Novel Regulators of GPCR Signaling in Hematopoietic Stem Cells

造血干细胞中 GPCR 信号传导的新型调节剂

基本信息

批准号：
10023156
负责人：
Christopher David Nevitt
金额：
$ 6.53万
依托单位：
ST. JUDE CHILDREN'S RESEARCH HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-16 至 2022-09-15
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10023156
关键词：
Address Aftercare Agonist Apoptosis Binding Biochemical Bioinformatics Biological Assay Biotin Blood Bone Marrow C-terminal CRISPR/Cas technology CXCR4 gene Cell Cycle Cell Transplantation Cell physiology Cells Cellular biology Chimera organism Chimeric Proteins Chimerism Complex Data Data Set Disease Dopamine Down-Regulation Educational process of instructing Educational workshop Electroporation Endocytosis Engraftment Environment Fellowship Flow Cytometry Frequencies G Protein-Coupled Receptor Signaling G-Protein-Coupled Receptors GTP-Binding Proteins Genes Hematological Disease Hematopoietic Hematopoietic Stem Cell Transplantation Hematopoietic System Hematopoietic stem cells Homing Human Immunofluorescence Immunologic K-562 Knock-out Label Laboratories Life Ligands Lysosomes Mass Spectrum Analysis Mentors Methods Modeling Molecular Mus Nervous system structure Opioid Outcome Output Pathway interactions Phenotype Postdoctoral Fellow Procedures Production Proteins Proteomics Recycling Regimen Regulation Resistance Resource Development Resources Role Saint Jude Children&apos s Research Hospital Sampling Savings Serum Signal Transduction Signaling Protein Sorting - Cell Movement Stem cell transplant Supporting Cell Tail Testing Time Training Transplantation Umbilical Cord Blood Work Writing Xenograft procedure career development chronic myeloid leukemia cell conditioning curative treatments experience human cord blood CD34+ cell improved in vivo insertion/deletion mutation interest knock-down leukemia treatment multipotent cell novel peripheral blood post-transplant protein transport receptor reconstitution self renewing cell small hairpin RNA success targeted sequencing therapeutic target trafficking training opportunity

项目摘要

Project Summary Hematopoietic stem and progenitor cells (HSPCs) are self-renewing, transplantable cells that support lifelong blood production. HSPC function and transplantation are tied to signal transduction through G-protein coupled receptors (GPCRs), particularly that of CXCR4 which regulates homing and niche retention. Our understanding of basic GPCR regulatory mechanisms in these cells are lacking. This is highlighted by our laboratories recent finding that two GPCR-trafficking proteins, GPRASP1 and GPRASP2, act as negative regulators of HSPC transplantation. Our preliminary data further indicate that these proteins limit HSPC quiescence and resistance to apoptosis in a CXCR4-dependent manner. GPRASP1 and -2 have been described in the nervous system to promote the lysosomal degradation and functional downregulation of opioid, dopamine, and other select receptors. The receptors regulated by GPRASP1 and -2 in HSCs are currently unknown. As a postdoctoral fellow in the McKinney-Freeman laboratory, I will investigate the molecular functions of GPRASP1 and -2 in HSPCs. In Aim 1, I will use biochemical methods to definethe impact of GPRASP1 and -2 on CXCR4 signaling. The impact of GPRASP1 and -2 on CXCR4 endocytosis, degradation, and downstream signaling outcomes after agonist stimulation will be analyzed in mouse HSPCs. In Aim 2, I will probe the role of GPRASP1 and -2 in transplantation of human HSPCs isolated from cord blood. Human HSPCs that have undergone gene editing by CRISPR/Cas9 nucleofection to knockout GPRASP1 or -2 and will be used in competitive xenotransplantation assays. By following the relative engraftment and blood production of wild type and knockout human HSPCs after xenotransplantation the impact of GPRASP proteins on this procedure will be determined. In Aim 3, I will use an unbiased proteomics approach to investigate the receptor targets of GPRASP1 and -2, as well as the downstream trafficking pathways associated with these proteins. Biotin proximity labeling will be performed using GPRASP-APEX fusion proteins in K562 chronic myelogenous leukemia cells both before and at multiple timepoints following agonist stimulation. Biotinylated samples will undergo Tandem Mass Tagging and multiplex mass spectrometry to determine the relative abundance of proteins across timepoints. This will allow for bioinformatic analysis of the receptor targets and downstream trafficking pathways associated with GPRASP1 and -2. All protein associations of interest will be validated by immunofluorescence in mouse HSPCs. These aims draw on my prior training as a biochemist but provide abundant training opportunities in the field of HSPC biology. The McKinney-Freeman laboratory and St. Jude are ideal environments in which to receive training in HSPC biology. Institutional resources provided, including state-of-the-art flow cytometry and proteomics cores, will greatly contribute to the success of this project. In addition, I will take advantage of numerous career development resources during my fellowship. These resources include scientific writing workshops, presentation and networking opportunities, and teaching and mentoring experiences.

项目摘要造血茎和祖细胞（HSPC）是自我更新的可移植细胞，可支持终身血液产生。 HSPC功能和移植通过G蛋白耦合而绑定到信号转导受体（GPCR），特别是调节归巢和利基保留率的CXCR4的受体。我们的理解这些细胞中缺乏基本的GPCR调节机制。我们的实验室最近强调了这一点发现两种GPCR贩运蛋白GPRASP1和GPRASP2充当HSPC的负调节剂移植。我们的初步数据进一步表明这些蛋白质限制了HSPC静止和抗性以CXCR4依赖性方式凋亡。神经系统中已经描述了GPRASP1和-2 促进阿片类药物，多巴胺和其他选择的溶酶体降解和功能下调受体。 HSC中由GPRASP1和-2调节的受体目前尚不清楚。作为博士后研究员在麦金尼 - 弗里曼实验室中，我将研究HSPC中GPRASP1和-2的分子功能。在AIM 1中，我将使用生化方法来定义GPRASP1和-2对CXCR4信号的影响。这 GPRASP1和-2对CXCR4内吞作用，降解和下游信号的影响激动剂刺激将在小鼠HSPC中进行分析。在AIM 2中，我将探讨GPRASP1和-2在从脐带血中分离出的人类HSPC的移植。经过基因编辑的人类HSPC crispr/cas9核对敲除gprasp1或-2，将用于竞争性异种移植测定。通过遵循野生型和敲除人类HSPC的相对植入和血液产生异种移植后，将确定GPRASP蛋白对此过程的影响。在AIM 3中，我会使用公正的蛋白质组学方法研究GPRASP1和-2的受体靶标，以及与这些蛋白质相关的下游运输途径。将使用生物素接近标签 K562中的Gprasp-Apex融合蛋白在多个之前和多个激动剂刺激后的时间点。生物素化样品将经过串联质量标记和多重质谱法确定跨时间的蛋白质相对丰度。这将允许与GPRASP1相关的受体靶标和下游运输途径的生物信息学分析和-2。所有感兴趣的蛋白质关联将通过小鼠HSPC中的免疫荧光来验证。这些目的借鉴了我作为生物化学家的先前培训，但在HSPC领域提供了丰富的培训机会生物学。麦金尼 - 弗里曼实验室和圣裘德是接受培训的理想环境 HSPC生物学。提供的机构资源，包括最先进的流式细胞术和蛋白质组学核心，将有助于该项目的成功。此外，我将利用许多职业在我的奖学金期间开发资源。这些资源包括科学写作研讨会，演讲以及网络机会，教学和指导经验。