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.
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