Molecular and cancer-related roles of ACLY exon 14

ACLY 外显子 14 的分子和癌症相关作用

• 批准号: 10230701
• 负责人: Julianna Supplee
• 金额: $ 4.6万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10230701
• 关键词: ATP Citrate (pro-S)-Lyase; Acetyl Coenzyme A; Acyl Coenzyme A; Agar; Alternative Splicing; Biochemical; Biogenesis; Biological; Biological Assay; Cancer cell line; Carcinogens; Cell Fractionation; Cell model; Cells; Cholesterol; Citrates; Code; Coenzyme A; Colorectal Cancer; Complex; Crosslinker; Cryoelectron Microscopy; Data; Disease; Epigenetic Process; Event; Exons; Fatty Acids; Fluorescence Microscopy; Fractionation; Gene Expression; Glucose; Goals; Histone Acetylation; In Vitro; Isotopes; Knockout Mice; Laboratories; Lead; Length; Ligation; Location; Lysine; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Mediator of activation protein; Membrane Proteins; Messenger RNA; Metabolic; Metabolic Diseases; Molecular; Mus; Mutate; Normal tissue morphology; Nuclear; Nutrient; Oxaloacetates; Pathway interactions; Phenotype; Phosphorylation; Phosphotransferases; Play; Primary carcinoma of the liver cells; Property; Protein Acetylation; Protein Isoforms; Proteins; Proteomics; RNA Splicing; Reaction; Recombinant Proteins; Regulation; Role; Serine; Source; Structure; Testing; The Cancer Genome Atlas; Therapeutic; Tissues; Translating; Translations; Variant; Western Blotting; Wild Type Mouse; base; cancer type; comparative; crosslink; data mining; detection of nutrient; epigenetic regulation; experimental study; genetic regulatory protein; insight; lipid biosynthesis; lipid metabolism; metabolomics; mimetics; mouse model; mutant; neoplastic cell; novel therapeutic intervention; protein protein interaction; tumor; tumor growth; tumor metabolism

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.

项目概要 该提案的目的是了解 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 以及这些事件如何与促进肿瘤生长的细胞途径联系起来。 ACLY 是细胞中葡萄糖衍生的非线粒体乙酰辅酶 A 的主要来源。胞质乙酰辅酶A是 脂肪酸和胆固醇合成的重要组成部分，核乙酰辅酶A参与表观遗传 通过组蛋白乙酰化进行调节。 ACLY 失调与癌症和其他代谢相关 尽管最近的数据表明 ACLY 剪接亚型也在疾病中发挥重要作用。 具体来说，优先使用全长 ACLY，而不是删除了外显子 14 的主要剪接变体 与正常组织相比，在许多肿瘤中表达。此外，外显子 14 有几个分子特征， 包括其在蛋白质表面的位置、无序结构、与假定的核定位的并置 序列和已知被磷酸化的丝氨酸 (S481)，这使得外显子 14 可能成为 ACLY 监管。总之，这些观察结果证明了我提出的研究的合理性，以确定外显子 14 在 ACLY 调节与癌症。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，这两种癌症类型均优先表达全长 ACLY 和 已知其从头脂肪生成增加，以研究这种表型。我的策略包括使用 荧光显微镜和细胞分级分离用于研究亚细胞定位、代谢组学和 蛋白质组学以确定代谢/表观遗传后果，以及集落形成测定和小鼠研究 评估 ACLY 亚型和 S481 突变体的促肿瘤特性（目标 1）。此外，我将 通过识别外显子 14 介导的生物化学和结构基础来表征这些调节 蛋白质-蛋白质与基因编码交联剂的相互作用，进行比较酶促和 热稳定性测定，并确定冷冻电子显微镜结构以与全长进行比较 ACLY 的结构（目标 2）。这些实验将共同解释外显子 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 包含如何支持肿瘤 增长，这可能会带来新的治疗策略。