MOLECULAR BASIS FOR ASSURANCE OF TYPE I COLLAGEN

在这项建议中，我们把注意力集中在定性问题上

In this proposal, we focus our attention on the problem of characterizing the cellular mechanisms for ensuring that collagen molecules with correct type I stoichiometry {pro alpha-1(I)2 pro alpha-2(I)} and folding result, and the role of a chaperone, Hsp47, in that process. the hypothesis set forth is that the rapid intracellular degradation of collagen is inversely related to the level of Hsp47 in the endoplasmic reticulum (ER). Hsp47 binds to the N-terminal of pro alpha-1 chains of type I collagen. This binding of the N-propeptide occurs at a very early stage, probably soon after pro alpha-1 chains are inserted into the ER. As such, Hsp47 is an important element in directing collagen chain selection and assembly. In the absence of Hsp47, nascent chains are seen to elongate, however, assembly of chains into heteropolymers is constrained in favor of [pro alpha-1(I)]3 homotrimers or nascent chains which are retained in the ER and degraded. The major degradative pathway employed for this latter cellular surveillance follows a pre-Golgi, non-lysomal route. Thus, selective degradation of unassembled, anomalous, or incompletely assembled heteropolymers plays a key role in disposing of excess collagen chains, thereby ensuring that only molecules with the correct stoichiometry reach the extracellular matrix. To test this hypothesis antisense oligonucleotides for Hsp47 will be employed such that complementary DNA or RNA sequences interact with mRNA to block translation of Hsp47. We will then demonstrate the collagen chain assembly into type I heterotrimers occurs when the process is directed by Hsp47 and that homotrimers [pro alpha-1(I)]3 and/or nascent chains predominate with diminished levels or absence of Hsp47. The latter will be shown to be degraded in a non- lysosomal pre-Golgi compartment. The information obtained in these studies will provide a better understanding of some of the fundamental mechanisms involving connective tissue injury and repair. Furthermore, elucidation of Hsp47's function during type I procollagen synthesis will provide an alternative means by which to modulate expression of collagen during health, disease, and repair. In particular, these studies will yield important information needed for establishing effective therapeutic procedures for the treatment of connective tissue disorders, particularly of ligaments, tendons, skin and oral mucosa.