Role of nuclear chaperones in genomic instability and carcinogenesis

核伴侣在基因组不稳定性和癌发生中的作用

• 批准号: 10817984
• 负责人: Sahiti Kuppa
• 金额: $ 8.86万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10817984
• 关键词: Affinity; Apoptosis; BRCA1 gene; BRCA2 gene; Base Sequence; Binding; Binding Proteins; Biochemical; Biophysics; Breast; Cell Cycle; Cell Cycle Arrest; Cell Cycle Regulation; Cell Death Induction; Cell Nucleus; Cell Signaling Process; Cells; Cervix Neoplasms; Chromosome Breakage; Clinical; Colorectal; Complex; Cryoelectron Microscopy; Cytoplasm; DNA; DNA Binding; DNA Damage; DNA Repair; DNA Repair Gene; DNA biosynthesis; DNA metabolism; DNA-Binding Proteins; Defect; Dissociation; Doctor of Philosophy; Eukaryota; Filament; Genes; Genetic; Genetic Recombination; Genomic Instability; Goals; Hela Cells; Homologous Gene; Human; Importins; Investigation; Knowledge; Laboratories; Lesion; Malignant Neoplasms; Metabolism; Modeling; Molecular Chaperones; Molecular Conformation; Mutation; Nuclear; Nuclear Import; Nuclear Pore Complex; Nucleoproteins; Organism; Orthologous Gene; PALB2 gene; Pancreas; Phase; Phenotype; Phosphotransferases; Post-Translational Protein Processing; Postdoctoral Fellow; Process; Prostate; Protein Binding Domain; Protein Isoforms; Proteins; RAD52 gene; Regulation; Research; Research Project Grants; Role; Saccharomyces cerevisiae; Signal Transduction; Single-Stranded DNA; Site; Stomach; Structure; Therapeutic; Training; Work; biological adaptation to stress; cancer cell; carcinogenesis; career; genome integrity; inhibitor; inhibitor therapy; insight; knock-down; medulloblastoma; novel; preservation; prevent; protein function; recruit; repaired; replication factor A; replication stress; response; restraint; skills; small molecule; therapeutic target; tool; tumor

SUMMARY Lesions, breaks, and errors in DNA are drivers of genomic instability resulting in a variety of cancers. The DNA damage response (DDR) is a signaling cascade that responds to breaks in the DNA and utilizes an array of DNA repair factors to correct the error and preserve genomic integrity. Cellular responses to DDR involve regulation of the cell cycle and signaling processes that either trigger DNA repair or programmed cell death. Proteins that function in DDR are shuttled into the nucleus in response to DNA damage. My PhD thesis work and the proposed goals center around understanding the mechanisms that regulate nuclear-cytoplasmic localization of DDR proteins. The F99 part of the proposal focuses on the regulation of Replication Protein A (RPA) by chaperone-like proteins. RPA is an essential single-stranded DNA (ssDNA) binding DDR factor that regulates all aspects of DNA metabolism including DNA replication, repair, and recombination. RPA is transported into the nucleus, recognizes, and binds ssDNA, and activates DDR by interacting with over three dozen RPA-interacting proteins (RIPs). How spurious RPA-RIP interactions are prevented in the cell in the absence of ssDNA has been a long-standing mystery. I have uncovered that Rtt105 (Regulator of Ty1 transposition 105), a chaperone-like protein, functions as a regulator by interacting with multiple domains of RPA and conformationally restraining the complex. This serves as an inhibitor of RPA-RIP interactions. Using sophisticated biophysical, biochemical, and structural tools I show that ssDNA binds to the RPA-Rtt105 complex and removes the restraints to promote recruitment of DDR factors. In higher eukaryotes, a protein called RPAIN (RPA-interacting protein) serves as the functional ortholog of Rtt105 and I will focus on deciphering its mechanism of action. In addition, using cryoEM, I will determine the structures of RPA bound to these chaperonelike proteins. In the K00 part of the proposal, I will focus on identifying chaperone-like proteins specific to other cancer-related DDR proteins such as BRCA1, BRCA2, RAD52, and PALB2. In addition, I will investigate the regulatory and signaling mechanisms that control nuclear-cytoplasmic distribution of DDR factors during DNA damage. Finally, using knowledge obtained from the biochemical and cellular studies I will develop targeted small molecule cancer therapeutic inhibitors to regulate DDR. The combined F99 and K00 training phases will provide me with the necessary skills towards an independent research career focused on generating targeted cancer therapeutics.

概括 DNA的病变，断裂和错误是基因组不稳定性的驱动因素，导致各种癌症。这 DNA损伤响应（DDR）是一个信号级联，可响应DNA中的断裂并使用阵列 DNA修复因子以纠正误差并保留基因组完整性。细胞对DDR的反应涉及 调节触发DNA修复或编程细胞死亡的细胞周期和信号传导过程。 在DDR中起作用的蛋白质响应DNA损伤而被穿梭到核​​中。我的博士学位论文工作 拟议的目标围绕理解调节核质质的机制 DDR蛋白的定位。该提案的F99部分侧重于复制蛋白A的调节 （RPA）通过伴侣样蛋白。 RPA是必不可少的单链DNA（ssDNA）结合DDR因子 调节DNA代谢的各个方面，包括DNA复制，修复和重组。 RPA是 运输到核中，识别和结合ssDNA，并通过与超过三个以上相互作用来激活DDR 十二个RPA相互作用蛋白（RIP）。如何在细胞中预防虚假的RPA-RIP相互作用 缺少ssDNA一直是一个长期的谜。我发现了RTT105（TY1的调节器 Transiposition 105），一种伴侣样蛋白，通过与RPA多个结构域相互作用作为调节剂 并在构象上限制复合物。这是RPA-RIP相互作用的抑制剂。使用 我显示的精致的生物物理，生化和结构工具，ssDNA与RPA-RTT105复合物结合 并消除限制以促进DDR因素的招募。在较高的真核生物中，一种称为rpain的蛋白质 （RPA相互作用蛋白）用作RTT105的功能直系同源 作用机理。另外，使用冷冻素，我将确定与这些伴侣（如蛋白）结合的RPA的结构。在提案的K00部分中，我将重点介绍特定于其他的类似伴侣的蛋白 与癌症相关的DDR蛋白，例如BRCA1，BRCA2，RAD52和PALB2。此外，我将调查 DNA期间DDR因子控制核胞质分布的调节和信号传导机制 损害。最后，使用从生化和细胞研究中获得的知识，我将开发目标 小分子癌治疗抑制剂调节DDR。联合F99和K00训练阶段将 为我提供必要的技能，致力于独立研究职业，专注于产生目标 癌症治疗学。