Enhancement of ubiquitination-dependent mitophagy by unconventionally-acting PROTACs

Enhancing mitophagy, a naturally-occurring cellular process for elimination of damaged mitochondria, holds great promise for the intervention of many human diseases. Proteolysis-targeting chimeras (PROTACs) are heterobifunctional molecules that induce ubiquitination and subsequent proteasome-mediated degradation of a target protein through simultaneously binding to the target protein and an E3 ubiquitin ligase. However, the narrow cavity of the proteasome prevents the degradation of mitochondria. Here we show that the E3 ubiquitin ligase MAP3K1, when recruited to the outer mitochondria membrane (OMM) protein TSPO by our PROTAC-designed molecules (termed “mitophagy-enhancing chimeras”, or MECs), induced extensive K63 ubiquitination of TSPO and other OMM proteins, reminiscent of the PINK1-activated Parkin, without triggering proteasome-mediated degradation of TSPO. Aided by NBR1 and Nur77, this increased K63 ubiquitination of OMM proteins triggered mitophagy exclusively for damaged mitochondria, leading to improved mitochondria function and diminished cellular ROS. With the capability to enhance mitophagy at low nanomolar concentrations, MECs effectively inhibited NLRP3 inflammasome activation, abrogated acetaminophen-induced acute liver injury and mitigated high-fat diet-induced obesity in mice. Our work provided a proof-of-concept for developing unconventionally-acting PROTACs to achieve degradation of damaged mitochondria and possibly other organelles. To assess MEC1 in a chronic disease, we first fed C57Bl/6 mice with either a high-fat diet (HFD) or normal diet (ND) for 2 months to verify the accelerated body weight gain by the HFD feeding, and then randomly divided the HFD mice to a control and a MEC1 treatment group, which received daily intraperitoneal (IP) injection of vehicle and 0.5 mg/kg MEC1, respectively.

增强线粒体自噬（mitophagy）——一种天然存在的清除受损线粒体的细胞过程——在多种人类疾病的干预中具有巨大应用前景。蛋白水解靶向嵌合体（Proteolysis-targeting chimeras, PROTACs）是一类双功能小分子，可同时结合靶蛋白与E3泛素连接酶，诱导靶蛋白发生泛素化并经蛋白酶体介导降解。然而，蛋白酶体的狭窄腔道无法实现线粒体的降解。本研究发现，通过我们设计的名为"增强线粒体自噬嵌合体（mitophagy-enhancing chimeras, MECs）"的PROTAC分子，将E3泛素连接酶MAP3K1招募至线粒体外膜（outer mitochondria membrane, OMM）蛋白TSPO后，可诱导TSPO及其他线粒体外膜蛋白发生广泛的K63位泛素化，这一过程类似PINK1激活的Parkin通路，但不会引发TSPO的蛋白酶体介导降解。在NBR1与Nur77的辅助下，线粒体外膜蛋白的K63位泛素化水平升高可特异性触发受损线粒体的自噬，进而改善线粒体功能并降低细胞内活性氧（ROS）水平。MECs在纳摩尔级低浓度下即可增强线粒体自噬，可有效抑制NLRP3炎症小体激活，消除对乙酰氨基酚诱导的急性肝损伤，并缓解高脂饮食诱导的小鼠肥胖。本研究为开发非常规作用机制的PROTACs以实现受损线粒体乃至其他细胞器的降解提供了概念验证。为评估MEC1在慢性疾病中的作用，我们首先将C57BL/6小鼠分为两组，分别饲喂高脂饮食（high-fat diet, HFD）与正常饮食（normal diet, ND），持续2个月以验证高脂饮食可加速小鼠体重增加；随后将高脂饮食喂养的小鼠随机分为对照组与MEC1处理组，分别每日腹腔内（intraperitoneal, IP）注射溶媒与0.5 mg/kg的MEC1。