Complexity of transcriptional regulation within the Rag locus: identification of a second Nwc promoter region within the Rag2 intron

Nwc represents a mysterious third evolutionarily conserved gene within the Rag locus. Here, we analyzed the phenotype of Nwctmpro1 mice, in which the Rag2 intragenic region containing the previously identified promoter responsible for initiating transcription of Nwc in all cells except lymphocytes was deleted by homologous recombination. Despite strong nonlymphocyte-specific inhibition of Nwc transcription which runs through the regulatory region of Rag genes, their expression remained suppressed, and no developmental, morphological, anatomical, functional, physiological, or cellular defects in Nwctmpro1 mice could be observed. However, careful analysis of the Rag2 intergenic region uncovered a second evolutionarily conserved Nwc promoter region from which a previously unknown Nwc transcript can be generated in nonlymphocytes of Nwctmpro1 and normal mice. The above results reveal an unexpected additional complexity of transcriptional regulation within the Rag/Nwc locus and show that strong inhibition of Nwc transcription in nonlymphoid cells is well tolerated. Complete inactivation of Nwc is necessary to get insight into its function at transcriptional and posttranscriptional levels

Increased Protein Stability and Interleukin-2 Production of a LAT(G131D)Variant With Possible Implications for T Cell Anergy

The adaptor LAT plays a crucial role in the transduction of signals coming from the TCR/CD3 complex. Phosphorylation of some of its tyrosines generates recruitment sites for other cytosolic signaling molecules. Tyrosine 132 in human LAT is essential for PLC-gamma activation and calcium influx generation. It has been recently reported that a conserved glycine residue preceding tyrosine 132 decreases its phosphorylation kinetics, which constitutes a mechanism for ligand discrimination. Here we confirm that a LAT mutant in which glycine 131 has been substituted by an aspartate (LAT(G131D)) increases phosphorylation of Tyr132, PLC-gamma activation and calcium influx generation. Interestingly, the LAT(G131D)mutant has a slower protein turnover while being equally sensitive to Fas-mediated protein cleavage by caspases. Moreover, J.CaM2 cells expressing LAT(G131D)secrete greater amounts of interleukin-2 (IL-2) in response to CD3/CD28 engagement. However, despite this increased IL-2 secretion, J.CaM2 cells expressing the LAT(G131D)mutant are more sensitive to inhibition of IL-2 production by pre-treatment with anti-CD3, which points to a possible role of this residue in the generation of anergy. Our results suggest that the increased kinetics of LAT Tyr132 phosphorylation could contribute to the establishment of T cell anergy, and thus constitutes an earliest known intracellular event responsible for the induction of peripheral tolerance

A Novel, LAT/Lck Double Deficient T Cell Subline J.CaM1.7 for Combined Analysis of Early TCR Signaling

Intracellular signaling through the T cell receptor (TCR) is essential for T cell development and function. Proper TCR signaling requires the sequential activities of Lck and ZAP-70 kinases, which result in the phosphorylation of tyrosine residues located in the CD3 ITAMs and the LAT adaptor, respectively. LAT, linker for the activation of T cells, is a transmembrane adaptor protein that acts as a scaffold coupling the early signals coming from the TCR with downstream signaling pathways leading to cellular responses. The leukemic T cell line Jurkat and its derivative mutants J.CaM1.6 (Lck deficient) and J.CaM2 (LAT deficient) have been widely used to study the first signaling events upon TCR triggering. In this work, we describe the loss of LAT adaptor expression found in a subline of J.CaM1.6 cells and analyze cis-elements responsible for the LAT expression defect. This new cell subline, which we have called J.CaM1.7, can re-express LAT adaptor after Protein Kinase C (PKC) activation, which suggests that activation-induced LAT expression is not affected in this new cell subline. Contrary to J.CaM1.6 cells, re-expression of Lck in J.CaM1.7 cells was not sufficient to recover TCR-associated signals, and both LAT and Lck had to be introduced to recover activatory intracellular signals triggered after CD3 crosslinking. Overall, our work shows that the new LAT negative J.CaM1.7 cell subline could represent a new model to study the functions of the tyrosine kinase Lck and the LAT adaptor in TCR signaling, and their mutual interaction, which seems to constitute an essential early signaling event associated with the TCR/CD3 complex.This research was funded by Consejeria de Salud de Andalucia, Junta de Andalucia (grant PI-0055-2017 to E.A.), and Fundacion Biomedica Cadiz Proyectos INIBICA 2019 (grant LI19/I14NCO15 to E.A. and M.M.A.-E.)

Editorial: Regulatory Mechanisms of Early Intracellular Signaling in T Lymphocytes

EA was funded by the Consejería de Salud de Andalucía, Junta de Andalucía (Grant PI-0055-2017 to EA), and Fundación Biomédica Cádiz Proyectos INIBICA 2019 (Grant LI19/I14NCO15). EBC was supported by John Fell Fund, and ERC AdG 670930 to Michael Dustin. AM was funded by the Leading Research Groups Support project obtained in 2019 (with the subsidy, increased for the period 2020–2025 in the amount of 2% of the subsidy referred to Art. 387 (3) of the Law of 20 July 2018 on Higher Education and Science, Poland).Ye

Progressive Ataxia, Memory Impairments, and Seizure Episodes in Spna2 R1098Q Mouse Variant Affecting Alpha II Spectrin’s Scaffold Stability

SPTAN1 spectrinopathies refer to a group of rare, inherited diseases associated with damage to non-erythrocytic α-II spectrin (α-II). They are linked to a range of mild to severe neuropathologies of the central and peripheral nervous systems, such as early infantile epileptic encephalopathy type 5, cerebellar ataxia, inherited peripheral neuropathy, and spastic paraplegia. Modeling human SPTAN1 encephalopathies in laboratory animals has been challenging partially because no haploinsufficiency-related phenotypes unfold in heterozygous Spna2 deficient mice nor stable transgenic lines of mice mimicking missense human SPTAN1 mutations have been created to date. Here, we assess the motor and memory performance of a dominant-negative murine Spna2 (SPTAN1) variant carrying a spontaneous point mutation replacing an arginine 1098 in the repeat 10th of α-II with the glutamine (R1098Q). By comparing groups of heterozygous R1098Q mice at different ages, we find evidence for progressive ataxia, and age-related deterioration of motor performance and muscle strength. We also document stress-induced, long-lasting seizure episodes of R1098Q mice and their poor performance in novel object recognition memory tests. Overall, we propose that the complexity of neuropathology-related phenotypes presented by the R1098Q mice recapitulates a number of symptoms observed in human patients carrying SPTAN1 mutations affecting α-II scaffold stability. This makes the R1098Q mice a valuable animal model for preclinical research

Identification of a novel protein encoded by third conserved gene within RAG locus

Recently, a third evolutionarily conserved gene, NWC, was discovered within the recombination activating gene (RAG) locus, known to contain the RAG1 and RAG2 genes. Here, we identify and characterize the murine endogenous NWC protein which has no homology to any known protein and is ubiquitously expressed. In the cell, the NWC protein which has been suggested to function as a transcriptional repressor, is found in the cytoplasm as well as in the nucleus