Mechanisms of Lrrk1 Regulation of Osteoclast Function

Lrrk1 调节破骨细胞功能的机制

基本信息

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
项目类别：
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

项目摘要

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（申请人提供）：富含亮氨酸重复激酶1（Lrrk1）属于ROCO家族蛋白，包含三个锚蛋白重复序列、七个富含亮氨酸重复序列、Roc的GTP酶样结构域（复杂蛋白的Ras）、COR（C- Roc 的末端）结构域，以及通过与 Roc 结构域结合的 GTP 调节的丝氨酸/苏氨酸激酶结构域。 Lrrk2 包含 ROCO 家族蛋白的大部分结构域和一个额外的 Lrrk2 特异性重复序列，但缺乏锚蛋白重复序列。 Lrrk1 和 Lrrk2 在多种组织中普遍表达。我们最近发现Lrrk1基因破坏的小鼠表现出由多核细胞功能障碍引起的严重骨硬化表型，而缺乏Lrrk2基因的小鼠则未能表现出明显的骨骼表型，这表明Lrrk1在骨细胞中具有Lrrk2无法补偿的独特结构和功能对于 Lrrk1 的丢失。对于破骨细胞中 Lrrk1 的信号通路知之甚少。我们这笔资助的重点是确定 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两个假设：1）Lrrk1通过其激酶激活调节破骨细胞功能； 2） Lrrk1 磷酸化其特定底物的丝氨酸/苏氨酸残基。为了验证假设1，设计实验通过体外坑试验来检查全长Lrrk1和截短Lrrk1对骨片上成熟破骨细胞骨吸收活性的结构和功能。人Lrrk1激酶结构域的同源模型用于对接小分子抑制剂，并且将在体外激酶和pi形成测定中测试类药物化合物的抑制作用。为了检验假设 2，实验设计使用磷酸丝氨酸/磷酸苏氨酸基序抗体通过免疫亲和纯化从野生型和 Lrrk1 缺陷破骨细胞中富集磷酸肽，并通过液相色谱和串联质谱法 (LC) 鉴定 Lrrk1 初始靶标。 - 质谱/质谱）。该应用结果将加深我们对Lrrk1信号调节破骨细胞功能和骨吸收的分子机制的理解，为骨质疏松等骨疾病的治疗和预防提供新的治疗策略。