title: A novel Zap70 mutation with reduced protein stability demonstrates the rate-limiting threshold for Zap70 in T-cell receptor signalling.
abstract: Loss of ζ-associated protein 70 (Zap70) results in severe immunodeficiency in humans and mice because of the critical role of Zap70 in T-cell receptor (TCR) signalling. Here we describe a novel mouse strain generated by N-ethyl-N-nitrosourea mutagenesis, with the reduced protein stability (rps) mutation in Zap70. The A243V rps mutation resulted in decreased Zap70 protein and a reduced duration of TCR-induced calcium responses, equivalent to that induced by a 50% decrease in catalytically active Zap70. The reduction of signalling through Zap70 was insufficient to substantially perturb thymic differentiation of conventional CD4 and CD8 T cells, although Foxp3(+) regulatory T cells demonstrated altered thymic production and peripheral homeostasis. Despite the mild phenotype, the Zap70(A243V) variant lies just above the functional threshold for TCR signalling competence, as T cells relying on only a single copy of the Zap70(rps) allele for TCR signalling demonstrated no intracellular calcium response to TCR stimulation. This addition to the Zap70 allelic series indicates that a rate-limiting threshold for Zap70 protein levels exists at which signalling capacity switches from nearly intact to effectively null.
authors.affiliation: Autoimmune Genetics Laboratory, VIB, Leuven, Belgium; Department of Microbiology and Immunology, University of Leuven, Leuven, Belgium.
authors.forename: Bénédicte
authors.initials: B
authors.lastname: Cauwe
authors.forename: Lei
authors.initials: L
authors.lastname: Tian
authors.forename: Dean
authors.initials: D
authors.lastname: Franckaert
authors.forename: Wim
authors.initials: W
authors.lastname: Pierson
authors.forename: Kim A
authors.initials: KA
authors.lastname: Staats
authors.forename: Susan M
authors.initials: SM
authors.lastname: Schlenner
authors.forename: Adrian
authors.initials: A
authors.lastname: Liston
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D020013:Calcium Signaling; D002454:Cell Differentiation; D004195:Disease Models, Animal; D005260:Female; D051858:Forkhead Transcription Factors / Q000378:metabolism; D020022:Genetic Predisposition to Disease; D006579:Heterozygote; D007700:Kinetics; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D055550:Protein Stability; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D016511:Severe Combined Immunodeficiency / Q000201:enzymology* / Q000235:genetics / Q000276:immunology; D013154:Spleen / Q000201:enzymology / Q000276:immunology; D016176:T-Lymphocyte Subsets / Q000201:enzymology* / Q000276:immunology; D060168:Thymocytes / Q000201:enzymology / Q000276:immunology; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000172:deficiency* / Q000235:genetics / Q000276:immunology / Q000378:metabolism
chemical_list: D051858:Forkhead Transcription Factors; C498833:Foxp3 protein, mouse; D011948:Receptors, Antigen, T-Cell; D051746:ZAP-70 Protein-Tyrosine Kinase; C497262:Zap70 protein, mouse
keywords: Foxp3; N-ethyl-N-nitrosourea mutagenesis; T cell; T-cell receptor; Zap70; protein stabilization