原生动物盘基网柄菌自噬相关蛋白ATG16敲除导致蛋白酶体活性降低的机制研究

Autophagy and ubiquitin proteasome system (UPS) are two major intracellular pathways for protein degradation and are crucial for normal protein and cell homeostasis. Extensive evidence has shown that there is a crosstalk between these two degradation pathways. In previous study, we showed that ATG16 knock out resulted in a significant decrease in proteasomal activity in Dictyostelium discoideum, however, the underlying mechanism needs more elucidations. To address how ATG16 connects autophagy and UPS, and the function of ATG16 in the crosstalk between autophagy and UPS, live imaging will be employed to monitor the colocalization of proteasomal subunits with autophagosome or autolysosome, and Western Blot will be performed to detect the generation of GFP monomers from GFP fusions in MG132-treated cells. In addition, the intact proteasomes (26S) from wild-type AX2 and ATG16 knock out cells will be isolated using Native-PAGE, followed by in-gel chymotrypsin-like proteasomal activity assay and by immunoblotting analysis with specific antibodies against proteasomal subunits. Furthermore, the accumulated proteins in ATG16 knock out cells will be identified using iTRAQ, and then these proteins which could result in a decrease in proteasomal activity but an increase in ubiquitinylated proteins upon overexpression will be screened. Taken together, this study will reveal the mechanism of decreased proteasomal activity in ATG16 knock out cells in three aspects: the autophagic degradation of inactivated proteasomes, the numbers and activity of intact proteasomes, and inhibitors of the proteasomes. Results of this project will provide: 1) an important evidence for direct interaction between autophagy and proteasome which mediated by ATG16; 2) new regulation model of proteostasis or homeostasis in Dictyostelium discoideum.

细胞稳态的维持是机体进行正常生命活动的必要条件。自噬和蛋白酶体系统作为细胞内两大主要降解途径，其精确调控对细胞稳态的维持是必需的。在原生动物盘基网柄菌中，前期研究发现，自噬相关蛋白ATG16敲除导致蛋白酶体活性降低，但其作用机制尚不清楚。本课题拟应用蛋白酶体抑制剂处理细胞，通过实时动态影像技术检测蛋白酶体与自噬体或自噬溶酶体的共定位，结合GFP单体切割实验检测蛋白酶体是否通过自噬降解；同时，在野生型和ATG16敲除细胞中，利用Native-PAGE分离蛋白酶体，并定量检测其活性和数量；最后，利用iTRAQ定量蛋白质组学技术筛选突变体细胞中的差异表达蛋白，通过生化、分子和细胞学研究，鉴定能够抑制蛋白酶体活性的相关蛋白。本课题通过深入探索ATG16缺失导致蛋白酶体活性降低的分子机制，一方面为了解盘基网柄菌中细胞稳态的维持机制提供重要的理论基础，另一方面也为自噬和蛋白酶体系统的交联提供新的证据。

细胞稳态的维持是机体进行正常生命活动的必要条件。自噬和蛋白酶体系统作为细胞内两大主要降解途径，其精确调控对细胞稳态的维持是必需的。在原生动物盘基网柄菌中，前期研究发现，自噬相关蛋白ATG16敲除导致蛋白酶体活性降低，但其作用机制尚不清楚。本研究通过构建AX2/RFP-PSMD1/PSMA4-GFP细胞株，通过免疫共沉淀实验证明PSMA4-GFP与蛋白酶体各亚基相互作用，说明PSMA4-GFP和RFP-PSMD1能够整合进蛋白酶体中，但是GFP切割实验显示PSMA4-GFP不通过自噬途径降解。运用TMT蛋白质组学技术分析了野生型AX2和自噬缺陷型ATG9¯，ATG16¯和ATG9¯/16¯细胞中的差异蛋白，GO分析表明，自噬缺陷导致细胞内脂质代谢，氧化磷酸化和活性氧清除过程受损，联合转录组学分析表明，自噬缺陷细胞中的差异蛋白并不是由于基因转录变化引起的，推测可能是由于自噬缺陷导致了蛋白质稳态失衡。TMT结果同时显示，自噬缺陷细胞中蛋白酶体各亚基的蛋白表达量没有发生显著变化，但是蛋白酶体组装的分子伴侣POMP在ATG9¯/16¯细胞中显著降低，预示ATG9¯/16¯细胞中的蛋白酶体数量减少。但是native-PAGE结果显示，ATG9¯/16¯细胞中的蛋白酶体数量相比野生型AX2是显著增加的，过表达GFP-POMP并不会增加蛋白酶体的数量和活性，也不会恢复ATG9¯/16¯细胞的发育缺陷，说明POMP并不是导致自噬缺陷细胞中蛋白酶体活性降低的原因。通过测定ATG9¯/16¯细胞中的蛋白酶体活性，发现无论是蛋白酶体的最高活性和蛋白酶体降解底物的速率，ATG9¯/16¯细胞都比AX2显著降低，说明自噬缺陷细胞ATG9¯/16¯中蛋白酶体活性的降低并不是由于蛋白酶体数量减少导致的，自噬缺陷细胞中应该存在一种机制抑制了其蛋白酶体的活性，该机制还需进一步研究。