able to reduce apoptosis induced by the BH3-only protein PUMA (p53 up-regulated modulator of apoptosis). Furthermore, we discovered that p73alpha is able to inhibit the pro-apoptotic effect of p73beta, demonstrating the existence of equilibrium between these two p73 isoforms. In conclusion, the reported overexpression of p73alpha in certain tumor types, and our findings that p73alpha exerts anti-apoptotic functions, indicate a potential oncogenic activity for p73.