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.

说明书(申请人提供)：富亮氨酸重复蛋白1(Lrrk1)属于ROCO家族蛋白，包含3个锚蛋白重复序列、7个富含亮氨酸重复序列、一个ROC(复合蛋白的RAS)的GTP酶样域、COR(ROC的C末端)结构域、以及一个受与ROC结构域结合的GTP调节的丝氨酸/苏氨酸激酶结构域。LRRK2含有大部分Roco家族蛋白结构域和一个额外的LRRK2特异性重复序列，但缺少ankryrin重复序列。Lrrk1和LRRK2在多种组织中广泛表达。我们最近发现，Lrrk1基因缺失的小鼠由于多核细胞功能障碍而表现出严重的骨化病表型，而LRRK2基因缺失的小鼠没有表现出明显的骨骼表型，这表明Lrrk1在骨细胞中具有LRRK2不能弥补Lrrk1缺失的独特结构和功能。破骨细胞中Lrrk1的信号转导途径知之甚少。我们在这项资助中的重点是确定Lrrk1 I破骨细胞的结构和功能，并确定潜在的Lrrk1激酶靶点和抑制剂。为此，我们在本研究中提出了两个假设：1)Lrrk1通过其激酶激活来调节破骨细胞的功能；2) Lrrk1使其特定底物的丝氨酸/苏氨酸残基磷酸化(S)。为了验证假设1，通过体外PIT实验检测了全长Lrrk1和截短型Lrrk1对成熟破骨细胞在骨片上骨吸收活性的结构和功能。人类Lrrk1蛋白激动域的同源模型被用来对接小分子抑制剂，类药物化合物的抑制将在体外的激酶和圆周率形成试验中进行测试。为了验证假设2，实验设计了通过免疫亲和纯化野生型和Lrrk1缺失的破骨细胞中的磷酸多肽，利用磷酸丝氨酸/磷酸苏氨酸基序抗体进行纯化，并用LC-MS/MS(LC-MS/MS)鉴定Lrrk1的初始靶标。这一应用的结果将加深我们对Lrrk1信号调节破骨细胞功能和骨吸收的分子机制的理解，并为骨质疏松等骨病的治疗和预防提供新的治疗策略。