STRUCTURE AND FUNCTION OF HYALURONAN SYNTHASES

描述（由申请人提供）：海洛烷（HA）是脊椎动物中普遍存在的细胞外基质组分，是细胞行为的有力调节剂，例如在细胞迁移、发育、癌症、伤口愈合和血管生成期间。越来越多的证据表明，HA的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至关重要。指导该项目的长期假设是，HA合成必须严格控制，以实现正常的稳态和健康。整个项目包含两个子项目，研究HA合成（由HA酶介导，我们是第一个克隆然后纯化的小组）和HA周转（由HA内吞受体介导）。因为这些子项目现在有了分子基础，并且已经大大扩展，所以它们被分成两个独立的R 01项目。目前的项目将继续研究链球菌和人类HA酶（HAS）。然而，HAS领域目前由于两个原因而停滞不前：没有人能够纯化活性天然人HAS，或者在存在其他两种HAS同种型的情况下评估一种单独HAS同种型的量或活性。因此，我们的战略，以推进该领域是使用链球菌HAS作为模型，以了解人类HAS的功能。我们的具体假设是，链球菌HAS的结构-功能分析将使我们了解人类HAS如何工作以及HA合成如何调节。我们将继续努力了解链球菌HAS，但也利用我们所了解的研究人类HAS。我们将采用生物化学，分子生物学和细胞生物学的技术来解决以下特定目标的多个假设。1)表征新合成HA还原端的新型HA-UDP键。2)确定几丁质-UDP寡聚体在HAS合成HA中的作用。3)鉴定激活seHAS的独特磷脂并测试其对人类HAS的影响。4)分析链球菌HAS的结构与功能关系。5)使用EPR光谱确定seHAS结构域的组织和运动。该项目的结果可能导致人类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.