Tunable Microchip Sorting of BRCA1 Nuclear Assemblies

 描述（由申请人提供）：乳腺癌易感蛋白BRCA 1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”癌症。 消极的”。三阴性肿瘤缺乏雌激素受体、孕激素受体和Her 2表达，这些是常用的药物靶点，以增强其他形式乳腺癌的治疗选择。因此，患有三阴性癌症的患者具有有限的治疗选择，并且在常规治疗后在更短的时间内复发。在正常情况下，BRCA 1蛋白作为肿瘤抑制因子，帮助纠正基因组DNA中的断裂，并确保新合成mRNA的保真度。这些调控过程中的缺陷导致基因组不稳定和肿瘤的发生。了解与BRCA 1突变相关的三阴性乳腺癌诱导的分子基础，可能有助于为患有这种侵袭性疾病的患者开发新的治疗选择。我们的总体目标是开发一种新的基于可调微芯片的策略，以研究参与核保护过程的BRCA 1蛋白组装体的结构属性，并研究BRCA 1蛋白中的缺陷如何影响这些必需蛋白组装体的形成。我们拟议的研究将提供一个多学科的机会，以弥合癌症生物学，材料科学和结构生物学中使用的技术，以解决涉及BRCA 1在基因调控事件中的作用的长期问题。可调的“亲和捕获”微芯片设备将用于从人类乳腺癌细胞中招募功能不同的BRCA 1相关蛋白复合物。我们将建立一个芯片上的分子分选技术，采用适配器分子特定的转录相关的复合物结合到DNA。分离的复合物将使用生物化学方法进行表征。然后，我们将利用冷冻电子显微镜（EM）来确定微芯片分选的BRCA 1蛋白组装体的3D结构。结合抗体标记和分子建模技术将使我们能够解释EM密度图，这将揭示BRCA 1相关组件之间的分子相互作用。从长远来看，我们的研究结果可以为设计新疗法提供结构模板，旨在调整乳腺癌和卵巢癌基底样亚型中的蛋白质-蛋白质相互作用。

 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.