The molecular basis for how acetyl-coenzyme A links metabolism to gene expression

乙酰辅酶 A 如何将代谢与基因表达联系起来的分子基础

• 批准号: 8996048
• 负责人: Gleb Bazilevsky
• 金额: $ 4.31万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8996048
• 关键词: ATP Citrate (pro-S)-Lyase; Acetyl Coenzyme A; Acetylation; Acetyltransferase; Acids; Adenosine Triphosphate; Affect; Automobile Driving; Binding; Biological Assay; Calorimetry; Cancerous; Cell Line; Cell physiology; Cells; Citrates; Communication; Complement; Complex; Coupled; DNA; Data; Dependence; Diet; Enzymes; Exhibits; Factor X; Gene Expression; Gene Targeting; Genes; Genetic Transcription; Glucose; Goals; Growth; Histone Acetylation; Histones; Human; In Vitro; Kinetics; Laboratories; Length; Link; Lysine; Malate Dehydrogenase; Mammalian Cell; Maps; Measures; Metabolism; Modification; Molecular; Mutagenesis; Mutation; Nature; Non-Histone Chromosomal Proteins; Phosphorylation; Production; Proliferating; Property; Proteins; Radioactive; Recombinants; Reporting; Roentgen Rays; Signal Pathway; Signal Transduction; Structure; Surface; Testing; Thermodynamics; Titrations; Transcriptional Activation; Western Blotting; X-Ray Crystallography; adipocyte differentiation; base; biophysical properties; cancer cell; cancer therapy; carcinogenesis; citrate lyase; cofactor; differential expression; enzyme activity; in vivo; mutant; p300/CBP-Associated Factor; protein complex; public health relevance; research study; response; stoichiometry; transcription factor; tumor growth

DESCRIPTION (provided by applicant): The goal of this proposal is to investigate how the essential metabolite acetyl-coenzyme A (Ac-CoA) links cellular metabolism to changes in gene expression. The lysine acetyltransferases (KATs) general control nonderepressible 5 (GCN5) and p300/CBP-associated factor (PCAF) are global transcription factors that function in multisubunit complexes to acetylate histone and non-histone proteins to regulate gene transcription. As a necessary cofactor for KAT activity, Ac-CoA cellular concentration is directly related to increased rates of GCN5 and PCAF activity, protein target acetylation, and the transcriptional activation of growth-promoting genes. In metazoans, Ac-CoA is produced from glucose-derived citrate by the enzyme adenosine triphosphate (ATP)-citrate lyase (ACLY). Our collaborator, Dr. Kathryn Wellen, has reported that ACLY links cellular metabolism to histone acetylation, with ACLY-dependent production of Ac-CoA driving increased histone acetylation. In addition, many actively-proliferating cells exhibit increased levels of ACLY, and the proliferation of cancer cells in particular is sensitive to the availability of glucose and proper ACLY function. ACLY also undergoes an increase in stability in response to elevated glucose levels and activation of growth-promoting signaling pathways by KAT-dependent transcription activation. Moreover, new unpublished data from the Wellen laboratory suggest that ACLY and PCAF form a complex in cells, complementing a recent report demonstrating that PCAF acetylates ACLY to promote ACLY stability and tumor growth. Yet, the nature of the communication between ACLY and GCN5/PCAF and how this communication may regulate the activity of each of the enzymes is not known. Based on these data, this proposal seeks to test the hypothesis that ACLY forms a direct complex with the GCN5 and PCAF to facilitate both ACLY acetylation and stability and GCN5 and PCAF acetylation activity towards histone substrates, to support the activation of growth promoting genes. This hypothesis will be investigated using in vitro pull-down assays on biochemically purified ACLY and GCN5/PCAF, in order to determine if they can bind directly to one another. The biophysical properties of the interaction will then be characterized with ITC, as well as the steady state kinetic parameters of the interacting enzymes by enzyme activity assays. Lastly, X-ray crystallography structure determination and mutagenesis experiments will provide the molecular basis for the interaction of ACLY with GCN5/PCAF. Lastly, this proposal will determine the in vivo consequence of the ACLY-GCN5/PCAF interaction by disrupting this interaction and interrogating mammalian cell lines for changes in global histone acetylation, the expression of specific metabolism and growth-promoting genes, and the ability of these cells to proliferate and differentiate. Together, these studies will delineate the molecular mechanism for how the AcCoA metabolite links cellular metabolism to gene expression and will provide new avenues for targeting ACLY function for cancer therapy.

描述（由申请人提供）：本提案的目标是研究必需代谢物乙酰辅酶 A (Ac-CoA) 如何将细胞代谢与基因表达的变化联系起来。赖氨酸乙酰转移酶 (KAT) 一般控制非阻抑性 5 (GCN5) 和 p300/CBP 相关因子 (PCAF) 是全局转录因子，在多亚基复合物中发挥作用，乙酰化组蛋白和非组蛋白，从而调节基因转录。作为 KAT 活性必需的辅助因子，Ac-CoA 细胞浓度与 GCN5 和 PCAF 活性的增加率、蛋白质靶标乙酰化以及生长促进基因的转录激活直接相关。在后生动物中，Ac-CoA 是通过三磷酸腺苷 (ATP)-柠檬酸裂解酶 (ACLY) 从葡萄糖衍生的柠檬酸产生的。我们的合作者 Kathryn Wellen 博士报告说，ACLY 将细胞代谢与组蛋白乙酰化联系起来，ACLY 依赖性的 Ac-CoA 产生可驱动组蛋白乙酰化增加。此外，许多活跃增殖的细胞表现出 ACLY 水平升高，并且癌细胞的增殖对葡萄糖的可用性和适当的 ACLY 功能尤其敏感。 ACLY 还随着葡萄糖水平升高而增加稳定性，并通过 KAT 依赖性转录激活激活生长促进信号通路。此外，Wellen 实验室未发表的新数据表明，ACLY 和 PCAF 在细胞中形成复合物，补充了最近的一份报告，证明 PCAF 乙酰化 ACLY 以促进 ACLY 稳定性和肿瘤生长。然而，ACLY 和 GCN5/PCAF 之间通讯的性质以及这种通讯如何调节每种酶的活性尚不清楚。基于这些数据，该提案试图检验这样的假设：ACLY 与 GCN5 和 PCAF 形成直接复合物，以促进 ACLY 乙酰化和稳定性以及 GCN5 和 PCAF 对组蛋白底物的乙酰化活性，以支持生长促进基因的激活。我们将使用生化纯化的 ACLY 和 GCN5/PCAF 的体外 Pull-down 测定来研究这一假设，以确定它们是否可以直接相互结合。然后，将通过 ITC 表征相互作用的生物物理特性，并通过酶活性测定来表征相互作用酶的稳态动力学参数。最后，X射线晶体学结构测定和诱变实验将为ACLY与GCN5/PCAF的相互作用提供分子基础。最后，该提案将通过破坏 ACLY-GCN5/PCAF 相互作用并询问哺乳动物细胞系整体组蛋白乙酰化的变化、特定代谢和生长促进基因的表达以及这些基因的能力来确定 ACLY-GCN5/PCAF 相互作用的体内后果。细胞增殖和分化。这些研究将共同​​描述 AcCoA 代谢物如何将细胞代谢与基因表达联系起来的分子机制，并将为针对癌症治疗的 ACLY 功能提供新途径。