TRAF6, a master activator of p53 transactivation, suppresses mitochondrial p53 and apoptosis
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus Homo sapiens
PROVIDER: GSE69730 | GEO | 2016/11/04
SECONDARY ACCESSION(S): PRJNA286309
REPOSITORIES: GEO
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Project description:Excessive generation and accumulation of highly reactive oxidizing molecules causes oxidative stress and oxidative damage to cellular components. Accumulating evidence indicates that autophagy diminishes oxidative damage in cells and maintains redox homeostasis by degrading and recycling intracellular damaged components. However, the molecular mechanisms underlying the activation of oxidative stress-mediated autophagic response remain elusive. Here we show that ubiquitin E3 ligase TNF receptor-associated factor 6 (TRAF6) and the deubiquitinase A20 coordinate to regulate ATG9A ubiquitination and autophagy activation in cells responding to oxidative stress. The reactive oxygen species (ROS)-dependent TRAF6-mediated non-proteolytic, K48/63-linked ubiquitination of ATG9A enhances its association with Beclin 1 and the assembly of class III phosphatidylinositol-3-kinase (PI3KC3/VPS34) UVRAG complex, thereby stimulating autophagy. Notably, depletion of ATG9A or expression of the ATG9A ubiquitination mutants markedly decreases ROS-induced VPS34 activation and autophagy. We further find that lipopolysaccharide (LPS)-induced ROS production also stimulates TRAF6-mediated ATG9A ubiquitination and enhances the assembly of the UVRAG-containing VPS34 complex in an ATG9A-dependent manner. Ablation of ATG9A causes aberrant TLR4 endosomal trafficking and decreased phosphorylation of IRF-3 in LPS-stimulated macrophages. Our findings provide important insight into how K48/K63-linked ubiquitination of ATG9A contributes to the regulation of oxidative stress-induced autophagy.
2022-02-24 | PXD030415 | Pride
Project description:As a critical cellular stress sensor, p53 mediates a variety of defensive processes including cell-cycle arrest, apoptosis, and senescence to prevent propagation of hyperproliferative cells or cells with a damaged genome, hence the formation of neoplasia. Transactivation of downstream genes plays an important while sometimes controversial role in regulating these cellular processes. To evaluate the dependence on transcriptional activation in p53’s activities, we generated genetically-modified mouse lines carrying mutations in the transactivation domains (TADs) of p53. These transactivatio-deficient mutants serve as unique reagents to probe the dependence on robust transactivation in p53-mediated cellular functions, as well as the underneath mechanisms. To identify genes differentially regulated by these p53 mutants, we performed gene expression profiling analysis on mouse embryonic fibroblast cells (MEFs) from these mice in the context of oncogenic Ras-induced premature cellular senescence. Mouse embryonic fibroblasts (MEFs) with different p53 genotypes were infected with retroviral H-Ras V12, which induces premature cellular senescence in p53 wild-type MEFs but not in p53 null MEFs. 5 genotypic groups of MEFs were used in the study: (i) p53L25Q/W26S, or "25,26", in which the first TAD (transactivation domain) of p53 is disrupted by the mutation, 5 biological samples; (ii) p53W53Q/F54S, or "53,54", in which the second TAD of p53 is disrupted by the mutation, 3 biological samples; (iii) p53L25Q/W26S/W53Q/F54S, or "QM", in which both TADs or p53 are disrupted, 3 biological samples; (iv) p53 wild-type, or "WT", 6 biological samples; (v) p53 null, or "Null", 6 biological samples.
2011-05-13 | E-GEOD-27901 | biostudies-arrayexpress
Project description:To investigate the genomic localization of NCoR/HDAC3/PGC1β complex and the enhancer/promoter activity in inflammatory genes, we used NCoR KO or PGC1β KO bone marrow-derived macrophages and Traf6 siRNA knockdown bone marrow-derived macrophages. We then performed chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for H3K27ac, NCoR, HDAC3, PGC1β, ERK1, p65, Fosl2, PU.1 and p300.
2023-12-21 | GSE247941 | GEO
Project description:The transcriptional error rate can be significantly increased by the presence of DNA lesions that instruct mis-insertion during transcription; a process referred to as transcriptional mutagenesis (TM). Herein, we determined the effect of O6-methylguanine (O6-meG) on transcription and subsequent transactivation activity of p53 in human lung H1299 cells. Levels of TM and effects on transactivation were determined by RNA-seq. Results showed that 47% of all p53 transcripts contained an uridine misincorporation opposite the lesion at 6 h post transfection, which was decreased to 18% at 24 h. TM at these levels reduced DNA binding activity of p53 to 21% and 80% compared to wild type p53, respectively. Gene expression data were analyzed to identify differentially expressed genes due to TM compared to the response of wild type p53. We show a temporal repression of transactivation of >100 high confidence p53 target genes including regulators of the cell cycle, DNA damage response and apoptosis. In addition, TM repressed the transcriptional downregulation by p53 of several negative regulators of proliferation and differentiation . Our work demonstrates that TM, even when restricting its effect to an individual transcription factor, has the potential to alter gene expression programs and diversify cellular phenotypes.
2019-10-16 | GSE138853 | GEO
Project description:Amyotrophic lateral sclerosis (ALS) is a fatal disease, characterized by the selective loss of motor neurons. Mutations in Cu/Zn superoxide dismutase (SOD1) are the second most common cause of ALS and it is now well accepted that they result in a gain of toxicity due to protein misfolding. We previously demonstrated in the SOD1G93A rat model that misfolded SOD1 exists as distinct conformers and deposits on mitochondrial subpopulations. Here, using SOD1G93A rats coupled with conformation-restricted antibodies for misfolded SOD1 (AMF7-63 and B8H10), we identified the interactomes of the mitochondrial pools of misfolded SOD1. This strategy identified binding partners that uniquely interacted with either AMF7-63- or B8H10-reactive SOD1 conformers as well as a high proportion of interactors common to both conformers. Of this latter set, we identified the E3 ubiquitin ligase TNF receptor-associated factor 6 (TRAF6) and determined that exposure of the SOD1 functional loops facilitates this interaction. However, this conformational criterion is not universally fulfilled by all SOD1 mutants and differentiates TRAF6-interacting from non-interacting SOD1 mutants. Functionally, TRAF6 stimulates mutant SOD1 polyubiquitination and aggregation. While TRAF6 E3 ubiquitin ligase activity is required for the former, it is dispensable for the latter, indicating that the polyubiquitination and aggregation of mutant SOD1 mediated by TRAF6 are independent events. We propose that misfolded SOD1 binding to TRAF6 may be relevant to ALS disease.
2020-02-24 | PXD015279 | Pride
Project description:Recent observations show that the single-cell response of p53 to ionizing radiation (IR) is “digital” in that it is the number of oscillations rather than the amplitude of p53 that shows dependence on the radiation dose. We present a model of this phenomenon. In our model, double-strand break (DSB) sites induced by IR interact with a limiting pool of DNA repair proteins, forming DSB–protein complexes at DNA damage foci. The persisting complexes are sensed by ataxia telangiectasia mutated (ATM), a protein kinase that activates p53 once it is phosphorylated by DNA damage. The ATM-sensing module switches on or off the downstream p53 oscillator, consisting of a feedback loop formed by p53 and its negative regulator, Mdm2. In agreement with experiments, our simulations show that by assuming stochasticity in the initial number of DSBs and the DNA repair process, p53 and Mdm2 exhibit a coordinated oscillatory dynamics upon IR stimulation in single cells, with a stochastic number of oscillations whose mean increases with IR dose. The damped oscillations previously observed in cell populations can be explained as the aggregate behavior of single cell
2020-05-13 | MODEL2005130001 | BioModels
Project description:Transcriptional profiling of the zebrafish embryonic host response to a systemic bacterial infection with Salmonella typhimurium (strain SL1027); comparison between traf6 knock-down and control morpholino treated embryos. Two-way factorial dual colour design with factor 1 'infection with Salmonella enterica serovar Typhimurium (S. typhimurium)' and factor 2 'morpholino knock-down of traf6' using a common reference made from the sample pool. All infection experiments were performed using mixed egg clutches of ABxTL strain zebrafish. Embryos injected with traf6 morpholino or a 5bp mismatch control morpholino were staged at 27 hours post fertilization (hpf) by morphological criteria and approximately 250 cfu of DsRed expressing Salmonella bacteria were injected into the caudal vein close to the urogenital opening. As a control an equal volume of PBS was likewise injected. Pools of 20-40 infected and control embryos were collected 8 hours post infection (hpi). For the microarray analysis, the whole procedure was preformed in triplicate on separate days. The triplicates are marked 1,2 and 3.
2010-08-01 | E-GEOD-20310 | biostudies-arrayexpress
Project description:The nuclear factor-kB (NF-kB) family of transcription factors is important for hematopoietic function, including development, maintenance, and differentiation of different hematopoietic lineages in response to cytokines and infection. Although ligand-independent or basal NF-kB signaling is required for HSC homeostasis in the absence of inflammation, the upstream tonic mediators of NF-kB signaling are not known. Herein we describe TNF receptor associated factor 6 (TRAF6) as an essential regulator of HSC homeostasis by preserving self-renewal and quiescence through basal activation of NF-kB. Hematopoietic-specific deletion of Traf6 resulted in impaired HSC self-renewal and fitness. Gene expression, RNA splicing, and molecular analyses of Traf6-deficient HSPC revealed changes in adaptive immune signaling, innate immune signaling, and NF-kB signaling, indicating that signaling via TRAF6 in the absence of cytokine stimulation and/or infection occurs in HSPC and is required for HSC function. In addition, we established that loss of NF-kB signaling is responsible for the major hematopoietic defects observed in Traf6-deficient HSPC as deletion of IKKb similarly resulted in impaired HSC self-renewal and fitness. Taken together, our observations position TRAF6 as an essential regulator of HSC homeostasis by maintaining a minimal threshold level of IKKb/NF-kB signaling.
2018-01-31 | GSE109050 | GEO
Project description:As a critical cellular stress sensor, p53 mediates a variety of defensive processes including cell-cycle arrest, apoptosis, and senescence to prevent propagation of hyperproliferative cells or cells with a damaged genome, hence the formation of neoplasia. Transactivation of downstream genes plays an important while sometimes controversial role in regulating these cellular processes. To evaluate the dependence on transcriptional activation in p53’s activities, we generated genetically-modified mouse lines carrying mutations in the transactivation domains (TADs) of p53. These transactivatio-deficient mutants serve as unique reagents to probe the dependence on robust transactivation in p53-mediated cellular functions, as well as the underneath mechanisms. To identify genes differentially regulated by these p53 mutants, we performed gene expression profiling analysis on mouse embryonic fibroblast cells (MEFs) from these mice in the context of oncogenic Ras-induced premature cellular senescence.
2011-05-13 | GSE27901 | GEO
Project description:Bortezomib (VelcadeM-BM-)) is widely used for the treatment of various human cancers, however, its mechanisms of action are not fully understood, particularly in myeloid malignancies. Bortezomib is a selective and reversible inhibitor of the proteasome. Paradoxically, we find that Bortezomib induces proteasome-independent degradation of TRAF6 protein, but not mRNA, in Myelodysplastic syndrome (MDS) and Acute Myeloid Leukemia (AML) cell lines and primary cells. The reduction in TRAF6 protein coincides with Bortezomib-induced autophagy, and subsequently with apoptosis in MDS/AML cells. RNAi-mediated knockdown of TRAF6 sensitized Bortezomib-sensitive and -resistant cell lines, underscoring the importance of TRAF6 in Bortezomib-induced cytotoxicity. Bortezomib-resistant cells expressing an shRNA targeting TRAF6 were resensitized to the cytotoxic effects of Bortezomib due to down-regulation of the proteasomal subunit alpha-1 (PSMA1). To uncover the molecular consequences following loss of TRAF6 in MDS/AML cells, we applied gene expression profiling and identified an apoptosis gene signature. Knockdown of TRAF6 in MDS/AML cell lines or patient samples resulted in rapid apoptosis and impaired malignant hematopoietic stem/progenitor function. In summary, we describe novel mechanisms by which TRAF6 is regulated through Bortezomib/autophagy-mediated degradation and by which it alters MDS/AML sensitivity to Bortezomib by controlling PSMA1 expression. TF-1 cells were transduced with lentivirus encoding shRNA targeting human TRAF6 and a negative control (pLKO) for 2.5 days. GFP positive cells were sorted for RNA exctration, labeling, and hybridization. A total of four samples were included, and two groups are assigned. Two replicates are for each group. Comparison comprises mRNA expression profile of TRAF6 knockdown (shT6) v.s. control vector (pLKO).
2012-06-14 | E-GEOD-38519 | biostudies-arrayexpress