Probing lipid-protein adduction with alkynyl surrogates: Application to Smith-Lemli-Opitz syndrome

Katherine Windsor, Thiago C. Genaro-Mattos, Hye Young H. Kim, Wei Liu, Keri A. Tallman, Sayuri Miyamoto, Zeljka Korade, Ned A. Porter

Research output: Contribution to journalArticle

18 Scopus citations

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 signifi- cantly 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.

Original languageEnglish (US)
Pages (from-to)2842-2850
Number of pages9
JournalJournal of Lipid Research
Volume54
Issue number10
DOIs
StatePublished - Oct 1 2013

Keywords

  • 7-dehydrocholesterol
  • Cholesterol
  • Lipid electrophiles
  • Lipid peroxidation
  • Oxysterol

ASJC Scopus subject areas

  • Biochemistry
  • Endocrinology
  • Cell Biology

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