RUI: Biochemical Analysis of Mastermind Dependent Activation of Notch Target Genes

智力上的功绩。Noch信号是一个重要且非常有意义的课题，因为它代表了在所有后生动物中发现的负责细胞生长、分化和维持的五个主要信号转导途径之一(Wnt/β-catenin、Hedgehog、受体酪氨酸激酶、核受体和Notch)。Notch信号的错误调节可能导致发育和疾病状态。在Notch受体的配体激活后，细胞内片段(NICD)移位到细胞核。NICD与Mam(主脑)蛋白和其他蛋白质形成一个三元复合体，以激活靶基因。在人类中，有三种Mam蛋白(Mam1-3)。每个MAM包含一个与NICD相互作用的氨基末端碱性区域和一个可能在转录激活中起作用的羧基末端酸性区域。Mam1的截短版本失去了这些羧基区域，但保留了基本结构域，当它们在模式生物中过度表达时，会产生显性的负表型。这个项目的总体目标是剖析Notch信号所必需的MAM蛋白质家族的分子元素，目的是在分子水平上深入了解其复杂性。酵母双杂交筛选和生化蛋白质-蛋白质下拉技术将被用来从组织培养细胞系中鉴定MAM相互作用的伙伴。假定的MAM相互作用蛋白的作用将通过分子和生物化学方法的组合来确定。该项目将使在圣何塞州立大学(SJSU)接受生物化学和分子生物学培训的本科生受益。就读上海州立大学的学生大多来自经济困难的家庭。这些学生中有许多来自代表性不足的学生或第一次上大学的学生。这个项目将为学生提供一个学习和进行研究的独特机会。学生将被鼓励思考和设计他们自己的实验，并将在州和全国会议上展示他们的结果。此外，为了尽可能多地接触到学生，在这个项目中开发的一些研究方法也将被纳入本科分子生物学实验课程。

Intellectual Merit. Notch signaling is an important and highly significant topic to understand because it represents one of the five major signal transduction pathways (Wnt/beta-catenin, Hedgehog, receptor tyrosine kinases, nuclear receptors, and Notch) found in all metazoan organisms responsible for growth, differentiation, and maintenance of cells. Misregulation of Notch signaling can result in developmental as well as disease states. After ligand activation of the Notch receptor, an intracellular segment (NICD) translocates to the nucleus. The NICD forms a ternary complex with the Mam (Mastermind) protein and other proteins to activate target genes. In humans, there are three Mam proteins (Mam1-3). Each Mam contains an amino terminal basic domain that interacts with NICD and a carboxy terminal acidic region that likely functions in transcriptional activation. Truncated versions of Mam1 that lose these carboxyl regions, but retain the basic domain, produce dominant negative phenotypes when they are over-expressed in model organisms. The overall goal of this project is to dissect the molecular elements of the Mam family of proteins that are necessary for Notch signaling with the goal of gaining insight into the complexity at the molecular level. Yeast two-hybrid screens and biochemical protein-protein pull down techniques will be employed to identify Mam-interacting partners from tissue culture cell lines. The role of putative Mam-interacting proteins will be determined though a combination of molecular and biochemical approaches.Broader Impacts. This project will benefit undergraduate students who will receive training in biochemistry and molecular biology at San José State University (SJSU). The majority of students attending SJSU are from economically disadvantaged families. A number of these students are from underrepresented or first time college attendees. This project will provide students with a unique opportunity to learn and conduct research. Students will be encouraged to think and design their own experiments and will present their results at both state and national conferences. Further, to reach as many students as possible, some of the research methods developed during this project will also be incorporated into an undergraduate molecular biology laboratory course.