Defining Genome Stability Mechanisms and their Regulation by SUMO and Ubiquitin

SUMO 和泛素定义基因组稳定性机制及其调控

• 批准号: 10687242
• 负责人: MICHAEL N BODDY
• 金额: $ 68.78万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10687242
• 关键词: Arsenic Trioxide; Binding; Biochemical; Biological; CRISPR/Cas technology; Cell Survival; Cells; Chromatin; Collaborations; Complex; DNA Damage; DNA Repair; DNA Repair Gene; DNA biosynthesis; DNA lesion; Data; Defect; Disease; Disparate; Environment; Eye; Genetic; Genome Stability; Goals; Health; Homeostasis; Knowledge; Label; Malignant Neoplasms; Maps; Mediating; Mediator; Mutation; Nuclear; Pathway interactions; Phase; Play; Process; Proteins; Proteome; Proteomics; Regulation; Research; Role; Signal Transduction; Site; Sumoylation Pathway; Telomere Pathway; Therapeutic; Therapeutic Effect; Therapeutic Intervention; Toxic effect; Ubiquitin; Virus Replication; biophysical techniques; cofactor; cohesin; combat; condensin; genetic manipulation; genome integrity; genotoxicity; insight; leukemia; novel; recruit; repaired; response; spatiotemporal; synergism; telomere; ubiquitin-protein ligase

Maintaining genetic integrity is crucial for cell viability and disease suppression. Consequently, cellular machinery such as the DNA damage response (DDR) has evolved to combat continual genotoxic insults. Whilst specific DNA repair mechanisms have been defined in great detail, understanding the spatiotemporal regulation of DDR factors in the cell remains a key challenge. Here, the covalent postranslational modifiers (PTMs) SUMO and ubiquitin play critical roles, both recruiting DDR proteins to DNA lesions, and then removing them as necessary to promote repair. Indeed, defects in the SUMO and ubiquitin pathways cause failed DDR orchestration, severe genetic instability, and disease. Therefore, the overarching goal of our research is to delineate SUMO and ubiquitin mediated mechanisms that maintain genome integrity, with an eye to identifying and exploiting potential therapeutic avenues. Our proposal centers on two factors, STUbL and SMC5/6, which integrate signaling through SUMO and ubiquitin to support key health-related processes. Of note, STUbL mediates the therapeutic effects of arsenic trioxide in leukemia, and SMC5/6 mutations cause severe disease. STUbL is an E3 ubiquitin ligase that selectively recognizes and ubiquitinates SUMOylated proteins to promote their degradation and/or extraction from chromatin. SMC5/6 is functionally related to cohesin and condensin but uniquely, can modify targets with SUMO and ubiquitin. To provide functional insights, we used proximity labeling to reliably map the proteomic environments of STUbL and SMC5/6 in key health-related settings such as: dysfunctional telomeres, DNA repair, and viral replication. Surprisingly, considerable overlap was identified between each proteome, creating further synergy and efficiency in our research. For example, the functions and targets of SMC5/6 and STUbL intersect in the “alternative lengthening of telomeres” (ALT) pathway used in ~15% of cancers. Thus, we would define STUbL and SMC5/6 roles in the elongation and “trimming” of telomeres through the novel ALT-specific targets and cofactors we identified. In addition, a wealth of recent data supports our hypothesis that STUbL controls SUMO pathway homeostasis, as well as specific targets, to support genome stability, DNA replication, and cell survival. Further analysis using genetic manipulation of the SUMO pathway (e.g. CRISPR/Cas9), mediators of SUMO chain toxicity, and new STUbL targets would establish this key paradigm in SUMO and ubiquitin pathway crosstalk. We also recently identified an SMC5/6 cofactor that binds SUMO and directs the complex to phase-separated ALT PML nuclear bodies, sites of viral replication, and likely DNA lesions, thereby unifying these seemingly disparate processes. Hence, we would define functions for SMC5/6 and its new SUMO binding cofactor in each of these processes to reveal common mechanisms. Overall, our collaborative teams' analysis of STUbL and SMC5/6 using proteomic, genetic, cell biological, biochemical, and biophysical methods would synergize to define key health-related mechanisms at the nexus of the SUMO and ubiquitin pathways; providing targets and guidance for therapeutic interventions.

保持遗传完整性对细胞活力和疾病抑制至关重要。因此，Cellular 诸如DNA损伤反应（DDR）的机制已经进化以对抗持续的遗传毒性损伤。 虽然已经非常详细地定义了特定的DNA修复机制，但理解时空 细胞中DDR因子的调节仍然是一个关键挑战。在这里，共价翻译后修饰语 （PTM）SUMO和泛素起着关键作用，都招募DDR蛋白到DNA损伤，然后去除 这是必要的，以促进修复。事实上，SUMO和泛素途径的缺陷导致DDR失败 协调，严重的遗传不稳定性和疾病。因此，我们研究的首要目标是 描述SUMO和泛素介导的维持基因组完整性的机制，着眼于识别 探索潜在的治疗途径我们的建议集中在两个因素，STUbL和SMC 5/6， 通过SUMO和泛素整合信号传导，以支持关键健康相关过程。注意，STUBL 介导三氧化二砷在白血病中的治疗作用，SMC 5/6突变导致严重疾病。 STUbL是一种E3泛素连接酶，其选择性识别并泛素化SUMO化蛋白，以促进 它们的降解和/或从染色质中提取。SMC 5/6与cohesin和condensin功能相关 但独特的是，可以用SUMO和遍在蛋白修饰靶点。为了提供功能性的见解，我们使用了接近性 标记以可靠地映射STUbL和SMC 5/6在关键健康相关环境中的蛋白质组环境， 例如：端粒功能障碍、DNA修复和病毒复制。令人惊讶的是，发现了相当多的重叠 在每个蛋白质组之间，在我们的研究中创造进一步的协同作用和效率。例如，函数 SMC 5/6和STUbL的靶点在所用的“端粒交替延长”（ALT）途径中相交， 约15%的癌症。因此，我们将定义STUbL和SMC 5/6在细胞的伸长和“修剪”中的作用。 端粒通过新的ALT特异性目标和辅因子，我们确定。此外，最近大量 数据支持我们的假设，即STUbL控制SUMO通路的稳态，以及特定的靶点， 支持基因组稳定性、DNA复制和细胞存活。进一步分析使用基因操作的 SUMO通路（例如CRISPR/Cas9）、SUMO链毒性的介导物和新的STUbL靶点将被发现。 在SUMO和泛素途径串扰中建立了这个关键范例。我们最近还确定了一个SMC 5/6 一种结合SUMO并将复合物导向相分离的ALT PML核体的辅因子， 复制和可能的DNA损伤，从而统一这些看似不同的过程。因此，我们将 定义SMC 5/6及其新的SUMO结合辅因子在这些过程中的功能，以揭示共同的 机制等总的来说，我们的合作团队使用蛋白质组学、遗传学、细胞学和生物学方法对STUbL和SMC 5/6进行的分析， 生物学、生物化学和生物物理学方法将协同作用，确定关键的健康相关机制， SUMO和泛素途径的联系;为治疗干预提供靶点和指导。