Mechanisms of Lrrk1 Regulation of Osteoclast Function

Lrrk1 调节破骨细胞功能的机制

• 批准号: 8769087
• 负责人: Weirong Xing
• 金额: $ 18.22万
• 依托单位: LOMA LINDA VETERANS ASSN RESEARCH & EDUC
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-18 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8769087
• 关键词: Albers-Schonberg disease; Ankyrin Repeat; Antibodies; Area; Biological Assay; Bone Diseases; Bone Resorption; Bone necrosis; C-terminal; Cartilage; Cell Lineage; Cells; Complex; Computers; Crystallography; DNA Sequence Rearrangement; Data; Defect; Development; Diagnosis; Docking; Drug Design; Epiphysial cartilage; Exhibits; Fracture; Functional disorder; Future; GTP Binding; Generations; Genes; Genetic Polymorphism; Goals; Grant; Guanosine Triphosphate Phosphohydrolases; Health; Homologous Gene; Homology Modeling; Human; In Vitro; Integrins; Knockout Mice; Lead; Length; Leucine-Rich Repeat; Libraries; Liquid Chromatography; Macrophage Colony-Stimulating Factor; Mediating; Molecular; Molecular Weight; Mus; Obstruction; Osteoclasts; Osteogenesis; Osteoporosis; Pathway interactions; Patients; Peptides; Peripheral; Pharmaceutical Preparations; Phenotype; Phosphorylation; Phosphotransferases; Play; Preclinical Drug Evaluation; Prevention; Process; Protein C; Protein Family; Protein-Serine-Threonine Kinases; Proteins; Public Health; Recombinant Proteins; Regulation; Resistance; Role; SRC gene; Series; Serine; Signal Pathway; Signal Transduction; Slice; Specificity; Structure; Substrate Interaction; TNFSF11 gene; Teriparatide; Testing; Therapeutic; Thick; Threonine; Tissues; Vertebral Bone; Western Blotting; base; bisphosphonate; bone; bone cell; bone loss; design; improved; in vitro testing; in vivo; inhibitor/antagonist; leucine-rich repeat kinase 2; long bone; macrophage; new therapeutic target; novel; novel strategies; novel therapeutics; osteoporosis with pathological fracture; protein-tyrosine kinase c-src; research study; response; seal; skeletal; small molecule; substantia spongiosa; tandem mass spectrometry; treatment strategy

DESCRIPTION (provided by applicant): Leucine rich repeat kinase 1 (Lrrk1) belongs to ROCO family proteins and contains three ankyrin repeat, seven leucine-rich repeat, a GTPase-like domain of Roc (Ras of complex proteins), COR (C-terminal of Roc) domain, and a serine/threonine kinase domain that is regulated by GTP binding to the Roc domain. Lrrk2 contains most domains of ROCO family protein and an extra Lrrk2 specific repeat, but lacks ankryrin repeat. Both Lrrk1 and Lrrk2 are ubiquitously expressed in multiple tissues. We recently found that mice with disruption of Lrrk1 gene displayed a severe phenotype of osteopetrosis caused by dysfunction of multinucleated cells while mice lacking Lrrk2 gene failed to show obvious skeletal phenotypes, indicating that Lrrk1 has a unique structure and function in bone cells that Lrrk2 cannot compensate for loss of Lrrk1. Little is known on the signaling pathways of Lrrk1 in osteoclasts. Our focus in this grant is to determine the structure and function of Lrrk1 i osteoclasts and identify potential Lrrk1 kinase targets and inhibitors. To this end, we propose two hypotheses in this study: 1) Lrrk1 regulates osteoclast functions via its kinase activation; 2) Lrrk1 phosphorylates serine/threonine residues of its specific substrate(s). To test the hypothesis 1, experiments are designed to examine the structure and function of full length Lrrk1 and truncated Lrrk1 on bone resorptive activity of mature osteoclasts on bone slices by in vitro pit assay. Homology models of human Lrrk1 kinase domain are used to dock small molecule inhibitors and the inhibition of drug-like compounds will be tested in vitro kinase and pi formation assays. To test the hypothesis 2, experiments are designed to enrich phosphor-peptides from wild type and Lrrk1 deficient osteoclasts via immunoaffinity purification by using phosphor-serine/phosphor-threonine motif antibodies, and identify Lrrk1 initial targets by liquid chromatography and tandem mass spectrometry (LC- MS/MS). The results of this application will advance our understanding of the molecular mechanisms of Lrrk1 signaling in regulating osteoclast function and bone resorption, and provide new therapeutic strategies for treatment and prevention of bone diseases such as osteoporosis.

描述（由申请人提供）：富含亮氨酸的重复序列激酶1（Lrrk 1）属于ROCO家族蛋白，包含3个锚蛋白重复序列、7个富含亮氨酸的重复序列、Roc的GTP酶样结构域（复合蛋白的Ras）、COR（Roc的C-末端）结构域和受与Roc结构域结合的GTP调节的丝氨酸/苏氨酸激酶结构域。Lrrk 2含有ROCO家族蛋白的大部分结构域和一个额外的Lrrk 2特异性重复序列，但缺乏锚蛋白重复序列。Lrrk 1和Lrrk 2在多种组织中普遍表达。我们最近发现Lrrk 1基因缺失的小鼠表现出严重的多核细胞功能障碍引起的骨硬化症表型，而Lrrk 2基因缺失的小鼠未能表现出明显的骨骼表型，表明Lrrk 1在骨细胞中具有独特的结构和功能，Lrrk 2不能弥补Lrrk 1的缺失。关于Lrrk 1在破骨细胞中的信号通路知之甚少。我们在这项资助的重点是确定Lrrk 1 i破骨细胞的结构和功能，并确定潜在的Lrrk 1激酶的目标和抑制剂。为此，我们提出了两个假设：1）Lrrk 1通过其激酶激活调节破骨细胞功能; 2） Lrrk 1磷酸化其特定底物的丝氨酸/苏氨酸残基。为了验证假设1，设计实验通过体外孔蚀试验来检测全长Lrrk 1和截短Lrrk 1的结构和功能对骨片上成熟破骨细胞的骨吸收活性的影响。使用人Lrrk 1激酶结构域的同源模型来对接小分子抑制剂，并且将在体外激酶和pi形成测定中测试药物样化合物的抑制。为了验证假设2，设计实验以通过使用磷酸-丝氨酸/磷酸-苏氨酸基序抗体经由免疫亲和纯化从野生型和Lrrk 1缺陷型破骨细胞富集磷酸-肽，并通过液相色谱和串联质谱（LC-MS/MS）鉴定Lrrk 1初始靶标。本研究的结果将有助于我们进一步了解Lrrk 1信号通路调控破骨细胞功能和骨吸收的分子机制，并为骨质疏松等骨疾病的治疗和预防提供新的治疗策略。