Upregulation of COX4-2 via HIF-1a stabilization in COX4-1 deficiency

Cytochrome-c-oxidase (COX) subunit 4 (COX4) plays important roles in the function, assembly and regulation of COX (mitochondrial respiratory complex 4), the terminal electron acceptor of the oxidative phosphorylation (OXPHOS) system. The principal COX4 isoform, COX4-1, is expressed in all tissues, whereas COX4-2 is mainly expressed in the lungs, or under hypoxia and other stress conditions. We have previously described a patient with a COX4-1 defect with a relatively mild clinical presentation compared to other primary COX deficiencies, and hypothesized that this could be the result of compensatory upregulation of COX4-2. To this end, COX4-1 was downregulated using a stable expression of COX4I1-targeting shRNAs in human foreskin fibroblasts (HFF), to mirror and compare it to the primary patient's cells. COX4-1, COX4-2 and HIF-1a were detected by immunocytochemistry. The mRNA transcripts of both COX4 isoforms and HIF-1 target genes were carried out by RT-qPCR. COX activity and OXPHOS function were measured by enzymatic and oxygen consumption assays, respectively. Pathways were analyzed by CEL-Seq2 and by RT-qPCR. Overall design: mRNA profiles of COX4I1 knockdown cells (5 plicates) vs. control (5 plicates)

细胞色素c氧化酶（Cytochrome-c-oxidase, COX）亚基4（COX4）在COX（线粒体呼吸链复合体4）——氧化磷酸化（OXPHOS）系统的末端电子受体——的功能、组装与调控中发挥关键作用。COX4的主要同工型COX4-1在所有组织中均有表达，而COX4-2主要在肺组织中表达，或在缺氧及其他应激条件下被诱导表达。本课题组此前曾报道1例携带COX4-1缺陷的患者，其临床表现相较于其他原发性COX缺陷症患者相对轻微，并据此提出假说：该表型可能源于COX4-2的代偿性上调。为验证该假说，本研究通过在人包皮成纤维细胞（HFF）中稳定表达靶向COX4I1的短发夹RNA（shRNA）以下调COX4-1的表达，以此模拟并对比该原发性患者的细胞表型。研究采用免疫细胞化学法检测COX4-1、COX4-2及缺氧诱导因子-1α（HIF-1α）的蛋白水平；通过逆转录实时定量聚合酶链反应（RT-qPCR）分析两种COX4同工型及HIF-1靶基因的mRNA转录本表达；分别采用酶学分析法与耗氧量检测法测定COX活性及OXPHOS功能；通过CEL-Seq2测序技术与RT-qPCR解析相关信号通路。实验整体设计：COX4I1敲低细胞组（5个重复样本）与对照组（5个重复样本）的mRNA表达谱。