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突变和细胞死亡，如果不加以控制，这种损伤可促进致癌。幸运的是，细胞可以通过调节基因表达来应对DNA损伤，以协调适当的DNA修复、复制、细胞周期和代谢。多年来对酿酒葡萄球菌和人类的研究表明，核糖核苷酸还原酶（RNR）活性以及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酿酒葡萄球菌Rnr1蛋白是RNR复合物的一个重要的大亚基，通过自噬被特异性地降解。蛋白质降解是调节生物过程的关键控制机制，也是DNA损伤反应的一个未被充分研究的组成部分。我们已经证明，Rnr1被特异性地包装到自噬体中，并在营养应激或雷帕霉素靶点（TOR）的药理抑制下被降解。我们还证明，当TOR被抑制时，自噬缺陷会促进Rnr1蛋白水平的增加和DNA损伤表型。自噬通常与体降解有关。我们的新研究结果强调了自噬对特定DNA损伤反应蛋白的靶向作用，并将营养感知与DNA损伤反应的优化联系起来。先前对人类的研究报道，自噬缺陷促进基因组不稳定。此外，mTOR抑制和自噬诱导与DNA损伤后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的等位基因缺失导致基因组不稳定，但自噬与基因组维持之间的机制尚不清楚。我们在酿酒酵母中的研究结果表明，自噬控制着Rnr1蛋白水平，我们认为自噬缺陷的人类细胞改变了DNA损伤反应蛋白的水平。DNA损伤反应蛋白的错误调控可以解释自噬缺陷细胞中观察到的基因组不稳定性和癌变的增加。我们假设自噬可以用来特异性地降解蛋白质，以优化DNA损伤反应。我们将在酿酒酵母中进行遗传、生化和蛋白质组学实验，以验证我们的假设，并确定允许自噬针对特定蛋白质进行降解的分子信号。

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.