Date of Award
lipids, cholesterol, lipid-modified proteins, histone H3 variants, fatty-acylation, az-chol
Lipids are essential components to all known life. They serve many functions from basic building blocks to covalent protein modification and are particularly involved in signaling. Many rare and widespread human health concerns involve lipids but despite their importance, their analysis either free or protein-bound has remained difficult for decades. Here, using bioorthogonal chemistry, we have developed a robust system to identify the proteins modified by lipids and advanced the ability to image cholesterol in cells and tissues. The unbiased proteomic analysis of fatty-acylated proteins using chemical reporters undertaken here revealed a greater diversity of lipid-modified proteins in mammalian cells than had previously been appreciated. Proteins targeted by a series of fatty acid chemical reporters ranging from myristic to stearic acid showed their involvement in a vast range of cellular processes from all cellular compartments. Many nuclear proteins were found, in particular histone H3 variants. Histones H3.1, H3.2 and H3.3 were demonstrated to be modified with fatty acid chemical reporters on the conserved cysteine 110, a novel site of S-acylation on histone H3.2. This newly discovered modification of histone H3 variants could have implications for nuclear organization and chromatin regulation. Cholesterol is an abundant sterol in the membranes of higher eukaryotes that is important in mammalian physiology and disease. This ancient lipid serves multiple essential functions in membranes and signaling, but the precise mechanisms of its trafficking and function as a protein PTM remain to be fully elucidated. The multiple techniques developed to visualize cholesterol often give conflicting results and can inaccurately depict cholesterol distribution due to structural perturbations of analogs or staining specificities. Here, using click chemistry, we developed the fluorescent visualization of cholesterol protein modification and cholesterol trafficking in vivo with 25-azidonorcholesterol (az-chol). Az-chol exhibits minimal structural perturbation, is covalently attached to proteins, incorporates into membranes like cholesterol, faithfully detects alterations in lipid trafficking and reveals an asymmetrical cholesterol distribution in the brain of the developing mouse embryo. The bioorthogonal chemical reporter az-chol provides a new, sensitive and versatile tool to investigate cholesterol distribution from individual proteins to whole organisms.
Wilson, John P., "Bioorthogonal Chemical Reporters Reveal Fatty-Acylation of Histone H3 Variants and Cholesterol Modification of Proteins and Trafficking in Cells" (2011). Student Theses and Dissertations. 136.