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

• 批准号: 9980928
• 负责人: MELANIE H. COBB
• 金额: $ 29.97万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-21 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9980928
• 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; Ensure; Exhibits; Fluorescence Resonance Energy Transfer; 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; Regulation; Research; Resistance; Role; Sampling; Signal Transduction; Syndrome; Testing; Time; Tissues; anaphase-promoting complex; diabetic; diabetic patient; enhancer-binding protein AP-2; exosome; experimental study; in vivo; inhibitor/antagonist; insulin regulation; insulin signaling; liver biopsy; mouse genetics; mutant; new therapeutic target; non-diabetic; novel; premature; prevent; receptor binding; reconstitution; recruit; single cell sequencing; transcriptome sequencing

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 胰岛素信号对动物生理的多个方面都是至关重要的。它的失调会导致胰岛素 抵抗综合征，如2型糖尿病。磁盘轴检查点可确保 染色体分离和防止非整倍体。关键的纺锤体检查点蛋白MAD2 而BubR1可以同时与Cdc20结合，将其从后期的启动子转化为 复合体/环体(APC/C)激活剂与称为有丝分裂的APC/C抑制复合体的一个亚单位 检查点复合体(MCC)。在检查点失活期间，MAD2的关键抑制因子p31comet 促进检查点失活和及时的染色体分离。最近，联合 在小鼠遗传学、细胞生物学、生物化学和单细胞基因组学方面，我们有 发现有丝分裂调节因子p31comet-MAD2-BubR1模块在胰岛素信号转导中的关键作用 通过调节胰岛素受体(IR)的内吞作用。在小鼠中，p31彗星的消融降低了 胰岛素结合之前的质膜，并导致多组织中胰岛素信号的缺陷和 代谢综合征。从机制上讲，MAD2通过规范的MAD2-相互作用直接与IR结合 Motif(MIM)。IR结合的MAD2促进依赖于BubR1的网状蛋白适配器AP2向IR的募集。 P31comet阻断MAD2-BubR1结合，阻止自发IR内吞作用。MAD2和BubR1 也是胰岛素刺激的IR内吞所必需的。有丝分裂调控因子之间的这种意想不到的联系 胰岛素信号提出了几个悬而未决的问题，我们希望在这项提案中解决这些问题。在……里面 目的1、进一步阐明胰岛素刺激IR内吞作用的机制及其调控。在……里面 特别是，我们将确定新发现的MAD2-BubR1机制如何与 先前描述了调节适当的IR内吞作用的机制。我们将确定如何实现这些目标 其机制受胰岛素信号的调节。在目标2中，我们将测试耐人寻味的假设 胰岛素信号相互调节纺锤体检查点。在初步结果中，我们创建了一个 敲入小鼠(Insr4A/4A)，突变的IR等位基因(4A)缺乏MAD2结合。IR 4A细胞具有 削弱了主轴检查点。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的检查点信号。在目标3中，我们将定义 IR与有丝分裂调节因子相互调节的生理功能 Insr4A/4A小鼠的表型。我们将测试是否有缺陷的红外线质膜定位 通过比较非糖尿病患者和非糖尿病患者肝活检组织中IR的定位，发现IR与2型糖尿病的发病有关 糖尿病患者。总的来说，拟议的研究将进一步阐明 有丝分裂调节因子和胰岛素信号之间意想不到的联系，并可能建立MAD2- BubR1-AP2模块作为治疗糖尿病的新靶点。

项目成果

TP53 promotes lineage commitment of human embryonic stem cells through ciliogenesis and sonic hedgehog signaling.

• DOI: 10.1016/j.celrep.2022.110395
• 发表时间: 2022-02-15
• 期刊: Cell reports
• 影响因子: 8.8
• 作者: Sivakumar S;Qi S;Cheng N;Sathe AA;Kanchwala M;Kumar A;Evers BM;Xing C;Yu H
• 通讯作者: Yu H

CRISPR-Cas9 screen in human embryonic stem cells to identify genes required for neural differentiation.

• DOI: 10.1016/j.xpro.2022.101682
• 发表时间: 2022-12-16
• 期刊: STAR PROTOCOLS
• 作者: Qi, Shutao;Sivakumar, Sushama;Yu, Hongtao
• 通讯作者: Yu, Hongtao

STAG2 promotes the myelination transcriptional program in oligodendrocytes.

• DOI: 10.7554/elife.77848
• 发表时间: 2022-08-12
• 期刊: ELIFE
• 影响因子: 7.7
• 作者: Cheng, Ningyan;Li, Guanchen;Kanchwala, Mohammed;Evers, Bret M.;Xing, Chao;Yu, Hongtao
• 通讯作者: Yu, Hongtao