The Importance of Prolyl 3-hydroxylation in recessive Osteogenesis Imperfecta

描述（由申请人提供）：本项目旨在研究隐性成骨不全症（OI）的分子发病机制，OI是一种伴有颅面异常的脆性骨病。Morello等人首先发现软骨相关蛋白（CRTAP）突变是导致VII型成骨不全（一种隐性成骨不全）的原因。此外，脯氨酸3-羟化酶（P3H1）突变已被确定为隐性OI[4]的病因。CRTAP和P3H1与亲环蛋白B （CypB）形成复合体，该复合体的功能是3-羟基化I型胶原[2]α - 1链上的单个脯氨酸残基。除了作为3-羟化酶的功能外，CypB还具有顺式/反式异构酶（PPIase）[9]的功能。存在于三螺旋区域的许多氨基酸残基都是顺式的，除非所有的键都是反式构象，否则胶原蛋白不能正常折叠。因此，PPIase的功能对于正确的胶原折叠是必不可少的。有趣的是，环孢素A对CypB的抑制导致胶原蛋白的过度修饰和胶原蛋白分泌的延迟——类似于在隐性成骨不全患者中观察到的胶原蛋白缺陷。最近的研究表明，3-羟基脯氨酸的存在可以稳定螺旋结构域[7]。然而，它的存在或缺失不太可能影响胶原的结构完整性，但它的缺失可能会破坏蛋白质-蛋白质相互作用，这是胶原蛋白正常折叠[7]所必需的。初步数据表明P3H1和CRTAP相互稳定。因此，当一个复杂成员突变时，另一个就退化了。由于所有已鉴定的CRTAP和P3H1突变均为半形或零型，因此尚不清楚3-羟基化功能和/或PPIase功能在隐性OI的分子发病机制中是否重要。因此，本项目旨在通过使P3H1羟化酶结构域失活而维持其蛋白结构，研究其对P3H1羟化酶活性的贡献及其与隐性OI发病机制的关系。为了解决P3H1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，并通过细胞培养进行修饰；2)通过研究含有点突变的敲入小鼠来评估3-羟化是否有助于骨质疏松症表型，该突变被证明是在保持P3H1复合物稳定性的同时最好地失活羟化酶功能。

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.