ABSTRACT
Polycomb repressive complex 2 (PRC2) consists of three core subunits, EZH2, EED and SUZ12, and plays pivotal roles in transcriptional regulation. The catalytic subunit EZH2 methylates histone H3 lysine 27 (H3K27), and its activity is further enhanced by the binding of EED to trimethylated H3K27 (H3K27me3). Small-molecule inhibitors that compete with the cofactor S-adenosylmethionine (SAM) have been reported. Here we report the discovery of EED226, a potent and selective PRC2 inhibitor that directly binds to the H3K27me3 binding pocket of EED. EED226 induces a conformational change upon binding EED, leading to loss of PRC2 activity. EED226 shows similar activity to SAM-competitive inhibitors in blocking H3K27 methylation of PRC2 target genes and inducing regression of human lymphoma xenograft tumors. Interestingly, EED226 also effectively inhibits PRC2 containing a mutant EZH2 protein resistant to SAM-competitive inhibitors. Together, we show that EED226 inhibits PRC2 activity via an allosteric mechanism and offers an opportunity for treatment of PRC2-dependent cancers.
Subject(s)
Antineoplastic Agents/pharmacology , Histones/metabolism , Lysine/metabolism , Polycomb Repressive Complex 2/antagonists & inhibitors , Sulfones/chemistry , Sulfones/pharmacology , Triazoles/chemistry , Triazoles/pharmacology , Allosteric Regulation/drug effects , Animals , Antineoplastic Agents/chemistry , Binding Sites/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Female , Histones/chemistry , Humans , Lysine/chemistry , Mice , Mice, Inbred BALB C , Mice, Nude , Models, Molecular , Molecular Structure , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/pathology , Polycomb Repressive Complex 2/chemistry , Polycomb Repressive Complex 2/metabolism , Structure-Activity Relationship , Sulfones/metabolism , Triazoles/metabolism , Tumor Cells, CulturedABSTRACT
Axon degeneration is supposed to be a therapeutic target for treating neurodegenerative diseases. Mauthner cells (M-cells) are ideal for studying axons in vivo because of their limited numbers, large size, and long axons. In this study, we labeled M-cells by single-cell electroporation with plasmids expressing DsRed2 or EGFP. Injury-induced axon degeneration in labeled M-cell was imaged under a confocal microscope, and we found that the Mauthner axons started to degenerate about 24 hr after lesion. The Wld(S) protein containing full-length Nmnat1 is well-known for its axon-protective function in many systems. Overexpression of Wld(S) in M-cells also greatly delayed axon degeneration in live zebrafish. Nmnat2 is the only Nmnat highly expressed in brain. Here we demonstrated that overexpression of Nmnat2 in M-cells significantly delayed axon degeneration in vivo, and disruption of the NAD synthesis activity of Nmnat2 markedly attenuated its axon-protective function. All these data show that injury-induced axon degeneration of M-cell has a mechanism similar to that in mammalians and would be a valuable model for studying axon degeneration in vivo.
Subject(s)
Axons/metabolism , Nerve Degeneration/metabolism , Nerve Tissue Proteins/metabolism , Nicotinamide-Nucleotide Adenylyltransferase/metabolism , Animals , Axons/pathology , Electroporation , HEK293 Cells , Humans , Immunoprecipitation , Microscopy, Confocal , Nerve Degeneration/genetics , Nerve Degeneration/pathology , Nerve Tissue Proteins/genetics , Nicotinamide-Nucleotide Adenylyltransferase/genetics , ZebrafishABSTRACT
SIRT1 plays an important role in adipogenesis, but how SIRT1 is regulated in adipogenesis is largely unknown. In this study, we show that both SIRT1 protein and mRNA levels were increased along with CCAAT/enhancer-binding protein alpha (C/EBPalpha) during adipocyte differentiation. C/EBPalpha, but not C/EBPalphap30, activated SIRT1 promoter in both HeLa cells and 3T3-L1 preadipocytes. Furthermore, C/EBPalpha upregulated SIRT1 mRNA and protein levels in HeLa cells and increased SIRT1 expression in a p53-independent manner in Soas2 cells. In preadipocytes, ectopic expression of C/EBPalpha upregulated SIRT1 protein level and knockdown of C/EBPalpha led to the decrease of SIRT1 protein level. Moreover, by promoter deletion analysis, gel shift assay and chromatin immunoprecipitation, we found that C/EBPalpha bound to the SIRT1 promoter at a consensus C/EBPalpha binding site. These data demonstrate that C/EBPalpha regulates SIRT1 expression during adipogenesis by directly binding to the SIRT1 promoter.
Subject(s)
Adipogenesis , CCAAT-Enhancer-Binding Protein-alpha/metabolism , Sirtuin 1/metabolism , 3T3-L1 Cells , Animals , CCAAT-Enhancer-Binding Protein-alpha/genetics , Cell Differentiation , Cell Line , Chromatin Immunoprecipitation , Gene Knockdown Techniques , HeLa Cells , Humans , Mice , Promoter Regions, Genetic , RNA, Messenger/metabolism , Sirtuin 1/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
Axon degeneration is an active program of self-destruction observed in many physiological and pathological settings. There are three Nicotinamide mononucleotide adenylyl transferase (Nmnat, EC2.7.7.1) in mammals. Overexpression of Nmnat1 or Nmnat3 can delay axon degeneration, while the role of Nmnat2 in axon degeneration remains largely unknown. Here we found that Nmnat2 was specifically and highly expressed in brain compared with Nmnat1 and Nmnat3. Furthermore, we found brain Nmnat2 was correlated with Alzheimer's disease in APPswe/PS1dE9 transgenic mice. Nmnat2 delayed Wallerian degeneration in cultured superior cervical ganglia (SCGs) from morphological changes, microtubule destruction and neurofilament degradation, mutation of the conserved enzyme activity site in Nmnat2 disrupted its enzyme activity as well as the axon-protective function. Our results demonstrate that the brain-specific Nmnat2 delays injury-induced axon degeneration dependent on its NAD synthesis activity. These findings provide new clues to further study the molecular mechanisms of axon degeneration and the related neurodegenerative diseases.
Subject(s)
Axons/metabolism , NAD/biosynthesis , Nicotinamide-Nucleotide Adenylyltransferase/metabolism , Superior Cervical Ganglion/metabolism , Wallerian Degeneration/metabolism , Animals , Axons/pathology , Cell Death/physiology , Cell Line , Cells, Cultured , Cytoprotection/physiology , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microtubules/metabolism , Neurofilament Proteins/metabolism , Nicotinamide-Nucleotide Adenylyltransferase/genetics , Rats , Rats, Sprague-Dawley , Superior Cervical Ganglion/pathology , Superior Cervical Ganglion/physiopathology , Time Factors , Wallerian Degeneration/genetics , Wallerian Degeneration/physiopathologyABSTRACT
Protein acetylation is increasingly recognized as an important post-translational modification. Although a lot of protein acetyltransferases have been identified, a few putative acetyltransferases are yet to be studied. In this study, we identified a novel protein acetyltransferase, Patt1, which belongs to GNAT family. Patt1 exhibited histone acetyltransferase activity and auto-acetylation activity. Deletion and mutation analysis of the predicted acetyltransferase domain in Patt1 showed that the conserved Glu139 was an important residue for its protein acetyltransferase activity. Furthermore, we found that Patt1 was highly expressed in liver and significantly downregulated in hepatocellular carcinoma tissues. In addition, we showed that overexpression of Patt1 enhanced the apoptosis of hepatoma cells dependent on its acetyltransferase activity, whereas knockdown of Patt1 significantly protected Chang liver cells from apoptosis. These data suggest that Patt1 might be involved in the development of hepatocellular carcinoma, and could be served as a potential therapy target for hepatocellular carcinoma.
Subject(s)
Carcinoma, Hepatocellular/enzymology , Histone Acetyltransferases/metabolism , Liver Neoplasms, Experimental/enzymology , Liver Neoplasms/enzymology , Liver/enzymology , Animals , Apoptosis/genetics , Carcinoma, Hepatocellular/pathology , Cloning, Molecular , Gene Expression Regulation, Neoplastic , HeLa Cells , Histone Acetyltransferases/genetics , Humans , Liver/pathology , Liver Neoplasms/pathology , Liver Neoplasms, Experimental/pathology , Male , Mice , Mice, Inbred C57BL , N-Terminal Acetyltransferase D , RNA, Small Interfering/geneticsABSTRACT
beta-Catenin plays an important role in development and tumorigenesis. However, the effect of a key acetyltransferase p300/CBP-associated factor (PCAF) on beta-catenin signaling is largely unknown. In this study, we found PCAF could increase the beta-catenin transcriptional activity, induce its nuclear translocation, and up-regulate its protein level by inhibiting its ubiquitination and improving its stability. Further studies showed that PCAF directly binds to and acetylates beta-catenin. The key ubiquitination sites Lys-19 and Lys-49 of beta-catenin were shown as the critical residues for PCAF-induced acetylation and stabilization. Knockdown of PCAF in colon cancer cells markedly reduced the protein level, transcriptional activity, and acetylation level of beta-catenin; promoted cell differentiation; inhibited cell migration; and repressed xenografted tumorigenesis and tumor growth in nude mice. All these data demonstrate that PCAF acetylates beta-catenin and regulates its stability, and they raise the prospect that therapies targeting PCAF may be of clinical use in beta-catenin-driven diseases, such as colon cancer.
Subject(s)
Protein Stability , Signal Transduction/physiology , beta Catenin/metabolism , p300-CBP Transcription Factors/metabolism , Acetylation , Animals , Cell Differentiation/physiology , Cell Line, Tumor , Cell Movement/physiology , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Humans , Lysine/metabolism , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Neoplasm Transplantation , Transcriptional Activation , Wnt Proteins/genetics , Wnt Proteins/metabolism , beta Catenin/genetics , p300-CBP Transcription Factors/geneticsABSTRACT
Resveratrol was reported to increase insulin sensitivity accompanied with the activation of AMP-activated protein kinase (AMPK), which is a key regulator of energy balance and an important drug target for type 2 diabetes. However, the effect of resveratrol structural analogs on AMPK activity and insulin sensitivity is still largely unknown. In this study, we analyzed the effect of several resveratrol structural analogs on AMPK activity in HepG2 cells, and combretastatin A-4 (CA-4) was identified as an activator of AMPK determined by its phosphorylation. AMPK activation was further confirmed by the phosphorylation of downstream acetyl-CoA carboxylase (ACC) and the decrease of upstream ATP level. Further investigation showed that CA-4 activates PPAR transcriptional activity in vitro with the luciferase reporter assay. In addition, we showed that CA-4 activated AMPK and downregulated gluconeogenic enzyme mRNA levels in liver, and improved the fasting blood glucose level in diabetic db/db mice. These results suggested that resveratrol analogs, such as CA-4, can function similarly as resveratrol and may provide important tools for improving insulin sensitivity.
Subject(s)
Diabetes Mellitus, Type 2/drug therapy , Glucose/metabolism , Multienzyme Complexes/metabolism , Protein Serine-Threonine Kinases/metabolism , Stilbenes/pharmacology , 3T3-L1 Cells , AMP-Activated Protein Kinases , Acetyl-CoA Carboxylase/metabolism , Adenosine Triphosphate/analysis , Animals , Enzyme Activation/drug effects , Humans , Mice , Mice, Inbred C57BL , Phosphorylation , Poly(ADP-ribose) Polymerases/geneticsABSTRACT
Insulin resistance is often characterized as the most critical factor contributing to the development of type 2 diabetes. SIRT1 has been reported to be involved in the processes of glucose metabolism and insulin secretion. However, whether SIRT1 is directly involved in insulin sensitivity is still largely unknown. Here we show that SIRT1 is downregulated in insulin-resistant cells and tissues and that knockdown or inhibition of SIRT1 induces insulin resistance. Furthermore, increased expression of SIRT1 improved insulin sensitivity, especially under insulin-resistant conditions. Similarly, resveratrol, a SIRT1 activator, enhanced insulin sensitivity in vitro in a SIRT1-dependent manner and attenuated high-fat-diet-induced insulin resistance in vivo at a dose of 2.5 mg/kg/day. Further studies demonstrated that the effect of SIRT1 on insulin resistance is mediated by repressing PTP1B transcription at the chromatin level. Taken together, the finding that SIRT1 improves insulin sensitivity has implications toward resolving insulin resistance and type 2 diabetes.
Subject(s)
Gene Expression Regulation , Insulin Resistance/genetics , Protein Tyrosine Phosphatase, Non-Receptor Type 1/antagonists & inhibitors , Protein Tyrosine Phosphatase, Non-Receptor Type 1/genetics , Sirtuins/physiology , Animals , Chromatin/metabolism , Down-Regulation , Insulin/pharmacology , Mice , Mice, Knockout , Protein Tyrosine Phosphatase, Non-Receptor Type 1/metabolism , RNA, Small Interfering/pharmacology , Resveratrol , Sirtuin 1 , Sirtuins/antagonists & inhibitors , Sirtuins/genetics , Stilbenes/pharmacologyABSTRACT
In general, SIRT1 is localized in nuclei. Here, we showed that endogenous and exogenous SIRT1 were both able to partially localize in cytoplasm in certain cell lines, and cytoplasm-localized SIRT1 was associated with apoptosis and led to increased sensitivity to apoptosis. Furthermore, we demonstrated that translocation of nucleus-localized SIRT1 from nuclei to cytoplasm was the main pathway leading to localization of SIRT1 in cytoplasm. In HeLa cells, wild type SIRT1 was completely localized in nuclei. By truncation of two predicted nuclear localization signals or fusion with an exogenous nuclear export signal, SIRT1 was partially localized in cytoplasm of HeLa cells and resulted in increased sensitivity to apoptosis. The apoptosis enhanced by cytoplasm-localized SIRT1 was independent of its deacetylase activity, but dependent on caspases. SIRT1 was distributed in cytoplasm at metaphase during mitosis, and overexpression of SIRT1 significantly augmented apoptosis for cells at metaphase. In summary, we found SIRT1 is able to localize in cytoplasm, and cytoplasm-localized SIRT1 enhances apoptosis.
