Molecular and cancer-related roles of ACLY exon 14

项目摘要 这项建议的目标是了解ATP选择性剪接和磷酸化的分子作用。 柠檬酸裂解酶(ACLY)外显子14以及这些事件如何连接到促进肿瘤生长的细胞途径。 ACLY是细胞内葡萄糖衍生的非线粒体乙酰辅酶A的主要来源。胞浆乙酰-辅酶A是一种 脂肪酸和胆固醇合成的基本构件，核乙酰辅酶A参与表观遗传学 通过组蛋白乙酰化进行调节。ACLY的失调与癌症和其他代谢相关 尽管最近的数据表明ACLY的剪接异构体也可以在疾病中发挥重要作用，但ACLY的剪接异构体在疾病中也发挥了重要作用。 具体地说，优先选择全长ACLY，而不是去除外显子14的主要剪接变体 与正常组织相比，在许多肿瘤组织中均有表达。此外，外显子14具有几个分子特征， 包括它在蛋白质表面的位置、无序结构、与假定的核定位的并列 序列，以及已知被磷酸化的丝氨酸(S481)，这使得外显子14可能是 ACLY规则。总之，这些观察结果证明了我提出的确定外显子14在人类基因组中的作用的研究的合理性。 ACLY规则与癌症。为了进行我的研究，我选择了肝细胞的细胞和小鼠模型 癌和结直肠癌，这两种癌症都是优先表达全长ACLY和 都已知增加了新生脂肪生成，以研究这一表型。我的策略包括使用 荧光显微镜和细胞分级研究亚细胞定位、代谢组学和 确定代谢/表观遗传后果的蛋白质组学，以及克隆形成试验和小鼠研究 目的1评价ACLY异构体和S481突变体的促癌作用。此外，我会 通过鉴定外显子14介导的这些调控的生化和结构基础 蛋白质-蛋白质与基因编码的交联物的相互作用，执行比较酶和 热稳定性分析，并与全长的冷冻电子显微镜结构进行比较 ACLY的结构(目标2)。总之，这些实验将解释外显子14的分子作用，如何 选择性剪接影响细胞乙酰辅酶A的利用，以及外显子14包涵体可能如何支持肿瘤 增长，这可能导致新的治疗策略。

Project Summary The goal of this proposal is to understand the molecular role of alternative splicing and phosphorylation of ATP- citrate lyase (ACLY) exon 14 and how these events may connect to cellular pathways that promote tumor growth. ACLY is the main source of glucose-derived, nonmitochondrial acetyl-CoA in the cell. Cytosolic acetyl-CoA is an essential building block for fatty acid and cholesterol synthesis, and nuclear acetyl-CoA participates in epigenetic regulation via histone acetylation. Dysregulation of ACLY is associated with cancer and other metabolic diseases, although recent data suggests that splice isoforms of ACLY can also play a major role in disease. Specifically, full-length ACLY, rather than a major splice variant with exon 14 removed, is preferentially expressed in many tumors compared to normal tissue. Moreover, exon 14 has several molecular features, including its location on the protein surface, disordered structure, juxtaposition to putative nuclear localization sequences, and a serine (S481) which is known to be phosphorylated, which make exon 14 a likely player in ACLY regulation. Together, these observations justify my proposed studies to determine the role of exon 14 in ACLY regulation and cancer. To carry out my studies, I have selected cell and mouse models of hepatocellular carcinoma and colorectal cancer, both of which are cancer types that preferentially express full-length ACLY and are known to have increased de novo lipogenesis, to study this phenotype. My strategy involves using fluorescence microscopy and cellular fractionation to study subcellular localization, metabolomics and proteomics to determine metabolic/epigenetic consequences, and colony formation assays and mouse studies to evaluate the tumor-promoting properties of ACLY isoforms and S481 mutants (Aim 1). Furthermore, I will characterize the biochemical and structural underpinnings of these modulations by identifying exon 14-mediated protein-protein interactions with genetically-encoded crosslinkers, performing comparative enzymatic and thermal stability assays, and determining cryo-electron microscopy structures for comparison with the full-length structure of ACLY (Aim 2). Together, these experiments will explain the molecular roles of exon 14, how alternative splicing impacts cellular acetyl-CoA utilization, and how exon 14 inclusion may be supporting tumor growth, which may lead to novel therapeutic strategies.