Probing the Role of Insulin-Like Growth Factor-Binding Protein 3 and Humanin in Regulating Hyaluronan Function

探讨胰岛素样生长因子结合蛋白 3 和护脑素在调节透明质酸功能中的作用

• 批准号: 9811032
• 负责人: HEDEEL I. GUY EVANS
• 金额: $ 44.55万
• 依托单位: EASTERN MICHIGAN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9811032
• 关键词: Affinity; Antibodies; Binding; Biochemical Process; Biological Assay; Biomedical Research; C-terminal; CD44 Antigens; CD44 gene; Carbohydrates; Cell Adhesion; Cell Death; Cell Line; Cell Survival; Cell physiology; Cells; Complex; Data; Disease; Education; Etiology; Glycosaminoglycans; Goals; Hyaluronan; Immobilization; In Vitro; Insulin-Like Growth Factor Binding Protein 3; Insulin-Like-Growth Factor I Receptor; Knowledge; Ligands; Mediating; Molecular; Peptides; Play; Protein Region; Proteins; Publishing; Regulation; Research Training; Role; Signal Transduction; Testing; base; career; cytotoxic; extracellular; glycosylation; graduate student; hands on research; human disease; humanin; insight; interest; mutant; novel; novel strategies; novel therapeutics; public health relevance; receptor; tool; undergraduate student

PROJECT SUMMARY Protein-peptide interactions are implicated in the etiology of many human diseases and known to play a crucial role in the undertaking of numerous cellular processes. Relatively little is known, however, about the role they play in regulating and fine-tuning carbohydrate signaling. Binding of Insulin-like growth factor-binding protein-3 (IGFBP-3) to the peptide, humanin, is well-studied and so is the interaction of the glycosaminoglycan, hyaluronan (HA), with its main receptor, CD44. However, while it is widely accepted that HA-receptor interaction is necessary for HA-induced effects, much remains to be unveiled about its basic operative mechanisms. The objective of this proposal is to investigate the physical and functional interactions between IGFBP-3 and humanin and their role in mediating HA-CD44 binding and signaling. Knowledge of this interplay and interlinkages involved is currently lacking. Accomplishing our aims in this proposal will provide needed insights into these linkages and will likely lead to novel understanding of the molecular mechanisms underlying fundamental protein- peptide-carbohydrate interactions. Our central hypothesis is that IGFBP-3 binds to HA and blocks its interaction with CD44, inhibiting cell survival. Humanin counteracts this effect by binding to IGFBP-3 thus disabling its binding to HA which is now free to interact with CD44. This hypothesis is based on the following observations: 1) humanin is known to bind residues 215-232 of mature IGFBP-3 in the C-terminal region of the protein. 2) This region of IGFBP-3 was shown earlier to bind certain glycosaminoglycans including HA. 3) Our recently published data showing that a) HA binds with a weaker affinity to this region of the protein than does humanin, b) either HA or humanin could bind to this IGFBP-3 segment, but not simultaneously, c) CD44 blocked HA binding to IGFBP-3, d) upon incubation of HA with CD44 and either IGFBP-3 protein or peptide, humanin was effective at binding and sequestering IGFBP-3 or peptide, thereby enabling access of CD44 to HA, e) while IGFBP-3 and humanin in the conditioned media can immuneprecipitate in a complex, the fraction of IGFBP-3 able to bind HA was not complexed with humanin, and f) IGFBP-3 exerts its cytotoxic effects on cell survival through a mechanism that depends on HA-CD44 interactions. Based on these observations, our specific aims are to: 1. Test the hypothesis that binding of IGFBP-3 to HA is inhibited by CD44 N-glycosylation but unaffected by CD44 reduction. 2. Test the hypothesis that there is a positive correlation between cell viability and the amount of IGFBP-3 bound to humanin in CD44-positive cell lines. 3. Test the hypothesis that while both IGFBP-3 and its peptide can disrupt HA-CD44 signaling, IGFBP-3, but not its peptide, can operate in an IGF-IR-dependent manner. Better understanding of these basic mechanisms will likely advance our knowledge of diseases resulting from dysregulation of protein-peptide-carbohydrate signaling. This R15 application provides an effective vehicle for introducing undergraduate and graduate students to an authentic and extensive hands-on research training at an early stage of their education and cultivates an interest in a career in biomedical research.

项目摘要 蛋白质-肽相互作用与许多人类疾病的病因学有关，并且已知在许多疾病中起关键作用。 在许多细胞过程中发挥作用。然而，人们对它们的作用知之甚少， 在调节和微调碳水化合物信号方面发挥作用。胰岛素样生长因子结合蛋白-3的结合 胰岛素样生长因子结合蛋白-3（IGFBP-3）与肽humanin之间的相互作用以及糖胺聚糖透明质酸之间的相互作用已得到充分研究 (HA)其主要受体为CD 44。然而，尽管广泛接受HA-受体相互作用是不可避免的。 虽然HA诱导效应的必要条件是多方面的，但其基本作用机制仍有待揭示。的 本研究的目的是探讨IGFBP-3与人胰岛素之间的物理和功能相互作用， 以及它们在介导HA-CD 44结合和信号传导中的作用。对这种相互作用和相互联系的了解 目前缺乏参与。实现我们在本提案中的目标将为这些问题提供必要的见解。 连接，并可能导致新的理解的分子机制的基础蛋白质- 肽-碳水化合物相互作用。我们的中心假设是IGFBP-3与HA结合并阻断其相互作用 CD 44抑制细胞存活Humanin通过与IGFBP-3结合来抵消这种作用，从而使其丧失功能。 结合HA，其现在自由地与CD 44相互作用。这一假设基于以下观察： 1)已知人胰岛素结合成熟IGFBP-3的C-末端区域的残基215-232。2)这 IGFBP-3的区域较早显示结合某些糖胺聚糖，包括HA。3)我们最近出版的 数据显示a）HA与蛋白质的该区域的结合亲和力比人源蛋白弱，B） HA或humanin可以结合该IGFBP-3区段，但不能同时结合。 d）在HA与CD 44和IGFBP-3蛋白或肽一起孵育后，humanin在 结合和螯合IGFBP-3或肽，从而使CD 44能够接近HA，e）而IGFBP-3和 条件培养基中的人素可以免疫沉淀成复合物，即IGFBP-3能够结合HA的部分 不与人球蛋白复合，和f）IGFBP-3通过与人球蛋白复合而对细胞存活发挥其细胞毒性作用。 依赖于HA-CD 44相互作用的机制。基于这些观察，我们的具体目标是：1。 检验IGFBP-3与HA的结合受CD 44 N-糖基化抑制但不受CD 44影响的假设 还原2.检验细胞活力与细胞内的蛋白质的量之间存在正相关性的假设。 IGFBP-3在CD 44阳性细胞系中与人蛋白结合。3.测试假设，虽然IGFBP-3及其 肽可以破坏HA-CD 44信号传导，IGFBP-3，但不是它的肽，可以在IGF-IR依赖性 方式更好地理解这些基本机制将可能促进我们对疾病的认识 由蛋白质-肽-碳水化合物信号传导失调引起。此R15应用程序提供了 有效的车辆介绍本科生和研究生一个真实的和广泛的动手 研究培训在他们的教育的早期阶段，并培养在生物医学研究的职业生涯的兴趣。