MOLECULAR BASIS FOR ASSURANCE OF TYPE I COLLAGEN

在这项建议中，我们将注意力集中在表征的问题上 确保胶原分子正确结合的细胞机制 类型I化学计量比{PROα-1(I)2 PROα-2(I)}和折叠结果， 以及伴侣Hsp47在这一过程中的作用。假设集 第四，胶原蛋白在细胞内的快速降解与之相反 与内质网中Hsp47的表达水平有关。热休克蛋白47 结合在I型胶原原α-1链的N端。这 N-前肽的结合发生在非常早期的阶段，可能很快 在原α-1链插入内质网后。因此，Hsp47是一种 指导胶原链选择和组装的重要因素。在……里面 然而，由于缺乏Hsp47，新生的链被认为是拉长的， 将链组装成杂聚体受到有利于[PRO]的限制 α-1(I)]3保留在内质网和内质网中的同源三聚体或新生链 降级了。后一种细胞的主要降解途径 监视遵循前高尔基体、非溶体的路线。因此，有选择性地 未组装、异常或未完全组装的降级 杂多聚合物在处理多余的胶原链方面起着关键作用， 从而确保只有具有正确化学计量比的分子才能到达 细胞外基质。为了检验这一假设的反义性 Hsp47的寡核苷酸将被使用，以使互补DNA或 RNA序列与mRNA相互作用，阻断Hsp47的翻译。我们会 然后演示胶原链组装成I型杂三聚体 当该过程由Hsp47指示并且同源三聚体[PRO Alpha-1(I)]3和/或新生的链占主导地位，水平或 Hsp47缺失。后者将被显示为在非 溶酶体前高尔基体隔室。从这些研究中获得的信息 将提供对一些基本机制的更好的理解 涉及结缔组织损伤和修复。此外，还澄清了 热休克蛋白47的S功能在I型前胶原合成过程中将提供一种 调节胶原蛋白表达的另一种方法 健康、疾病和修复。特别是，这些研究将产生 建立有效治疗方法所需的重要信息 结缔组织疾病的治疗程序，特别是 韧带、肌腱、皮肤和口腔粘膜。

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.