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In vivo functional mapping of the conserved protein domains within murine Themis1.

Zvezdova E, Lee J, El-Khoury D, Barr V, Akpan I, Samelson L, Love PE - Immunol. Cell Biol. (2014)

Bottom Line: Themis1 orthologs contain three conserved domains: a proline-rich region (PRR) that binds to the ubiquitous cytosolic adapter Grb2, a nuclear localization sequence (NLS), and two copies of a novel cysteine-containing globular (CABIT) domain.In the present study, we evaluated the functional importance of each of these motifs by retroviral reconstitution of Themis1(-/-) progenitor cells.The results demonstrate an essential requirement for the PRR and NLS motifs but not the conserved CABIT cysteines for Themis1 function.

View Article: PubMed Central - PubMed

Affiliation: Section on Cellular and Developmental Biology, Program on Genomics of Differentiation, Eunice Kennedy Shriver, National Institute of Child Health and Human Development, Bethesda, MD, USA.

ABSTRACT
Thymocyte development requires the coordinated input of signals that originate from numerous cell surface molecules. Although the majority of thymocyte signal-initiating receptors are lineage-specific, most trigger 'ubiquitous' downstream signaling pathways. T-lineage-specific receptors are coupled to these signaling pathways by lymphocyte-restricted adapter molecules. We and others recently identified a new putative adapter protein, Themis1, whose expression is largely restricted to the T lineage. Mice lacking Themis1 exhibit a severe block in thymocyte development and a striking paucity of mature T cells revealing a critical role for Themis1 in T-cell maturation. Themis1 orthologs contain three conserved domains: a proline-rich region (PRR) that binds to the ubiquitous cytosolic adapter Grb2, a nuclear localization sequence (NLS), and two copies of a novel cysteine-containing globular (CABIT) domain. In the present study, we evaluated the functional importance of each of these motifs by retroviral reconstitution of Themis1(-/-) progenitor cells. The results demonstrate an essential requirement for the PRR and NLS motifs but not the conserved CABIT cysteines for Themis1 function.

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Effect of Themis1 or Themis1 mutant protein expression on Themis+/+ thymocyte development. Lethally irradiated C57BL6/J (CD45.1+) mice were reconstituted with lineage depleted Themis1+/+ bone marrow cells (CD45.2+) that had been infected with a bicistronic EGFP expressing retroviral vector (Empty vector) or the same vector encoding either wild type Themis1 cDNA (WT) or mutant Themis1 proteins: Themis-ΔPRR (PRR), Themis-ΔNLS (NLS) or Themis-ΔCys (Cys1/2). Eight weeks post-reconstitution, bone marrow chimeras sacrificed and thymocytes and splenocytes were analysed by flow cytometry. a. Intracellular staining for Themis1 in CD45.2+ EGFP+ CD4+CD8+ (DP) thymocytes from bone marrow chimeric mice. Grey histograms represent Themis1 expression in EGFP− DP thymocytes. Green histograms represent expression of Themis1 in EGFP+ DP thymocytes, expressing the indicated Themis1 proteins. b. Representative flow cytometry analysis of CD45.2+ EGFP+ thymocytes from bone marrow chimeras. Upper plots show CD4 versus CD8 profiles, middle histograms show percent of TCRβ+ cells. Bottom plots show percent of mature CD45.2+ EGFP+ TCRβhiCD24lo thymocytes. Plots in right column show Themis1+/+ thymocytes infected with empty retroviral vector for reference. c. Percentage of mature (TCRβhiCD24lo) CD45.2+ EGFP+ CD4-SP and CD8-SP thymocytes in the indicated bone marrow chimeras (n=6 each). N.S., not significant (Two-tailed T-test, unequal variance). All comparisons are to vector only.
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Figure 5: Effect of Themis1 or Themis1 mutant protein expression on Themis+/+ thymocyte development. Lethally irradiated C57BL6/J (CD45.1+) mice were reconstituted with lineage depleted Themis1+/+ bone marrow cells (CD45.2+) that had been infected with a bicistronic EGFP expressing retroviral vector (Empty vector) or the same vector encoding either wild type Themis1 cDNA (WT) or mutant Themis1 proteins: Themis-ΔPRR (PRR), Themis-ΔNLS (NLS) or Themis-ΔCys (Cys1/2). Eight weeks post-reconstitution, bone marrow chimeras sacrificed and thymocytes and splenocytes were analysed by flow cytometry. a. Intracellular staining for Themis1 in CD45.2+ EGFP+ CD4+CD8+ (DP) thymocytes from bone marrow chimeric mice. Grey histograms represent Themis1 expression in EGFP− DP thymocytes. Green histograms represent expression of Themis1 in EGFP+ DP thymocytes, expressing the indicated Themis1 proteins. b. Representative flow cytometry analysis of CD45.2+ EGFP+ thymocytes from bone marrow chimeras. Upper plots show CD4 versus CD8 profiles, middle histograms show percent of TCRβ+ cells. Bottom plots show percent of mature CD45.2+ EGFP+ TCRβhiCD24lo thymocytes. Plots in right column show Themis1+/+ thymocytes infected with empty retroviral vector for reference. c. Percentage of mature (TCRβhiCD24lo) CD45.2+ EGFP+ CD4-SP and CD8-SP thymocytes in the indicated bone marrow chimeras (n=6 each). N.S., not significant (Two-tailed T-test, unequal variance). All comparisons are to vector only.

Mentions: To determine if Themis-ΔPRR, Themis-ΔNLS1 or Themis-ΔCys effect thymocyte development in the presence of wild-type Themis1, we infected bone marrow progenitors from wild-type (B6-CD45.1) mice with retrovirus encoding wild-type Themis1 or the three Themis1 mutants. Interestingly, expression of either WT Themis1 or Themis-ΔCys resulted in a consistent though not statistically significant increase in CD4-SP and CD8-SP T thymocytes and peripheral CD4-SP and CD8-SP T cells (Figs. 5&6). These results are consistent with data obtained with Themis1 transgenic mice (data not shown) and suggest that T cell maturation can be enhanced by augmentation of Themis1 expression by either WT Themis1 or Themis-ΔCys. Expression of Themis-ΔPRR or Themis-ΔNLS1 in wild-type bone marrow progenitors did not appreciably enhance or diminish T cell maturation indicating that neither protein exerted dominant-negative effect on thymocyte development in the presence of WT Themis1 (Figs. 5&6).


In vivo functional mapping of the conserved protein domains within murine Themis1.

Zvezdova E, Lee J, El-Khoury D, Barr V, Akpan I, Samelson L, Love PE - Immunol. Cell Biol. (2014)

Effect of Themis1 or Themis1 mutant protein expression on Themis+/+ thymocyte development. Lethally irradiated C57BL6/J (CD45.1+) mice were reconstituted with lineage depleted Themis1+/+ bone marrow cells (CD45.2+) that had been infected with a bicistronic EGFP expressing retroviral vector (Empty vector) or the same vector encoding either wild type Themis1 cDNA (WT) or mutant Themis1 proteins: Themis-ΔPRR (PRR), Themis-ΔNLS (NLS) or Themis-ΔCys (Cys1/2). Eight weeks post-reconstitution, bone marrow chimeras sacrificed and thymocytes and splenocytes were analysed by flow cytometry. a. Intracellular staining for Themis1 in CD45.2+ EGFP+ CD4+CD8+ (DP) thymocytes from bone marrow chimeric mice. Grey histograms represent Themis1 expression in EGFP− DP thymocytes. Green histograms represent expression of Themis1 in EGFP+ DP thymocytes, expressing the indicated Themis1 proteins. b. Representative flow cytometry analysis of CD45.2+ EGFP+ thymocytes from bone marrow chimeras. Upper plots show CD4 versus CD8 profiles, middle histograms show percent of TCRβ+ cells. Bottom plots show percent of mature CD45.2+ EGFP+ TCRβhiCD24lo thymocytes. Plots in right column show Themis1+/+ thymocytes infected with empty retroviral vector for reference. c. Percentage of mature (TCRβhiCD24lo) CD45.2+ EGFP+ CD4-SP and CD8-SP thymocytes in the indicated bone marrow chimeras (n=6 each). N.S., not significant (Two-tailed T-test, unequal variance). All comparisons are to vector only.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4414023&req=5

Figure 5: Effect of Themis1 or Themis1 mutant protein expression on Themis+/+ thymocyte development. Lethally irradiated C57BL6/J (CD45.1+) mice were reconstituted with lineage depleted Themis1+/+ bone marrow cells (CD45.2+) that had been infected with a bicistronic EGFP expressing retroviral vector (Empty vector) or the same vector encoding either wild type Themis1 cDNA (WT) or mutant Themis1 proteins: Themis-ΔPRR (PRR), Themis-ΔNLS (NLS) or Themis-ΔCys (Cys1/2). Eight weeks post-reconstitution, bone marrow chimeras sacrificed and thymocytes and splenocytes were analysed by flow cytometry. a. Intracellular staining for Themis1 in CD45.2+ EGFP+ CD4+CD8+ (DP) thymocytes from bone marrow chimeric mice. Grey histograms represent Themis1 expression in EGFP− DP thymocytes. Green histograms represent expression of Themis1 in EGFP+ DP thymocytes, expressing the indicated Themis1 proteins. b. Representative flow cytometry analysis of CD45.2+ EGFP+ thymocytes from bone marrow chimeras. Upper plots show CD4 versus CD8 profiles, middle histograms show percent of TCRβ+ cells. Bottom plots show percent of mature CD45.2+ EGFP+ TCRβhiCD24lo thymocytes. Plots in right column show Themis1+/+ thymocytes infected with empty retroviral vector for reference. c. Percentage of mature (TCRβhiCD24lo) CD45.2+ EGFP+ CD4-SP and CD8-SP thymocytes in the indicated bone marrow chimeras (n=6 each). N.S., not significant (Two-tailed T-test, unequal variance). All comparisons are to vector only.
Mentions: To determine if Themis-ΔPRR, Themis-ΔNLS1 or Themis-ΔCys effect thymocyte development in the presence of wild-type Themis1, we infected bone marrow progenitors from wild-type (B6-CD45.1) mice with retrovirus encoding wild-type Themis1 or the three Themis1 mutants. Interestingly, expression of either WT Themis1 or Themis-ΔCys resulted in a consistent though not statistically significant increase in CD4-SP and CD8-SP T thymocytes and peripheral CD4-SP and CD8-SP T cells (Figs. 5&6). These results are consistent with data obtained with Themis1 transgenic mice (data not shown) and suggest that T cell maturation can be enhanced by augmentation of Themis1 expression by either WT Themis1 or Themis-ΔCys. Expression of Themis-ΔPRR or Themis-ΔNLS1 in wild-type bone marrow progenitors did not appreciably enhance or diminish T cell maturation indicating that neither protein exerted dominant-negative effect on thymocyte development in the presence of WT Themis1 (Figs. 5&6).

Bottom Line: Themis1 orthologs contain three conserved domains: a proline-rich region (PRR) that binds to the ubiquitous cytosolic adapter Grb2, a nuclear localization sequence (NLS), and two copies of a novel cysteine-containing globular (CABIT) domain.In the present study, we evaluated the functional importance of each of these motifs by retroviral reconstitution of Themis1(-/-) progenitor cells.The results demonstrate an essential requirement for the PRR and NLS motifs but not the conserved CABIT cysteines for Themis1 function.

View Article: PubMed Central - PubMed

Affiliation: Section on Cellular and Developmental Biology, Program on Genomics of Differentiation, Eunice Kennedy Shriver, National Institute of Child Health and Human Development, Bethesda, MD, USA.

ABSTRACT
Thymocyte development requires the coordinated input of signals that originate from numerous cell surface molecules. Although the majority of thymocyte signal-initiating receptors are lineage-specific, most trigger 'ubiquitous' downstream signaling pathways. T-lineage-specific receptors are coupled to these signaling pathways by lymphocyte-restricted adapter molecules. We and others recently identified a new putative adapter protein, Themis1, whose expression is largely restricted to the T lineage. Mice lacking Themis1 exhibit a severe block in thymocyte development and a striking paucity of mature T cells revealing a critical role for Themis1 in T-cell maturation. Themis1 orthologs contain three conserved domains: a proline-rich region (PRR) that binds to the ubiquitous cytosolic adapter Grb2, a nuclear localization sequence (NLS), and two copies of a novel cysteine-containing globular (CABIT) domain. In the present study, we evaluated the functional importance of each of these motifs by retroviral reconstitution of Themis1(-/-) progenitor cells. The results demonstrate an essential requirement for the PRR and NLS motifs but not the conserved CABIT cysteines for Themis1 function.

Show MeSH
Related in: MedlinePlus