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: 9125794
• 负责人: Gleb Bazilevsky
• 金额: $ 4.36万
• 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/9125794
• 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; Health; Histone Acetylation; Histones; Human; In Vitro; Kinetics; Laboratories; Length; Link; Lyase; 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; cofactor; differential expression; enzyme activity; in vivo; knock-down; mutant; non-histone protein; p300/CBP-Associated Factor; protein complex; 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）：本提案的目的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变化联系起来。赖氨酸乙酰基转移酶（KATs）一般控制非去阻遏蛋白5（GCN 5）和p300/CBP相关因子（PCAF）是全局性转录因子，其在多亚基复合物中起作用以乙酰化组蛋白和非组蛋白来调节基因转录。作为KAT活性的必要辅因子，Ac-CoA细胞浓度与GCN 5和PCAF活性的增加速率、蛋白质靶乙酰化和促生长基因的转录激活直接相关。在后生动物中，Ac-CoA通过酶三磷酸腺苷（ATP）-柠檬酸裂解酶（ACLY）从葡萄糖衍生的柠檬酸盐中产生。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的产生驱动了组蛋白乙酰化的增加。此外，许多活跃增殖的细胞表现出增加的ACLY水平，并且癌细胞的增殖特别对葡萄糖的可用性和适当的ACLY功能敏感。ACLY还经历了响应于升高的葡萄糖水平和通过KAT依赖性转录激活的促生长信号传导途径的激活的稳定性增加。此外，来自Wellen实验室的新的未发表数据表明，ACLY和PCAF在细胞中形成复合物，补充了最近的一份报告，该报告表明PCAF乙酰化ACLY以促进ACLY稳定性和肿瘤生长。然而，ACLY和GCN 5/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，该提议试图检验以下假设：ACLY与GCN 5和PCAF形成直接复合物以促进ACLY乙酰化和稳定性以及GCN 5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。将使用体外下拉试验对生物化学纯化的ACLY和GCN 5/PCAF进行研究，以确定它们是否可以直接相互结合。然后，相互作用的生物物理特性将用ITC表征，以及通过酶活性测定相互作用的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为ACLY与GCN 5/PCAF的相互作用提供了分子基础。最后，该提案将通过破坏这种相互作用并询问哺乳动物细胞系的全局组蛋白乙酰化、特定代谢和生长促进基因的表达以及这些细胞增殖和分化的能力来确定ACLY-GCN 5/PCAF相互作用的体内后果。总之，这些研究将描述AcCoA代谢产物如何将细胞代谢与基因表达联系起来的分子机制，并将为靶向ACLY功能的癌症治疗提供新的途径。