蛋白激酶RIP1对于细胞自噬的代谢调控机制研究

Autophagy is an evolutionarily conserved mechanism to maintain the cellular homeostasis in physiological and pathological conditions. Dysregulation in metabolism has played a critical role in regulating autophagy, and is closely associated with many diseases such as cancer, neurodegeneration. Protein kinase RIP1 is a crucial regulator of cell survival and death, and it has been reported that RIP1 also regulates autophagy. However, the regulatory mechanism is still unclear. Here, we aim to systematically study the regulatory mechanism of RIP1 on autophagy and cellular metabolism. Our preliminary metabolomics data showed that the level of aspartate and its metabolism were significantly elevated in RIP1-/- cells, accompanied by an increase of ATP production. Therefore, we hypothesized that RIP1 may regulate autophagy through modulating aspartate metabolism. We believe this project will not only broaden our vision on the regulatory mechanisms of autophagy and the function of RIP1, but also shed light on the new target for drug discovery of autophagy and RIP1 related diseases.

自噬对于维持细胞存活和机体发挥正常功能十分关键，而代谢异常往往引发自噬异常，导致多种重大疾病如肿瘤、神经退行性疾病等。蛋白激酶RIP1是凋亡和坏死的关键调控因子，对自噬也有重要调节作用，是治疗多种疾病的重要靶标。但目前RIP1对于自噬的调控机制尚不清楚，阻碍了以RIP1为靶点治疗自噬异常相关疾病的发展。申请人拟以RIP1缺失小鼠为模型，运用代谢组学技术分析调控自噬的代谢通路，结合细胞生物学和基因表达分析等手段探索RIP1调节自噬的代谢机制。预实验发现RIP1缺失小鼠的自噬水平较野生型明显降低，且RIP1缺失会导致天冬氨酸和ATP代谢水平改变，进而调节自噬水平。据此假设RIP1通过调控天冬氨酸代谢而调节自噬，拟开展实验研究其中的代谢调控机理和信号传导的分子机制。本研究开创性地从代谢的角度研究RIP1的功能，探索RIP1调控的代谢对自噬的作用机制，为治疗自噬异常引起相关疾病提供新的思路。

RIPK1是细胞死亡和存活的关键调节因子。Ripk1基因缺失促进小鼠出生前存活，但抑制新生小鼠存活,以上现象的机制尚不清楚。代谢调节和自噬对新生儿存活至关重要。哺乳动物出生后未哺乳前这一时期处于严重饥饿状态，细胞自噬在出生后饥饿时期发挥重要功能，提示RIPK1可能通过调控细胞自噬在饥饿应激中发挥重要作用。代谢调控对于饥饿条件下细胞及机体维持能量稳态及存活非常关键。我们的代谢组学分析显示Ripk1缺乏增加了新生小鼠和饥饿条件下哺乳动物细胞中的天冬氨酸水平。天冬氨酸累积促进TCA-氧化磷酸化能量代谢，造成饥饿条件下ATP水平的异常升高，抑制了饥饿诱导的AMPK/ULK1通路,最终导致饥饿诱导的细胞自噬失败。转录组分析发现了RIPK1缺失显著下调了多个天冬氨酸分解代谢酶。转录因子预测分析结果显示SP1是天冬氨酸分解代谢酶的共同调控因子。后续分子机制研究揭示RIPK1缺失下调转录因子SP1的磷酸化，抑制了SP1的核定位，从而导致SP1转录活性降低。此项研究发现了蛋白激酶RIPK1的一个新的代谢调控功能，解释了RIPK1缺失导致的出生致死表型的分子机制，为RIPK1、自噬及饥饿应激异常引起相关疾病的分子机制研究和治疗提供了新的思路。