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对于动物生理的多个方面至关重要。它的失调会导致胰岛素抗药性综合征，例如2型糖尿病。主轴检查站确保了染色体分离和针对非整倍性的守卫。钥匙主轴检查点蛋白mad2 和bubr1可以轻松地与cdc20结合，从促进的盘子转换为复合物/循环体（APC/C）激活剂至APC/C抑制性复合物的亚基，称为有丝分裂检查点复合体（MCC）。在检查点灭活期间，MAD2的关键抑制剂P31COMET 促进检查点失活和及时的染色体分离。最近，合并小鼠遗传学，细胞生物学，生物化学和单细胞基因组学的方法，我们有发现了胰岛素信号传导中有丝分裂调节剂的p31COMET – MAD2 -BUBR1模块的关键作用通过调节性胰岛素受体（IR）内吞作用。在鼠标中，p31Comet消融会减少IR 在胰岛素结合之前的质膜，并在多个时间内引起胰岛素信号不良，并且代谢综合征。从机械上讲，MAD2通过规范的MAD2相互作用直接与IR结合主题（MIM）。 IR-BOND MAD2最爱BUBR1依赖于网状蛋白适配器AP2对IR的募集。 p31COMET阻止MAD2-BUBR1关联并防止自发性红外内吞作用。 MAD2和BUBR1 胰岛素刺激的IR内吞作用也需要。有丝分裂调节器之间的意外联系胰岛素信号引发了我们希望在此提案中解决的几个杰出问题。 AIM 1，我们将进一步阐明胰岛素刺激的IR内吞作用的机理和调节。在特别是，我们将确定新发现的MAD2 – BUBR1机制如何与先前描述的机制介导了适当的IR内吞作用。我们将确定如何机制受胰岛素信号传导调节。在AIM 2中，我们将检验一个有趣的假设胰岛素信号传导相互调节主轴检查点。在初步结果中，我们创建了一个具有突变的IR等位基因（4A）缺乏MAD2结合的敲入小鼠（INSR4A/4A）。 IR 4A细胞具有削弱的主轴检查点。我们将确定IR促进主轴的机制检查点通过细胞和体外重构实验信号传导。在AIM 3中，我们将定义 IR和有丝分裂调节剂之间相互调控的生理功能通过检查 INSR4A/4A小鼠的表型。我们将测试有缺陷的红外质膜定位通过比较非糖尿病和肝脏活检中的IR定位来促进2型糖尿病糖尿病患者。总的来说，拟议的研究将进一步阐明有丝分裂调节剂与胰岛素信号传导之间的意外联系，并可能建立MAD2 – BUBR1 – AP2模块是治疗糖尿病的新型治疗靶标。