Molecular and cancer-related roles of ACLY exon 14

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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.