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Mitotic Checkpoint Regulators in Insulin Signaling

胰岛素信号传导中的有丝分裂检查点调节剂

基本信息

批准号：
9363756
负责人：
HONGTAO YU
金额：
$ 29.97万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-21 至 2021-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9363756
关键词：
Ablation Address Affect Alleles Aneuploidy Animals Binding Biochemistry Biological Assay Blood Glucose Cell division Cell membrane Cells Cellular biology Chromosome Segregation Clathrin Clathrin Adaptors Collection Complex Development Diabetes Mellitus Diet Disease Elements Endocytosis Energy Transfer Ensure Exhibits Genetic Genomics Human IRS1 gene Immune checkpoint inhibitor In Vitro Insulin Insulin Receptor Insulin Resistance Knock-in Mouse Leucine Link MXD1 gene Maps Mediating Metabolic Metabolic Diseases Metabolic syndrome Metabolism Mitogen-Activated Protein Kinases Mitosis Mitotic Mitotic Checkpoint Molecular Target Mus Mutate Non-Insulin-Dependent Diabetes Mellitus Obesity Pathway interactions Patients Phenotype Physiological Physiology Proteins Publishing Recombinant Proteins Recruitment Activity Regulation Research Resistance Role Sampling Signal Transduction Syndrome Testing Time Tissues Transcription Factor AP-2 Alpha anaphase-promoting complex diabetic diabetic patient exosome experimental study in vivo inhibitor/antagonist insulin signaling liver biopsy mutant new therapeutic target non-diabetic novel premature prevent receptor binding reconstitution single cell sequencing transcriptome sequencing

项目摘要

Project Summary Insulin signaling is critical for multiple facets of animal physiology. Its dysregulation causes insulin resistance syndromes, such as type 2 diabetes. The spindle checkpoint ensures the fidelity of chromosome segregation and guards against aneuploidy. The key spindle checkpoint proteins Mad2 and BubR1 can simultaneously bind to Cdc20, converting it from an anaphase promoting complex/cyclosome (APC/C) activator to a subunit of an APC/C-inhibitory complex called the mitotic checkpoint complex (MCC). During checkpoint inactivation, a critical inhibitor of Mad2, p31comet promotes checkpoint inactivation and timely chromosome segregation. Recently, combining approaches in mouse genetics, cell biology, biochemistry, and single-cell genomics, we have discovered a critical role of the p31comet–Mad2–BubR1 module of mitotic regulators in insulin signaling through regulating insulin receptor (IR) endocytosis. In the mouse, p31comet ablation diminishes IR at the plasma membrane prior to insulin binding and causes defective insulin signaling in multiple tissues and metabolic syndrome. Mechanistically, Mad2 directly binds to IR through a canonical Mad2-interacting motif (MIM). IR-bound Mad2 facilitates BubR1-dependent recruitment of the clathrin adaptor AP2 to IR. p31comet blocks Mad2-BubR1 association and prevents spontaneous IR endocytosis. Mad2 and BubR1 are also required for insulin-stimulated IR endocytosis. This unexpected link between mitotic regulators and insulin signaling raises several outstanding questions that we wish to address in this proposal. In Aim 1, we will further elucidate the mechanism and regulation of insulin-stimulated IR endocytosis. In particular, we will determine how the newly discovered Mad2–BubR1 mechanism cooperates with previously described mechanisms to mediate proper IR endocytosis. We will establish how these mechanisms are regulated by insulin signaling. In Aim 2, we will test the intriguing hypothesis that insulin signaling reciprocally regulates the spindle checkpoint. In preliminary results, we have created a knock-in mouse (Insr4A/4A) with mutated IR alleles (4A) deficient for Mad2 binding. IR 4A cells have a weakened spindle checkpoint. We will determine the mechanisms by which IR promotes spindle checkpoint signaling through cellular and in vitro reconstitution experiments. In Aim 3, we will define the physiological functions of the mutual regulation between IR and mitotic regulators by examining the phenotypes of the Insr4A/4A mouse. We will test whether defective IR plasma membrane localization contributes to type 2 diabetes by comparing IR localization in liver biopsies from non-diabetic and diabetic patients. Collectively, the proposed research will further clarify the mechanism and function of the unexpected link between mitotic regulators and insulin signaling, and may establish the Mad2– BubR1–AP2 module as a novel therapeutic target for treating diabetes.

项目摘要胰岛素信号传导对动物生理学的多个方面至关重要。它的失调会导致胰岛素抵抗综合征，如2型糖尿病。主轴检查点确保了染色体分离和防止非整倍体。关键纺锤体检查点蛋白Mad 2 BubR 1可以同时与Cdc 20结合，将其从促进细胞分裂的后期转化为促进细胞分裂的后期。在一个实施方案中，APC/C激活剂与APC/C抑制复合物的亚基（称为有丝分裂抑制复合物）结合。检查点复合体（MCC）。在检查点失活期间，Mad 2的关键抑制剂p31彗星促进检查点失活和及时的染色体分离。最近，结合在小鼠遗传学，细胞生物学，生物化学和单细胞基因组学的方法，我们有发现了有丝分裂调节因子p31 comet-Mad 2-BubR 1模块在胰岛素信号传导中的关键作用通过调节胰岛素受体（IR）的内吞作用。在小鼠中，p31彗星消融减少了IR在在胰岛素结合之前破坏质膜，并导致多种组织中胰岛素信号传导缺陷，代谢综合征在机制上，Mad 2通过典型的Mad 2相互作用蛋白直接与IR结合。基序（MIM）。IR结合的Mad 2促进网格蛋白衔接子AP 2向IR的BubR 1依赖性募集。 p31 comet阻断Mad 2-BubR 1结合并阻止自发IR内吞作用。Mad 2和BubR 1 也是胰岛素刺激的IR内吞作用所必需的。有丝分裂调控因子之间的联系而胰岛素信号传导提出了几个我们希望在本提案中解决的突出问题。在目的1：进一步阐明胰岛素刺激的胰岛素抵抗内吞作用的机制和调控。在特别是，我们将确定新发现的Mad 2-BubR 1机制如何与先前描述的介导适当IR内吞作用的机制。我们将确定这些如何这些机制是由胰岛素信号调节的。在目标2中，我们将测试一个有趣的假设，胰岛素信号转导通路调节纺锤体检查点。在初步结果中，我们创建了一个具有突变的IR等位基因（4A）的敲入小鼠（Insr 4A/4A），其缺乏Mad 2结合。IR 4A细胞具有弱化纺锤体检查点。我们将确定IR促进纺锤体的机制通过细胞和体外重建实验检测点信号传导。在目标3中，我们将定义 IR和有丝分裂调节因子之间相互调节的生理功能。 Insr 4A/4A小鼠的表型。我们将测试是否有缺陷的IR质膜定位通过比较非糖尿病患者和糖尿病患者总的来说，这项研究将进一步阐明有丝分裂调节因子和胰岛素信号之间的意外联系，并可能建立Mad 2- BubR 1-AP 2模块作为治疗糖尿病的新靶点