Regulation of T cell ligand discrimination by tuning the phosphorylation kinetics of Zap70 substrates

通过调节 Zap70 底物的磷酸化动力学来调节 T 细胞配体辨别

• 批准号: 9720665
• 负责人: Wan-Lin Lo
• 金额: $ 16.2万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-14 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9720665
• 关键词: Adaptive Immune System; Adaptor Signaling Protein; Affect; Amino Acids; Antigen Receptors; Antigens; Aspartate; Autoantigens; Autoimmune Responses; Binding; Biochemical; Biological; CD3 Antigens; Calcium; Calcium Signaling; Career Transition Award; Cell physiology; Cellular biology; Characteristics; Charge; Collaborations; Complement; Data; Discrimination; Docking; Dose; Engineering; Event; Exhibits; Foundations; Future; Glutamates; Glycine; Goals; Homeostasis; Human; Immunologics; Kinetics; Knock-in; Knock-in Mouse; Knowledge; LCP2 gene; Ligands; Link; Lymphocyte-Specific p56LCK Tyrosine Protein Kinase; Maintenance; Mediating; Modeling; Modification; Molecular; Mus; Mutagenesis; Mutate; Mutation; National Institute of Allergy and Infectious Disease; Outcome; Pathway interactions; Peptide/MHC Complex; Peripheral; Pharmaceutical Preparations; Phospholipase; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Positioning Attribute; Proteins; Research; Research Personnel; Research Project Grants; Role; Scaffolding Protein; Scanning; Self Tolerance; Sensitivity and Specificity; Series; Short Interspersed Nucleotide Elements; Signal Pathway; Signal Transduction; Signaling Protein; Site; Speed; T cell differentiation; T cell regulation; T cell response; T-Cell Activation; T-Cell Development; T-Cell Immunologic Specificity; T-Lymphocyte; Testing; Therapeutic; Thymocyte Development; Thymus Gland; Tyrosine; ZAP-70 Gene; base; career; cell mediated immune response; chimeric antigen receptor; improved; in vivo; innovation; insight; mathematical algorithm; mutant; novel; preference; recruit; release of sequestered calcium ion into cytoplasm; response

PROJECT SUMMARY/ABSTRACT This NIAID K22 Career Transition Award focuses on the long-term research goals of the candidate, Dr. Wan- Lin Lo, to become an independent investigator and to acquire data for a future R01 application. This proposal focuses on the examination of T cell ligand discrimination capability through manipulating phosphorylation efficiency of Zap70 substrates. Upon TCR stimulation, the activated T cell kinase Lck phosphorylates tyrosines on CD3 and z-chains, leading to the recruitment, phosphorylation and activation of the kinase Zap70. Activated Zap70 subsequently phosphorylates multiple sites within the adaptors LAT and SLP76. Phospho-tyrosines on LAT and SLP76 become docking sites for additional signaling proteins, resulting in LAT- or SLP76-based signalosomes. There are five Zap70 substrate tyrosines in LAT, and three in SLP76. Each of these tyrosine substrates is dedicated to specifically interact with distinct proteins that are involved in divergent downstream pathways. For example, phosphorylation of LAT Y132 is the only tyrosine that can couple TCR signals to the PLCg1 pathway and the resultant calcium increase. Previously, it was thought that phosphorylation of Zap70 tyrosine substrates was strictly a signal propagation step that diversifies or amplifies signals. However, Dr. Lo’s preliminary data showed that, at least for LAT Y132, Zap70-mediated phosphorylation of this site has greater significance. Zap70-mediated phosphorylation of LAT Y132 has uniquely slow phosphorylation kinetics. This is because the amino acid preceding Y132 is a glycine, whereas other Zap70 substrates contain a negatively charged amino acid at this position which complements Zap70’s positively charged docking site. By replacing this glycine with a negatively charged residue, Dr. Lo observed faster phosphorylation of Y132, and yet this allowed T cells to inappropriately react to weakly binding self-antigens. Dr. Lo hypothesizes that LAT Y132 has evolved to serve as a unique TCR signal bottleneck to support a proper degree of T cell ligand discrimination. To test this hypothesis, she will explore whether the Y132-phosphorylation-PLCg1 bottleneck is really uniquely suited for ligand discrimination by examining whether the phosphorylation kinetics of other Zap70 tyrosine substrates can have similar effects on T cell’s ability to discriminate self and non-self (Aim I). She will also slow down the phosphorylation speed of other Zap70 substrates, by mutating the preceding negatively charged residue to a glycine. This mutagenesis approach will impose “Y132-like” slow kinetics on other Zap70 tyrosine substrates, to test whether these nodes can also serve as kinetic bottlenecks and affect ligand discrimination. She will also evaluate T cell development and peripheral T cell function in a LAT G135D knock-in mouse (glycine to glutamate mutation preceding Y136, homologous to human G131-Y132), to determine how disruption of this kinetic threshold impacts thymic T cell selection and T cell tolerance in vivo (Aim II). The obtained data will provide insights into how T cell ligand discrimination is regulated and will establish the foundation of new research projects for Dr. Lo’s independent scientific career.

项目摘要/摘要 NIAID K22职业过渡奖的重点是候选人Wan-博士的长期研究目标 Lin lo，成为一名独立研究者并获取未来R01应用程序的数据。这个建议 专注于通过操纵磷酸化来检查T细胞配体歧视能力 ZAP70底物的效率。 TCR刺激后，活化的T细胞激酶LCK磷酸化酪氨酸 在CD3和Z链上，导致激酶ZAP70的募集，磷酸化和激活。活性 ZAP70随后磷酸化适配器LAT和SLP76中的多个位点。磷酸酪氨酸 LAT和SLP76成为其他信号蛋白的对接站点，导致基于LAT-或SLP76 LAT中有五个ZAP70底物酪氨酸，SLP76中有3个。这些酪氨酸中的每一个 底物专门与在下游发散的不同蛋白质专门相互作用 途径。例如，LAT Y132的磷酸化是唯一可以将TCR信号与TCR信号相对的酪氨酸 PLCG1途径和最终的钙增加。以前，据认为ZAP70的磷酸化 酪氨酸底物严格来说是一个信号传播步骤，该步骤多样化或放大器信号。但是，Lo博士 初步数据表明，至少对于LAT Y132，ZAP70介导的磷酸化具有更大的 意义。 ZAP70介导的LAT Y132的磷酸化具有唯一的慢速磷酸化动力学。这是 因为Y132之前的氨基酸是甘氨酸，而其他ZAP70底物则含有负含量 在这个位置充电的氨基酸，完成了ZAP70积极充电的对接站点。通过更换 该甘氨酸具有负电荷的住所，LO博士观察到Y132的磷酸化速度更快，但 允许T细胞不适当地对弱结合的自抗原反应。 Lo博士假设LAT Y132有 演变为作为独特的TCR信号瓶颈，以支持适当程度的T细胞配体歧视。 为了检验这一假设，她将探讨Y132-磷酸化-plcg1瓶颈是否真的是独特的 通过检查其他ZAP70酪氨酸的磷酸化动力学是否适合配体歧视 底物可能会对T细胞区分自我和非自我的能力有类似的影响（AIM I）。她也会放慢 通过突变前带负电荷的其他ZAP70底物的磷酸化速度 残留到甘氨酸。这种诱变方法将在其他ZAP70酪氨酸上施加“ Y132样”慢动力学 底物测试这些节点是否也可以用作动力学瓶颈并影响配体歧视。 她还将评估LAT G1​​35D敲入小鼠中T细胞的发育和周围T细胞功能 （Y136之前，与人G131-Y132同源的甘氨酸到谷氨酸突变），以确定如何 这种动力学阈值的破坏会影响胸腺T细胞的选择和体内T细胞耐受性（AIM II）。这 获得的数据将提供有关如何调节T细胞配体歧视的见解，并将建立 Lo博士独立科学职业的新研究项目的基础。