SCFβ-TrCP调控MFN1泛素化降解介导线粒体动力学和细胞命运

The fine-tuned mitochondrial fusion and fission dynamics are crucial for the health and physiological functions of mitochondria. MFN1, a dynamin-related GTPase, is essential for mitochondrial fusion. Accumulating evidence has showed that MFN1 stability or its activity can be regulated at post- transcriptional or post-translational levels, however, its regulation is still poorly understood. Our preliminary data showed that 1) Neddylation pathway inhibitor MLN4924 treatment upregulates MFN1 expression and induces mitochondrial fission-to-fusion conversion; 2) mitochondrial located SCFβ-TrCP E3 ubiquitin ligase interacts with MFN1 and negatively regulates its expression through ubiquitination and degradation. Moreover, SCFβ-TrCP E3 ubiquitin ligase regulates mitochondrial dynamics and function; and 3) knockdown of the F-box protein β-TrCP of SCFβ-TrCP E3 ubiquitin ligase upregulates MFN1 expression and increases autophagy after amino acid starvation, while MFN1 knockdown suppresses autophagy. Therefore, we propose that the mitochondrial localized SCFβ-TrCP E3 ubiquitin ligase mediates MFN1 ubiquitination and degradation, thus regulates mitochondrial dynamics and function, and consequently mediates cell fate. To approve this hypothesis, we will explore 1) how SCFβ-TrCP E3 ubiquitin ligase regulates mitochondrial structure and function; 2) what are the underlying mechanisms of MFN1 ubiquitination and degradation by SCFβ-TrCP E3 ubiquitin ligase; and 3) how cell fate is determined by SCFβ-TrCP E3 ubiquitin ligase through the regulation of MFN1. Our proposed study will advance our understanding in the regulation of MFN1 and provide new insights into mitochondrial dynamics and cell fate. Moreover, the project will provide a new foundation for mitochondrial dynamics in physiological and pathological conditions.

MFN1为介导线粒体融合的关键蛋白，对线粒体动力学及其功能十分重要，但MFN1表达或活性调控目前尚未完全清楚。我们发现类泛素化通路抑制剂MLN4924上调MFN1表达，诱导线粒体从分裂向融合状态转换；E3泛素连接酶SCFβ-TrCP可定位于线粒体，并同MFN1相互作用；改变SCFβ-TrCP表达会影响MFN1表达、线粒体动力学及其功能；敲减SCFβ-TrCP上调MFN1表达，增加氨基酸饥饿诱导的细胞自噬，而再敲减MFN1则抑制自噬。因此，我们推测SCFβ-TrCP通过介导MFN1泛素化降解，调控线粒体动力学及其功能，影响细胞命运。我们将在本课题中进一步研究SCFβ-TrCP对线粒体动力学及其功能的影响，探索SCFβ-TrCP泛素化降解MFN1的机制，分析SCFβ-TrCP对MFN1的调控在细胞命运决定中的作用，以揭示MFN1表达调控机制，为明确线粒体动力学变化的生理病理学意义提供理论基础。

拟素化修饰异常激活与细胞能量代谢失调在多种肿瘤中已被检测到，但拟素化(neddylaton)修饰是否及如何调控细胞代谢，目前仍不明确。在本项目中，我们发现：1) 使用拟素化修饰抑制剂MLN4924处理细胞，线粒体发生从分裂向融合的转换，且呈时间及药物浓度依赖性增加；2）MLN4924处理后，细胞线粒体融合调控因子MFN1表达显著增加，MFN2表达有少量增加。另外，线粒体分裂调控因子DRP1表达在MLN4924处理后没有显著变化，但其在线粒体分布减少，细胞质分布增加；3）细胞中敲减MFN1后，MLN4924诱导的线粒体融合被完全抑制，敲减MFN2后约70%的线粒体融合被抑制，过表达DRP1抑制约50%的线粒体融合；4）MFN1含有SCFꞵ-TrCP E3泛素连接酶经典结合基序，ꞵ-TrCP1通过泛素化降解途径负向调控MFN1的表达水平及稳定性；5）非靶向代谢组学检测发现拟素化抑制后，细胞代谢发生显著性变化，其中，线粒体功能受到抑制，而糖酵解功能被激活；6）线粒体功能检测发现MLN4924促进细胞的氧气消耗速率、线粒体DNA拷贝数及线粒体ROS产生，抑制线粒体膜电位。另外，MLN4924促进PKM2的四聚化形成使其激活，进而介导MLN4924诱导的糖酵解；7）相比单药使用，联合MLN4924及氧化磷酸化抑制剂metformin或PKM2抑制剂shikonin，显著性抑制肿瘤细胞的体内和体外增殖。综上所述，本研究揭示了拟素化修饰对细胞能量代谢的调控，并且为抗肿瘤联合用药提供了理论及实验基础。