STRUCTURE AND FUNCTION OF HYALURONAN SYNTHASES

描述（由申请人提供）：脊椎动物组织基质含有透明质酸（HA），它调节细胞行为（例如在发育，癌症，伤口愈合和血管生成过程中）。人类每天的HA周转量为克，积累的数据表明正常HA周转量过程的改变在疾病发病机制中可能是至关重要的。此外，小于正常水平的透明质酸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。尚不清楚小的透明质酸是否在体内通过降解或合成产生。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。透明质酸是由透明质酸合成酶（HAS）合成的，在本项目下由首席研究员首次克隆。许多疾病，包括出生缺陷、关节炎、溃疡性伤口和某些癌症，可能是由HAS功能改变引起的，或者是由HAS功能改变促进的。我们的目标是了解三种人类血凝素同工酶如何制造血凝素，调节血凝素大小，以及血凝素如何通过膜转移到基质或作为血凝素“外衣”的细胞表面的分子细节。进展的一个主要障碍是，去污剂对人类同工酶不起作用，而天然的、活性的人类HAS还不能在细胞中进行特异性免疫纯化或定位。我们将利用最近关于链球菌HAS脂质依赖性的发现来建立条件，以洗涤剂溶解人类HAS1、2和3并保持活性。我们的策略是利用链球菌HAS的结构-功能分析来阐明I类HAS家族的共同机制，以了解人类HAS的功能以及如何纯化活性人类HAS。这个更新期的一个主要项目重点是确定细菌和人类HASs是否通过孔或abc转运体机制，或两者兼有，将HA从细胞质溶胶输出到细胞表面或ECM。两种孔隙假说有待验证，一种假说是HAS本身产生了一个蛋白质内孔隙，另一种假说是HAS在生物合成过程中通过其孔隙将HA进行矢量转运。我们还将测试三种分子预测的ABC转运体假说的HA出口。以下具体目标涉及多个假设。1)表征HAS的孔隙活性。2)确定HAS是否将HA向跨膜转运。3)确定HA合成机制的关键特征。4)验证abc转运体输出HA假说的三个分子预测。5)克服阻碍人类活性哈斯化合物纯化的两个障碍。公共卫生相关性：许多疾病，包括出生缺陷、关节炎、溃疡性伤口和某些癌症，都可能由体内透明质酸（HA）合成的改变引起，或由其促进。透明质酸是由透明质酸合成酶合成的，该合成酶是在该项目下由首席研究员首次克隆的。了解三种人类血凝素合成酶如何发挥作用的细节将使我们和其他人能够制定策略来确定这些酶的调节剂和抑制剂（例如改变血凝素活性，血凝素大小或细胞定位），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.