Structural Analysis of the TCR-CD3 Complex and TCR Signaling
Structural Analysis of the TCR-CD3 Complex and TCR Signaling
批准号:
9251684
负责人:
Roy A Mariuzza
金额:
$70.04万
依托单位国家:
美国
项目类别:
财政年份:
2016
资助国家:
美国
项目状态:
已结题
起止时间:
2016-05-01 至 2018-04-30
关键词:
AddressAffinityAntigensAntiviral AgentsArchitectureAutoimmune ProcessBindingBinding ProteinsBiologicalBiological AssayCD3 AntigensCRISPR/Cas technologyCell LineCell physiologyCell surfaceCellsChemicalsComplexCoupledCrystallizationDataDevelopmentDirected Molecular EvolutionDockingElementsEpitopesEventFlow CytometryGoalsHLA-A2 AntigenHLA-DR4 AntigenHealthHeteronuclear NMRHuman T-lymphotropic virus 1Immune responseImmunologyIn VitroIndividualLengthLibrariesLigandsLigationLinkMHC Class I GenesMHC Class II GenesMHC binding peptideMalignant NeoplasmsMapsMature T-LymphocyteMeasurableMediatingMicrobeMolecularMolecular ConformationMolecular ImmunologyMultiprotein ComplexesMusMutagenesisMutationMyelin Basic ProteinsNMR SpectroscopyPeptide/MHC ComplexPeptidesPhysiologicalPlayProcessReceptor SignalingRelaxationResearch PersonnelResolutionRetroviral VectorRoentgen RaysRoleSignal PathwaySignal TransductionSiteStructureSurfaceSystemT-Cell ActivationT-Cell DevelopmentT-Cell ReceptorT-LymphocyteTaxesTechnologyTestingTimeValidationVariantViral AntigensX-Ray CrystallographyYeastsadaptive immunitybasedesigndimerextracellulargenome editingmutantnovel strategiesreceptorreconstitution
中文摘要
英文摘要
DESCRIPTION (provided by applicant): The T cell receptor (TCR)-CD3 complex is composed of a diverse TCRαβ heterodimer noncovalently associated with the invariant CD3 dimers CD3εγ, CD3εδ, and CD3ζζ. The TCR mediates peptide-MHC (pMHC) recognition, while the CD3 molecules transduce activation signals to the T cell. Whereas much is known about downstream T cell signaling pathways, the mechanism whereby TCR engagement by pMHC initiates signaling remains a mystery. Our goal is to understand the early events of TCR signaling by obtaining information on: 1) the spatial organization of the TCR-CD3 complex; and 2) possible allosteric changes in TCR conformation and/or dynamics upon binding pMHC that are relayed to CD3. These studies will be undertaken through a highly integrated and comprehensive project that combines multiple approaches, technologies, and investigators. To assure the generality of our findings, we will study both an MHC class I-restricted TCR (A6) and an MHC class II-restricted TCR (MS2-3C8). A6 recognizes a viral antigen from HTLV-1 bound to HLA-A2; MS2-3C8 recognizes a self-peptide from myelin basic protein bound to HLA-DR4. Our Specific Aims are: 1. Structure determination of the wild-type TCR-CD3 complex by NMR. Although the extracellular regions of CD3 are known to interact with the extracellular regions of the TCR, all attempts to crystallize TCR-CD3 complexes have been thwarted by the very low affinity of TCR-CD3 interactions in solution. We will employ NMR chemical shift perturbation and PRE to determine binding epitopes between CD3 and TCR. These data will be used to determine a structure for the wild-type TCR-CD3 complex. We will address whether pMHC binding alters TCR-CD3 interactions, possibly triggering T cell signaling. 2. Structural analysis of affinity- matured TCR-CD3 complexes by X-ray crystallography. We will attempt to overcome the weak association between TCR and CD3 ectodomains by in vitro directed evolution using yeast surface display to stabilize TCR-CD3 complexes for crystallization. Information on binding epitopes from NMR will be used to design TCR mutant libraries by focusing mutagenesis on those regions that contact CD3 (or vice versa). 3. Biological validation of the TCR-CD3 structure. We will validate TCR-CD3 interfaces identified by NMR or X-ray crystallography by evaluating the effects of structure-based mutations in TCR-CD3 interfaces on complex assembly, cell surface expression, signaling, and T cell function and development. The TCR-CD3 complex will be reconstituted using 2A-linked retroviral vectors to generate T cells or retrogenic mice expressing defined combinations of TCR and CD3 components. 4. Structural and dynamics analysis of free and pMHC-bound states of TCRαβ ectodomains. We will address the allosteric change hypothesis by carrying out solution NMR analysis of full-length TCR ectodomains in free form and bound to pMHC, for both TCRs A6 and MS2- 3C8. We will investigate whether pMHC ligation induces changes in TCR conformation and/or dynamics and, if so, relate these changes to a possible mechanism for TCR triggering involving interactions with CD3.
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Structural Basis for T Cell Recognition of SARS-CoV-2
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批准号:10592711
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项目类别:
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资助金额:$23.27万
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财政年份:2023
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负责人:Roy A Mariuzza
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依托单位:
Structure, Function and Mechanistic Analysis of LAG3
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批准号:10542350
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资助金额:$71.2万
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财政年份:2019
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负责人:Roy A Mariuzza
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依托单位:
Structure, Function and Mechanistic Analysis of LAG3
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批准号:10317043
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项目类别:
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资助金额:$71.2万
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财政年份:2019
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负责人:Roy A Mariuzza
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依托单位:
Structure, Function and Mechanistic Analysis of LAG3
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批准号:10078854
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项目类别:
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资助金额:$71.2万
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财政年份:2019
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负责人:Roy A Mariuzza
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依托单位:
Structure and Activation of a Multiprotein Signaling Complex
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批准号:10238118
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项目类别:
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资助金额:$69.2万
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财政年份:2017
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负责人:Roy A Mariuzza
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依托单位:
Structure and Activation of a Multiprotein Signaling Complex
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批准号:9752433
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项目类别:
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资助金额:$70.18万
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财政年份:2017
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负责人:Roy A Mariuzza
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依托单位:
Structure and Activation of a Multiprotein Signaling Complex
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批准号:9448073
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项目类别:
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资助金额:$71.1万
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财政年份:2017
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负责人:Roy A Mariuzza
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依托单位:
Structural Analysis of the TCR-CD3 Receptor Complex
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批准号:8512173
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项目类别:
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资助金额:$22.8万
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财政年份:2013
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负责人:Roy A Mariuzza
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依托单位:
Structural Analysis of the TCR-CD3 Receptor Complex
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批准号:8605510
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项目类别:
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资助金额:$19.0万
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财政年份:2013
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负责人:Roy A Mariuzza
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依托单位:
Solution structure and dynamics of TCR in free and peptide-MHC-bound states
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批准号:8301177
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项目类别:
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资助金额:$22.8万
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财政年份:2012
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负责人:Roy A Mariuzza
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依托单位:
Solution structure and dynamics of TCR in free and peptide-MHC-bound states
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批准号:8416301
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项目类别:
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资助金额:$19.0万
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财政年份:2012
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负责人:Roy A Mariuzza
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依托单位:
CRYSTAL STRUCTURES OF TCR AND MHC AND CD4 COMPLEX
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批准号:8363340
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项目类别:
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资助金额:$0.23万
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财政年份:2011
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负责人:Roy A Mariuzza
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依托单位:
Recombinant Antibodies from the Sea Lamprey
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批准号:7707212
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项目类别:
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资助金额:$22.5万
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财政年份:2009
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负责人:Roy A Mariuzza
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依托单位:
STRUCT BAS FOR PEPTIDOGLYCAN RECOGNITION BY HUMAN PEPTIDOGLYCAN RECOGNITION
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批准号:7955594
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项目类别:
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资助金额:$1.17万
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财政年份:2009
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负责人:Roy A Mariuzza
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依托单位:
Recombinant Antibodies from the Sea Lamprey
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批准号:7898573
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项目类别:
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资助金额:$18.75万
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财政年份:2009
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负责人:Roy A Mariuzza
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依托单位:
Recognition of Self and Mutated Self by Autoimmune and Tumor-Specific TCRs
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批准号:7474448
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项目类别:
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资助金额:$40.5万
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财政年份:2008
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负责人:Roy A Mariuzza
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依托单位:
Recognition of Self and Mutated Self by Autoimmune and Tumor-Specific TCRs
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批准号:8274773
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项目类别:
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资助金额:$38.47万
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财政年份:2008
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负责人:Roy A Mariuzza
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依托单位:
Recognition of Self and Mutated Self by Autoimmune and Tumor-Specific TCRs
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批准号:7880834
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项目类别:
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资助金额:$38.86万
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财政年份:2008
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负责人:Roy A Mariuzza
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依托单位:
Recognition of Self and Mutated Self by Autoimmune and Tumor-Specific TCRs
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批准号:8099588
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项目类别:
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资助金额:$38.47万
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财政年份:2008
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负责人:Roy A Mariuzza
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依托单位:
COMPLEX OF PHOSPHOLIPASE C GAMMA1, GADS AND AN SLP-76 MOTIF
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批准号:7726245
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项目类别:
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资助金额:$0.4万
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财政年份:2008
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负责人:Roy A Mariuzza
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依托单位:
海外基金