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: 10405414
• 负责人: Wan-Lin Lo
• 金额: $ 10.8万
• 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 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10405414
• 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 这个NIAID K22职业转型奖侧重于候选人的长期研究目标，万博士- Lin Lo，成为一名独立的研究者，并为未来的R 01申请获取数据。这项建议 重点是通过操纵磷酸化来检查T细胞配体识别能力 Zap 70底物的效率。在TCR刺激后，活化的T细胞激酶Lck使酪氨酸磷酸化 在CD 3和z链上，导致激酶Zap 70的募集、磷酸化和活化。激活 Zap 70随后磷酸化衔接子LAT和SLP 76内的多个位点。磷酸酪氨酸 LAT和SLP 76成为另外的信号蛋白的对接位点，导致基于LAT或SLP 76的蛋白质结合。 信号体LAT中有5个Zap 70底物酪氨酸，SLP 76中有3个。这些酪氨酸 底物专门与不同的蛋白质相互作用，这些蛋白质参与不同的下游反应。 途径。例如，LAT Y132的磷酸化是唯一可以将TCR信号偶联至TCR的酪氨酸。 PLCg 1途径和由此产生的钙增加。以前，人们认为Zap 70的磷酸化 酪氨酸底物严格地说是使信号多样化或放大的信号传播步骤。然而，罗医生的 初步数据显示，至少对于LAT Y132，Zap 70介导的该位点的磷酸化具有更大的 意义Zap 70介导的LAT Y132磷酸化具有独特的缓慢磷酸化动力学。这是 因为Y132之前的氨基酸是甘氨酸，而其他Zap 70底物含有负的 在这个位置上的带电荷的氨基酸，其补充Zap 70的带正电荷的对接位点。通过更换 这种带有负电荷残基的甘氨酸，Lo博士观察到Y132的磷酸化速度更快，然而， 使T细胞对弱结合的自身抗原产生不适当的反应。Lo博士假设LAT Y132具有 进化为充当独特的TCR信号瓶颈以支持适当程度的T细胞配体辨别。 为了验证这一假设，她将探索Y132-磷酸化-PLCg 1瓶颈是否真的是唯一的 适用于通过检测其他Zap 70酪氨酸磷酸化动力学 底物可以对T细胞区分自身和非自身的能力具有类似的作用（目的I）。她也会慢下来 降低其他Zap 70底物的磷酸化速度， 残基转化为甘氨酸。这种诱变方法将对其他Zap 70酪氨酸激酶施加“Y132样”缓慢动力学。 底物，以测试这些节点是否也可以作为动力学瓶颈，并影响配体歧视。 她还将评估LAT G135 D基因敲入小鼠的T细胞发育和外周T细胞功能 （Y136之前的甘氨酸到谷氨酸的突变，与人G131-Y132同源），以确定 这种动力学阈值的破坏影响胸腺T细胞选择和体内T细胞耐受性（Aim II）。的 获得的数据将提供关于T细胞配体识别如何调节的见解，并将建立 为卢博士的独立科研生涯奠定新的研究项目基础。