细胞自噬介导PMT1基因调节酵母细胞寿命的作用机制研究

As a scavenger, autophagy plays a pivotal role in maintaining the intracellular homeostasis, which is involved in a variety of biological processes, including cellular senescence and lifespan modulation. Our previous study has revealed that protein O-mannosyltransferase1 deletion strain (pmt1△) significantly extended the replicative lifespan (RLS) of yeast cells. Moreover, the data of quantitative PCR unveiled that the mRNA expression levels of autophagy related genes 8 (ATG8) was significantly up-regulated in pmt1△ strain; and ATG8 deficiency shortened RLS of pmt1△ strain, indicating the potential role of autophagy in the effect of PMT1 on regulating the yeast’s lifespan. Nevertheless, the mechanism of PMT1 deficiency on regulating the yeast’s life remains to be explored. In the present project, we will fully investigated into the function of PMT1 in autophagy, and further identify differentially expressed autophagy-related genes in pmt1△ strain utilizing mRNA microarray. Additionally, we will probe into whether these differentially expressed autophagy-related genes impact on the yeast’s lifespan through silencing or enforced expression of these genes. Finally, in order to fully reveal the molecular mechanism of PMT1 deficiency extending lifespan, double gene deficiency (autophagy-related genes and PMT1) will be constructed to reverse the effect of PMT1 deficiency on the yeast’s lifespan. Taken together, this project will not only fully elucidate the mechanism of PMT1 deficiency regulating the cell’s lifespan, but also deepen the understanding the molecular mechanism of aging and senile diseases.

自噬作为细胞内的“清道夫”，在细胞衰老和寿命调节过程中的作用日益受到关注。我们前期研究揭示，蛋白质 O-甘露糖转移酶1（PMT1）基因缺失菌株（pmt1△）可显著延长酵母细胞的复制性寿命，尽管如此，PMT1基因影响酵母寿命的机制依然有待深入探讨。本项目前期结果显示：自噬相关基因ATG8在pmt1△菌株中表达显著上调，并且缺失ATG8基因可明显缩短pmt1△酵母细胞的复制性寿命，提示PMT1基因可能通过影响自噬从而参与调节细胞寿命。基于此，本项目拟进一步探讨PMT1基因在细胞自噬反应中的作用；利用高通量芯片筛选和鉴定自噬相关基因在pmt1△菌株中的表达情况；通过功能获得与缺失等实验明确差异表达的自噬相关基因对酵母寿命的影响；最后研究细胞自噬在pmt1△菌株延长酵母寿命中的作用，从而深入揭示pmt1△菌株影响寿命的分子机制。研究结果对深化认识细胞自噬在衰老和衰老相关疾病中的作用具有重要意义。

自噬参与维持细胞内环境稳态，在细胞衰老和寿命调节等多种生物过程中起着至关重要的作用。我们前期研究揭示，蛋白质 O-甘露糖转移酶1（PMT1）基因缺失菌株（pmt1△）可显著延长酵母细胞的复制性寿命。本课题利用高通量芯片筛选和实时RT-PCR鉴定缺失PMT1基因诱导细胞自噬相关基因ATG5、ATG8和ATG13表达上调；进一步缺氮培养基诱导细胞自噬反映，研究发现pmt1△菌株中GFP-Atg8p表达较弱，GFP表达较强，提示缺失酵母PMT1基因诱导细胞自噬表达上调。通过构建自噬标志基因ATG8缺失和过表达菌株，研究Atg8p在酵母细胞寿命的作用，结果发现缺失ATG8基因缩短酵母细胞复制性寿命，减弱其分裂增殖能力，提示Atg8p参与酵母细胞寿命的调控。另外，研究发现缺失ATG8基因明显缩短pmt1△酵母细胞的复制性寿命，提示细胞自噬在pmt1△菌株延长酵母寿命中的有一定的作用，但细胞自噬影响pmt1△菌株寿命的具体分子机制仍旧有待进一步探讨。综上，细胞自噬在野生型酵母细胞和pmt1△菌株寿命调控中有一定作用，研究结果对深化认识细胞自噬在衰老和衰老相关疾病中的作用具有重要意义。