Department of Life Sciences, Pohang University of Science and Technology, Pohang, Gyeongbuk, South Korea.
Department of Life Sciences, Korea University, Seoul, South Korea.
J Biol Chem. 2024 Oct;300(10):107731. doi: 10.1016/j.jbc.2024.107731. Epub 2024 Aug 30.
Nα-terminal acetylation in eukaryotic proteins creates specific degradation signals (Ac/N-degrons) targeted for ubiquitin-mediated proteolysis via the Ac/N-degron pathway. Despite the identification of key components of the Ac/N-degron pathway over the past 15 years, the precise recognition domain (Ac/N domain) remains unclear. Here, we defined the Ac/N domain of the endoplasmic reticulum MARCHF6 E3 ubiquitin ligase through a systematic analysis of its cytosol-facing regions using alanine-stretch mutagenesis, chemical crosslinking-based co-immunoprecipitation-immunoblotting, and split-ubiquitin assays in human and yeast cells. The Ac/N domain of MARCHF6 exhibits preferential binding specificity to Nα-terminally acetylated proteins and peptides over their unacetylated counterparts, mediating the degradation of Ac/N-degron-bearing proteins, such as the G-protein regulator RGS2 and the lipid droplet protein PLIN2. Furthermore, abolishing the recognition of Ac/N-degrons by MARCHF6 stabilized RGS2 and PLIN2, thereby increasing the resistance to ferroptosis, an iron-dependent lipid peroxidation-mediated cell death. These findings provide mechanistic and functional insights into how MARCHF6 serves as a rheostatic modulator of ferroptosis by recognizing Ac/N-degron substrates via its Ac/N domain and non-Ac/N-degron substrates via distinct recognition sites.
真核蛋白 N 端乙酰化可创建特定的降解信号（Ac/N 降解结构域），通过 Ac/N 降解结构域途径靶向泛素介导的蛋白水解。尽管在过去 15 年中已经鉴定出 Ac/N 降解结构域途径的关键成分，但精确的识别结构域（Ac/N 结构域）仍不清楚。在这里，我们通过对人源和酵母细胞中细胞质面向区域进行丙氨酸延伸诱变、基于化学交联的共免疫沉淀-免疫印迹和分裂泛素测定的系统分析，定义了内质网 MARCHF6 E3 泛素连接酶的 Ac/N 结构域。MARCHF6 的 Ac/N 结构域对 N 端乙酰化蛋白和肽具有优先的结合特异性，而对其未乙酰化的对应物则没有，从而介导 Ac/N 降解结构域蛋白的降解，如 G 蛋白调节剂 RGS2 和脂滴蛋白 PLIN2。此外，通过 MARCHF6 消除对 Ac/N 降解结构域的识别可稳定 RGS2 和 PLIN2，从而增加对铁依赖性脂质过氧化介导的细胞死亡即铁死亡的抗性。这些发现为 MARCHF6 通过其 Ac/N 结构域识别 Ac/N 降解结构域底物，以及通过独特的识别位点识别非 Ac/N 降解结构域底物，从而作为铁死亡的动态调节因子提供了机制和功能方面的见解。
