BASIS FOR STRESS TOLERANCE IN OSTEOLIGAMENT CELLS

在这个提议中提出的假设是，colligin/hsp 47是其中之一， 为压力提供分子基础的细胞蛋白质 宽容 因此，我们认为colligin/hsp 47属于一个普遍存在的 蛋白质家族（分子伴侣），其作用是 介导其他蛋白质折叠和组装成寡聚体 结构. 由于colligin/hsp 47的主要底物已被证明是 各种类型的胶原蛋白，hsp 47被认为具有短暂的暴露于 前胶原在合成过程中，发生的解折叠和重折叠， 从内质网的运输，并在恢复过程中的压力 例如热休克。 为了验证这些假设，并进一步增加 我们将利用组织培养， Western印迹分析，北方印迹分析，蛋白结合， 免疫细胞化学和电子显微镜，以完成以下任务 三年内的具体目标。 首先，我们将验证 colligin/hsp 47与细胞内胶原蛋白相互作用，并决定其 与进化和完成的新生前α-1 I型链结合。 其次，我们将确定时间和房室表达的 colligin/hsp 47与前胶原I。 接下来，我们将确定 胶原蛋白/hsp 47-胶原蛋白的结合或解离需要输入 的能量，并与原胶原链合成的速率相称。 最后，我们将证明存在序列的策略布局 在与HSP 47异常持续相关的前胶原中。 此外，与这些网站的关联/分离发生在 与前胶原I折叠相容的顺序方式， 寡聚化，并对应于伴侣蛋白的作用。 这些研究将使人们更好地了解一些 涉及韧带和肌腱损伤的基本机制。 此外，本发明还提供了一种方法， 这些研究将提供重要信息， 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。

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.