Probes for protein adduction in cholesterol biosynthesis disorders: Alkynyl lanosterol as a viable sterol precursor

Keri A. Tallman, Hye Young H Kim, Zeljka Korade, Thiago Cardoso Genaro De Mattos, Phillip A. Wages, Wei Liu, Ned A. Porter

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

The formation of lipid electrophile-protein adducts is associated with many disorders that involve perturbations of cellular redox status. The identities of adducted proteins and the effects of adduction on protein function are mostly unknown and an increased understanding of these factors may help to define the pathogenesis of various human disorders involving oxidative stress. 7-Dehydrocholesterol (7-DHC), the immediate biosynthetic precursor to cholesterol, is highly oxidizable and gives electrophilic oxysterols that adduct proteins readily, a sequence of events proposed to occur in Smith-Lemli-Opitz syndrome (SLOS), a human disorder resulting from an error in cholesterol biosynthesis. Alkynyl lanosterol (a-Lan) was synthesized and studied in Neuro2a cells, Dhcr7-deficient Neuro2a cells and human fibroblasts. When incubated in control Neuro2a cells and control human fibroblasts, a-Lan completed the sequence of steps involved in cholesterol biosynthesis and alkynyl-cholesterol (a-Chol) was the major product formed. In Dhcr7-deficient Neuro2a cells or fibroblasts from SLOS patients, the biosynthetic transformation was interrupted at the penultimate step and alkynyl-7-DHC (a-7-DHC) was the major product formed. When a-Lan was incubated in Dhcr7-deficient Neuro2a cells and the alkynyl tag was used to ligate a biotin group to alkyne-containing products, protein-sterol adducts were isolated and identified. In parallel experiments with a-Lan and a-7-DHC in Dhcr7-deficient Neuro2a cells, a-7-DHC was found to adduct to a larger set of proteins (799) than a-Lan (457) with most of the a-Lan protein adducts (423) being common to the larger a-7-DHC set. Of the 423 proteins found common to both experiments, those formed from a-7-DHC were more highly enriched compared to a DMSO control than were those derived from a-Lan. The 423 common proteins were ranked according to the enrichment determined for each protein in the a-Lan and a-7-DHC experiments and there was a very strong correlation of protein ranks for the adducts formed in the parallel experiments.

Original languageEnglish (US)
Pages (from-to)182-190
Number of pages9
JournalRedox Biology
Volume12
DOIs
StatePublished - Aug 1 2017

Fingerprint

Lanosterol
Biosynthesis
Sterols
Cholesterol
Proteins
Fibroblasts
Smith-Lemli-Opitz Syndrome
Experiments
Oxidative stress
Alkynes
Biotin
Dimethyl Sulfoxide
7-dehydrocholesterol
Oxidation-Reduction
Oxidative Stress

Keywords

  • 7-dehydrocholesterol
  • Alkynyl sterols
  • Cholesterol
  • DHCR7
  • GC-MS
  • HPLC-MS
  • Smith-Lemli-Opitz Syndrome

ASJC Scopus subject areas

  • Organic Chemistry
  • Clinical Biochemistry

Cite this

Probes for protein adduction in cholesterol biosynthesis disorders : Alkynyl lanosterol as a viable sterol precursor. / Tallman, Keri A.; Kim, Hye Young H; Korade, Zeljka; Cardoso Genaro De Mattos, Thiago; Wages, Phillip A.; Liu, Wei; Porter, Ned A.

In: Redox Biology, Vol. 12, 01.08.2017, p. 182-190.

Research output: Contribution to journalArticle

Tallman, Keri A. ; Kim, Hye Young H ; Korade, Zeljka ; Cardoso Genaro De Mattos, Thiago ; Wages, Phillip A. ; Liu, Wei ; Porter, Ned A. / Probes for protein adduction in cholesterol biosynthesis disorders : Alkynyl lanosterol as a viable sterol precursor. In: Redox Biology. 2017 ; Vol. 12. pp. 182-190.
@article{09b65ea1abbb457fbdb235d1975d4cba,
title = "Probes for protein adduction in cholesterol biosynthesis disorders: Alkynyl lanosterol as a viable sterol precursor",
abstract = "The formation of lipid electrophile-protein adducts is associated with many disorders that involve perturbations of cellular redox status. The identities of adducted proteins and the effects of adduction on protein function are mostly unknown and an increased understanding of these factors may help to define the pathogenesis of various human disorders involving oxidative stress. 7-Dehydrocholesterol (7-DHC), the immediate biosynthetic precursor to cholesterol, is highly oxidizable and gives electrophilic oxysterols that adduct proteins readily, a sequence of events proposed to occur in Smith-Lemli-Opitz syndrome (SLOS), a human disorder resulting from an error in cholesterol biosynthesis. Alkynyl lanosterol (a-Lan) was synthesized and studied in Neuro2a cells, Dhcr7-deficient Neuro2a cells and human fibroblasts. When incubated in control Neuro2a cells and control human fibroblasts, a-Lan completed the sequence of steps involved in cholesterol biosynthesis and alkynyl-cholesterol (a-Chol) was the major product formed. In Dhcr7-deficient Neuro2a cells or fibroblasts from SLOS patients, the biosynthetic transformation was interrupted at the penultimate step and alkynyl-7-DHC (a-7-DHC) was the major product formed. When a-Lan was incubated in Dhcr7-deficient Neuro2a cells and the alkynyl tag was used to ligate a biotin group to alkyne-containing products, protein-sterol adducts were isolated and identified. In parallel experiments with a-Lan and a-7-DHC in Dhcr7-deficient Neuro2a cells, a-7-DHC was found to adduct to a larger set of proteins (799) than a-Lan (457) with most of the a-Lan protein adducts (423) being common to the larger a-7-DHC set. Of the 423 proteins found common to both experiments, those formed from a-7-DHC were more highly enriched compared to a DMSO control than were those derived from a-Lan. The 423 common proteins were ranked according to the enrichment determined for each protein in the a-Lan and a-7-DHC experiments and there was a very strong correlation of protein ranks for the adducts formed in the parallel experiments.",
keywords = "7-dehydrocholesterol, Alkynyl sterols, Cholesterol, DHCR7, GC-MS, HPLC-MS, Smith-Lemli-Opitz Syndrome",
author = "Tallman, {Keri A.} and Kim, {Hye Young H} and Zeljka Korade and {Cardoso Genaro De Mattos}, Thiago and Wages, {Phillip A.} and Wei Liu and Porter, {Ned A.}",
year = "2017",
month = "8",
day = "1",
doi = "10.1016/j.redox.2017.02.013",
language = "English (US)",
volume = "12",
pages = "182--190",
journal = "Redox Biology",
issn = "2213-2317",
publisher = "Elsevier BV",

}

TY - JOUR

T1 - Probes for protein adduction in cholesterol biosynthesis disorders

T2 - Alkynyl lanosterol as a viable sterol precursor

AU - Tallman, Keri A.

AU - Kim, Hye Young H

AU - Korade, Zeljka

AU - Cardoso Genaro De Mattos, Thiago

AU - Wages, Phillip A.

AU - Liu, Wei

AU - Porter, Ned A.

PY - 2017/8/1

Y1 - 2017/8/1

N2 - The formation of lipid electrophile-protein adducts is associated with many disorders that involve perturbations of cellular redox status. The identities of adducted proteins and the effects of adduction on protein function are mostly unknown and an increased understanding of these factors may help to define the pathogenesis of various human disorders involving oxidative stress. 7-Dehydrocholesterol (7-DHC), the immediate biosynthetic precursor to cholesterol, is highly oxidizable and gives electrophilic oxysterols that adduct proteins readily, a sequence of events proposed to occur in Smith-Lemli-Opitz syndrome (SLOS), a human disorder resulting from an error in cholesterol biosynthesis. Alkynyl lanosterol (a-Lan) was synthesized and studied in Neuro2a cells, Dhcr7-deficient Neuro2a cells and human fibroblasts. When incubated in control Neuro2a cells and control human fibroblasts, a-Lan completed the sequence of steps involved in cholesterol biosynthesis and alkynyl-cholesterol (a-Chol) was the major product formed. In Dhcr7-deficient Neuro2a cells or fibroblasts from SLOS patients, the biosynthetic transformation was interrupted at the penultimate step and alkynyl-7-DHC (a-7-DHC) was the major product formed. When a-Lan was incubated in Dhcr7-deficient Neuro2a cells and the alkynyl tag was used to ligate a biotin group to alkyne-containing products, protein-sterol adducts were isolated and identified. In parallel experiments with a-Lan and a-7-DHC in Dhcr7-deficient Neuro2a cells, a-7-DHC was found to adduct to a larger set of proteins (799) than a-Lan (457) with most of the a-Lan protein adducts (423) being common to the larger a-7-DHC set. Of the 423 proteins found common to both experiments, those formed from a-7-DHC were more highly enriched compared to a DMSO control than were those derived from a-Lan. The 423 common proteins were ranked according to the enrichment determined for each protein in the a-Lan and a-7-DHC experiments and there was a very strong correlation of protein ranks for the adducts formed in the parallel experiments.

AB - The formation of lipid electrophile-protein adducts is associated with many disorders that involve perturbations of cellular redox status. The identities of adducted proteins and the effects of adduction on protein function are mostly unknown and an increased understanding of these factors may help to define the pathogenesis of various human disorders involving oxidative stress. 7-Dehydrocholesterol (7-DHC), the immediate biosynthetic precursor to cholesterol, is highly oxidizable and gives electrophilic oxysterols that adduct proteins readily, a sequence of events proposed to occur in Smith-Lemli-Opitz syndrome (SLOS), a human disorder resulting from an error in cholesterol biosynthesis. Alkynyl lanosterol (a-Lan) was synthesized and studied in Neuro2a cells, Dhcr7-deficient Neuro2a cells and human fibroblasts. When incubated in control Neuro2a cells and control human fibroblasts, a-Lan completed the sequence of steps involved in cholesterol biosynthesis and alkynyl-cholesterol (a-Chol) was the major product formed. In Dhcr7-deficient Neuro2a cells or fibroblasts from SLOS patients, the biosynthetic transformation was interrupted at the penultimate step and alkynyl-7-DHC (a-7-DHC) was the major product formed. When a-Lan was incubated in Dhcr7-deficient Neuro2a cells and the alkynyl tag was used to ligate a biotin group to alkyne-containing products, protein-sterol adducts were isolated and identified. In parallel experiments with a-Lan and a-7-DHC in Dhcr7-deficient Neuro2a cells, a-7-DHC was found to adduct to a larger set of proteins (799) than a-Lan (457) with most of the a-Lan protein adducts (423) being common to the larger a-7-DHC set. Of the 423 proteins found common to both experiments, those formed from a-7-DHC were more highly enriched compared to a DMSO control than were those derived from a-Lan. The 423 common proteins were ranked according to the enrichment determined for each protein in the a-Lan and a-7-DHC experiments and there was a very strong correlation of protein ranks for the adducts formed in the parallel experiments.

KW - 7-dehydrocholesterol

KW - Alkynyl sterols

KW - Cholesterol

KW - DHCR7

KW - GC-MS

KW - HPLC-MS

KW - Smith-Lemli-Opitz Syndrome

UR - http://www.scopus.com/inward/record.url?scp=85014101322&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85014101322&partnerID=8YFLogxK

U2 - 10.1016/j.redox.2017.02.013

DO - 10.1016/j.redox.2017.02.013

M3 - Article

C2 - 28258022

AN - SCOPUS:85014101322

VL - 12

SP - 182

EP - 190

JO - Redox Biology

JF - Redox Biology

SN - 2213-2317

ER -