Department of Biochemistry and Molecular Biology, the University of Texas McGovern Medical School, Houston, Texas, USA.
University of California at San Diego, La Jolla, California, USA.
FASEB J. 2022 May;36(5):e22246. doi: 10.1096/fj.202101500RR.
Sickling is the central pathogenic process of sickle cell disease (SCD), one of the most prevalent inherited hemolytic disorders. Having no easy access to antioxidants in the cytosol, elevated levels of reactive oxygen species (ROS) residing at the plasma membrane in sickle red blood cells (sRBCs) easily oxidize membrane proteins and thus contribute to sickling. Although the ubiquitin-proteasome system (UPS) is essential to rapidly clear ROS-damaged membrane proteins and maintain cellular homeostasis, the function and regulatory mechanism of the UPS for their clearance in sRBCs remains unidentified. Elevated levels of polyubiquitinated membrane-associated proteins in human sRBCs are reported here. High throughput and untargeted proteomic analyses of membrane proteins immunoprecipitated by ubiquitin antibodies detected elevated levels of ubiquitination of a series of proteins including cytoskeletal proteins, transporters, ROS-related proteins, and UPS machinery components in sRBCs. Polyubiquitination of membrane-associated catalase was increased in sRBCs, associated with decreased catalase activity and elevated ROS. Surprisingly, shuttling of p97 (ATP-dependent valosin-containing chaperone protein), a key component of the UPS to shuttle polyubiquitinated proteins from the membrane to cytosol for proteasomal degradation, was significantly impaired, resulting in significant accumulation of p97 along with polyubiquitinated proteins in the membrane of human sRBCs. Functionally, inhibition of p97 directly promoted accumulation of polyubiquitinated membrane-associated proteins, excessive ROS levels, and sickling in response to hypoxia. Overall, we revealed that p97 dysfunction underlies impaired UPS and contributes to oxidative stress in sRBCs.
镰状化是镰状细胞病（SCD）的中心发病过程，SCD 是最常见的遗传性溶血性疾病之一。由于在细胞质中无法轻易获得抗氧化剂，因此位于镰状红细胞（sRBC）质膜中的活性氧（ROS）水平升高，容易氧化膜蛋白，从而导致镰状化。虽然泛素-蛋白酶体系统（UPS）对于快速清除 ROS 损伤的膜蛋白并维持细胞内稳态至关重要，但 UPS 清除 sRBC 中这些蛋白的功能和调节机制仍未确定。这里报道了人 sRBC 中多聚泛素化膜相关蛋白的水平升高。通过泛素抗体免疫沉淀的膜蛋白的高通量和非靶向蛋白质组学分析检测到，sRBC 中一系列蛋白的泛素化水平升高，包括细胞骨架蛋白、转运蛋白、ROS 相关蛋白和 UPS 机械组件。sRBC 中膜结合过氧化氢酶的多聚泛素化增加，与过氧化氢酶活性降低和 ROS 升高相关。令人惊讶的是，p97（ATP 依赖性含有缬氨酸的伴娘蛋白）的穿梭，UPS 将多聚泛素化蛋白从膜转运到细胞质进行蛋白酶体降解的关键组成部分，显著受损，导致 p97 与多聚泛素化蛋白一起在人 sRBC 的膜中大量积累。功能上，p97 的抑制直接促进了多聚泛素化膜相关蛋白、过量 ROS 水平和缺氧时镰状化的积累。总体而言，我们揭示了 p97 功能障碍是 UPS 受损的基础，并导致 sRBC 中的氧化应激。
