Identification of Proteins Important for Male Osteoporosis

鉴定对男性骨质疏松症重要的蛋白质

• 批准号: 8143422
• 负责人: HONG-WEN DENG
• 金额: $ 62万
• 依托单位: TULANE UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-18 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8143422
• 关键词: Accounting; Address; Affect; Age; Antibodies; Biochemical; Biology; Blood specimen; Bone Density; Bone Marrow; Bone Resorption; Candidate Disease Gene; Caucasians; Caucasoid Race; Cell Nucleus; Cells; China; Cluster Analysis; Complement; Complex; Cytoskeleton; Cytosol; DNA; DNA Microarray Chip; Detection; Diagnosis; Diagnostic; Disease; Dissection; Epidemiologic Studies; Epidemiology; Etiology; Female; Foundations; Fracture; Funding; Gender; Gene Proteins; Genes; Genetic; Genetic Polymorphism; Genome; Genomics; Goals; Grant; Health; Heritability; Hydrophobicity; In Vitro; Informatics; Ions; Isoelectric Point; Link; Liquid Chromatography; Membrane; Membrane Proteins; Mesenchymal Stem Cells; Messenger RNA; Metabolic Pathway; Molecular; Molecular Genetics; Molecular Profiling; Molecular Weight; Nebraska; Negative Finding; Nuclear; Osteoblasts; Osteoclasts; Osteogenesis; Osteoporosis; Outcome; Pathway interactions; Peptides; Phenotype; Population; Positioning Attribute; Prevention; Principal Investigator; Prognostic Marker; Property; Protein Precursors; Proteins; Proteome; Proteomics; Public Health; Recruitment Activity; Risk; Sampling; Scanning; Science; Specificity; TNFRSF5 gene; Testing; Tobacco; Trauma; United States National Institutes of Health; Validation; Variant; Western Blotting; Woman; Work; base; bone; bone loss; bone mass; bone metabolism; cohort; design; functional genomics; genetic linkage; genetic pedigree; genome wide association study; genome-wide; hip bone; in vivo; innovation; insight; mRNA Expression; male; men; monocyte; mortality; multidisciplinary; nano; novel; osteoclastogenesis; osteogenic; osteoporosis with pathological fracture; peripheral blood; precursor cell; programs; protein expression; research study; sex; tandem mass spectrometry; two-dimensional

DESCRIPTION (provided by applicant): Identification of Proteins Important for Male Osteoporosis Osteoporosis is a major public health problem, mainly characterized by low bone mineral density (BMD). Variation of BMD is largely genetically determined (heritability of > 60%). Some BMD genes/genomic regions are gender specific. Although women have lower BMD than men, men suffer significantly higher mortality rate upon osteoporotic fractures. However, studies on osteoporosis have largely been focused on women. Few genetic epidemiological studies and no proteomic epidemiological study on osteoporosis have been performed on men. Bone marrow mesenchymal stem cells (BMMSCs) and peripheral blood monocytes (PBMs) are precursors for osteoblasts (bone formation cells) and osteoclasts (bone resorption cells), respectively. Proteomics is a powerful state-of-the-art strategy in genetic dissection of complex diseases, such as osteoporosis. However, a major problem affecting the power of current most proteomic studies is the limited detection of low abundance proteins and proteins with extreme isoelectric point, molecular weight, and hydrophobicity, especially membrane proteins. A NOVEL approach to this problem is subcellular proteome extraction to stepwise isolate proteins from membrane, cytosol, nucleus, and cytoskeleton E fractions followed by sensitive 2D-nanoLC-ESI-MS/MS for fractioning and identifying significant proteins. Our hypothesis is that changes in the protein expression profiles in BMMSCs and PBMs underlie molecular mechanisms of BMD variation and are associated with osteoporosis in men. Our major goals here are to identify proteins differentially expressed in BMMSCs and PBMs in men with high vs. low BMD and thus identify proteins (and their genes) associated with male osteoporosis in BMMSCs and PBMs. We will recruit 120 otherwise healthy Caucasian males at peak bone mass ages of 25-50, including 60 subjects with low and 60 with high BMD (age matched population bottom or top 20% respectively in terms of BMD). Half of the sample (30 low vs. 30 high BMD subjects) will serve as the "discovery cohort" and the other half (30 low vs. 30 high BMD subjects) will serve as the "replication cohort". We will take fresh bone marrow and peripheral blood samples from each male subject, as we do in our ongoing NIH SCOR projects for female subjects. BMMSCs and PBMs will be isolated by subcellular proteome extraction for membrane proteins together with proteins of cytosolic, nuclear, and cytoskeletal fractions. Proteomic profiling experiments and analyses will be performed on the isolated protein samples from the discovery E cohort using 2D-nanoLC-ESI-MS/MS . Significant differentially expressed proteins identified will be verified by Western blotting using samples from the replication cohort. The major results (particularly those obtained from PBMs) of this study may be used to design customary diagnostic protein antibody chips and/or protein markers for prognosis of male osteoporosis. In combination with our ongoing projects for identifying risk genes of osteoporosis through genome-wide DNA association scan and genome-wide mRNA expression study of osteogenic cells, this study will powerfully and efficiently identify genes and some of their functions for male osteoporosis. PUBLIC HEALTH RELEVANCE: Osteoporosis is a disease with severe bone loss and a significantly increased risk of low trauma fractures. Male osteoporosis, compared with female osteoporosis, has higher mortality rate upon fractures. This project aims to identify proteins that are important to male osteoporosis. The findings will provide essential scientific basis for effective prevention, diagnosis and treatment of the disease.

描述（由申请人提供）：对男性骨质疏松症重要的蛋白质的鉴定骨质疏松症是一个主要的公共卫生问题，主要特征是骨矿物质密度（BMD）低。 BMD 的变异很大程度上是由基因决定的（遗传力 > 60%）。一些 BMD 基因/基因组区域具有性别特异性。尽管女性的骨密度低于男性，但男性骨质疏松性骨折的死亡率却明显更高。然而，骨质疏松症的研究主要集中在女性。对男性骨质疏松症的遗传流行病学研究和蛋白质组流行病学研究很少。骨髓间充质干细胞（BMMSC）和外周血单核细胞（PBM）分别是成骨细胞（骨形成细胞）和破骨细胞（骨吸收细胞）的前体。蛋白质组学是复杂疾病（例如骨质疏松症）基因解析的一种强大的最先进策略。然而，影响当前大多数蛋白质组学研究能力的一个主要问题是对低丰度蛋白质和具有极端等电点、分子量和疏水性的蛋白质，特别是膜蛋白的检测有限。解决这个问题的一种新颖方法是亚细胞蛋白质组提取，逐步从膜、细胞质、细胞核和细胞骨架 E 级分中分离蛋白质，然后使用敏感的 2D-nanoLC-ESI-MS/MS 进行分级和鉴定重要蛋白质。我们的假设是，BMMSC 和 PBM 中蛋白质表达谱的变化是 BMD 变异的分子机制的基础，并且与男性骨质疏松症相关。我们的主要目标是鉴定高 BMD 与低 BMD 男性 BMMSC 和 PBM 中差异表达的蛋白质，从而鉴定 BMMSC 和 PBM 中与男性骨质疏松症相关的蛋白质（及其基因）。我们将招募 120 名骨量峰值年龄为 25-50 岁的健康白人男性，其中包括 60 名低 BMD 受试者和 60 名高 BMD 受试者（就 BMD 而言，年龄匹配的人群分别处于底部或顶部 20%）。一半样本（30 名低 BMD 受试者与 30 名高 BMD 受试者）将作为“发现队列”，另一半（30 名低 BMD 受试者与 30 名高 BMD 受试者）将作为“复制队列”。我们将从每位男性受试者身上采集新鲜骨髓和外周血样本，就像我们在正在进行的 NIH SCOR 女性受试者项目中所做的那样。 BMMSC 和 PBM 将通过亚细胞蛋白质组提取来分离膜蛋白以及细胞质、细胞核和细胞骨架部分的蛋白质。将使用 2D-nanoLC-ESI-MS/MS 对发现 E 队列中分离的蛋白质样品进行蛋白质组分析实验和分析。鉴定出的显着差异表达蛋白将使用来自复制队列的样本通过蛋白质印迹法进行验证。本研究的主要结果（特别是从 PBM 获得的结果）可用于设计用于男性骨质疏松症预后的常规诊断蛋白抗体芯片和/或蛋白标记物。结合我们正在进行的通过成骨细胞全基因组DNA关联扫描和全基因组mRNA表达研究来识别骨质疏松症风险基因的项目，这项研究将有力而有效地识别男性骨质疏松症的基因及其一些功能。公众健康相关性：骨质疏松症是一种骨质流失严重且低度创伤性骨折风险显着增加的疾病。与女性骨质疏松症相比，男性骨质疏松症的骨折死亡率更高。该项目旨在鉴定对男性骨质疏松症重要的蛋白质。研究结果将为该病的有效预防、诊断和治疗提供重要的科学依据。