Structural Basis of Cell Signaling by BRCT Domains

描述（由申请人提供）：我们的长期目标是了解含BRCT结构域蛋白在细胞信号传导中的功能。BRCT基序首先在乳腺癌蛋白BRCA 1中被鉴定为串联对，后来在其他参与DNA修复和细胞周期控制的蛋白质中被鉴定为串联对。我们发现BRCT结构域是一个磷蛋白结合基序。虽然BRCA 1利用BRCT串联重复序列，但其他蛋白质，包括拓扑异构酶II结合蛋白、TopBP 1和DNA连接酶IIIalpha，通过BRCT结构域的同源二聚体识别其磷酸化靶标。也有强有力的证据表明，NBS 1（奈梅亨断裂综合征）蛋白通过串联的BRCT和FHA结构域结合磷酸化的组蛋白H2 AX。这些观察结果表明，BRCT蛋白通过不同偶联的BRCT结构域结合磷酸化的靶标-即，BRCT串联重复序列、BRCT同源二聚体、BRCT-FHA串联结构域-表明至少存在3种磷蛋白识别模式。没有BRCT/磷蛋白复合物的3D结构。为了了解磷蛋白识别是如何实现的，我们将使用NMR光谱，微量热和生物化学技术来确定3D结构，并探测蛋白质-磷酸肽复合物的相互作用的能量学，说明所有3种BRCT结合模式。我们还将测试磷酸肽结合调节其他BRCT介导的相互作用的假设。我们的具体目标是： 目标1.探索BRCA 1与解旋酶BACH 1和CtIP辅阻遏物的磷酸化肽的相互作用，以阐明BRCT串联重复序列识别磷蛋白的分子机制。 目标2.探讨TopBP 1与E2 F1转录因子磷酸化肽段的相互作用，以阐明BRCT同源二聚体识别磷蛋白的分子机制。 目标3。探讨NBS 1与磷酸化组蛋白H2 AX肽的相互作用，以阐明FHA-BRCT串联结构域识别磷蛋白的分子机制。 目标4。研究与DNA连接酶IIIa结合的磷蛋白对BRCT介导的与DNA修复蛋白XRCC 1相互作用的可能调节作用。

DESCRIPTION (provided by applicant): Our long-term goal is to understand the function of BRCT domain-containing proteins in cellular signaling. The BRCT motif was first identified as a tandem pair in the breast cancer protein BRCA1, and later in other proteins involved in DNA repair and cell cycle control. We showed that the BRCT domain is a phosphoprotein binding motif. While BRCA1 utilizes a BRCT tandem repeat, other proteins, including topoisomerase II binding protein, TopBP1, and DNA ligase IIIalpha, recognize their phosphorylated targets by means of a homodimer of BRCT domains. There is also strong evidence that NBS1 (Nijmegen Breakage Syndrome) protein binds phosphorylated histone H2AX through a tandem of BRCT and FHA domains. These observations that BRCT proteins bind phosphorylated targets through differently coupled BRCT domains - i.e., BRCT tandem repeats, BRCT homodimer, BRCT-FHA tandem domains - indicate that at least 3 modes of phosphoprotein recognition exist. There is no 3D structure of a BRCT/phosphoprotein complex. To understand how phosphoprotein recognition is achieved, we will use NMR spectroscopy, microcalorimetry and biochemistry techniques to determine the 3D structures, and probe the energetics of interaction of protein-phosphopeptide complexes illustrating all 3 BRCT binding modes. We will also test the hypothesis that phosphopeptide binding modulates other BRCT-mediated interactions. Our specific aims are: Aim 1. Probe the interaction of BRCA1 with phosphorylated peptides from the helicase BACH1 and CtIP corepressor to elucidate the molecular mechanism of phosphoprotein recognition by a BRCT tandem repeat. Aim 2. Probe the interaction between TopBP1 and a phosphorylated peptide from E2F1 transcription factor to elucidate the molecular mechanism of phosphoprotein recognition by a BRCT homodimer. Aim 3. Probe the interaction of NBS1 with a phosphorylated histone H2AX peptide to elucidate the molecular mechanism of phosphoprotein recognition by FHA-BRCT tandem domains. Aim 4. Investigate a possible regulatory effect of phosphoprotein binding to DNA ligase IIIa on its BRCT-mediated interaction with DNA repair protein XRCC1.