Promyelocytic Leukemia Protein (PML) Requirement for Interferon-induced Global Cellular SUMOylation.
Ontology highlight
ABSTRACT:
SUBMITTER: Maroui MA
PROVIDER: S-EPMC5986244 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:This proteomics dataset was generated to study the changes in SUMO site abundances between control cells and Interferon-alpha treated cells using SILAC. Cells were first fractionated into the cytoplasmic and nuclear fractions. SUMOylated proteins were then purified from these extracts by Ni-NTA via the 6xHIS tag on the SUMO3 construct. The proteins were then digested with trypsin and the peptides desalted on HLB cartridges. The SUMOylated peptides, which harbor an NQTGG remnant after tryptic digestion, were enriched with our anti-NQTGG antibody. The resulting peptides were injected on the orbitrap fusion instrument. The .raw files were analyzed with maxqunt.
| MSV000081525 | MassIVE
Project description:Promyelocytic leukemia protein (PML) is a tumor suppressor acting as the organizer of subnuclear structures called PML nuclear bodies (NBs). Both covalent modification of PML by the small ubiquitin-like modifier (SUMO) and non-covalent binding of SUMO to the PML SUMO binding domain (SBD) are necessary for PML NB formation and maturation. PML sumoylation and proteasome-dependent degradation induced by the E3 ubiquitin ligase, RNF4, are enhanced by the acute promyelocytic leukemia therapeutic agent, arsenic trioxide (As2O3). Here, we established a novel bioluminescence resonance energy transfer (BRET) assay to dissect and monitor PML/SUMO interactions dynamically in living cells upon addition of therapeutic agents. Using this sensitive and quantitative SUMO BRET assay that distinguishes PML sumoylation from SBD-mediated PML/SUMO non-covalent interactions, we probed the respective roles of covalent and non-covalent PML/SUMO interactions in PML degradation and interaction with RNF4. We found that, although dispensable for As2O3-enhanced PML sumoylation and RNF4 interaction, PML SBD core sequence was required for As2O3- and RNF4-induced PML degradation. As confirmed with a phosphomimetic mutant, phosphorylation of a stretch of serine residues, contained within PML SBD was needed for PML interaction with SUMO-modified protein partners and thus for NB maturation. However, mutation of these serine residues did not impair As2O3- and RNF4-induced PML degradation, contrasting with the known role of these phosphoserine residues for casein kinase 2-promoted PML degradation. Altogether, these data suggest a model whereby sumoylation- and SBD-dependent PML oligomerization within NBs is sufficient for RNF4-mediated PML degradation and does not require the phosphorylation-dependent association of PML with other sumoylated partners.
| S-EPMC2713554 | biostudies-literature
Project description:The HMGA2 architectural protein functions in a variety of cellular processes, such as cell growth, transcription regulation, neoplastic transformation, and progression. Up-regulation of HMGA2 protein is observed in many tumors and is associated with advanced cancers with poor prognoses. Although the expression and biochemical properties of HMGA2 protein are regulated by microRNA and phosphorylation, it is unknown whether HMGA2 activity can also be regulated by SUMOylation, and that is what is investigated in this report. We identified HMGA2 as a SUMOylation target and showed that the expression of wild-type HMGA2, but not SUMOylation-defective HMGA2(2K/R), selectively lowered the steady-state level of PML protein. Consequently, the HMGA2-elicited PML down-regulation rendered a reduction in the average number of PML nuclear bodies per cell and the volume of PML assembled per PML nuclear body. Using small interfering RNA to suppress endogenous ubiquitin expression and proteasome inhibitor to repress ubiquitin-mediated protein degradation, we showed that HMGA2 confers PML down-regulation through ubiquitin-proteasome-dependent protein degradation. Importantly, arsenic trioxide treatment stimulated HMGA2 SUMOylation, leading to the formation of HMGA2 nuclear foci surrounding PML nuclear bodies and the stimulation of PML degradation. Collectively, our results unveil a previously unrecognized effect by HMGA2 on the modulation of PML protein level, providing a novel mechanism underlying HMGA2 function and underscoring the molecular basis for oncogenic progression by HMGA2.
| S-EPMC2862987 | biostudies-literature
Project description:Promyelocytic leukemia protein (PML) is a tumor suppressor that is highly expressed in vascular endothelium and inflamed tissues, yet its role in inflammation-associated cytokine-regulated angiogenesis and underlying mechanism remains largely unclear. We show that tumor necrosis factor ? (TNF?) and interferon ? (IFN?) stimulate PML expression while suppressing EC network formation and migration, two key events during angiogenesis. By a knockdown approach, we demonstrate that PML is indispensable for TNF?- and IFN?-mediated inhibition of EC network formation. We further demonstrate that signal transducer and activator of transcription 1 (STAT1) binds PML promoter and that is an important regulator of PML expression. Knockdown of STAT1 reduces endogenous PML and blocks TNF?- and IFN?-induced PML accumulation and relieves TNF?- and IFN?-mediated inhibition of EC network formation. Our data also indicate that PML regulates EC migration, in part, by modulating expression of downstream genes, such as negatively regulating integrin ?1 (ITGB1). In addition, knockdown of STAT1 or PML alleviates TNF?- and IFN?-mediated inhibition of ITGB1 expression. Antibody blockade demonstrates that ITGB1 is functionally important for PML- and STAT1-regulated EC migration. Taken together, our data provide novel mechanistic insights that PML functions as a negative regulator in EC network formation and migration.
| S-EPMC3390613 | biostudies-literature
Project description:Dysregulation of PML, a significant tumor suppressor is linked with cancers of different histological origins, with a decreased expression observed with a higher tumor grade. This necessitates studying the mechanisms to maintain a stable expression of PML. However much less is known about the transcriptional regulation of PML, more so in the context of breast carcinoma. ERβ has emerged as a critical factor in understanding breast cancer, especially since a huge proportion of breast cancers are ERα- and thus insensitive to tamoxifen therapy. This study aims to uncover an unidentified mechanism of PML gene regulation and its stabilization in breast cancer via ERβ signalling and the impact on cellular apoptosis. We found that clinical expression of PML positively correlates with that of ERβ both in normal and breast carcinoma samples and inversely correlates with markers of cellular proliferation, hinting towards a possible mechanistic interdependence. Both mRNA and protein expression of PML were increased in response to ERβ overexpression on multiple human breast cancer cell lines. Mechanistically, luciferase reporter assays and chromatin-immunoprecipitation assays demonstrated that ERβ can interact with the PML promoter via ERE and AP1 sites to enhance its transcription. ERβ induced stable PML expression causes a decline of its target protein Survivin and simultaneously provides a stable docking platform leading to stabilisation of its target Foxo3a, further causing transcriptional upregulation of pro-apoptotic factors p21 and p27. Immunohistochemical analyses of cancer and normal breast tissues and functional assays conducted corroborated the findings. Collectively, our study identifies ERβ signalling as a novel mechanism for PML gene regulation in ERα- breast cancer. It also reveals bi-directional downstream effect in which 'ERβ-PML-(Foxo3a/Survivin)' network acts as a therapeutic axis by suppressing cellular survival and promoting cellular apoptosis in breast carcinoma.
| S-EPMC6736969 | biostudies-literature
Project description:Previous studies have suggested that most papillomaviruses enter the host cell via clathrin-dependent receptor-mediated endocytosis but have not addressed later steps in viral entry. To examine these events, we followed the localization of L2 and packaged DNA after entry of infectious virions or L1/L2 pseudovirions. Confocal microscopic analyses of HeLa cells showed a time-dependent uncoating of capsids in cytoplasmic vesicles and the accumulation of both L2 and viral DNA at distinct nuclear domains identified as nuclear domain 10 (ND10). Both L2 and the pseudogenome had a punctate distribution and localized to ND10 in promyelocytic leukemia protein (PML)-expressing cells, whereas L2 had a diffuse nuclear distribution in PML-/- cells. The number of pseudovirus-infected cells was an order of magnitude higher in the PML+ cells compared with the PML-/- cells, and viral genome transcription after infection with authentic bovine papillomavirus virions was similarly elevated in PML+ cells. The results identify a role for PML in the enhancement of viral infectivity in the early part of the life cycle. We propose a model in which L2 chaperones the viral genome to ND10 to efficiently initiate viral transcription.
| S-EPMC521143 | biostudies-other
Project description:Cardiac glycosides (CGs), inhibitors of Na+/K+-ATPase (NKA), used clinically to treat heart failure, have garnered recent attention as potential anti-cancer and anti-viral agents. A high-throughput phenotypic screen designed to identify modulators of promyelocytic leukemia protein (PML) nuclear body (NB) formation revealed the CG gitoxigenin as a potent activator of PML. We demonstrate that multiple structurally distinct CGs activate the formation of PML NBs and induce PML protein SUMOylation in an NKA-dependent fashion. CG effects on PML occur at the post-transcriptional level, mechanistically distinct from previously described PML activators and are mediated through signaling events downstream of NKA. Curiously, genomic deletion of PML in human cancer cells failed to abrogate the cytotoxic effects of CGs and other apoptotic stimuli such as ceramide and arsenic trioxide that were previously shown to function through PML in mice. These findings suggest that alternative pathways can compensate for PML loss to mediate apoptosis in response to CGs and other apoptotic stimuli.
| S-EPMC4816303 | biostudies-literature
Project description:BackgroundEnterovirus 71 (EV71) usually infects infants causing hand-foot-mouth disease (HFMD), even fatal neurological disease like aseptic meningitis. Effective drug for preventing and treating EV71 infection is unavailable currently. EV71 3C mediated the cleavage of many proteins and played an important role in viral inhibiting host innate immunity. Promyelocytic leukemia (PML) protein, the primary organizer of PML nuclear bodies (PML-NBs), can be induced by interferon and is involved in antiviral activity. PML inhibits EV71 replication, and EV71 infection reduces PML expression, but the molecular mechanism is unclear.MethodsThe cleavage of PMLIII and IV was confirmed by co-transfection of EV71 3C protease and PML. The detailed cleavage sites were evaluated further by constructing the Q to A mutant of PML. PML knockout cells were infected with EV71 to identify the effect of cleavage on EV71 replication. Immunofluorescence analysis to examine the interference of EV71 3C on the formation of PML-NBs.ResultsEV71 3C directly cleaved PMLIII and IV. Furthermore, 3C cleaved PMLIV at the sites of Q430-A431 and Q444-S445 through its protease activity. Overexpression of PMLIV Q430A/Q444A variant exhibited stronger antiviral potential than the wild type. PMLIV Q430A/Q444A formed normal nuclear bodies that were not affected by 3C, suggesting that 3C may impair PML-NBs production via PMLIV cleavage and counter its antiviral activities. PML, especially PMLIV, which sequesters viral proteins in PML-NBs and inhibits viral production, is a novel target of EV71 3C cleavage.ConclusionsEV71 3C cleaves PMLIV at Q430-A431 and Q444-S445. Cleavage reduces the antiviral function of PML and decomposes the formation of PML-NBs, which is conducive to virus replication.
| S-EPMC8686290 | biostudies-literature
Project description:The promyelocytic leukemia (PML) gene, a tumor suppressor inactivated in acute promyelocytic leukemia (APL), regulates apoptosis induced by DNA damage. However, the molecular mechanisms by which PML modulates apoptosis following genotoxic stress are only partially elucidated. PML is essential for p53-dependent induction of programmed cell death upon gamma-irradiation through PML-nuclear body (NB)-mediated control of p53 acetylation. Here, we show that PML selectively regulates proapoptotic transcription factors upon different types of DNA damage. We find that Pml inactivation protects fibroblasts from UV-induced apoptosis in a p53-independent manner. We demonstrate that c-Jun is required for UV-induced apoptosis and that PML is essential for both c-Jun transcriptional activation and DNA binding upon UV radiation. We find that PML physically interacts with c-Jun and that upon UV radiation the PML-NBs reorganize into novel nuclear microspeckled structures (UV-NBs), where PML and c-Jun dynamically accumulate. These data identify a novel PML-dependent pathway for c-Jun transcriptional activation and induction of apoptosis in response to DNA damage and shed new light on the role of PML in tumor suppression.
| S-EPMC1895009 | biostudies-literature
Project description:The multitasking promyelocytic leukemia (PML) protein was originally recognized as a tumor-suppressive factor, but more recent evidence has implicated PML in tumor cell prosurvival actions and poor patient prognosis in specific cancer settings. Here, we report that inducible PMLIV expression inhibits cell proliferation as well as self-renewal and impairs cell cycle progression of breast cancer cell lines in a reversible manner. Transcriptomic profiling identified a large number of PML-deregulated genes associated with various cell processes. Among them, cell cycle- and division-related genes and their cognitive regulators are highly ranked. In this study, we focused on previously unknown PML targets, namely the Forkhead transcription factors. PML suppresses the Forkhead box subclass M1 (FOXM1) transcription factor at both the RNA and protein levels, along with many of its gene targets. We show that FOXM1 interacts with PMLIV primarily via its DNA-binding domain and dynamically colocalizes in PML nuclear bodies. In parallel, PML modulates the activity of Forkhead box O3 (FOXO3), a factor opposing certain FOXM1 activities, to promote cell survival and stress resistance. Thus, PMLIV affects the balance of FOXO3 and FOXM1 transcriptional programs by acting on discrete gene subsets to favor both growth inhibition and survival. Interestingly, PMLIV-specific knockdown mimicked ectopic expression vis-à-vis loss of proliferative ability and self-renewal, but also led to loss of survival ability as shown by increased apoptosis. We propose that divergent or similar effects on cell physiology may be elicited by high or low PMLIV levels dictated by other concurrent genetic or epigenetic cancer cell states that may additionally account for its disparate effects in various cancer types.
| S-EPMC6547613 | biostudies-literature