SAM Domains

描述（由申请人提供）：无菌α基序（SAM）结构域是真核细胞中最常见的蛋白质模块之一。它们存在于许多关键调控蛋白、支架蛋白和转录因子中，其中包括许多与遗传疾病有关的蛋白质。对分离系统的重点研究表明，SAM结构域是多种蛋白质-蛋白质相互作用的伙伴，可以形成同质低聚物、异质低聚物、同质聚合物、异质聚合物和与其他大分子的配合物。然而，绝大多数SAM结构域在功能上仍未被表征，这给我们对重要生物学途径和疾病状态的理解留下了重大空白。在这里，我们寻求利用我们在SAM结构域结构和功能方面积累的大量专业知识，从全局上阐明人类基因组中SAM结构域的功能。目标一：鉴定人类SAM聚合物。每个人- sam结构域都将表达为与增压GFP的融合，以保持聚合物的溶解度。SAM结构域将通过天然凝胶和/或凝胶过滤色谱来筛选聚集体的形成。如果发现它们形成聚集体，它们将被电子显微镜或原子力显微镜进一步检查，以区分聚集体和真正的聚合物。目的二：确定所有人类同质和异质sam相互作用，发现聚合物调节剂和新的共聚物。我们将使用分裂DHFR分析来识别相互结合的sam。确定的相互作用将用Aim i的GFP融合物进行生化验证。我们还希望发现新的SAM聚合物调节剂和可能的SAM共聚物。我们相信这里提出的以发现为导向的方法是一种有效的方法，可以最大限度地利用我们之前NIH资助的进步，造福他人。

DESCRIPTION (provided by applicant): Sterile Alpha Motif (SAM) domains are among the most common protein modules in eukaryotic cells. They are found in many key regulatory proteins, scaffolding proteins, and transcription factors, which include many proteins involved in genetic diseases. Focused investigation of isolated systems has revealed that SAM domains are versatile protein-protein interaction partners, forming homo-oligomers, hetero-oligomers, homo- polymers, hetero-polymers and complexes with other macromolecules. Nevertheless, the vast majority of SAM domains remain functionally uncharacterized, leaving major gaps in our understanding of important biological pathways and disease states. Here we seek to leverage the considerable expertise we have developed with SAM domain structure and function to globally illuminate the functions of SAM domains in the human genome. The aims are: Aim I: Identify human SAM polymers. Every human-SAM domain will be expressed as a fusion to supercharged GFP to maintain polymer solubility. The SAM domains will be screened for aggregate formation by native gels and/or gel filtration chromatography. If they are found to form aggregates, they will be further examined by electron microscopy or AFM to distinguish aggregates from true polymers. Aim II: Identify all human homo- and hetero-SAM interactions discover polymer regulators and new copolymers. We will use a split DHFR assay to identify SAMs that bind to one another. Identified interactions will be verified biochemically with our GFP fusions from Aim I. We also hope to discover new SAM polymer regulators and possibly SAM copolymers. We believe the discovery-oriented approach proposed here is an efficient way to maximize the utility of our prior NIH funded advances for the benefit of others. PUBLIC HEALTH RELEVANCE: Cellular functions require precise communication between proteins. Disease occurs when these exchanges go awry. This project seeks to identify and characterize the interactions of a protein binding module found in about a hundred human proteins, including proteins directly involved in autism, leukemia, cleft palate, immune disorders, deafness and blindness. Thus, this project could illuminate the mechanisms of diverse disease states.