Hsp90 Stabilizes SIRT1 Orthologs in Mammalian Cells and C. elegans.
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ABSTRACT:
SUBMITTER: Nguyen MT
PROVIDER: S-EPMC6274930 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:SIRT1, the closest mammalian homolog of yeast Sir2, is an NAD(+)-dependent deacetylase with relevant functions in cancer, aging, and metabolism among other processes. SIRT1 has a diffuse nuclear localization but is recruited to the PML nuclear bodies (PML-NBs) after PML upregulation. However, the functions of SIRT1 in the PML-NBs are unknown. In this study we show that primary mouse embryo fibroblasts lacking SIRT1 contain reduced PML protein levels that are increased after reintroduction of SIRT1. In addition, overexpression of SIRT1 in HEK-293 cells increases the amount of PML protein whereas knockdown of SIRT1 reduces the size and number of PML-NBs and the levels of PML protein in HeLa cells. SIRT1 stimulates PML sumoylation in vitro and in vivo in a deacetylase-independent manner. Importantly, the absence of SIRT1 reduces the apoptotic response of vesicular stomatitis virus-infected cells and favors the extent of this PML-sensitive virus replication. These results show a novel function of SIRT1 in the control of PML and PML-NBs.
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Project description:BackgroundAccurate determination of orthology is central to comparative genomics. For vertebrates in particular, very large gene families, high rates of gene duplication and loss, multiple mechanisms of gene duplication, and high rates of retrotransposition all combine to make inference of orthology between genes difficult. Many methods have been developed to identify orthologous genes, mostly based upon analysis of the inferred protein sequence of the genes. More recently, methods have been proposed that use genomic context in addition to protein sequence to improve orthology assignment in vertebrates. Such methods have been most successfully implemented in fungal genomes and have long been used in prokaryotic genomes, where gene order is far less variable than in vertebrates. However, to our knowledge, no explicit comparison of synteny and sequence based definitions of orthology has been reported in vertebrates, or, more specifically, in mammals.ResultsWe test a simple method for the measurement and utilization of gene order (local synteny) in the identification of mammalian orthologs by investigating the agreement between coding sequence based orthology (Inparanoid) and local synteny based orthology. In the 5 mammalian genomes studied, 93% of the sampled inter-species pairs were found to be concordant between the two orthology methods, illustrating that local synteny is a robust substitute to coding sequence for identifying orthologs. However, 7% of pairs were found to be discordant between local synteny and Inparanoid. These cases of discordance result from evolutionary events including retrotransposition and genome rearrangements.ConclusionsBy analyzing cases of discordance between local synteny and Inparanoid we show that local synteny can distinguish between true orthologs and recent retrogenes, can resolve ambiguous many-to-many orthology relationships into one-to-one ortholog pairs, and might be used to identify cases of non-orthologous gene displacement by retroduplicated paralogs.
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Project description:ObjectiveGlycogen synthase kinase-3β (GSK3β) has been recognized as a suppressor of Wnt/β-catenin signaling, which is critical for the stemness maintenance of breast cancer stem cells. However, the regulatory mechanisms of GSK3β protein expression remain elusive.MethodsCo-immunoprecipitation and mass spectral assays were performed to identify molecules binding to GSK3β, and to characterize the interactions of GSK3β, heat shock protein 90 (Hsp90), and co-chaperones. The role of PGK1 in Hsp90 chaperoning GSK3β was evaluated by constructing 293T cells stably expressing different domains/mutants of Hsp90α, and by performing a series of binding assays with bacterially purified proteins and clinical specimens. The influences of Hsp90 inhibitors on breast cancer stem cell stemness were investigated by Western blot and mammosphere formation assays.ResultsWe showed that GSK3β was a client protein of Hsp90. Hsp90, which did not directly bind to GSK3β, interacted with phosphoglycerate kinase 1 via its C-terminal domain, thereby facilitating the binding of GSK3β to Hsp90. GSK3β-bound PGK1 interacted with Hsp90 in the "closed" conformation and stabilized GSK3β expression in an Hsp90 activity-dependent manner. The Hsp90 inhibitor, 17-AAG, rather than HDN-1, disrupted the interaction between Hsp90 and PGK1, and reduced GSK3β expression, resulting in significantly reduced inhibition of β-catenin expression, to maintain the stemness of breast cancer stem cells.ConclusionsOur findings identified a novel regulatory mechanism of GSK3β expression involving metabolic enzyme PGK1-coupled Hsp90, and highlighted the potential for more effective cancer treatment by selecting Hsp90 inhibitors that do not affect PGK1-regulated GSK3β expression.
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Project description:p62/SQSTM1 is frequently up-regulated in many cancers including hepatocellular carcinoma. Highly expressed p62 promotes hepato-carcinogenesis by activating many signaling pathways including Nrf2, mTORC1, and NFκB signaling. However, the underlying mechanism for p62 up-regulation in hepatocellular carcinoma remains largely unclear. Herein, we confirmed that p62 was up-regulated in hepatocellular carcinoma and its higher expression was associated with shorter overall survival in patients. The knockdown of p62 in hepatocellular carcinoma cells decreased cell growth in vitro and in vivo. Intriguingly, p62 protein stability could be reduced by its acetylation at lysine 295, which was regulated by deacetylase Sirt1 and acetyltransferase GCN5. Acetylated p62 increased its association with the E3 ligase Keap1, which facilitated its poly-ubiquitination-dependent proteasomal degradation. Moreover, Sirt1 was up-regulated to deacetylate and stabilize p62 in hepatocellular carcinoma. Additionally, Hepatocyte Sirt1 conditional knockout mice developed much fewer liver tumors after Diethynitrosamine treatment, which could be reversed by the re-introduction of exogenous p62. Taken together, Sirt1 deacetylates p62 at lysine 295 to disturb Keap1-mediated p62 poly-ubiquitination, thus up-regulating p62 expression to promote hepato-carcinogenesis. Therefore, targeting Sirt1 or p62 is a reasonable strategy for the treatment of hepatocellular carcinoma.
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