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Analysis of the Functional Roles of a Novel G-alpha Nucleotide Cycle

新型 G-α 核苷酸循环的功能作用分析

基本信息

批准号：
9773524
负责人：
JOHN H KEHRL
金额：
$ 50.7万
依托单位：
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/9773524
关键词：
4D Imaging ANGPTL2 gene Actins Adopted Adoptive Transfer Animal Model Anti-inflammatory Antibodies Autophagocytosis Binding Biochemical Bioinformatics Bone Marrow CD31 Antigens Caenorhabditis elegans Cell Nucleus Cell Size Cell division Cells Centrosome Cholinesterase Inhibitors Collaborations Complex Confocal Microscopy Cytokinesis Cytoskeleton Data Defect Development Disease Dissociation Drosophila genus Dynein ATPase Endothelial Cells Ensure Exhibits F-Actin Filopodia Fluorescence Resonance Energy Transfer G Actin GPSM1 gene GPSM2 gene GTP Binding GTP-Binding Protein alpha Subunits, Gs GTP-Binding Proteins GTPase-Activating Proteins Generations Genetic study Glues Guanine Nucleotide Dissociation Inhibitors Guanine Nucleotide Exchange Factors Guanosine Triphosphate Heterogeneity Heterotrimeric GTP-Binding Proteins Human Immune In Vitro Inflammasome Inflammatory Intercellular Junctions Intravenous infusion procedures Iowa Knock-out Label Lymphocyte Malignant Neoplasms Mammalian Cell Mammals Mediating Metaphase Microscopy Microtubules Mitosis Mitotic Mitotic Spindle Apparatus Mitotic spindle Modeling Monitor Mus National Institute of Allergy and Infectious Disease Normal Cell Nuclear Nucleotides Pathogenesis Pertussis Toxin Phagocytosis Phenotype Play Process Protein Isoforms Proteins RGS Domain RGS Proteins RGS3 gene Regulation Resistance Role Signal Pathway Signal Transduction Signaling Protein Site Source Structure Time Tissues United States National Institutes of Health Universities WAVE protein cell cortex chromosome movement daughter cell functional plasticity genetic regulatory protein imaging platform imaging system in vivo inhibitor/antagonist insight life history lymph nodes macrophage molecular modeling novel novel therapeutics polymerization prevent protein complex protein function protein protein interaction receptor reconstitution recruit segregation stem temporal measurement venule

项目摘要

These studies have focused on the role of Gi-proteins and their regulators in mitosis, autophagy, lysosomal function, macrophage function, and actin dynamics. In model organisms such as Caenorhabditis elegans and Drosophila receptor-independent heterotrimeric G protein function is vital for the orientation of mitotic spindle, generation of microtubule pulling force, aster-induced cytokinesis, and centration of the nucleus-centrosome complex. This new paradigm is now being extended to mammalian cells. We and others have shown that Gi proteins and their regulators such as AGS3, LGN, and RGS14 localize in centrosomes, at the mitotic cell cortex, and at the midbody region. At these sites AGS3, LGN, and RGS14 likely bind Galphai proteins and function similar to G beta/gamma subunits. We have shown a role for a non-GPCR activator of Gi protein termed Ric-8A in human cell division. Ric-8A expression occurs in most human cells including high levels in lymphocytes. We have evidence that Ric-8A is important for recruiting a signaling complex to the metaphase cell cortex consisting of NuMA, LGN, dynein, p150 glued, and Galphai1. Interference with the localization of this complex caused defects in mitotic spindle orientation and normal cell division. In non-canonical G-protein signaling, Galphai associates with guanine nucleotide dissociation inhibitors (GDI) other than Gbeta/gamma. Wave proteins, which help regulate the actin cytoskeleton have a domain that resembles a GoLoco motif and Wave1 has been shown to bind Galphai. We have observed that Galphai protein can adopt a filamentous-like structure, which coordinates dynamically with actin polymerization during the developments of filopodia and lamellipodia. Galphai partially co-localizes with WAVE1 and Arp2/3 both by confocal microscopy and electon microscopy. FRET studies are consistent with a close physical interaction between actin and the Galphai protein. The GTP-bound form of the protein recruits more WAVE1 and Arp2/3 to cell protrusion regions than does the GDP-bound form. Modeling protein-protein interactions suggests that the GDP-bound form of this G protein would likely competes with G-actin for binding to the WH2 domain (part of VCA domain) of WAVE1 and WAVE2 protein. We have established collaborations with Phillip Cruz (NIAID, NIH) to assist with bioinformatics and molecular modeling of the interactions of Galphai with the WAVE regulatory complex proteins and with Baoyn Chen (Iowa State University) to examine the interactions of Galphai proteins with the WAVE regulatory complex via direct biochemical studies. To better understand the role of actin regulation in vivo we have established a novel four-dimensional imaging platform to precisely determine the profile and dynamics of lymphocyte transmigration in vivo. This 4D imaging system allows for advanced spatial and temporal resolution. By labeling the lymph node vasculature with fluorescently-labeled antibody against PECAM-1 we documented that lymphocytes predominated crossed high endothelia venules (HEVs) by migrating through endothelial cell junctions. Furthermore, we observed real-time HEV pocket formation. To monitor F-actin dynamics we have used LifeAct-GFP bone-marrow reconstituted mice as a source of lymphocytes for adoptive transfer. Since the cells very rapidly access the HEVs following intravenous infusion, we can treat the cells prior to transfer with various inhibitors of actin polymerization. We have found that ARP2/3 and formin inhibitors curtail lymphocyte actin dynamics in the HEVs and largely prevent transmigration. Macrophages exist as innate immune subsets that exhibit phenotypic heterogeneity and functional plasticity. Their phenotypes are dictated by inputs from the tissue microenvironment. G-protein are essential in transducing signals from the microenvironment. We use genetically modified mice to investigate the role of Galphai2 in inflammasome activity and macrophage polarization. Galphai2 in murine bone marrow-derived macrophages (BMDMs) regulates inflammasome activity independent of inflammasome activated (NLRP3, AIM2, and NLRC4). This regulation stems from the biased polarity of BMDMs. We determined that BMDMs with excess Galphai2 signaling have a tendency towards classically activated pro-inflammatory (M1) phenotype, Galphai2 deficient are biased towards alternatively activated anti-inflammatory (M2) phenotype. Long-term, but not short-term inhibition of Gi with pertussis toxin recapitulates the knockout phenotype, indicating that the inflammatory changes are built into the macrophage life history. This data indicates that excess Galphi2 signaling promotes an M1 macrophage phenotype, while Galphai2 signaling deficiency promotes an M2 phenotype. Understanding Galphai2-mediated effects on macrophage polarization may bring to light insights regarding disease pathogenesis and the reprogramming of macrophages for the development of novel therapeutics.

这些研究集中于Gi蛋白及其调节剂在有丝分裂、自噬、溶酶体功能、巨噬细胞功能和肌动蛋白动力学中的作用。在模式生物如秀丽隐杆线虫和果蝇中，受体独立的异源三聚体G蛋白功能对于有丝分裂纺锤体的定向、微管拉力的产生、紫菀诱导的胞质分裂和核-中心体复合物的集中是至关重要的。这种新的模式现在正在扩展到哺乳动物细胞。我们和其他人已经表明，Gi蛋白及其调节因子，如AGS 3，LGN和RGS 14定位在中心体，在有丝分裂细胞皮质，并在中间体区域。在这些位点，AGS 3、LGN和RGS 14可能结合Galphai蛋白，并且功能类似于G β/γ亚基。我们已经显示了Gi蛋白的非GPCR激活剂Ric-8A在人类细胞分裂中的作用。Ric-8A表达发生在大多数人细胞中，包括淋巴细胞中的高水平。我们有证据表明，Ric-8A是重要的招募信号复合物的中期细胞皮质组成的NuMA，LGN，动力蛋白，p150胶合，和Galphai 1。干扰该复合物的定位引起有丝分裂纺锤体方向和正常细胞分裂的缺陷。在非经典G蛋白信号传导中，Galphai与除G β/γ以外的鸟嘌呤核苷酸解离抑制剂（GDI）缔合。帮助调节肌动蛋白细胞骨架的Wave蛋白具有类似于GoLoco基序的结构域，并且已经显示Wave 1结合Galphai。我们已经观察到，Galphai蛋白可以采取一个类似的结构，动态协调肌动蛋白聚合过程中的丝状伪足和片状伪足的发展。 Galphai与WAVE 1和Arp 2/3的共聚焦显微镜和电子显微镜均显示部分共定位。FRET研究与肌动蛋白和Galphai蛋白之间的密切物理相互作用一致。GTP结合形式的蛋白质比GDP结合形式招募更多的WAVE 1和Arp 2/3到细胞突起区域。蛋白质-蛋白质相互作用的建模表明，GDP结合形式的G蛋白可能会与G-肌动蛋白竞争结合WAVE 1和WAVE 2蛋白的WH 2结构域（VCA结构域的一部分）。我们与菲利普克鲁兹（NIAID，NIH）建立了合作关系，以协助Galphai与WAVE调控复合物蛋白相互作用的生物信息学和分子建模，并与Baoyn Chen（爱荷华州州立大学）建立了合作关系，以通过直接的生物化学研究来检查Galphai蛋白与WAVE调控复合物的相互作用。为了更好地了解肌动蛋白在体内的调节作用，我们建立了一个新的四维成像平台，以精确地确定淋巴细胞在体内的迁移的轮廓和动力学。这种4D成像系统允许先进的空间和时间分辨率。通过用荧光标记的抗PECAM-1抗体标记淋巴结脉管系统，我们证明了淋巴细胞通过内皮细胞连接迁移而占主导地位。此外，我们观察到实时HEV口袋形成。为了监测F-肌动蛋白动力学，我们使用LifeAct-GFP骨髓重建小鼠作为过继转移的淋巴细胞来源。由于细胞在静脉输注后非常迅速地接近HEV，我们可以在转移前用各种肌动蛋白聚合的抑制剂处理细胞。我们已经发现，ARP 2/3和ARP 4抑制剂减少了HEV中的淋巴细胞肌动蛋白动力学，并在很大程度上阻止了迁移。巨噬细胞作为先天免疫亚群存在，表现出表型异质性和功能可塑性。它们的表型由来自组织微环境的输入决定。G蛋白在微环境信号转导中起重要作用。我们使用基因修饰的小鼠来研究Galpha 12在炎性小体活性和巨噬细胞极化中的作用。鼠骨髓源性巨噬细胞（BMDM）中的Galpha 12调节炎性小体活性，而不依赖于炎性小体活化（NLRP 3、AIM 2和NLRC 4）。这种调节源于BMDM的偏置极性。我们确定具有过量Galpha 12信号传导的BMDM具有经典活化的促炎（M1）表型的趋势，Galpha 12缺陷偏向于交替活化的抗炎（M2）表型。用百日咳毒素对Gi的长期而非短期抑制再现了敲除表型，表明炎症变化被构建到巨噬细胞生命史中。该数据表明，过量的Galphi 2信号传导促进M1巨噬细胞表型，而Galphi 2信号传导缺陷促进M2表型。了解Galpha 12介导的对巨噬细胞极化的影响可能会带来关于疾病发病机制和巨噬细胞重编程的见解，以开发新的治疗方法。