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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要该 NIAID K22 职业转型奖重点关注候选人 Wan 博士的长期研究目标- Lin Lo，成为一名独立调查员并为未来的 R01 应用获取数据。专注于通过操纵磷酸化来检查 T 细胞配体辨别能力 Zap70 底物的效率在 TCR 刺激下，激活的 T 细胞激酶 Lck 磷酸化酪氨酸。 CD3 和 z 链上，导致激酶 Zap70 的募集、磷酸化和激活。 Zap70 随后磷酸化接头 LAT 和 SLP76 上的多个位点。 LAT 和 SLP76 成为其他信号蛋白的对接位点，从而产生基于 LAT 或 SLP76 的 LAT 中有 5 个 Zap70 底物酪氨酸，SLP76 中有 3 个。底物专门与参与不同下游的不同蛋白质发生特异性相互作用例如，LAT Y132 的磷酸化是唯一可以将 TCR 信号耦合到 TCR 信号的酪氨酸。 PLCg1途径和由此产生的钙增加以前被认为是Zap70的磷酸化。严格来说，酪氨酸底物是使信号多样化或放大的信号传播步骤。初步数据表明，至少对于LAT Y132，Zap70介导的该位点的磷酸化具有更大的 Zap70 介导的 LAT Y132 磷酸化具有独特的缓慢磷酸化动力学。因为 Y132 之前的氨基酸是甘氨酸，而其他 Zap70 底物含有阴性该位置上的带电氨基酸通过替换补充了 Zap70 的带正电对接位点。 Lo博士观察到Y132的磷酸化速度更快，但这种甘氨酸带有带负电的残基允许 T 细胞对 LAT Y132 具有的弱结合自身抗原做出不适当的反应。进化成为独特的 TCR 信号瓶颈，支持适当程度的 T 细胞配体辨别。为了检验这个假设，她将探索 Y132-磷酸化-PLCg1 瓶颈是否真的是独一无二的通过检查其他 Zap70 酪氨酸的磷酸化动力学是否适合进行配体辨别底物对 T 细胞区分自我和非自我的能力也有类似的影响（目标 I）。通过突变前面的带负电的底物，降低其他 Zap70 底物的磷酸化速度这种诱变方法将对其他 Zap70 酪氨酸施加“Y132 样”缓慢动力学。底物，以测试这些节点是否也可以充当动力学瓶颈并影响配体辨别。她还将评估 LAT G135D 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，与人类 G131-Y132 同源），以确定如何该动力学阈值的破坏会影响胸腺 T 细胞的选择和体内 T 细胞的耐受性 (Aim II)。获得的数据将提供关于 T 细胞配体歧视如何调节的见解，并将建立为卢博士的独立科学事业奠定新的研究项目。