Role of the Leprecan Genes in Skeletal Formation

妖精基因在骨骼形成中的作用

基本信息

批准号：
8709813
负责人：
ROY MORELLO
金额：
$ 32.52万
依托单位：
UNIV OF ARKANSAS FOR MED SCIS
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-17 至 2017-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8709813
关键词：
2-oxoglutarate 3-dioxygenase proline Alleles Amino Acid Sequence Amino Acids Attention Binding Biological Biological Assay Bone Marrow COL1A1 gene COL1A2 gene Calvaria Cartilage Cell Differentiation process Cellular biology Co-Immunoprecipitations Collagen Collagen Gene Collagen Type I Complex Connective Tissue Connective Tissue Diseases Data Defect Development Disease Enzymes Etiology Family member Fibrillar Collagen Gene Family Gene Mutation Gene Proteins Genes Genetic Homeostasis Human Hydroxylation In Vitro Knockout Mice Link Location LoxP-flanked allele Mass Spectrum Analysis Mediating Modification Molecular Mus Mutant Strains Mice Mutate Mutation Osteoblasts Osteoclasts Osteogenesis Osteogenesis Imperfecta Pattern Peptide Sequence Determination Phenotype Post-Translational Protein Processing Probability Process Production Protein Family Proteins Regulation Role Rough endoplasmic reticulum Site Skeletal Development Skeletal bone Skeleton Skin Synaptonemal Complex Testing Tissues Ursidae Family Work base bone bone cell bone mass cyclophilin B human disease insight member mineralization mutant novel osteogenic paralogous gene progenitor protein complex research study skeletal skeletal dysplasia success

项目摘要

DESCRIPTION (provided by applicant): Our studies of the CRTAP gene, encoding cartilage associated protein, and the discovery that its mutations cause recessive osteogenesis imperfecta (OI) have led to the identification of additional non-collagen gene mutations responsible for this disease. Most of these mutations occur in rER resident proteins that are involved in fibrillar collagen modification, folding and assembly. Importantly, while some of these processes are relatively well understood others, such as prolyl 3-hydroxylation, have remained quite unexplored and deserve more attention. Because prolyl 3-hydroxylation is found in various types of collagens, both fibrillar and non- fibrillar, the identification of the molecular componens responsible for this modification and how they work together in different tissues will provide important clues about tissue-specific matrix molecules production and novel mechanisms of disease. Based on previous characterization of the prolyl 3-hydroxylation complex, containing Crtap, P3h1 and CypB, we now propose to determine the role of Sc65, a novel member of the Leprecan gene family and the most closely related protein to Crtap. Our preliminary data show that Sc65 is a rER protein, highly expressed in the skeleton and when mutated causes a low bone mass phenotype. We hypothesize that Sc65 mediates interactions with the other prolyl 3-hydroxylase enzymes, P3h2 and/or P3h3, contributes to collagen post-translational modification at sites that are distinct from those modified by the Crtap complex and, when mutated, causes the development of OI. To test these hypotheses, the following specific aims are proposed: 1) Comprehensively characterize the skeletal phenotype of mice with a homozygous gene- trap insertion in the Sc65 gene using micro-CT, bone histomorphometry, collagen mass spectrometry and FTIRI; 2) Determine effects of Sc65 mutation on bone cell differentiation using primary calvarial osteoblasts and ex-vivo bone-marrow osteogenic and osteoclastogenic cultures from Sc65 mutant and WT control mice; 3) Determine the sub-cellular localization of the Sc65 protein and identify its interacting partners by co- immunoprecipitation and protein sequencing; and 4) Generate a conditional knock-out mouse by creation of a Sc65 floxed allele, inactivate it in bone and characterize its skeletal phenotype. Successful completion of this work will 1) elucidate a novel role for Sc65 in skeletal development and bone homeostasis, 2) identify Sc65 protein interactors which will provide insight into the molecular mechanisms involved in collagen prolyl 3- hydroxylation, 3) enhance understanding of the Leprecan genes in skeletal development and other tissues, and 4) provide further insight into the role of these non-collagenous proteins in the etiology of human connective tissue disease, including OI.

描述（由申请人提供）：我们对CRTAP基因的研究，编码软骨相关的蛋白质以及发现其突变导致隐性成骨的发现Imprimenta（OI）导致鉴定出了额外的非胶原基因突变，负责该疾病。这些突变中的大多数发生在与原纤维胶原蛋白修饰，折叠和组装有关的居民蛋白中。重要的是，其中一些过程相对较充分地理解了其他过程，例如3-羟基化，仍然没有探索，值得更多的关注。由于在各种类型的胶原蛋白（纤维纤维和非光纤）中发现了脯氨酰3-羟基化，因此鉴定负责这种修饰的分子成分以及它们如何在不同组织中一起工作的分子成分将提供有关组织特异性基质分子生产和疾病的新机制的重要线索。基于先前的3-羟基化复合物的表征，其中包含CRTAP，P3H1和CYPB，我们现在建议确定SC65的作用，SC65是Leprecan基因家族的新成员，以及与CRTAP最密切相关的蛋白质。我们的初步数据表明，SC65是一种RER蛋白，在骨骼中高度表达，而突变会导致低骨质量表型。我们假设SC65介导了与其他3-羟化酶P3H2和/或P3H3的相互作用，在与CRTAP复合物修饰的位点上的胶原蛋白变化后修饰有助于胶原蛋白，并在突变时导致OI的发展。为了检验这些假设，提出了以下特定目的：1）全面表征小鼠在SC65基因中使用微CT，骨骼组态形态，胶原蛋白质谱法和FTIRI中纯合基因插入小鼠的骨骼表型； 2）确定SC65突变对骨细胞分化的影响，使用钙化成骨细胞和前体骨肉体骨骼成骨和骨质碎片培养物，来自SC65突变体和WT对照小鼠的影响； 3）确定SC65蛋白的亚细胞定位，并通过免疫沉淀和蛋白质测序来识别其相互作用的伴侣； 4）通过创建SC65 flox的等位基因，在骨骼中灭活并表征其骨骼表型，从而产生有条件的敲除小鼠。这项工作的成功完成将1）阐明SC65在骨骼发育和骨骼稳态中的新作用，2）确定SC65蛋白相互作用者，将为胶原蛋白3-羟基化所涉及的分子机制提供深入了解，3）增强对脑骨骼发育和其他组织中的LEPRECAN基因的理解，并在skelet and-bectues中的理解，以及其他洞察力，并在其他方面提供了这些洞察力，这些局限性不得不洞察到这些洞察力，这些洞察力是洞察力的，这些洞察力是洞察力的，这些洞察力是这些洞察力的洞察力。人类结缔组织疾病的病因，包括OI。