SAM Domains

描述(申请人提供)：不育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的调控蛋白、支架蛋白和转录因子中，其中包括许多与遗传病有关的蛋白质。对分离系统的集中研究表明，SAM结构域是多功能的蛋白质-蛋白质相互作用伙伴，形成均低聚物、杂低聚物、均聚物、异聚物以及与其他大分子的络合物。然而，绝大多数SAM结构域在功能上仍未确定，在我们对重要的生物学途径和疾病状态的理解上留下了重大的空白。在这里，我们试图利用我们在SAM结构域结构和功能方面开发的大量专业知识来全面阐明人类基因组中SAM结构域的功能。目的是：目的一：鉴定人类SAM聚合物。每个人-SAM结构域都将被表达为与增压GFP的融合，以保持聚合物的溶解性。SAM结构域将通过天然凝胶和/或凝胶过滤层析来筛选聚集体的形成。如果发现它们形成聚集体，将通过电子显微镜或AFM进一步检查，以区分聚集体和真正的聚合物。目的II：鉴定所有人类同源和异源SAM相互作用，发现聚合物调节剂和新的共聚物。我们将使用分裂的DHFR分析来鉴定相互结合的SAM。已确定的相互作用将通过我们来自AIM I的GFP融合进行生化验证。我们还希望发现新的SAM聚合物调节剂，并可能发现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式，可以最大限度地利用我们之前由NIH资助的预付款，造福于他人。 与公共健康相关：细胞功能需要蛋白质之间的精确沟通。当这些交流出现问题时，疾病就会发生。该项目旨在识别和表征在大约100种人类蛋白质中发现的蛋白质结合模块的相互作用，这些蛋白质包括与自闭症、白血病、腭裂、免疫紊乱、耳聋和失明直接相关的蛋白质。因此，这个项目可以阐明不同疾病状态的机制。

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.