Elucidating binding modes of BRCT-modules

阐明 BRCT 模块的结合模式

基本信息

批准号：
8280924
负责人：
Jamaine S Davis
金额：
$ 14.4万
依托单位：
MEHARRY MEDICAL COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-02-15 至 2017-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8280924
关键词：
Binding Biochemical C-terminal Cancer Etiology Cell Survival Complex Computer software Crystallization DNA Damage DNA Repair Data Defect Fostering Genome Stability Goals Health In Vitro Individual Investigation Knowledge Length Maintenance Malignant Neoplasms Mediating Mission Molecular Nuclear Protein Outcome Pathway interactions Peptides Phosphopeptides Phosphoserine Play Positioning Attribute Predisposition Protein Binding Proteins Public Health Regulation Repair Complex Research Resolution Roentgen Rays Role Serine Signal Transduction Specificity Structure Substrate Specificity Surface Plasmon Resonance Testing Threonine Transactivation Work analytical ultracentrifugation base improved in vivo innovation insight irradiation killings malignant breast neoplasm neoplastic cell novel preference programs protein protein interaction research study response screening tumor

项目摘要

DESCRIPTION (provided by applicant): There is a fundamental gap in our understanding of how mechanisms of phosphoserine binding modules by BRCT domains enable the assembly of multiprotein DNA repair complexes. Overlooking this gap is an important problem because, until it is solved, understanding how defects in the regulation of DNA repair can potentially cause cancer will remain obscure. The long-term goal is to understand how full-length PTIP regulates the DNA damage response pathway. The objective of this particular application is to provide a molecular description of how PTIP-(BRCT)4 interactions help to regulate DNA repair. The central hypothesis is that (BRCT)4, is able to recognize a broader spectrum of phosphopeptide sequence motifs, that were initially determined, and it can mediate interactions with other BRCT-domain containing proteins. This hypothesis has been formulated based on preliminary data obtained in the applicant's lab. The rationale for the proposed research is that, once it is known how PTIP interactions relay DNA damage signals to regulate DNA repair in tumor cells, we can identify agents that disrupt these interactions thereby making them more sensitive to chemo- and irradiation therapies. This hypothesis will be tested by investigation of three aims: 1) determination of the X-ray crystal structure of the tandem pairs of C-terminal BRCT domains; 2) determination of the structural basis of substrate specificity of the tandem pair of BRCT domains; 3) determination of the mechanism of substrate preference of the tandem pairs of BRCT domains. Within the first aim, native crystals diffract to 2.8 A and the heavy atom derivative diffracts to 3.2 A. Automated software programs will be used to solve the structure. Within the second aim, we have purified sufficient amounts of protein for biochemical and structural investigations and will begin crystallization screening and direct peptide binding experiments using surface Plasmon resonance (SPR). Lastly, within the third aim, peptide arrays will be conducted to identify novel sequences recognized by (BRCT)4, and confirmed with SPR. This approach is innovative, in my opinion, because it will be the first comprehensive study in the field focused on the importance of four consecutive BRCT domains. The proposed research is significant, because it will elucidate the differences in binding mechanisms among BRCT domains, and fundamentally expand our understanding of protein recognition with BRCT-domain containing proteins. Ultimately, this knowledge can identify agents that disrupt BRCT domain interactions and thereby make tumor cells more sensitive to killing by chemo- and irradiation therapies.

描述（由申请人提供）：对于 BRCT 结构域的磷酸丝氨酸结合模块如何实现多蛋白 DNA 修复复合物的组装，我们的理解存在根本性的差距。忽视这一差距是一个重要的问题，因为在这个问题得到解决之前，了解 DNA 修复调节缺陷如何可能导致癌症仍然是个谜。长期目标是了解全长 PTIP 如何调节 DNA 损伤反应途径。这一特定应用的目的是提供 PTIP-(BRCT)4 相互作用如何帮助调节 DNA 修复的分子描述。中心假设是 (BRCT)4 能够识别最初确定的更广泛的磷酸肽序列基序，并且它可以介导与其他含有 BRCT 结构域的蛋白质的相互作用。该假设是根据申请人实验室获得的初步数据制定的。这项研究的基本原理是，一旦了解 PTIP 相互作用如何传递 DNA 损伤信号以调节肿瘤细胞中的 DNA 修复，我们就可以识别破坏这些相互作用的药物，从而使它们对化疗和放疗更加敏感。该假设将通过三个目标的研究来检验：1）确定 C 端 BRCT 结构域串联对的 X 射线晶体结构； 2)确定BRCT结构域串联对的底物特异性的结构基础； 3)确定BRCT结构域串联对的底物偏好机制。在第一个目标中，天然晶体衍射至 2.8 A，重原子衍生衍射至 3.2 A。将使用自动化软件程序来求解结构。在第二个目标中，我们已经纯化了足够量的蛋白质用于生化和结构研究，并将开始使用表面等离子共振（SPR）进行结晶筛选和直接肽结合实验。最后，在第三个目标中，将进行肽阵列来识别 (BRCT)4 识别的新序列，并通过 SPR 进行确认。在我看来，这种方法是创新的，因为它将是该领域第一个专注于四个连续 BRCT 领域重要性的综合研究。这项研究意义重大，因为它将阐明 BRCT 结构域之间结合机制的差异，并从根本上扩展我们对含有 BRCT 结构域的蛋白质的蛋白质识别的理解。最终，这些知识可以识别破坏 BRCT 结构域相互作用的药物，从而使肿瘤细胞对化疗和放射疗法的杀伤更加敏感。