SUMO-dependent Regulation of Ubiquitin Ligases in Genomic Stability

描述（由申请人提供）：维持基因完整性对于细胞活力和抑制人类疾病（包括癌症）至关重要。因此，DNA损伤反应（DDR）已经进化到对抗基因组每天遇到的无数威胁，例如环境中产生的基因毒素或细胞代谢的副产品。鉴于DDR在疾病抑制中的重要作用，人们对其进行了深入的研究，并鉴定出了一组定义明确的核心DDR蛋白。然而，同样重要的问题是，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。到目前为止的研究已经揭示了一个复杂的级联翻译后修饰（PTMs），如磷酸化，泛素化和sumo化，驱动DDR蛋白动力学；但在大多数情况下，关键的机械细节是缺失的。我们通过建立我们最近发现的(i) SUMO靶向泛素连接酶（STUbL）来解决这一关键缺陷，该酶选择性地泛素化SUMO链修饰的蛋白质以产生双PTM信号（ii）分子分离酶Cdc48(p97)-Ufd1-Npl4 （Cdc48- un）对这种SUMO泛素信号的意外识别。STUbL活性与Cdc48-UN分离酶“马达”的功能耦合表明，SUMO和泛素对DDR蛋白进行动态控制的新机制，即通过选择性地从DNA损伤中提取SUMO-泛素共修饰蛋白，偶联或不偶联蛋白酶体降解。与此一致的是，灭活定位于DNA损伤的STUbL或Cdc48-UN会破坏正常的DDR蛋白动力学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来确定STUbL和Cdc48-UN在DDR中的具体联合目标和机制。在Aim 1中，我们将描述STUbL和Cdc48-UN在重塑拓扑异构酶1 (Top1)-DNA加合物的蛋白质组中的共同功能，这对这种高度遗传毒性病变的修复至关重要。在Aim 2中，我们将确定STUbL和Cdc48-UN促进裂变酵母中去保护端粒引发的DDR的合作机制。在人类中，这种端粒去保护是自发发生的，例如在衰老过程中，可能是致瘤性的。在Aim 3中，举例说明了裂变酵母开创性分析的强度（Aim 2），我们揭示了STUbL也促进了去保护哺乳动物端粒的DDR。我们将定义该DDR中的关键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裂变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同实现。结果将提供SUMO和泛素信号如何在DNA损伤中整合以重塑蛋白质组，从而促进DDR的机制见解。此外，由于STUbL介导三氧化二砷对白血病的治疗作用，p97 （Cdc48）也被靶向治疗，我们的研究结果将对此类疗法的开发和改进具有重要指导意义。

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.