title: Probing lipid-protein adduction with alkynyl surrogates: application to Smith-Lemli-Opitz syndrome.
abstract: Lipid modifications aid in regulating (and misregulating) protein function and localization. However, efficient methods to screen for a lipid's ability to modify proteins are not readily available. We present a strategy to identify protein-reactive lipids and apply it to a neurodevelopmental disorder, Smith-Lemli-Opitz syndrome (SLOS). Alkynyl surrogates were synthesized for polyunsaturated fatty acids, phospholipids, cholesterol, 7-dehydrocholesterol (7-DHC), and a 7-DHC-derived oxysterol. To probe for protein-reactive lipids, we used click chemistry to biotinylate the alkynyl tag and detected the lipid-adducted proteins with streptavidin Western blotting. In Neuro2a cells, the trend in amount of protein adduction followed known rates of lipid peroxidation (7-DHC >> arachidonic acid > linoleic acid >> cholesterol), with alkynyl-7-DHC producing the most adduction among alkynyl lipids. 7-DHC reductase-deficient cells, which cannot properly metabolize 7-DHC, exhibited significantly more alkynyl-7-DHC-protein adduction than control cells. Model studies demonstrated that a 7-DHC peroxidation product covalently modifies proteins. We hypothesize that 7-DHC generates electrophiles that can modify the proteome, contributing to SLOS's complex pathology. These probes and methods would allow for analysis of lipid-modified proteomes in SLOS and other disorders exhibiting 7-DHC accumulation. More broadly, the alkynyl lipid library would facilitate exploration of lipid peroxidation's role in specific biological processes in numerous diseases.
authors.affiliation: Departments of Chemistry, Vanderbilt University, Nashville, TN; and.
authors.forename: Katherine
authors.initials: K
authors.lastname: Windsor
authors.forename: Thiago C
authors.initials: TC
authors.lastname: Genaro-Mattos
authors.forename: Hye-Young H
authors.initials: HY
authors.lastname: Kim
authors.forename: Wei
authors.initials: W
authors.lastname: Liu
authors.forename: Keri A
authors.initials: KA
authors.lastname: Tallman
authors.forename: Sayuri
authors.initials: S
authors.lastname: Miyamoto
authors.forename: Zeljka
authors.initials: Z
authors.lastname: Korade
authors.forename: Ned A
authors.initials: NA
authors.lastname: Porter
mesh_terms: D045744:Cell Line, Tumor; D045304:Cytochromes c / Q000737:chemistry / Q000378:metabolism; D003684:Dehydrocholesterols / Q000737:chemistry / Q000378:metabolism; D005231:Fatty Acids, Unsaturated / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D054878:Lipoylation; D010084:Oxidation-Reduction; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000378:metabolism; D019082:Smith-Lemli-Opitz Syndrome / Q000175:diagnosis / Q000378:metabolism*
chemical_list: D003684:Dehydrocholesterols; D005231:Fatty Acids, Unsaturated; D010743:Phospholipids; D020543:Proteome; D045304:Cytochromes c; C016705:7-dehydrocholesterol
keywords: 7-dehydrocholesterol; cholesterol; lipid electrophiles; lipid peroxidation; oxysterol