Structure-Function studies of human hyaluronidases

描述（由申请人提供）：本项目重点研究人透明质酸酶-1（hHyal-1）和透明质酸酶-2（hHyal-2）在介导细胞外基质多糖透明质酸（HA）所承担的生物学功能中的作用。透明质酸是将细胞结合在一起的“胶水”。为了使细胞移入或移出基质，HA必须被hHyals片段化。受损组织的修复需要细胞在基质内移动，异常细胞的逃逸（转移）也是如此。HA片段被邻近细胞检测到，并且通过信号转导途径发生细胞内反应。接收到的信号取决于透明质酸产生的HA片段的大小。hHyal-1和hHyal-2在组织炎症和癌症中起着关键作用，因此是开发新型抗癌和抗炎疗法的极好靶点。直到最近，由于与活性N-糖基化hHyals的高产率生产相关的技术挑战，这些酶的结构-功能研究还不可能。最近在Herzberg实验室开发了一种制备hHyal-1和hHyal-2的方法，并确定了hHyal-1的晶体结构。现阶段将采用体外方法准确确定hHyal-1和hHyal-2的催化功能和抑制作用，以及它们与蛋白质伴侣的相互作用，所述蛋白质伴侣是透明质酸结合蛋白CD 44、由hHyal-2调节的罗恩受体酪氨酸激酶和激活罗恩并利用hHyal-2附着于宿主细胞的Jaagsiekte绵羊逆转录病毒包膜蛋白。本提案中提出的研究计划将在原子水平上阐明透明质酸酶控制的复杂细胞过程的分子机制，并将为新疗法的开发提供结构基础。公共卫生相关性：透明质酸酶-1和透明质酸酶-2通过其控制胞外多糖透明质酸（HA）周转的酶活性对胞外基质的完整性和功能至关重要。HA周转是微妙平衡的，透明质酸酶活性升高导致炎性疾病和癌症进展和侵袭。该项目旨在表征人透明质酸酶及其与细胞伴侣蛋白的体外相互作用，以阐明其体内功能，并为透明质酸酶作为抗癌和抗炎药物靶标的开发奠定基础。

DESCRIPTION (provided by applicant): This project focuses on the roles of human hyaluronidase-1 (hHyal-1) and hyaluronidase-2 (hHyal-2) in the mediation of the biological functions assumed by the extracellular matrix polysaccharide hyaluronan (HA). HA serves as the "glue" that binds cells together. In order for cells to move in or out of the matrix, HA must be fragmented by hHyals. Repair of injured tissue requires cell movement within the matrix, as does the escape of an abnormal cell (metastasis). HA fragments are detected by neighboring cells, and an intracellular response occurs via signal transduction pathways. The signal received is dependent on the size of the HA fragments as produced by the Hyals. Of particular medical importance, hHyal-1 and hHyal-2 play key roles in tissue inflammation and in cancer, and therefore are excellent targets for the development of novel anti-cancer and anti-inflammatory therapeutics. Until recently, structure-function studies of these enzymes have not been possible owing to the technical challenge associated with high yield production of the active N-glycosylated hHyals. A method for hHyal-1 and hHyal-2 preparation has been recently developed in the Herzberg lab and the crystal structure of hHyal-1 has been determined. The stage is now set to employ in vitro methods to accurately define hHyal-1 and hHyal-2 catalytic function and inhibition, and their interactions with protein partners, CD44 - the hyaluronan biding protein, RON receptor tyrosine kinase that is regulated by hHyal-2, and Jaagsiekte sheep retrovirus envelope protein that activates RON and uses hHyal-2 to attach to the host cell. The research plan set forth in this proposal will illuminate at the atomic level the molecular mechanisms underlying the complex cellular processes controlled by the hyaluronidases and will provide the structural foundation for the development of new therapeutics. PUBLIC HEALTH RELEVANCE: Hyaluronidase-1 and hyaluronidase-2 are crucial to the integrity and functioning of the extracellular matrix through their enzymatic activity that controls the turnover of the extracellular polysaccharide hyaluronan (HA). The HA turnover is delicately balanced and heightened hyaluronidase activity leads to inflammatory diseases and cancer progression and invasion. This project seeks to characterize the human hyaluronidases and their interactions with cellular partner proteins in vitro to shed light on their in vivo function and to lay the foundation for development of the hyaluronidases as anti cancer and anti inflammatory drug targets.