NRF-1调控PINK1/Parkin介导的线粒体自噬对应激条件下细胞适应的影响

Mitophagy mediated by PINK1/Parkin plays an essential role in mitochondrial quality control which is conducive for cells to maintain normal physiological functions and contributes to cellular adaptation to stress. Although there has been considerable progress in the elucidation of various aspects of PINK1 and Parkin post-translational modifications such as phosphorylation activation, stability and degradation, the transcriptional regulation of PINK1 and Parkin remains unclear. The nuclear respiratory factor-1 (NRF-1) serves as a transcription factor that activates the expression of a wide range of nuclear genes essential for mitochondrial biogenesis and function, including mitochondrial respiratory complex subunits. Recent studies have identified the existence of the NRF-1 binding consensus sequence in the proximal promoter regions of more than two thousand genes, suggesting more potential functions of NRF-1 might be discovered. Our studies have identified for the first time that NRF-1 affected mitophagy in a variety of cells. We also discovered the putative NRF-1 core binding sequences in the 5’-regulator region of pink1 and parkin gene and the binding of NRF-1 to pink1 and parkin gene was verified by ChIP. When NRF-1 expression was changed either by RNA interference or by pathological stimuli, the mRNA and protein levels of pink1 and parkin were affected which indicated the positive regulation of pink1 and parkin transcription by NRF-1. These results put forward a logical hypothesis that NRF-1 can affect mitophagy through the positive regulation of PINK1 and Parkin expression and involves in mitochondria quality control, promoting cellular adaptation and survival under stress conditions. The study will suggest a new founction of NRF-1 in mitochondria quality control and a novel mechanism of mitophagy, also extend our understanding on mechanisms of cellular adaptation to stress conditions.

PINK1/Parkin介导的线粒体自噬在线粒体质量控制中发挥重要作用，有利于维持细胞的正常生理功能，促进细胞对多种应激的适应。目前对于PINK1和Parkin转录水平的调控机制还未阐明。NRF-1是公认的调控线粒体呼吸链蛋白表达的核转录因子，但最新研究提示NRF-1可能具有更多的潜在功能。我们的研究首次发现NRF-1对线粒体自噬有明显影响。进一步研究揭示，pink1和parkin基因5’-调控区有潜在的NRF-1结合位点，能发生具有功能性的实际结合，对转录有正调控作用。另外，应激条件下NRF-1的表达变化能够明显影响PINK1和Parkin的表达。故假设NRF-1能通过调控PINK1和Parkin的表达影响线粒体自噬，直接参与线粒体质量控制，对细胞在应激下的适应发挥重要作用。本研究将发现NRF-1参与线粒体质量控制的新功能，提出线粒体自噬调控的新机制，拓展对应激条件下细胞适应机制的认识。

核呼吸因子1（Nuclear respiratory factor 1, NRF-1）是调控核基因编码的线粒体呼吸链相关蛋白表达的转录因子之一。本项目组通过ChIP-Seq技术发现3045个基因的启动子区含有NRF-1的共有结合序列，这些理论上NRF-1的靶基因，其功能不仅与线粒体相关，还涉及RNA代谢、细胞周期、DNA损伤修复、激素调节、炎症反应等，提示NRF-1可能具有广泛的生理功能。本项目首次研究了NRF-1对线粒体自噬相关基因PINK1和Parkin的转录调控作用，并探讨了NRF-1对PINK1/Parkin介导的线粒体自噬的影响。结果表明，NRF-1能够结合于人PINK1和Parkin基因的启动子区，对PINK1和Parkin基因的表达发挥正调控作用。另外，敲降不同细胞NRF-1的表达，细胞的线粒体自噬能力明显减弱，提示NRF-1可以通过调控PINK1/Parkin介导的线粒体自噬，参与线粒体的质量控制。我们还发现，敲降NRF-1的表达，线粒体的更新速率明显变慢。这说明NRF-1可能在线粒体生物合成和线粒体清除中都发挥了关键的调控作用，对线粒体的更新至关重要。本项目还研究了间歇性低氧（Intermittent Hypoxia，IH）对NRF-1表达的影响，并探讨了PINK1/Parkin介导的线粒体自噬对IH抗细胞氧化应激损伤的影响，明确了NRF-1的调控作用。结果发现，适度的IH对OGD/R、AMA以及H2O2诱导的肝细胞氧化应激损伤都具有保护作用。IH增强了PINK1/Parkin介导的线粒体自噬能力，直接参与了IH诱导的细胞保护作用。IH诱导线粒体自噬能力的增强与PINK1和Parkin的表达上调有关，NRF-1在其中发挥了关键的调控作用。本项目还进一步的拓展了对NRF-1功能的研究,发现NRF-1能够直接调控Star基因，介导低氧诱导的雄性大鼠睾酮合成下调；还能直接调控p65基因，对慢性阻塞性肺疾病条件下NFκB通路的激活发挥重要的调控作用。通过本项目的开展，首次明确了NRF-1在线粒体质量控制中的作用，拓宽了对线粒体网络动态调控的认识。另外，本项目的研究成果还有助于进一步深入阐明NRF-1的生物学功能，加深对一些重要病理生理过程的理解。