STRUCTURE AND FUNCTION OF HYALURONAN SYNTHASES

描述（申请人提供）：透明质酸（HA）是脊椎动物中普遍存在的细胞外基质成分，是细胞行为的强大调节剂，例如在细胞迁移，发育，癌症，伤口愈合和血管生成过程中。越来越多的证据表明，HA正常合成和降解的改变在关节炎和癌症等疾病的发病机制中可能是至关重要的。指导该项目的长期假设是，为了实现正常的体内平衡和健康，必须严格控制透明质酸的合成。整个项目包含两个子项目，研究了HA合成（由HA合成酶介导，我们是第一个克隆和纯化的小组）和HA周转（由HA受体介导的内吞作用）。因为这些子项目现在已经有了分子基础，并且已经大大扩展，所以它们被分成两个独立的R01项目。目前的项目将继续研究链球菌和人类HA合成酶（HASs）。然而，由于两个原因，目前HAS领域停滞不前：没有人能够纯化活性的天然人类HAS，或者在其他两个存在的情况下评估单个HAS异构体的数量或活性。因此，我们推进该领域的策略是使用链球菌HASs作为模型来理解人类HASs的功能。我们的具体假设是，对链球菌HASs的结构-功能分析将使我们了解人类HASs是如何工作的以及HA的合成是如何被调节的。我们将继续努力了解链球菌的HASs，但也将利用我们所学到的来研究人类的HASs。我们将运用生物化学、分子生物学和细胞生物学的技术来解决以下具体目标中的多个假设。1)表征新合成HA还原端的新型HA- udp联动。2)确定几丁质- udp低聚物在HA合成中的作用。3)鉴定活化seHAS的独特磷脂，并测试其对人类HASs的影响。4)分析链球菌HASs的结构-功能关系。5)利用EPR光谱法确定seHAS结构域的组织和运动。这个项目的结果可能会导致人类HAS蛋白在各种病理和疾病中的作用的首次调查，也可能导致链球菌疾病新药的开发。

DESCRIPTION (provided by applicant): Hyaluronan (HA), which is a ubiquitous extracellular matrix component in vertebrates, is a powerful modulator of cell behavior, e.g. during cell migration, development, cancer, wound healing and angiogenesis. Accumulating evidence suggests that alterations in the normal synthesis and degradation of HA can be critical in the pathogenesis of diseases such as arthritis and cancer. The long-term hypothesis guiding this project is that HA synthesis must be tightly controlled in order to achieve normal homeostasis and health. The overall project has contained two sub-projects that have investigated HA synthesis (mediated by HA synthases, which we were the first group to clone and then to purify) and HA turnover (mediated by the HA Receptor for Endocytosis). Because these sub-projects now have a molecular basis and have greatly expanded, they are being split into two separate R01 projects. The current project will continue to study the streptococcal and human HA synthases (HASs). However, the HAS field is presently stalled for two reasons: no one has been able to purify an active native human HAS, or to assess the amount or activity of one individual HAS isoform in the presence of the other two. Consequently, our strategy to advance the field is to use the streptococcal HASs as a model to understand the functions of the human HASs. Our specific hypothesis is t hat structure-function analyses of the streptococcal HASs will allow us to understand how the human HASs work and how HA synthesis is regulated. We will continue efforts to understand the streptococcal HASs, hut also use what we learn to study the human HASs. We will employ techniques in biochemistry, molecular biology and cell biology to address multiple hypotheses in the following specific aims. 1) To characterize the novel HA-UDP linkage at the reducing end of newly synthesized HA. 2) To determine the role of chitin-UDP oligomers in HA synthesis by HAS. 3) To identify the unique phospholipids that activate seHAS and test their effects on human HASs. 4) To analyze structure-function relationships in the streptococcal HASs. 5) To determine the organization and motion of seHAS domains using EPR spectroscopy. Results from this project could lead to the first investigations of the roles of human HAS proteins in various pathologies and diseases and could also lead to the development of new drugs for streptococcal diseases.