SUMO-dependent Regulation of Ubiquitin Ligases in Genomic Stability

描述(申请人提供)：保持遗传完整性对于细胞存活和抑制包括癌症在内的人类疾病都是至关重要的。因此，DNA损伤反应(DDR)已经进化为对抗基因组每天遇到的无数威胁，例如环境中产生的遗传毒素或作为细胞新陈代谢的副产品。鉴于DDR在疾病抑制中的根本重要性，它一直是密集研究的主题，从而确定了一组明确的核心DDR蛋白。然而，同样重要的问题，即这些核心DDR因子如何被招募到DNA损伤中，并从DNA损伤中清除，以促进DNA修复，这些问题在很大程度上仍然没有答案。到目前为止，研究已经揭示了一系列复杂的翻译后修饰(PTM)，例如磷酸化、泛素化和SUMO化，它们驱动了DDR蛋白质的动力学；但在大多数情况下，关键的机制细节缺乏。我们解决了我们提案中的这一关键缺陷，方法是建立在我们最近发现的(I)相扑靶向泛素连接酶(STUbL)的基础上，该酶选择性泛素化相扑链修饰的蛋白质以产生双重PTM信号，以及(Ii)分子分离酶CDC48(P97)-Ufd1-Npl4(CDC48-UN)意外地识别这种相扑泛素信号。STUbL活性与CDC48-UN分离酶“马达”的这种功能性偶联为SUMO和泛素动态控制DDR蛋白提供了一种新的机制，即通过选择性地从DNA损伤中提取SUMO-泛素共修饰的蛋白，连接或不连接到蛋白酶体的降解。与此一致，失活STUbL或CDC48-UN，这两种都定位于DNA损伤，扰乱正常的DDR蛋白质动态，并导致严重的基因组不稳定。在此，我们建议通过我们的三个协同具体目标，确定STUbL和CDC48-UN在复员方案中的具体联合目标和机制。在目标1中，我们将描述STUbL和CDC48-UN在重塑拓扑异构酶1(Top1)-DNA加合物蛋白质组中的共同功能，这对修复这种高度遗传毒性的损伤至关重要。在目标2中，我们将确定STUbL和CDC48-UN促进裂解酵母中去保护端粒引发DDR的协同机制。在人类中，这样的端粒失去保护是自发发生的，例如在衰老过程中，并可能导致肿瘤。在目标3中，举例说明了在分裂酵母中的开创性分析的力量(目标2)，我们揭示了STUbL也促进了去保护的哺乳动物端粒的DDR。我们将在此DDR中定义关键STUbL目标，并确定它们是否也是p97(CDC48)目标。这些重点目标将通过在分裂酵母和哺乳动物细胞中使用创新的遗传、细胞生物学、生化和蛋白质组学方法来协作实现。研究结果将为相扑和泛素信号如何在DNA损伤处整合以重塑蛋白质组，从而促进DDR提供机械性的见解。此外，由于STUbL介导了三氧化二砷对白血病的治疗效果，而p97(CDC48)也是疾病治疗的靶点，我们的结果将对此类治疗的发展和改进起到至关重要的指导作用。

DESCRIPTION (provided by applicant): Maintenance of genetic integrity is critical for both cell viability and the suppression of human disease, including cancer. Therefore, the DNA damage response (DDR) has evolved to combat the myriad threats that the genome encounters on a daily basis e.g. genotoxins arising in the environment or as byproducts of cellular metabolism. Given the fundamental importance of the DDR in disease suppression, it has been the subject of intense investigation, resulting in the identification of a well-defined set of core DDR protein. However, the equally important questions of how these core DDR factors are recruited to, and cleared from, DNA lesions to promote DNA repair remains largely unanswered. Studies so far have revealed a complex cascade of posttranslational modifications (PTMs) e.g. phosphorylation, ubiquitination and sumoylation that drives DDR protein dynamics; but in most cases, key mechanistic detail is missing. We address this critical deficiency in our proposal, by building on our recent discoveries of (i) a SUMO-targeted ubiquitin ligase (STUbL) that selectively ubiquitinates SUMO chain modified proteins to generate a dual PTM signal and (ii) the unanticipated recognition of this SUMO-ubiquitin signal by the molecular segregase Cdc48(p97)-Ufd1-Npl4 (Cdc48-UN). This functional coupling of STUbL activity to the Cdc48-UN segregase "motor" suggests a novel mechanism for the dynamic control of DDR proteins by SUMO and ubiquitin i.e. through the selective extraction of SUMO-ubiquitin co-modified proteins from DNA lesions, coupled or not to proteasomal degradation. Consistent with this, inactivating STUbL or Cdc48-UN, both of which localize to DNA lesions, disrupts normal DDR protein dynamics and causes severe genome instability. Herein, we propose to define the specific joint targets and mechanisms of STUbL and Cdc48-UN in the DDR through our three synergistic Specific Aims. In Aim 1, we will delineate co-functions for STUbL and Cdc48-UN in remodeling the proteome at topoisomerase 1 (Top1)-DNA adducts, which is crucial for the repair of this highly genotoxic lesion. In Aim 2, we will determine the cooperative mechanisms of STUbL and Cdc48-UN that promote the DDR elicited by de-protected telomeres in fission yeast. In humans, such telomere de-protection occurs spontaneously e.g. during ageing, and can be tumorigenic. In Aim 3, exemplifying the strength of pioneering analyses in fission yeast (Aim 2), we reveal that STUbL also promotes the DDR at de-protected mammalian telomeres. We will define the key STUbL targets in this DDR and determine if they are also p97 (Cdc48) targets. These focused Aims will be achieved collaboratively using innovative genetic, cell biological, biochemical and proteomic approaches in both fission yeast and mammalian cells. Results will provide mechanistic insight into how SUMO and ubiquitin signaling is integrated at DNA lesions to remodel the proteome, and thereby promote the DDR. Moreover, as STUbL mediates the therapeutic effect of arsenic trioxide in leukemia, and p97 (Cdc48) is also being targeted in disease therapy, our results will critically guide the development and improvement of such therapies.