Ubiquitin ligase Siah2 regulates RevErb? degradation and the mammalian circadian clock.
ABSTRACT: Regulated degradation of proteins by the proteasome is often critical to their function in dynamic cellular pathways. The molecular clock underlying mammalian circadian rhythms relies on the rhythmic expression and degradation of its core components. However, because the tools available for identifying the mechanisms underlying the degradation of a specific protein are limited, the mechanisms regulating clock protein degradation are only beginning to be elucidated. Here we describe a cell-based functional screening approach designed to quickly identify the ubiquitin E3 ligases that induce the degradation of potentially any protein of interest. We screened the nuclear hormone receptor RevErb? (Nr1d1), a key constituent of the mammalian circadian clock, for E3 ligases that regulate its stability and found Seven in absentia2 (Siah2) to be a key regulator of RevErb? stability. Previously implicated in hypoxia signaling, Siah2 overexpression destabilizes RevErb?/?, and siRNA depletion of Siah2 stabilizes endogenous RevErb?. Moreover, Siah2 depletion delays circadian degradation of RevErb? and lengthens period length. These results demonstrate the utility of functional screening approaches for identifying regulators of protein stability and reveal Siah2 as a previously unidentified circadian clockwork regulator that mediates circadian RevErb? turnover.
Project description:The time-dependent degradation of core circadian clock proteins is essential for the proper functioning of circadian timekeeping mechanisms that drive daily rhythms in gene expression and, ultimately, an organism's physiology. The ubiquitin proteasome system plays a critical role in regulating the stability of most proteins, including the core clock components. Our laboratory developed a cell-based functional screen to identify ubiquitin ligases that degrade any protein of interest and have started screening for those ligases that degrade circadian clock proteins. This screen identified Spsb4 as a putative novel E3 ligase for RevErb?. In this article, we further investigate the role of Spsb4 and its paralogs in RevErb? stability and circadian rhythmicity. Our results indicate that the paralogs Spsb1 and Spsb4, but not Spsb2 and Spsb3, can interact with and facilitate RevErb? ubiquitination and degradation and regulate circadian clock periodicity.
Project description:Pulmonary inflammatory responses lie under circadian control; however, the importance of circadian mechanisms in the underlying fibrotic phenotype is not understood. Here, we identify a striking change to these mechanisms resulting in a gain of amplitude and lack of synchrony within pulmonary fibrotic tissue. These changes result from an infiltration of mesenchymal cells, an important cell type in the pathogenesis of pulmonary fibrosis. Mutation of the core clock protein REVERB? in these cells exacerbated the development of bleomycin-induced fibrosis, whereas mutation of REVERB? in club or myeloid cells had no effect on the bleomycin phenotype. Knockdown of REVERB? revealed regulation of the little-understood transcription factor TBPL1. Both REVERB? and TBPL1 altered integrin?1 focal-adhesion formation, resulting in increased myofibroblast activation. The translational importance of our findings was established through analysis of 2 human cohorts. In the UK Biobank, circadian strain markers (sleep length, chronotype, and shift work) are associated with pulmonary fibrosis, making them risk factors. In a separate cohort, REVERB? expression was increased in human idiopathic pulmonary fibrosis (IPF) lung tissue. Pharmacological targeting of REVERB? inhibited myofibroblast activation in IPF fibroblasts and collagen secretion in organotypic cultures from IPF patients, thus suggesting that targeting of REVERB? could be a viable therapeutic approach.
Project description:The nuclear receptor REVERB? is a core component of the circadian clock and proposed to be a dominant regulator of hepatic lipid metabolism. Using antibody-independent ChIP-sequencing of REVERB? in mouse liver, we reveal a high-confidence cistrome and define direct target genes. REVERB?-binding sites are highly enriched for consensus RORE or RevDR2 motifs and overlap with corepressor complex binding. We find no evidence for transcription factor tethering and DNA-binding domain-independent action. Moreover, hepatocyte-specific deletion of Reverb? drives only modest physiological and transcriptional dysregulation, with derepressed target gene enrichment limited to circadian processes. Thus, contrary to previous reports, hepatic REVERB? does not repress lipogenesis under basal conditions. REVERB? control of a more extensive transcriptional program is only revealed under conditions of metabolic perturbation (including mistimed feeding, which is a feature of the global Reverb? -/- mouse). Repressive action of REVERB? in the liver therefore serves to buffer against metabolic challenge, rather than drive basal rhythmicity in metabolic activity.
Project description:A functioning ubiquitin proteasome system (UPS) is essential for a number of diverse cellular processes and maintenance of overall cellular homeostasis. The ability of proteasome inhibitors, such as Velcade, to promote extrinsic apoptotic effects illustrates the importance of the ubiquitin proteasome system in the regulation of death receptor signaling. Here, we set out to define the UPS machinery, particularly the E3 ubiquitin ligases, that repress apoptosis through the extrinsic pathway. A cell-based genome-wide E3 ligase siRNA screen was established to monitor caspase-8 activity following the addition of TRAIL.Data from the high-throughput screen revealed that targeting the RING-finger containing E3 ligase Siah2 as well as the signaling platform molecule POSH (SH3RF1) conferred robust caspase-8 activation in response to TRAIL stimulus. Silencing Siah2 or POSH in prostate cancer cells led to increased caspase activity and apoptosis in response to both TRAIL and Fas ligand. The E3 activity of Siah2 was responsible for mediating apoptosis resistance; while POSH protein levels were critical for maintaining viability. Further characterization of Siah2 revealed it to function downstream of early death receptor events in the apoptotic pathway. The observed apoptosis resistance provides one biological explanation for the induction of Siah2 and POSH reported in lung and prostate cancer, respectively. Expanding on an initial yeast-two-hybrid screen we have confirmed a physical interaction between E3 ligases Siah2 and POSH. Utilizing a yeast-two-hybrid mapping approach we have defined the spacer region of POSH, more specifically the RPxAxVxP motif encompassing amino acids 601-607, to be the site of Siah2 binding.The data presented here define POSH and Siah2 as important mediators of death receptor mediated apoptosis and suggest targeting the interaction of these two E3 ligases is a promising novel cancer therapeutic strategy.
Project description:Circadian biology regulates inflammatory responses in mice via the clock protein REVERBα, resulting in altered mortality and morbidity. The influence of this immune-modulation pathway in humans is unclear, but may affect outcomes after transplant. We sought to determine whether the circadian clock affects primary graft dysfunction after lung transplantation, and the role of the clock protein REVERBα. In this study we investigated the action of a synthetic REVERB ligand, (GSK4112) in human monocyte-derived macrophages.
Project description:The circadian clock relies on regulated degradation of clock proteins to maintain rhythmicity. Despite this, we know few components that mediate protein degradation. This is due to high levels of functional redundancy within plant E3 ubiquitin ligase families. In order to overcome this issue and discover E3 ubiquitin ligases that control circadian function, we generated a library of transgenic Arabidopsis plants expressing dominant-negative 'decoy' E3 ubiquitin ligases. We determined their effects on the circadian clock and identified dozens of new potential regulators of circadian function. To demonstrate the potency of the decoy screening methodology to overcome redundancy and identify <i>bona fide</i> clock regulators, we performed follow-up studies on <i>MAC3A</i> (<i>PUB59</i>) and <i>MAC3B</i> (<i>PUB60</i>). We show that they redundantly control circadian period by regulating splicing. This work demonstrates the viability of ubiquitin ligase decoys as a screening platform to overcome genetic challenges and discover E3 ubiquitin ligases that regulate plant development.
Project description:The RING finger E3 ubiquitin ligase Siah2 is implicated in control of diverse cellular biological events, including MAPK signaling and hypoxia. Here we demonstrate that Siah2 is subject to regulation by the deubiquitinating enzyme USP13. Overexpression of USP13 increases Siah2 stability by attenuating its autodegradation. Consequently, the ability of Siah2 to target its substrates prolyl hydroxylase 3 and Spry2 (Sprouty2) for ubiquitin-mediated proteasomal degradation is attenuated. Conversely, inhibition of USP13 expression with corresponding shRNA decreases the stability of both Siah2 and its substrate Spry2. Thus, USP13 limits Siah2 autodegradation and its ubiquitin ligase activity against its target substrates. Strikingly, the effect of USP13 on Siah2 is not mediated by its isopeptidase activity: mutations in its ubiquitin-binding sequences positioned within the ubiquitin-specific processing protease and ubiquitin-binding domains, but not within putative catalytic sites, abolish USP13 binding to and effect on Siah2 autodegradation and targeted ubiquitination. Notably, USP13 expression is attenuated in melanoma cells maintained under hypoxia, thereby relieving Siah2 inhibition and increasing its activity under low oxygen levels. Significantly, on melanoma tissue microarray, high nuclear expression of USP13 coincided with high nuclear expression of Siah2. Overall, this study identifies a new layer of Siah2 regulation mediated by USP13 binding to ubiquitinated Siah2 protein with a concomitant inhibitory effect on its activity under normoxia.
Project description:Circadian rhythm serves an essential role in numerous physiological functions. Circadian oscillations are organized by circadian clock components at the molecular level. The precision of the circadian clock is controlled by transcriptional-translational negative feedback loops, as well as post-translational modi?cations of clock proteins, including ubiquitination; however, the influence of E3 ligases on clock protein ubiquitination requires further investigation. The results of co-immunoprecipitation and immunofluorescent localization, indicated that the endoplasmic reticulum transmembrane E3 ubiquitin ligase HRD1, encoded by the synoviolin 1 gene, interacted with brain and muscle ARNT-like 1 (BMAL1) and enhanced BMAL1 protein ubiquitination. In addition, the results of western blotting and reverse transcription-quantitative PCR suggested that HRD1 promoted K48-associated polyubiquitination of BMAL1 and thus mediated its degradation via the ubiquitin-proteasome system. Furthermore, gene knockdown and gene overexpression assays revealed that HRD1-dependent degradation of BMAL1 protein regulated the expression of BMAL1 target genes and the amplitude of circadian oscillations in mammalian cells. The findings of the current study indicate that HRD1 may influence the regulation of circadian rhythm via modulation of BMAL1 stability.
Project description:Moderate reductions in peroxisome proliferator-activated receptor (PPAR)? levels control insulin sensitivity as effectively as activation of PPAR? in adipocytes by the thiazolidinediones. That observation suggests that PPAR? activity can be regulated by modulating the amount of PPAR? protein in adipocytes. Activation of PPAR? in adipocytes is linked to changes in PPAR? protein levels via increased degradation of PPAR? proteins by the ubiquitin proteasome system. Identification of the ubiquitin ligase or ligases that recognize ligand bound PPAR? is an essential step in determining the physiological significance of the relationship between activation and ubiquitin-dependent degradation of PPAR?. Using an RNA interference-based screen, we identified five RING (really interesting new gene)-type ubiquitin ligases that alter PPAR? protein levels in adipocytes. Here, we demonstrate that Drosophila seven-in-absentia homolog 2 (Siah2), a mammalian homolog of Drosophila seven-in-absentia, regulates PPAR? ubiquitylation and ligand-dependent activation of PPAR? in adipocytes. We also demonstrate that Siah2 expression is up-regulated during adipogenesis and that PPAR? interacts with Siah2 during adipogenesis. In addition, Siah2 is required for adipogenesis. These data suggest that modulation of PPAR? protein levels by the ubiquitin ligase Siah2 is essential in determining the physiological effects of PPAR? activation in adipocytes.
Project description:The RING finger ubiquitin ligase Siah2 controls the stability of various substrates involved in stress and hypoxia responses, including the PHD3, which controls the stability of HIF-1alpha. In the present study we determined the role of Siah2 phosphorylation in the regulation of its activity toward PHD3. We show that Siah2 is subject to phosphorylation by p38 MAPK, which increases Siah2-mediated degradation of PHD3. Consistent with these findings, MKK3/MKK6 double-deficient cells, which cannot activate p38 kinases, exhibit impaired Siah2-dependent degradation of PHD3. Phosphopeptide mapping identified T24 and S29 as the primary phospho-acceptor sites. Phospho-mutant forms of Siah2 (S29A or T24A/S29A) exhibit impaired degradation of PHD3, particularly after hypoxia. Conversely, a phospho-mimic form of Siah2 (T24E/S29D) exhibits stronger degradation of PHD3, compared with wild type Siah2. Whereas phospho-mutant Siah2 exhibits weaker association with PHD3, phospho-mimic Siah2 associates as well as wild type and is localized within the perinuclear region, suggesting that phosphorylation of Siah2 affects its subcellular localization and, consequently, the degree of its association with PHD3. In all, our findings reveal the phosphorylation of Siah2 by p38 and the implications of such phosphorylation for Siah2 activity toward PHD3.