COMPUTATIONAL ANALYSIS OF PHYLOGENETIC RELATIONSHIP OF TAGAP - T CELL ACTIVATION RHO GTPASE ACTIVATING PROTEIN IN SELECTED MAMMALIAN SPECIES

Abstract Rho GTPases are molecular switches controlling a broad range of cellular processes in lymphocytes including activation, immune response and gene transcription. They rapidly cycle between a guanosine diphosphate (GDP)-bound and guanosine triphosphate (GTP)-bound state, and the GTP-bound state is the active conformation that initiates downstream signaling pathways via effector proteins. We identified a novel Rho effector, RTKN2, that is highly expressed in lymphocytes, particulary freshly isolated CD4 T-cells; and is switched off in activated T-cells. When the protein was over-expressed in an unrelated cell line, it conferred resistance to apoptosis, and this resistance was maintained using the media alone from the over-expressing cells. In the current study we investigated genes relating to high or low expression of RTKN2 in lymphocytes. In human samples, RTKN2 expression was down regulated in PHA activated T-cells and it correlated with the expression of Zap-70, a Rho-guanine nucleotide exchange factor (GEF). We then utilized a real time cytokine array (Superarray Bioscience Corporation) to compare high versus low RTKN2 expression levels in the same population of lymphocytes. The real time array linked high levels of RTKN2 with cytokines BMP8b, (a TGF-beta family protein) and IL-16 (a modulator of T cell activation, and an inhibitor of HIV replication relationship), and this observation was confirmed in a series of T-cell samples. Further, we studied the effect of down regulation of RTKN2 in primary lymphocytes, using short hairpin (sh)RNA plasmid that also expressed GFP. Transfected GFP-positive cells had lower survival than cells transfected with scrambled (sh)RNA, and also demonstrated increased sensitivity to the the induction of apoptosis by oxysterols. These findings indicate that RTKN2 plays a role in survival of T-lymphocytes and this may be mediated through the secretion of cytokines.

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Short Disordered Epitope of CRTAM Ig-Like V Domain as a Potential Target for Blocking Antibodies

International Journal of Molecular Sciences ◽

10.3390/ijms21228798 ◽

2020 ◽

Vol 21 (22) ◽

pp. 8798

Author(s):

Julio Angel Vázquez-Martínez ◽

Miguel Angel Gómez-Lim ◽

Edgar Morales-Ríos ◽

Jorge Alberto Gonzalez-y-Merchand ◽

Vianney Ortiz-Navarrete

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cd4 T Cells ◽

Cell Activation ◽

Nk Cell ◽

Polyclonal Antibodies ◽

Mammalian Species ◽

Efficient Production ◽

Dynamics Simulations

Class-I Restricted T Cell-Associated Molecule (CRTAM) is a protein that is expressed after T cell activation. The interaction of CRTAM with its ligand, nectin-like 2 (Necl2), is required for the efficient production of IL-17, IL-22, and IFNγ by murine CD4 T cells, and it plays a role in optimal CD8 T and NK cell cytotoxicity. CRTAM promotes the pro-inflammatory cytokine profile; therefore, it may take part in the immunopathology of autoimmune diseases such as diabetes type 1 or colitis. Thus, antibodies that block the interaction between CRTAM and Necl2 would be useful for controlling the production of these inflammatory cytokines. In this work, using bioinformatics predictions, we identified three short disordered epitopes (sDE1-3) that are located in the Ig-like domains of murine CRTAM and are conserved in mammalian species. We performed a structural analysis by molecular dynamics simulations of sDE1 (QHPALKSSKY, Ig-like V), sDE2 (QRNGEKSVVK, Ig-like C1), and sDE3 (CSTERSKKPPPQI, Ig-like C1). sDE1, which is located within a loop of the contact interface of the heterotypic interaction with Nectl2, undergoes an order–disorder transition. On the contrary, even though sDE2 and sDE3 are flexible and also located within loops, they do not undergo order–disorder transitions. We evaluated the immunogenicity of sDE1 and sDE3 through the expression of these epitopes in chimeric L1 virus-like particles. We confirmed that sDE1 induces polyclonal antibodies that recognize the native folding of CRTAM expressed in activated murine CD4 T cells. In contrast, sDE3 induces polyclonal antibodies that recognize the recombinant protein hCRTAM-Fc, but not the native CRTAM. Thus, in this study, an exposed disordered epitope in the Ig-like V domain of CRTAM was identified as a potential site for therapeutic antibodies.

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The Epitopic Dissection of the CD2 Defined Molecule: Relationship of the Second Workshop Antibodies in Terms of Reactivities with Leukocytes, Rosette Blocking Properties, Induction of Positive Modulation of the Molecule, and Triggering T Cell Activation

Leukocyte Typing II ◽

10.1007/978-1-4613-8587-5_4 ◽

1986 ◽

pp. 53-66 ◽

Cited By ~ 8

Author(s):

Alain Bernard ◽

Philippe Brottier ◽

Eric Georget ◽

Virginia Lepage ◽

Laurence Boumsell

Keyword(s):

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Relationship Of

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GTPase-activating protein Rasal1 associates with ZAP-70 of the TCR and negatively regulates T-cell tumor immunity

Nature Communications ◽

10.1038/s41467-019-12544-4 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 5

Author(s):

Youg Raj Thaker ◽

Monika Raab ◽

Klaus Strebhardt ◽

Christopher E. Rudd

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Immunity ◽

T Cell Activation ◽

Cell Activation ◽

T Cell Responses ◽

Negative Regulator ◽

Cd4 T Cell ◽

Gtpase Activating Protein ◽

Cell Responses

Abstract Immunotherapy involving checkpoint blockades of inhibitory co-receptors is effective in combating cancer. Despite this, the full range of mediators that inhibit T-cell activation and influence anti-tumor immunity is unclear. Here, we identify the GTPase-activating protein (GAP) Rasal1 as a novel TCR-ZAP-70 binding protein that negatively regulates T-cell activation and tumor immunity. Rasal1 inhibits via two pathways, the binding and inhibition of the kinase domain of ZAP-70, and GAP inhibition of the p21ras-ERK pathway. It is expressed in activated CD4 + and CD8 + T-cells, and inhibits CD4 + T-cell responses to antigenic peptides presented by dendritic cells as well as CD4 + T-cell responses to peptide antigens in vivo. Furthermore, siRNA reduction of Rasal1 expression in T-cells shrinks B16 melanoma and EL-4 lymphoma tumors, concurrent with an increase in CD8 + tumor-infiltrating T-cells expressing granzyme B and interferon γ-1. Our findings identify ZAP-70-associated Rasal1 as a new negative regulator of T-cell activation and tumor immunity.

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