STRUCTURE AND FUNCTION OF HYALURONAN SYNTHASES

描述(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期间)。人类每天的HA周转克，不断积累的数据表明，正常HA周转过程中的变化在疾病发病机制中可能是关键的。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学活性。目前尚不清楚小分子HA在体内是通过降解还是合成产生的。指导这个项目的长期假设是，为了实现正常的动态平衡和健康，必须严格控制HA的合成。HA是由HA合成酶(HAS)合成的，它是由首席研究员在本项目下首次克隆的。许多疾病，包括出生缺陷、关节炎、溃烂伤口，以及一些癌症，都可能是由HAS功能改变引起的或由其促进。我们的目标是了解这3种人类同工酶如何制造HA，调节HA的大小，以及HA如何通过膜转移到基质或作为HA“外套”到细胞表面的分子细节。进展的一个主要障碍是洗涤剂使人的同工酶失活，而天然的、有活性的人还不能被特异性地免疫纯化或定位在细胞中。我们将利用最近关于链球菌HAS的脂质依赖性的发现来建立条件，以保持活性的洗涤剂增溶人类HAS1、2和3。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家族中常见的机制，以了解人类HASS是如何发挥作用的，以及如何纯化活性人类Has。在这一更新期间的一个主要项目重点是确定细菌和人类HASS是通过毛孔还是ABC转运蛋白机制，还是两者兼而有之，将HA从胞浆输出到细胞表面或ECM。需要检验的两个孔假设是，HAS本身创建了蛋白质内的孔，并在生物合成过程中通过孔向外转移HA。我们还将检验ABC转运体假说对HA输出的三个分子预测。以下具体目标涉及多个假设。1)表征HAS的孔道活性。2)确定Has是否跨膜向外转运HA。3)确定HAS合成HA机制的关键特征。4)验证ABC转运蛋白输出HA这一假设的三个分子预测。5)克服两个阻碍活性人HASS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，可能是由于体内透明质酸(HA)合成的改变而引起或促进的。HA是由HA合成酶合成的，该合酶是由首席研究员在该项目下首次克隆的。了解这三种人HA合成酶的功能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HA活性、HA大小或细胞定位)，这些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.