Tallman Keri A, Kim Hye-Young H, Korade Zeljka, Genaro-Mattos Thiago C, Wages Phillip A, Liu Wei, Porter Ned A
Department of Chemistry and Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, TN 37235, United States.
Vanderbilt Kennedy Center for Research on Human Development, Vanderbilt University, Nashville, TN 37235, United States; Department of Psychiatry, Vanderbilt University, Nashville, TN 37235, United States.
Redox Biol. 2017 Aug;12:182-190. doi: 10.1016/j.redox.2017.02.013. Epub 2017 Feb 24.
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.
脂质亲电试剂 - 蛋白质加合物的形成与许多涉及细胞氧化还原状态扰动的疾病有关。加合蛋白质的身份以及加合对蛋白质功能的影响大多未知，对这些因素的进一步了解可能有助于确定各种涉及氧化应激的人类疾病的发病机制。7-脱氢胆固醇（7-DHC）是胆固醇的直接生物合成前体，极易氧化，会生成易于与蛋白质加合的亲电氧化甾醇，这一系列事件被认为发生在史密斯-勒米-奥皮茨综合征（SLOS）中，这是一种由胆固醇生物合成错误导致的人类疾病。炔基羊毛甾醇（α-Lan）在Neuro2a细胞、Dhcr7缺陷型Neuro2a细胞和人成纤维细胞中合成并进行了研究。当在对照Neuro2a细胞和对照人成纤维细胞中孵育时，α-Lan完成了胆固醇生物合成所涉及的一系列步骤，炔基胆固醇（α-Chol）是形成的主要产物。在Dhcr7缺陷型Neuro2a细胞或SLOS患者的成纤维细胞中，生物合成转化在倒数第二步被中断，炔基-7-DHC（α-7-DHC）是形成的主要产物。当α-Lan在Dhcr7缺陷型Neuro2a细胞中孵育，并使用炔基标签将生物素基团连接到含炔烃的产物上时，蛋白质 - 甾醇加合物被分离并鉴定出来。在Dhcr7缺陷型Neuro2a细胞中对α-Lan和α-7-DHC进行的平行实验中，发现α-7-DHC与比α-Lan（457种）更多的一组蛋白质（799种）加合，大多数α-Lan蛋白质加合物（423种）与更大的α-7-DHC组共有。在两个实验中发现的423种共有蛋白质中，与二甲基亚砜对照相比，由α-7-DHC形成的蛋白质比由α-Lan衍生的蛋白质富集程度更高。根据在α-Lan和α-7-DHC实验中为每种蛋白质确定的数据富集程度对这423种共有蛋白质进行排名，并且在平行实验中形成的加合物的蛋白质排名具有非常强的相关性。
