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.

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包含大多数Roco家族蛋白的结构域和额外的LRRK2特异性重复，但缺乏Ankryrin重复。 LRRK1和LRRK2均普遍表达在多个组织中。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表型，而缺乏LRRK2基因的小鼠表现出明显的骨骼表观型，表明LRRK1在LRRK1中没有LRRK1在骨细胞中具有独特的结构和在骨骼中的功能，而LRRK2无法补偿LRRRK2。在破骨细胞中LRRK1的信号传导途径上几乎没有知名度。我们在这笔赠款上的重点是确定LRRK1 I破骨细胞的结构和功能，并确定潜在的LRRK1激酶靶标和抑制剂。为此，我们在这项研究中提出了两个假设：1）LRRK1通过其激酶激活调节破骨细胞的功能； 2） LRRK1磷酸化其特异性底物的丝氨酸/苏氨酸残基。为了检验假设1，设计实验旨在检查全长LRRK1的结构和功能，并通过体外凹坑测定法对骨切片成熟破骨细胞的骨吸收活性进行截短的LRRK1。人LRRK1激酶结构域的同源性模型用于对接小分子抑制剂，并且将在体外激酶和PI形成测定中测试类似药物样化合物的抑制作用。为了检验假设2，设计实验是为了通过使用磷酸 - 塞氨酸/磷酸 - 硫代硫代基氨酸基元抗体的免疫接触纯化来富集野生型和LRRK1缺乏骨质的磷酸肽，并通过液体色谱和Tandem质量谱（MSS/MSS/mss）识别LRRK1初始靶标，并识别LRRK1初始靶标。该应用的结果将促进我们对调节破骨细胞功能和骨吸收的LRRK1信号传导的分子机制的理解，并为治疗和预防骨质疾病（例如骨质疏松症）提供新的治疗策略。