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 的损伤、断裂和错误是基因组不稳定的驱动因素，导致多种癌症。这 DNA 损伤反应 (DDR) 是一种信号级联反应，可响应 DNA 中的断裂并利用阵列 DNA 修复因子来纠正错误并保持基因组完整性。细胞对 DDR 的反应涉及 细胞周期和信号传导过程的调节，触发 DNA 修复或程序性细胞死亡。 在 DDR 中发挥作用的蛋白质会因 DNA 损伤而穿梭进入细胞核。我的博士论文工作 拟议的目标围绕了解调节核细胞质的机制 DDR蛋白的定位。该提案的F99部分重点关注复制蛋白A的调控 (RPA) 由分子伴侣样蛋白引起。 RPA 是一种重要的单链 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 转运到细胞核中，识别并结合单链DNA，并通过与超过三个相互作用来激活DDR 十几种 RPA 相互作用蛋白 (RIP)。如何防止细胞中虚假的 RPA-RIP 相互作用 ssDNA的缺失一直是个谜。我发现 Rtt105（Ty1 的调节器） 转座 105) 是一种类伴侣蛋白，通过与 RPA 的多个结构域相互作用而发挥调节作用 并在构象上限制复合物。这是 RPA-RIP 相互作用的抑制剂。使用 复杂的生物物理、生化和结构工具 我证明 ssDNA 与 RPA-Rtt105 复合物结合 并消除促进复员方案人员招募的限制。在高等真核生物中，一种称为 RPAIN 的蛋白质 （RPA 相互作用蛋白）作为 Rtt105 的功能直系同源物，我将重点破译其 作用机制。此外，我将使用冷冻电镜确定与这些伴侣蛋白结合的 RPA 的结构。在该提案的 K00 部分，我将重点关注识别其他特定的伴侣蛋白 癌症相关的 DDR 蛋白，例如 BRCA1、BRCA2、RAD52 和 PALB2。另外，我会调查 DNA 过程中控制 DDR 因子核细胞质分布的调控和信号传导机制 损害。最后，利用从生化和细胞研究中获得的知识，我将开发有针对性的 调节 DDR 的小分子癌症治疗抑制剂。 F99 和 K00 联合训练阶段将 为我提供独立研究职业所需的技能，重点是产生有针对性的研究 癌症治疗。