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。羟基磷灰石是将细胞粘合在一起的“粘合剂”。为了使细胞移入或移出基质，HA必须被hHyals碎片化。受损组织的修复需要细胞在基质中移动，就像异常细胞(转移)的逃逸一样。HA片段被邻近的细胞检测到，细胞内的反应通过信号转导途径发生。接收到的信号取决于Hyals产生的HA碎片的大小。具有特殊医学意义的hHyal-1和hHyal-2在组织炎症和癌症中发挥关键作用，因此是开发新型抗癌和抗炎治疗药物的极佳靶点。直到最近，由于高产量生产活性N-糖基化hHyals相关的技术挑战，这些酶的结构-功能研究是不可能的。最近在Herzberg实验室发展了一种制备hHyal-1和hHyal-2的方法，并测定了hHyal-1的晶体结构。该阶段现在可以使用体外方法来准确地确定hHyal-1和hHyal-2的催化功能和抑制，以及它们与蛋白质伙伴、CD44-透明质酸结合蛋白、受hHyal-2调控的RON受体酪氨酸激酶和激活RON并使用hHyal-2附着到宿主细胞的Jaagsiekte绵羊逆转录病毒包膜蛋白的相互作用。这项建议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背后的分子机制，并将为新疗法的开发提供结构基础。与公共健康相关：透明质酸酶-1和透明质酸酶-2对细胞外基质的完整性和功能至关重要，它们的酶活性控制细胞外多糖透明质酸(HA)的周转。HA的周转微妙地平衡，透明质酸酶活性升高会导致炎症性疾病和癌症的进展和侵袭。本项目旨在研究人透明质酸酶及其与细胞伴侣蛋白的体外相互作用，以阐明它们在体内的功能，并为透明质酸酶作为抗癌和抗炎药物靶点的开发奠定基础。

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.