Structure of Sclerostin Protein Complexes

硬化素蛋白复合物的结构

• 批准号: 8232023
• 负责人: RAJIV KUMAR
• 金额: $ 17.74万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8232023
• 关键词: Affect; Affinity; Aging; Alkaline Phosphatase; American; Amino Acids; Anabolic Agents; Apoptosis; Binding; Binding Proteins; Bone Density; C-terminal; Caspase; Cell Differentiation process; Cell Proliferation; Cell division; Complex; Crystallization; Crystallography; Cystine; Data; Development; Disease; Docking; Embryo; Extracellular Domain; Extracellular Matrix Proteins; Fibroblasts; Fracture; Genes; Health; Human; Knowledge; LDL-Receptor Related Protein 1; Link; Mammalian Cell; Mediating; Medical; Mesenchymal Stem Cells; Molecular; Molecular Conformation; Morbidity - disease rate; Mutation; Names; Osteoblasts; Osteoclasts; Osteocytes; Osteogenesis; Osteoporosis; Pathway interactions; Patients; Pharmaceutical Preparations; Phenotype; Play; Postmenopause; Proteins; Reporting; Role; Sclerosis; Signal Transduction; Site; Skeleton; Solutions; Structure; Surface Plasmon Resonance; Syndrome; Teriparatide; Therapeutic; Van Buchem disease; Vascular Endothelial Cell; Woman; Xenopus; angiogenesis; base; bone; bone mass; bone morphogenetic protein 6; bone morphogenetic protein receptors; bone quality; cell motility; cyr61 protein; design; insight; lipoprotein receptor related protein 5; men; mineralization; mutant; protein complex; public health relevance; receptor; receptor binding; small molecule

DESCRIPTION (provided by applicant): The objective of this R21 application is to investigate at the atomic level, the mechanism of action of sclerostin, an osteocyte-derived, secreted, cystine-knot protein that inhibits bone formation by examining how sclerostin interacts with proteins that play an essential role in mediating its activity. Because the disruption of sclerostin expression or activity increases bone mass insights developed through our studies may be useful in devising treatments for osteoporosis, a disease affecting 10 million Americans. Bone morphogenetic protein 6 (BMP6), cysteine-rich protein 61 (Cyr61) and low-density-lipoprotein receptor-related protein 5 (LRP5) are key sclerostin-associating proteins reported to mediate sclerostin activity. No structural data exists for these protein complexes. Moreover, preliminary docking studies suggest that the conformations observed in two sclerostin NMR solution-structures must be altered to affect binding to these three proteins. We hypothesize that in contrast to its highly disordered structure in the solution-state, the "loop 2" region of sclerostin (amino acids G86-R109) becomes highly structured when sclerostin binds proteins such as BMP6, Cyr61 and LRP5 whose functions it modulates. If true, sclerostin activity might be antagonized by allosteric modulation and by targeting its interaction site(s), through the use of orally effective, small molecules that would generate new bone in patients. In Aim 1 we will determine the molecular mechanism of sclerostin interaction with BMP6; in Aim 2 we will determine the molecular mechanism of sclerostin interaction with Cyr61 C-terminal domain and in Aim 3 we will identify crystallization conditions for the sclerostin complex with LRP5 extracellular domain. In Aims 1-2, our proposed mechanism for sclerostin interaction will be probed by x-ray crystallography followed by analysis of binding-affinities of structure-guided mutants through the use of surface plasmon resonance. In Aim 3 conditions for crystallizing sclerostin-LRP5 1st ?-propeller complexes will be established. Significance: Osteoporosis is a significant medical health problem associated with fractures and considerable morbidity, prevalent particularly in aging and post-menopausal women. While effective bone anti-resorptive drugs are available for osteoporosis treatment, they have little effect on bone formation. The only anabolic agent available now is teriparatide that must be administered parenterally. Sclerostin is an osteocyte derived protein that inhibits bone formation. Antagonizing its function is a potential therapeutic strategy to increase high quality bone. Our studies will allow an atomic-level understanding of the interface between sclerostin and three protein partners that mediate sclerostin function. We anticipate this knowledge would provide a strong basis for development of antagonistic drugs that can be administrated orally, especially if structural results suggest sclerostin might be amenable to allosteric modulation by small-molecules. PUBLIC HEALTH RELEVANCE: Orally effective drugs that build bone are required for the treatment of osteoporosis. By determining the structures of complexes of sclerostin (a bone inhibitory protein) with other proteins with which it interacts, we will be able to obtain the information that will allow the design of such drugs. This effort will greatly help women and men with osteoporosis and osteoporosis-related fractures.

描述（由申请人提供）：该 R21 申请的目的是在原子水平上研究硬化蛋白的作用机制，硬化蛋白是一种骨细胞衍生的分泌型胱氨酸结蛋白，通过检查硬化蛋白如何与蛋白质相互作用来抑制骨形成在调解其活动中发挥重要作用。由于硬化蛋白表达或活性的破坏会增加骨量，因此通过我们的研究得出的见解可能有助于设计骨质疏松症的治疗方法，骨质疏松症是一种影响 1000 万美国人的疾病。骨形态发生蛋白 6 (BMP6)、富含半胱氨酸的蛋白 61 (Cyr61) 和低密度脂蛋白受体相关蛋白 5 (LRP5) 是据报道介导硬化素活性的关键硬化素相关蛋白。这些蛋白质复合物不存在结构数据。此外，初步对接研究表明，必须改变在两种硬化素 NMR 溶液结构中观察到的构象才能影响与这三种蛋白质的结合。我们假设，与其在溶液状态下的高度无序结构相反，当硬化素结合其功能调节的 BMP6、Cyr61 和 LRP5 等蛋白质时，硬化素的“环 2”区域（氨基酸 G86-R109）变得高度结构化。如果属实，硬化素活性可能会被变构调节和靶向其相互作用位点所拮抗，通过使用口服有效的小分子，在患者体内产生新骨。在目标 1 中，我们将确定硬化蛋白与 BMP6 相互作用的分子机制；在目标 2 中，我们将确定硬化蛋白与 Cyr61 C 末端结构域相互作用的分子机制，在目标 3 中，我们将确定硬化蛋白与 LRP5 胞外结构域复合物的结晶条件。在目标 1-2 中，我们提出的硬化素相互作用机制将通过 X 射线晶体学来探测，然后通过使用表面等离振子共振来分析结构引导突变体的结合亲和力。在目标 3 中，将建立硬化蛋白-LRP5 第一个 β-螺旋桨复合物结晶的条件。 意义：骨质疏松症是一个与骨折和相当高的发病率相关的重大医疗健康问题，尤其在老龄化和绝经后妇女中普遍存在。虽然有效的骨抗吸收药物可用于骨质疏松症的治疗，但它们对骨形成的影响很小。目前唯一可用的合成代谢药物是必须肠胃外给药的特立帕肽。硬化蛋白是一种骨细胞衍生的蛋白质，可抑制骨形成。拮抗其功能是增加高质量骨的潜在治疗策略。我们的研究将从原子水平上了解硬化素和介导硬化素功能的三种蛋白质伙伴之间的界面。我们预计这一知识将为开发可口服给药的拮抗药物提供坚实的基础，特别是如果结构结果表明硬化素可能适合小分子的变构调节。 公共卫生相关性：治疗骨质疏松症需要口服有效的骨质增强药物。通过确定硬化素（一种骨抑制蛋白）与其相互作用的其他蛋白质的复合物的结构，我们将能够获得允许设计此类药物的信息。这项努力将极大地帮助患有骨质疏松症和骨质疏松症相关骨折的女性和男性。

项目成果

Thrombopoietin promotes NHEJ DNA repair in hematopoietic stem cells through specific activation of Erk and NF-κB pathways and their target, IEX-1.

血小板生成素通过特异激活 Erk 和 NF-κB 途径及其靶标 IEX-1，促进造血干细胞中的 NHEJ DNA 修复。

• 发表时间: 2014-01-23
• 影响因子: 20.3
• 作者: de Laval, Bérengère;Pawlikowska, Patrycja;Barbieri, Daniela;Besnard;Cico, Alba;Kumar, Rajiv;Gaudry, Murielle;Baud, Véronique;Porteu, Françoise
• 通讯作者: Porteu, Françoise