核受体Nur77介导的线粒体自噬的功能与机制研究

Mitochondrion is susceptible to damage from inflammation, a process that is implicated in the development of numerous inflammatory diseases, cancer and aging. Maintaining mitochondrial fidelity is therefore critical to elicit the correct signaling responses to maintain cellular homeostasis. Mitophagy, a selective form of autophagy to remove damaged mitochondria, has been identified as a plausible mechanism for mitochondrial quality control through undefined mechanisms. We previously discovered that Nur77, an orphan member of the nuclear receptor superfamily, could migrate from the nucleus to mitochondria to modulate apoptosis. In our effort to identify small molecule ligands for inducing Nur77 mitochondrial targeting, we recently found that celastrol, a potent anti-inflammatory triterpenoid quinine-methide isolated from the root of Tripterygium wilfordii, the traditional Chinese medicine known as “Thunder God Vine” could bind to Nur77, promoting its translocation to mitochondria where it interacts with tumor necrosis factor receptor-associated factor 2 (TRAF2). The interaction results in not only the inhibition of TRAF2 polyubiquitination and NF-κB activation but also the induction of Nur77 polyubiquitination that primes damaged mitochondria for autophagy through interaction with p62/SQSTM1. These results let us to hypothesize that Nur77-TRAF2 interaction mediates the coupling of inflammation and mitophagy and represents an important mechanism for mitochondrial quality control. We propose to address our hypothesis by studying the role of celastrol and its induction of Nur77 interaction with TRAF2 in promoting mitophagy and inhibiting inflammation using different in vitro and animal models. Results obtained will establish a new mitophagic pathway relevant to the development of cancer and inflammatory diseases and provide important insight into the mechanism of celastrol action.

炎症引起的线粒体损伤与许多炎症性疾病、癌症和衰老的发生发展密切相关，清除损伤线粒体、维持自身稳态尤为重要。线粒体自噬是线粒体质量控制的有效方式。本课题组前期报道了核受体Nur77迁移到线粒体调节细胞凋亡。最近的筛选发现具有抗炎作用的雷公藤红素可与Nur77结合，诱导其线粒体定位并与TRAF2相互作用，从而抑制TRAF2泛素化和NF-κB的激活，诱导Nur77的泛素化，促进泛素化Nur77与自噬接头蛋白p62/SQSTM1相互作用，诱导线粒体自噬抑制炎症。因此我们假设Nur77-TRAF2相互作用将炎症和线粒体自噬紧密联系在一起，是线粒体质量控制的重要机制。本项目将从分子、细胞、整体动物等多个层面研究雷公藤红素诱导的Nur77-TRAF2相互作用在线粒体自噬抑制炎症中的作用。本项目的成功开展将阐明一条与癌症和炎症性疾病密切相关的新型线粒体自噬通路；并阐明抗炎药物雷公藤红素的作用机制。

线粒体（mitochondrion）是细胞中的一个重要细胞器, 是细胞进行有氧呼吸及制造能量的主要场所。 近年来研究证明, 线粒体还在细胞死亡、免疫和炎症等众多生物学进程中发挥重要作用。因而, 线粒体损伤会影响细胞的正常功能，导致导致众多肿瘤、炎症性疾病和衰老的发生发展。通过线粒体自噬清除损伤的线粒体是治疗多种疾病包括肿瘤的新型及有效途径。然而, 如何清理损伤线粒体还不清楚, 阻碍了线粒体药物的研发, 是国际上的一个研究热点。本项目研究结果发现具有抗炎作用的雷公藤红素可与Nur77结合，诱导其线粒体定位并与TRAF2相互作用，从而抑制TRAF2泛素化和NF-κB的激活，诱导Nur77的泛素化，促进泛素化Nur77与自噬接头蛋白p62/SQSTM1相互作用，诱导线粒体自噬抑制炎症。该成果不仅揭示了一条全新的线粒体自噬通路, 同时也为开发雷公藤红素奠定了基础。同时, 我们研究了雷公籐红素与Nur77的结合模式, 发现了雷公藤红素与可逆性共价及非共价的模式特异性地结合到Nur77蛋白的表面位点上，雷公藤红素先以特异性的非共价键结合, 将雷公藤红素的6号位碳原子暴露于Nur77的第515号位的半胱氨酸，进而形成可逆的共价键，这种特异性的结合赋予了雷公藤红素与Nur77, 为开发新型高效低毒的雷公藤红素提供了方向。我们发现了新型有效的雷公藤红素合成方法, 设计并合成了一系列雷公雷红素衍生物,并开展了构效关系研究, 发现6号位迈克尔加成形成的共价键对于雷公藤红素靶向Nur77发挥抗炎活性至关重要，同时13号位的羰基成脂或者成酰胺后，雷公藤红素的凋亡活性将会增强，在此基础上发现了多个优化的雷公藤红素衍生物。为了进一步解决雷公藤红素毒副作用问题, 我们开发了由EpCAM适配体、聚乙二醇和树突状大分子组成的生物偶联物，将雷公藤红素特异性地转移到EpCAM丰富的肿瘤中，提高了其抗肿瘤疗效并减轻毒性。在该项目的支持下, 发表了15篇文章, 圆满的完成了预定目标,建议给予滚动支持。