The Importance of Prolyl 3-hydroxylation in recessive Osteogenesis Imperfecta

描述（由申请人提供）：本项目的目标是研究隐性成骨骨不全（OI）的分子发病机制-一种颅面异常的脆骨疾病。Morello et.等人首先鉴定出Carcinoma Associated Protein（CRTAP）中的突变是VII型OI（一种隐性OI）的病因[3]。此外，脯氨酰3-羟化酶（P3 H1）突变已被确定为隐性OI的病因[4]。CRTAP和P3 H1与亲环蛋白B（CypB）形成复合物，该复合物的功能是3-羟基化I型胶原α 1链上的单个脯氨酸残基[2]。除了作为3-羟化酶的功能外，CypB还作为顺式/反式异构酶（PPI酶）[9]。三螺旋区中存在的许多氨基酸残基为顺式形式，除非所有键均为反式构象，否则胶原蛋白将无法正确折叠[11]。因此，PPIase的功能对于正确的胶原蛋白折叠至关重要。有趣的是，环孢菌素A对CypB的抑制导致过度修饰的胶原蛋白和胶原蛋白分泌的延迟-类似于在隐性OI患者中观察到的胶原蛋白缺陷[19]。最近，数据表明，3-羟脯氨酸的存在可以稳定螺旋结构域[7]。然而，它的存在或不存在不太可能影响胶原蛋白的结构完整性，但它的缺乏可能会破坏适当胶原蛋白折叠所必需的蛋白质-蛋白质相互作用[7]。初步数据表明，P3 H1和CRTAP相互稳定。因此，当一个复杂的成员是突变的，另一个是降解。由于所有确定的CRTAP和P3 H1突变是亚型或无效的，目前还不清楚是否3-羟基化功能和/或PPIase功能是重要的隐性OI的分子发病机制。因此，本项目旨在研究P3 H1羟化酶活性的贡献，以及它如何通过在保持蛋白质结构的同时使羟化酶结构域失活而与隐性OI的发病机制相关。为了解决P3 H1羟化酶活性的贡献，提出了以下实验：1）研究3-羟化酶失活对胶原分泌的影响，以及通过细胞培养进行修饰，和2）通过研究敲除，在含有点突变的小鼠中，证明最好地抑制羟化酶功能，同时保持P3 H1复合物的稳定性。 公共卫生相关性：该项目剖析了P3 H1蛋白的酶活性在胶原蛋白修饰复合物中的作用，该复合物在胶原蛋白的加工中起两种作用。该项目的结果将影响我们对胶原蛋白加工调节的理解，并将揭示导致隐性OI的分子异常。

DESCRIPTION (provided by applicant): The goal of this project is to study the molecular pathogenesis of recessive osteogenesis imperfecta (OI)- a brittle bone disease with craniofacial abnormalities. Morello et. al. first identified mutations in Cartilage Associated Protein (CRTAP) as causative of OI Type VII, a recessive OI[3]. Additionally, mutations to prolyl 3- hydroxylase (P3H1) have been identified as causative of recessive OI[4]. CRTAP and P3H1 form a complex with cyclophilin B (CypB) and this complex functions to 3-hydroxylate a single proline residue on the alpha 1 chain of type I collagen[2]. In addition to its function as a 3-hydroxylase, CypB functions as a cis/trans isomerase (PPIase)[9]. Many of the amino acid residues present in the triple helix region are in the cis form and collagen will not fold properly unless all bonds are in the trans conformation[11]. Thus, the function of PPIase is essential for proper collagen folding. Interestingly, the inhibition of CypB by cyclosporin A results in overmodified collagen and a delay in collagen secretion- similar to the collagen defects observed in recessive OI patints[19]. Recently, data suggest that the presence of 3-hydroxyproline may stabilize the helix domain[7]. However, its presence or absence is not likely to affect the structural integrity of collagen, but its absence may disrupt protein-protein interactions integral for proper collagen folding[7]. Preliminary data demonstrate that P3H1 and CRTAP stabilize each other. Thus, when one complex member is mutant, the other is degraded. Since all identified CRTAP and P3H1 mutations are hypomorphic or null, it is unclear whether the 3- hydroxylation function and/or the PPIase function are important in the molecular pathogenesis of recessive OI. Thus, this project aims to study the contribution on P3H1 hydroxylase activity and how it pertains to the pathogenesis of recessive OI by inactivating the hydroxylase domain while maintaining the protein structure. To address the contribution of P3H1 hydroxylase activity, the following experiments are proposed: 1)study the consequences of 3-hydroxylase inactivation on collagen secretion, and modification by cell culture, and 2) assess whether 3-hydroxylation contributes to an osteoporotic phenotype by studying knock-in mice containing the point mutation demonstrated to best inactivate the hydroxylase function while maintaining the stability of the P3H1 complex. Public Health Relevance: This project dissects the role of the enzymatic activity of the P3H1 protein within a collagen modifying complex that serves two roles in the processing of collagen. The results of this project will impact our understanding of the regulation of collagen processing and will shed light on the molecular abnormalities leading to recessive OI.