Targeted Degradation of DNA Damage Response Proteins by Autophagy

描述（由申请人提供）：导致DNA损伤的环境暴露可促进诱变和细胞死亡，如果不加以控制，这种损伤可促进致癌作用。幸运的是，细胞可以通过调节基因表达来协调适当的DNA修复，复制，细胞周期和代谢来应对DNA损伤。在S工作多年。酿酒酵母和人类已经证明，核糖核苷酸还原酶（RNR）活性以及因此dNTP水平的调节是DNA损伤后的关键控制点。在我们最近的论文中，我们使用了系统生物学和基因靶向方法来证明S。酿酒酵母Rnr1蛋白是RNR复合物的重要大亚基，通过自噬特异性降解。蛋白质降解是调节生物过程的关键控制机制，也是DNA损伤反应的一个未充分研究的组成部分。我们已经表明，Rnr1被特异性包装到自噬体中，并响应于营养应激或雷帕霉素靶点（TOR）的药理学抑制而降解。我们还证明，当TOR被抑制时，自噬缺陷促进Rnr1蛋白水平增加和DNA损伤表型。自噬通常参与整体降解。我们的新结果强调了通过自噬靶向特定的DNA损伤反应蛋白，并且它们还将营养感测与DNA损伤反应的优化联系起来。以前的人类研究报告说，自噬缺陷促进基因组不稳定性。此外，mTOR抑制和自噬诱导与DNA损伤后的p53和共济失调-毛细血管扩张突变（ATM）信号传导有关。人类自噬基因Beclin 1的等位基因丢失导致基因组不稳定，但将自噬与基因组维持联系起来的机制还不清楚。我们在S.酿酒酵母证明自噬控制Rnr1蛋白水平，我们认为自噬缺陷的人类细胞具有改变的DNA损伤反应蛋白水平。DNA损伤反应蛋白的错误调节可以解释在自噬缺陷细胞中观察到的基因组不稳定性增加和致癌作用。我们假设自噬可用于特异性降解蛋白质以优化DNA损伤反应。我们将进行遗传，生物化学和蛋白质组学为基础的实验，在S。酿酒酵母来测试我们的假设，并定义允许自噬靶向特定蛋白质降解的分子信号。 公共卫生相关性：我们的研究将为自噬激活时DNA损伤反应的调节提供深入了解，自噬是双重环境压力的代表。具体来说，我们将定义的机制，核糖核苷酸还原酶和其他DNA损伤反应蛋白是专门针对降解的自噬。最终，我们的工作将表征一种新的蛋白质靶向机制，并促进癌症预防和癌症治疗应用。

DESCRIPTION (provided by applicant): Environmental exposures that cause DNA damage can promote mutagenesis and cell death, and if left unchecked this damage can promote carcinogenesis. Fortunately, cells can respond to DNA damage by regulating gene expression to coordinate proper DNA repair, replication, cell cycle and metabolism. Years of work in S. cerevisiae and humans have demonstrated that ribonucleotide reductase (RNR) activity, and thus regulation of dNTP levels, is a key control point after DNA damage. In our recent paper we have used systems biology and gene targeted approaches to demonstrate that the S. cerevisiae Rnr1 protein, an essential large subunit of the RNR complex, is specifically degraded via autophagy. Protein degradation is a key control mechanism that can regulate biological processes, and an understudied component of the DNA damage response. We have shown that Rnr1 is specifically packaged into an autophagosome and degraded in response to nutrient stress or pharmacological inhibition of target of rapamycin (TOR). We have also demonstrated that defects in autophagy promote increased Rnr1 protein levels and a DNA damage phenotype when TOR is inhibited. Autophagy is usually involved in bulk degradation. Our novel results highlight targeting of a specific DNA damage response protein by autophagy, and they also connect nutrient sensing to optimization of the DNA damage response. Previous studies in humans report that defects in autophagy promote genome instability. Additionally, mTOR inhibition and the induction of autophagy have been linked to p53 and Ataxia-telangiectasia mutated (ATM) signaling after DNA damage. Allelic loss of the human autophagy gene Beclin 1 leads to genome instability, but the mechanism connecting autophagy to genome maintenance is not well understood. Our findings in S. cerevisiae demonstrate that autophagy controls Rnr1 protein levels and we suggest that human cells defective in autophagy have altered levels of DNA damage response proteins. Mis-regulation of DNA damage response proteins would account for the increased genome instability and carcinogenesis observed in autophagy deficient cells. We hypothesize that autophagy can be used to specifically degrade proteins to optimize the DNA damage response. We will perform genetic, biochemical and proteomic based experiments in S. cerevisiae to test our hypothesis and to define the molecular signals that allow autophagy to target specific proteins for degradation. PUBLIC HEALTH RELEVANCE: Our studies will provide insight into the regulation of the DNA damage response when autophagy is activated, a condition representative of duel environmental stresses. Specifically, we will define the mechanism by which ribonucleotide reductase and other DNA damage response proteins are specifically targeted for degradation by autophagy. Ultimately our work will characterize a novel protein targeting mechanism and promote cancer prevention and cancer therapeutic applications.