BASIS FOR STRESS TOLERANCE IN OSTEOLIGAMENT CELLS

骨韧带细胞的应激耐受性基础

• 批准号: 3222446
• 负责人: JOHN J SAUK
• 金额: $ 17.68万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-09-01 至 1994-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3222446
• 关键词: Golgi apparatus; SDS polyacrylamide gel electrophoresis; adenosinetriphosphatase; animal tissue; binding proteins; bioenergetics; chemical association; chick embryo; collagen; electron microscopy; fibroblasts; gelatin; gene expression; immunocytochemistry; immunoprecipitation; intracellular transport; ligaments; northern blottings; physiologic stressor; polysomes; procollagen; protein biosynthesis; protein folding; protein sequence; protein structure function; protein transport; stress proteins; tendons; thin layer chromatography; tissue /cell culture; western blottings

The hypothesis set forth in this proposal is that colligin/hsp47 is one of the cellular proteins that serves to provide the molecular basis for stress tolerance. As such, we propose that colligin/hsp47 belongs to a ubiquitous family of proteins, (molecular chaperones), whose proposed role is to mediate the folding and assembly of other proteins into oligomeric structures. Since colligin/hsp47's major substrate has been shown to be various collagen types, hsp47 is seen to have a transient exposure to procollagens during synthesis, the unfolding and refolding that occurs with transport from the endoplasmic reticulum, and during recovery from stresses such as heat shock. In order to test these hypotheses and add further to our understanding of stress tolerance we will utilize tissue culture, Western blot analysis, Northern blot analyses, protein binding, immunocytochemistry and electron microscopy to accomplish the following specific aims during a 3-year period. First we will verify that colligin/hsp47 interacts with intracellular collagen and determine it's binding to evolving and completed nascent pro-alpha-1 type I chains. Second, we will determine the temporal and compartmental expression of colligin/hsp47 with procollagen I. Next, we will determine whether the association or disassociation of colligin/hsp47-collagens requires an input of energy and is commensurate with the rate of procollagen chain synthesis. Finally, we will prove that there exists a strategic placement of sequences in procollagen of unusually persistent association with hsp47. Furthermore, that the association/disassociation from these sites occurs in a sequential manner that is compatible with procollagen I folding and oligomerization, and corresponding with the action of a chaperone protein. These studies will provide a better understanding of some of the fundamental mechanisms involving ligament and tendon injury. In addition, these studies will provide important information needed for establishing effective therapeutic procedures for the treatment of connective tissue disorders, particularly those of ligaments and tendons.

该提案中提出的假设是 colligin/hsp47 是其中之一 为压力提供分子基础的细胞蛋白 宽容。 因此，我们认为 colligin/hsp47 属于普遍存在的 蛋白质家族（分子伴侣），其提议的作用是 介导其他蛋白质折叠和组装成寡聚体 结构。 由于 Colligin/hsp47 的主要底物已被证明是 各种胶原蛋白类型，hsp47 被认为短暂暴露于 前胶原在合成过程中发生的展开和重折叠 从内质网运输，以及从压力恢复期间 如热休克。 为了检验这些假设并进一步补充 我们对压力耐受性的理解我们将利用组织培养， 蛋白质印迹分析、北方印迹分析、蛋白质结合、 免疫细胞化学和电子显微镜来完成以下任务 3年期间的具体目标。 首先我们将验证 colligin/hsp47 与细胞内胶原蛋白相互作用并确定其 与进化和完整的新生 pro-alpha-1 I 型链结合。 其次，我们将确定 colligin/hsp47 与前胶原 I。接下来，我们将确定是否 Colligin/hsp47-胶原蛋白的结合或解离需要输入 能量并与原胶原链合成速率相称。 最后，我们将证明存在序列的策略布局 与 hsp47 异常持久相关的前胶原。 此外，与这些站点的关联/解除关联发生在 与前胶原 I 折叠兼容的顺序方式 寡聚化，并与伴侣蛋白的作用相对应。 这些研究将有助于更好地理解一些 涉及韧带和肌腱损伤的基本机制。 此外， 这些研究将为建立 治疗结缔组织的有效治疗方法 疾病，特别是韧带和肌腱的疾病。