Molecular Biology of Hyaluronan Biosynthesis

透明质酸生物合成的分子生物学

基本信息

批准号：
8816855
负责人：
Jochen Zimmer
金额：
$ 27.55万
依托单位：
UNIVERSITY OF VIRGINIA
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-01-01 至 2018-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8816855
关键词：
Acetylglucosamine Affect Alginates Amber Anabolism Architecture Autoimmune Diseases Binding Biochemical Biological Assay Biological Models Biological Process Biotechnology Carcinoma Cartilage Catalysis Cell Adhesion Cell Communication Cell Differentiation process Cell Surface Receptors Cell membrane Cell-Cell Adhesion Cells Cellulose Charge Chemicals Chlorella Co-Immunoprecipitations Collaborations Complex Connective and Soft Tissue Crystallization Data Deposition Detergents Development Engineering Enzymes Escherichia coli Event Extracellular Matrix Glucuronic Acids Human Human body Hyaluronan Imaging Techniques Immunosuppressive Agents In Vitro Inflammatory Laboratories Lead Length Life Link Lipid Bilayers Lipids Malignant Neoplasms Maps Mediating Medicine Membrane Membrane Biology Membrane Lipids Membrane Proteins Methods Microbial Biofilms Microfilaments Modeling Molecular Molecular Biology Molecular Weight Mono-S Nature Phase Phenylalanine Photobleaching Physiological Physiological Processes Physiology Polymers Polysaccharides Positioning Attribute Process Property Proteins Protocols documentation Publishing Reaction Regulation Research Resistance Resolution Rheumatoid Arthritis Robotics Side Signal Transduction Site Skin Streptococcus Structure Techniques Terminator Codon Transferase Transmembrane Domain UV Radiation Exposure Universities Vertebrates Viral Virginia Virus X-Ray Crystallography base cell motility chemical property crosslink dodecyldimethylamine oxide extracellular glycosyltransferase hyaluronan synthase 1 member membrane synthesis mutant physical property proteoliposomes public health relevance reconstitution research study rho GTP-Binding Proteins stoichiometry sugar three dimensional structure

项目摘要

DESCRIPTION (provided by applicant): The extracellular matrix in vertebrates provides structural support to the cell, aids in osmo-regulation, and is particularly important in mediating cell-cell interactions in soft connective tissues, such as cartilage and skin. A major component of the extracellular matrix is hyaluronan (HA), which is an extracellular linear polysaccharide containing alternating N-acetylglucosamine (NAG) and glucuronic acid (GA) residues. HA affects many physiological processes, from cell adhesion and migration to cell differentiation and embryological development. Because of its broad impact on human physiology, a large number of pathological conditions, including many forms of cancer, autoimmune diseases, inflammatory processes, and rheumatoid arthritis, correlate with altered expression levels of HA. On a molecular level, HA is produced inside the cell by the membrane-embedded hyaluronan synthase (HAS). HAS is a remarkable enzyme. It not only catalyzes the synthesis of HA from UDP-activated substrates, but it also transports the growing polymer across the cell membrane to deposit it within the extracellular matrix. In order to accomplish this task, HAS has to fulfill several functions. The enzyme binds the substrates UDP-NAG and -GA, it catalyzes the glycosyl transfer reaction to form HA, and it translocates the growing polymer across the cell membrane through a pore formed by its own transmembrane region. To understand how HA exerts its physiological function and to produce HA polymers with defined properties for biomedical applications, we must first unravel how HAS synthesizes HA and how it deposits the polymer in the extracellular matrix. To this end, we propose three aims that will reveal the assembly of biologically active HAS subunits in native lipid membranes, will identify the interactions between HAS and the translocating HA polymer, and will allow us to determine the structure of HAS by X-ray crystallography. First, we will combine co-immunoprecipitation studies with chemical cross-linking and photobleaching techniques to visualize HAS oligomers in native membranes. The low-resolution structural data will then be integrated with high-resolution structures of monomeric HAS to reconstruct the native, membrane-embedded HAS oligomer. Second, the interactions of HAS with the translocating HA polymer will be mapped by introducing UV-inducible cross-linkers into the TM-region of HAS. Cross-linking during HA translocation will identify positions that are in close proximity to the polysaccharide, thus delineating the physico-chemical properties of the HA transmembrane channel. Third, biochemical and low resolution structural data will be integrated with a high-resolution structure of HAS obtained by X-ray crystallography. Determining the structure of HAS both in a detergent-solubilized but also in a membrane-embedded state will reveal the architecture and oligomeric form of the synthase, allowing us to delineate the mechanism by which this marvelous enzyme synthesizes one of the most abundant extracellular polysaccharides in the human body.

描述（由申请人提供）：脊椎动物的细胞外基质为细胞提供结构支持，有助于渗透压调节，并且在介导软结缔组织（例如软骨和皮肤）中的细胞与细胞相互作用。的一个主要组成部分细胞外基质是透明质酸（HA），它是一种细胞外线性多糖，含有交替的N-乙酰氨基葡萄糖（NAG）和葡萄糖醛酸（GA）残基。 HA 影响许多生理过程，从细胞粘附和迁移到细胞分化和胚胎发育。由于其对人类生理学的广泛影响，许多病理状况，包括多种形式的癌症、自身免疫性疾病、炎症过程和类风湿性关节炎，都与 HA 表达水平的改变相关。在分子水平上，HA 由膜嵌入的透明质酸合酶 (HAS) 在细胞内产生。 HAS 是一种非凡的酶。它不仅催化 UDP 激活的底物合成 HA，而且还将生长的聚合物运输穿过细胞膜，将其沉积在细胞外基质内。为了完成这项任务，HAS 必须完成几个功能。该酶结合底物UDP-NAG和-GA，催化糖基转移反应形成HA，并通过其自身跨膜区域形成的孔将生长的聚合物跨细胞膜移位。为了了解HA如何发挥其生理功能并生产具有特定特性的HA聚合物用于生物医学应用，我们必须首先阐明HAS如何合成HA以及它如何将聚合物沉积在细胞外基质中。为此，我们提出了三个目标，即揭示生物活性 HAS 亚基在天然脂质膜中的组装，识别 HAS 与易位 HA 聚合物之间的相互作用，并使我们能够通过 X 射线晶体学确定 HAS 的结构。首先，我们将免疫共沉淀研究与化学交联和光漂白技术相结合，以可视化天然膜中的 HAS 寡聚物。然后，低分辨率结构数据将与单体 HAS 的高分辨率结构整合，以重建天然的膜嵌入 HAS 寡聚物。其次，通过将 UV 诱导交联剂引入 HAS 的 TM 区域，可以绘制 HAS 与易位 HA 聚合物的相互作用。 HA 易位过程中的交联将识别靠近多糖的位置，从而描绘出 HA 跨膜通道的物理化学特性。第三，生化和低分辨率结构数据将与通过X射线晶体学获得的HAS高分辨率结构相结合。确定去污剂溶解和膜嵌入状态下的 HAS 结构将揭示合酶的结构和寡聚形式，使我们能够描述这种奇妙的酶合成最丰富的细胞外多糖之一的机制。人体。