PDF(Pubmed)
Abstract：
Recent genome-wide association studies have identified a missense variant p.A165T in mitochondrial amidoxime-reducing component 1 (mARC1) that is strongly associated with protection from all-cause cirrhosis and improved prognosis in nonalcoholic steatohepatitis. The precise mechanism of this protective effect is unknown. Substitution of alanine 165 with threonine is predicted to affect mARC1 protein stability and to have deleterious effects on its function. To investigate the mechanism, we have generated a knock-in mutant mARC1 A165T and a catalytically dead mutant C273A (as a control) in human hepatoma HepG2 cells, enabling characterization of protein subcellular distribution, stability, and biochemical functions of the mARC1 mutant protein expressed from its endogenous locus. Compared to WT mARC1, we found that the A165T mutant exhibits significant mislocalization outside of its traditional location anchored in the mitochondrial outer membrane and reduces protein stability, resulting in lower basal levels. We evaluated the involvement of the ubiquitin proteasome system in mARC1 A165T degradation and observed increased ubiquitination and faster degradation of the A165T variant. In addition, we have shown that HepG2 cells carrying the MTARC1 p.A165T variant exhibit lower N-reductive activity on exogenously added amidoxime substrates in vitro. The data from these biochemical and functional assays suggest a mechanism by which the MTARC1 p.A165T variant abrogates enzyme function which may contribute to its protective effect in liver disease.
摘要：
最近的全基因组关联研究已经确定了线粒体胺肟减少成分1（mARC1）中的错义变体p.A165T，其与非酒精性脂肪性肝炎（NASH）的全因肝硬化保护和预后改善密切相关。这种保护作用的确切机制是未知的。预测用苏氨酸取代丙氨酸165会影响mARC1蛋白的稳定性，并对其功能产生有害影响。为了研究机制，我们在人肝癌HepG2细胞中产生了敲入突变体mARC1A165T和催化死亡突变体C273A（作为对照），能够表征蛋白质亚细胞分布，稳定性,和从其内源性基因座表达的mARC1突变蛋白的生化功能。与野生型（WT）mARC1相比，我们发现A165T突变体在其锚定在线粒体外膜的传统位置之外表现出明显的错误定位，并降低了蛋白质的稳定性，导致较低的基础水平。我们评估了泛素蛋白酶体系统在mARC1A165T降解中的参与，并观察到A165T变体的泛素化增加和降解更快。此外,我们已经表明，携带MTARC1p.A165T变体的HepG2细胞在体外对外源添加的胺肟底物表现出较低的N-还原活性。来自这些生化和功能测定的数据表明MTARC1p.A165T变体消除酶功能的机制，这可能有助于其在肝病中的保护作用。
