STRUCTURE AND FUNCTION OF HYALURONAN SYNTHASES

描述（由申请方提供）：脊椎动物组织基质含有透明质酸（HA），可调节细胞行为（例如，在发育、癌症、伤口愈合和血管生成期间）。人类每天的HA周转量为克，积累的数据表明，正常HA周转过程的改变在疾病发病机制中可能是至关重要的。此外，小于正常的HA在炎症反应和癌细胞对化疗药物的敏感性方面具有意想不到的生物活性。目前尚不清楚小HA是否通过降解或合成在体内产生。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。HA由HA合成酶（HAS）合成，HAS首先由主要研究者在本项目下克隆。许多疾病，包括出生缺陷、关节炎、溃疡性伤口和一些癌症，可能是由改变的HAS功能引起的，或由改变的HAS功能促进的。我们的目标是了解3种人HAS同工酶如何产生HA、调节HA大小以及HA如何通过膜转移到基质或作为HA“涂层”转移到细胞表面的分子细节。进展的一个主要障碍是去污剂使人同工酶失活，并且天然的、活性的人HAS还不能被特异性免疫纯化或定位在细胞中。我们将使用最近的研究结果的脂依赖性的链球菌HAS建立条件，洗涤剂溶解人HAS 1，2和3保留的活动。我们的策略是使用链球菌HAS的结构-功能分析来阐明I类HAS家族内的共同机制，以了解人类HAS的功能以及如何纯化活性人类HAS。在此更新期间的一个主要项目重点是确定细菌和人类HAS是否通过孔或ABC转运蛋白机制或通过两者将HA从细胞溶质输出到细胞表面或ECM。待测试的两个孔假说是HAS本身产生蛋白质内孔，并且HAS在生物合成期间通过其孔载体地易位HA。我们也将检验ABC-转运蛋白假说对HA输出的三个分子预测.以下具体目标涉及多种假设。1)表征HAS的孔活性。2)确定HAS是否跨膜载体转运HA。3)确定HAS合成HA机制的关键特征。4)为了测试ABC转运蛋白输出HA的假设的三个分子预测。5)克服了两个阻碍活性人HAS纯化的障碍。公共卫生相关性：许多疾病，包括出生缺陷、关节炎、溃疡伤口和一些癌症，可能是由体内透明质酸（HA）合成的改变引起或促进的。HA是由主要研究者在本项目下首次克隆的HA酶合成的。了解这三种人HA酶的功能细节将使我们和其他人能够开发策略来鉴定这些酶的调节剂和抑制剂（例如改变HA活性，HA大小或细胞定位），这些酶可能是临床应用的候选药物，特别是在癌症，转移和链球菌疾病中。

DESCRIPTION (provided by applicant): Vertebrate tissue matrices contain hyaluronan (HA), which modulates cell behavior (e.g. during development, cancer, wound healing and angiogenesis). Humans turnover grams of HA daily, and accumulating data suggest that alterations in the normal HA-turnover process can be critical in disease pathogenesis. Also, HA that is smaller than normal has unexpected biological activities in inflammatory responses and cancer-cell sensitivity to chemotherapeutics. It is not yet known if small HA arises in vivo by degradation or synthesis. The long-term hypothesis guiding this project is that HA synthesis must be tightly controlled in order to achieve normal homeostasis and health. HA is synthesized by HA synthase (HAS), first cloned under this project by the principal investigator. Many diseases, including birth defects, arthritis, ulcerating wounds, and some cancers may arise from, or be promoted by, altered HAS function. Our goal is to under- stand the molecular details of how the 3 human HAS isozymes make HA, regulate HA size, and how HA is transferred through membranes to the matrix or to the cell surface as an HA "coat". A major obstacle to progress is that detergents inactive the human isozymes, and native, active human HAS cannot yet be specifically immuno-purified or localized in cells. We will use recent findings about the lipid-dependence of streptococcal HAS to establish conditions to detergent-solubilize human HAS1, 2 and 3 with retention of activity. Our strategy is to use structure-function analyses of streptococcal HAS to elucidate common mechanisms within the Class I HAS family, in order to understand how the human HASs function and how to purify active human HAS. A major project focus during this renewal period is to determine if bacterial and human HASs export HA from the cytosol to the cell surface or ECM by a Pore or ABC-transporter mechanism, or by both. Two Pore Hypotheses to be tested are that HAS itself creates an intra-protein pore, and that HAS translocates HA vectorally through its pore during biosynthesis. We will also test three molecular predictions of the ABC- transporter Hypothesis for HA export. The following specific aims address multiple hypotheses. 1) To characterize the pore activity of HAS. 2) To determine if HAS translocates HA vectorally across membranes. 3) To determine key features of the mechanism of HA synthesis by HAS. 4) To test three molecular predictions of the hypothesis that ABC-transporters export HA. 5) To overcome two obstacles that hinder purification of active human HASs. Public Health Relevance: Many diseases, including birth defects, arthritis, ulcerating wounds, and some cancers may arise from, or be promoted by, altered synthesis of hyaluronan (HA) in the body. HA is synthesized by HA synthases, which were first cloned under this project by the principal investigator. Knowing details about how the three human HA synthases function will enable us and others to develop strategies to identify modulators and inhibitors of these enzymes (e.g. to alter HA activity, HA size or cellular localization) that might be drug candidates for clinical applications, particularly in cancer, metastasis, and streptococcal diseases.