BASIS FOR STRESS TOLERANCE IN OSTEOLIGAMENT CELLS

这项建议中提出的假设是，胶原素/hsp47是 为压力提供分子基础的细胞蛋白质 宽容。因此，我们认为Coligin/hsp47属于一种普遍存在的 蛋白质家族，(分子伴侣)，其建议的作用是 调节其他蛋白质的折叠和组装成低聚物 结构。自从胶原素/hsp47‘S以来，主要底物已被证明是 各种胶原蛋白类型，hsp47被认为有短暂的接触 合成过程中的前胶原蛋白，发生在 内质网运输和应激恢复过程 比如热休克。为了检验这些假说并进一步补充 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，Northern印迹分析，蛋白质结合， 免疫细胞化学和电子显微镜完成以下工作 3年内的具体目标。首先，我们将核实 胶原素/hsp47与细胞内胶原相互作用并确定其 结合到不断进化和完成的新生前-α-1 I型链。 第二，我们将确定时间和隔间的表达 胶原蛋白/hsp47与前胶原I，接下来，我们将确定是否 胶原素/hsp47-胶原蛋白的结合或解离需要输入 并与前胶原链合成的速度相称。 最后，我们将证明序列存在一个策略性位置 在异常持久的与hsp47相关的前胶原中。 此外，与这些站点的关联/解除关联发生在 与I型前胶原折叠兼容的顺序方式和 齐聚作用，与伴侣蛋白的作用相对应。 这些研究将使我们更好地了解一些 涉及韧带和肌腱损伤的基本机制。此外, 这些研究将提供所需的重要信息 结缔组织治疗的有效治疗方法 紊乱，尤指韧带和肌腱的紊乱。

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.