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

• 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
• 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' 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

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.

项目概要 造血干细胞和祖细胞 (HSPC) 是自我更新、可移植的细胞，可支持终生 血液的产生。 HSPC 功能和移植通过 G 蛋白偶联与信号转导相关 受体 (GPCR)，特别是调节归巢和生态位保留的 CXCR4 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 转运蛋白 GPRASP1 和 GPRASP2 充当 HSPC 的负调节因子 移植。我们的初步数据进一步表明这些蛋白质限制 HSPC 的静止和抵抗 以 CXCR4 依赖性方式导致细胞凋亡。 GPRASP1 和 -2 在神经系统中被描述为 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 GPRASP1 和 -2 调节的受体目前尚不清楚。作为一名博士后 在McKinney-Freeman实验室，我将研究GPRASP1和-2在HSPC中的分子功能。 在目标 1 中，我将使用生化方法来定义 GPRASP1 和 -2 对 CXCR4 信号传导的影响。这 GPRASP1 和 -2 对 CXCR4 内吞作用、降解和下游信号转导结果的影响 将在小鼠 HSPC 中分析激动剂刺激。在目标 2 中，我将探讨 GPRASP1 和 -2 在 从脐带血中分离出人类 HSPC 进行移植。经过基因编辑的人类 HSPC CRISPR/Cas9 核转染可敲除 GPRASP1 或 -2，并将用于竞争性异种移植 化验。通过跟踪野生型和敲除人类 HSPC 的相对植入和血液生产 异种移植后，将确定 GPRASP 蛋白对此过程的影响。在目标 3 中，我将 使用无偏见的蛋白质组学方法来研究 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。生物素邻近标记将使用 K562慢性粒细胞白血病细胞在多次治疗前和多次治疗后的GPRASP-APEX融合蛋白 激动剂刺激后的时间点。生物素化样品将进行串联质量标记和多重分析 质谱法测定不同时间点蛋白质的相对丰度。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分析 和-2。所有感兴趣的蛋白质关联都将通过小鼠 HSPC 中的免疫荧光进行验证。这些 目标借鉴我之前作为生物化学家的培训，但在 HSPC 领域提供丰富的培训机会 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， 将为该项目的成功做出巨大贡献。另外，我会利用众多的职业优势 在我的研究期间开发资源。这些资源包括科学写作研讨会、演示 以及社交机会以及教学和指导经验。