The Importance of Prolyl 3-hydroxylation in recessive Osteogenesis Imperfecta

描述(申请人提供)：本项目的目标是研究隐性成骨不全(OI)的分子发病机制，OI是一种伴有头面部异常的脆性骨骼疾病。莫雷洛等人。艾尔首次确定软骨相关蛋白(CRTAP)突变是OI VII型的原因，OI是一种隐性OI[3]。此外，3-羟基脯氨酸酶(P3H1)的突变已被确认为隐性OI的原因[4]。CRTAP和P3H1形成与亲环素B(CypB)的复合体，该复合体的功能是将I型胶原的α1链上的单个脯氨酸残基3-羟基化[2]。除了作为3-羟基酶的功能外，CypB还具有顺式/反式异构酶(PPIase)的功能[9]。存在于三螺旋区域的许多氨基酸残基是顺式的，除非所有的键都是反式构象，否则胶原不会正确折叠[11]。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抑制CypB会导致胶原过度修饰和胶原分泌延迟--类似于隐性OI中观察到的胶原缺陷[19]。最近，有数据表明，3-羟基脯氨酸的存在可能稳定螺旋结构域[7]。然而，它的存在或缺失不太可能影响胶原的结构完整性，但它的缺失可能会扰乱正确的胶原折叠所必需的蛋白质-蛋白质相互作用[7]。初步数据表明，P3H1和CRTAP相互稳定。因此，当一个复杂的成员是突变的，另一个是降解的。由于所有已鉴定的CRTAP和P3H1突变都是亚型或零突变，目前尚不清楚3-羟化功能和/或PPIase功能是否在隐性OI的分子发病机制中起重要作用。因此，本项目旨在研究P3H1羟基酶活性的贡献，以及它如何通过在保持蛋白质结构的同时失活羟基酶结构域来参与隐性OI的发病。为了解决P3H1羟基酶活性的贡献，建议进行以下实验：1)研究3-羟基酶失活对胶原分泌的影响，并通过细胞培养进行修饰，以及2)通过研究包含点突变的敲入小鼠来评估3-羟化是否导致骨质疏松症表型，该点突变被证明在保持P3H1复合体稳定性的同时最好地灭活了羟基酶功能。

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.