Tunable Microchip Sorting of BRCA1 Nuclear Assemblies

 描述(申请人提供)：乳腺癌易感蛋白BRCA1的突变与家族性乳腺癌和卵巢癌密切相关，这些癌症被归类为“三重”。 否定的“。三阴性肿瘤缺乏雌激素受体、孕激素受体和Her2表达，这些都是通常用于增强其他形式乳腺癌治疗选择的药物靶点。因此，患有三阴性癌症的患者治疗选择有限，在常规治疗后较短的时间内死于复发。在正常情况下，BRCA1蛋白作为肿瘤抑制因子，帮助纠正基因组DNA的断裂，并确保新合成的mRNA的保真度。这些调控过程中的缺陷会导致基因组的不稳定和肿瘤的发生。了解与BRCA1突变相关的三阴性乳腺癌诱导的分子基础将大大有助于为患有这种侵袭性疾病的患者开发新的治疗方案。我们的总体目标是开发一种新的基于微芯片的可调策略，以研究核保护过程中涉及的BRCA1蛋白质组件的结构属性，并研究BRCA1蛋白质中的缺陷如何影响这些基本蛋白质组件的形成。我们拟议的研究将提供一个多学科的机会，将癌症生物学、材料科学和结构生物学中使用的技术联系起来，以解决涉及BRCA1在基因调控事件中的角色的长期存在的问题。可调的“亲和力捕捉”微芯片设备将用于从人类乳腺癌细胞中招募功能不同的BRCA1相关蛋白质复合体。我们将建立一种芯片上的分子分类技术，该技术使用与DNA结合的转录相关复合体特有的适配分子。分离的复合体将使用生化方法进行表征。然后，我们将利用低温电子显微镜(EM)来确定微芯片分选的BRCA1蛋白组件的3D结构。抗体标记和分子建模的组合技术将使我们能够解释EM密度图，该图将揭示BRCA1相关组件之间的分子相互作用。从长远来看，我们的发现可以为旨在调整乳腺癌和卵巢癌基底细胞样亚型的蛋白质-蛋白质相互作用的新疗法的设计提供一个结构模板。

 DESCRIPTION (provided by applicant): Mutations in the breast cancer susceptibility protein, BRCA1, are heavily implicated in familial breast and ovarian cancers that are classified as "triple negative". Triple negative tumors lack estrogen receptors, progesterone receptors and Her2 expression that are commonly used drug targets to enhance treatment options for other forms of breast cancer. Thus, patients afflicted with triple negative cancers have limited treatment options and succumb to recurrence in less time following conventional therapy. Under normal conditions, the BRCA1 protein acts as a tumor suppressor, helping correct breaks in genomic DNA and ensure fidelity in newly synthesized mRNA. Defects in these regulatory processes lead to genomic instability and to tumor initiation. Understanding the molecular basis for triple negative breast cancer induction related to BRCA1 mutations could significantly contribute to the development of new treatment options for patients afflicted with this aggressive disease. Our overall goal is to develop a new tunable microchip-based strategy to study the structural attributes of BRCA1 protein assemblies involved in nuclear protective processes -- and to examine how defects in the BRCA1 protein can impact the formation of these essential protein assemblies. Our proposed research will provide a multi-disciplinary opportunity to bridge technologies used in cancer biology, material science, and structural biology to address long-standing questions involving the role BRCA1 in gene regulatory events. Tunable "Affinity Capture" microchip devices will be used recruit functionally distinct BRCA1- associated protein complexes from human breast cancer cells. We will establish an on-chip molecular sorting technique that employs adaptor molecules specific for transcription-related complexes bound to DNA. Isolated complexes will be characterized using biochemical methodologies. We will then utilize cryo-Electron Microscopy (EM) to determine the 3D architecture of the microchip-sorted BRCA1 protein assemblies. Combined techniques of antibody labeling and molecular modeling will permit us to interpret EM density maps that will reveal the molecular interactions among the BRCA1-associated assemblies. Long-term, our findings could provide a structural template for the design of new therapies aimed at tuning protein-protein interactions in basal-like subtypes of breast and ovarian cancers.