The Choline Metabolite-Interacting Proteome of Mammalian Cells: Discovery and Significance

Abstract

Choline is an essential nutrient for mammalian cells that acts as a substrate for Kennedy pathway, wherein, upon cellular internalization, it is sequentially converted into phosphocholine and cytidine 5'-diphosphocholine to ultimately synthesize the choline lipids, phosphatidylcholine and sphingomyelin. Our understanding of the cellular functions of these metabolites, independent of their roles as intermediates of the Kennedy pathway remains limited. The elucidation of these extended functions of choline metabolites also has important implications for cancer biology as elevated cellular levels of these metabolites is an established hallmark of cancer. Towards this goal, in my doctoral thesis, I identified the choline metabolite and lipid-interacting proteome of mammalian cells by utilizing a chemoproteomic approach that entailed the development of a bifunctional choline probe adept at metabolically labeling them with the alkyne affinity tag and the diazirine photocrosslinkable group. The design of this probe was guided by the results of my structure-function studies, wherein, lipidomics and cellular imaging experiments revealed that the choline lipid biosynthetic machinery can accommodate remarkably diverse choline analogs as precursors. Additionally, these studies resulted in the serendipitous discovery of a novel choline lipid headgroup remodeling mechanism involving sequential dealkylation and methylation. Quantitative proteomics studies on mammalian cells metabolically labeled with the bifunctional choline probe resulted in the identification of 674 functionally diverse protein hits, several of which are attractive drug targets and play important roles in cancer progression. The functional relevance of choline metabolite-protein interactions was investigated by performing binding studies on one of the protein hits, the attractive anticancer target, p32. These studies revealed that phosphocholine specifically inhibits the binding of p32 to several of its endogenous ligands as well as to the promising anticancer agent, Lyp-1. Furthermore, I also discovered that choline and phosphocholine act as allosteric activators of several enzymes belonging to the Kreb’s cycle and the glycolysis pathway. Collectively, my doctoral research provides fundamental insights into choline lipid biology and establishes choline metabolites as multifunctional cellular agents that play important roles in cellular metabolism and disease physiology by modulating protein function.