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: 8783415
• 负责人: Gleb Bazilevsky
• 金额: $ 4.27万
• 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/8783415
• 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; 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; 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基本代谢物乙酰辅酶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(KATS)、一般控制不可降压因子5(GCN5)和p300/CBP相关因子(PCAF)是以多亚单位复合体形式参与乙酰化组蛋白和非组蛋白调控基因转录的全球性转录因子。作为KAT活性必需的辅因子，Ac-CoA的细胞浓度直接关系到GCN5和PCAF活性的增加，蛋白质靶标乙酰化，以及促生长基因的转录激活。在后生动物中，Ac-CoA是由葡萄糖衍生的柠檬酸在三磷酸腺苷(ATP)-柠檬酸裂解酶(ACLY)的作用下产生的。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依赖的Ac-CoA的产生驱动组蛋白乙酰化的增加。此外，许多活跃的增殖细胞表现出ACLY水平的增加，尤其是癌细胞的增殖对葡萄糖的供应和适当的ACLY功能非常敏感。ACLY还经历了对葡萄糖水平升高的响应的稳定性增加，并通过依赖Kat的转录激活激活了促进生长的信号通路。此外，来自Wellen实验室的新的未发表的数据表明，ACLY和PCAF在细胞内形成复合体，补充了最近的一份报告，该报告表明PCAF乙酰化ACLY以促进ACLY的稳定性和肿瘤生长。然而，ACLY和GCN5/PCAF之间的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，这一建议试图验证这样的假设，即ACLY与GCN5和PCAF形成直接复合体，促进ACLY的乙酰化和稳定性，以及GCN5和PCAF对组蛋白底物的乙酰化活性，以支持促生长基因的激活。这一假设将通过对生化纯化的ACLY和GCN5/PCAF进行体外下拉试验来研究，以确定它们是否可以直接相互结合。然后，用ITC表征相互作用的生物物理性质，并通过酶活性测定来确定相互作用酶的稳态动力学参数。最后，X射线晶体结构测定和诱变实验将为ACLY与GCN5/PCAF相互作用提供分子基础。最后，这项建议将通过中断ACLY-GCN5/PCAF相互作用并询问哺乳动物细胞系全局组蛋白乙酰化、特定代谢和促进生长基因的表达以及这些细胞的增殖和分化能力的变化，来确定ACLY-GCN5/PCAF相互作用的体内后果。总之，这些研究将阐明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，并将为癌症治疗提供靶向ACLY功能的新途径。