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A peptide derived from the Wiskott-Aldrich syndrome (WAS) protein-interacting protein (WIP) restores WAS protein level and actin cytoskeleton reorganization.

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Presentation on theme: "A peptide derived from the Wiskott-Aldrich syndrome (WAS) protein-interacting protein (WIP) restores WAS protein level and actin cytoskeleton reorganization."— Presentation transcript:

1 A peptide derived from the Wiskott-Aldrich syndrome (WAS) protein-interacting protein (WIP) restores WAS protein level and actin cytoskeleton reorganization in lymphocytes from patients with WAS mutations that disrupt WIP binding  Michel J. Massaad, PhD, Narayanaswamy Ramesh, PhD, Severine Le Bras, PhD, Silvia Giliani, PhD, Lucia D. Notarangelo, MD, Waleed Al-Herz, MD, Luigi D. Notarangelo, MD, Raif S. Geha, MD  Journal of Allergy and Clinical Immunology  Volume 127, Issue 4, Pages e2 (April 2011) DOI: /j.jaci Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 nWIP interacts with and stabilizes WASP. A, Sequence and position of nWIP. B, Co-immunoprecipitation of WASP with WIP and nWIP in Jurkat T cells. Membranes were Western-blotted with antibodies to EGFP, WASP, and actin. The experiment is representative of 3 independent experiments with similar results. IP, Immunoprecipitation; WB, Western blot. C, Representative intracellular WASP staining in mouse T cells expressing EGFP, EGFP-WIP, or EGFP-nWIP. D, Histogram overlay showing the level of WASP in the gated EGFP+ populations. Bar graph shows the means ± SEMs of WASP levels from 3 independent experiments. Max, Maximum. Journal of Allergy and Clinical Immunology  , e2DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 WAS mRNA and protein levels in patient cells. A, Quantitative real-time PCR of WAS mRNA from EBV-B cells of patients with WAS/XLT and controls. Results are expressed as fold change from the average of WAS mRNA levels in controls. B, Representative Western blot of WASP and WIP in lysates of EBV-B cells from patients with WAS/XLT and 3 controls. C, Bar graph represents the means ± SEMs of WASP levels from 3 different experiments, expressed as fold change compared with the controls. Ctrls, Controls. Journal of Allergy and Clinical Immunology  , e2DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 Stabilization of WASP in EBV-B cells from WAS/XLT patients with the D77G and N204fs mutations. Representative WASP staining in EBV-B cells from patients D77G and N204fs expressing EGFP, EGFP-WIP, or EGFP-nWIP. Shown are the dot plots and the histogram overlay of WASP MFI in their gated EGFP+ populations. Ctrl, Control; Max, maximum. Journal of Allergy and Clinical Immunology  , e2DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 Average WASP increase in EBV-B cells from WAS/XLT patients. Bar graphs represent the means ± SEMs of WASP levels in the EGFP+ populations from 3 independent experiments for each patient. ND, Not detected; NS, not significant. Journal of Allergy and Clinical Immunology  , e2DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 Spreading of T cells from patients with mutations in WASP. A, Phalloidin staining of the actin cytoskeleton of T cells from control (Ctrl), and patients with L39P and D77G mutations (2 each) stimulated with immobilized anti-CD3. Bar = 10 μm. B, Pooled results represent the means ± SEMs of the percentage of spread cells from 3 controls and 2 patients (2 for each mutation). NS, not significant. Journal of Allergy and Clinical Immunology  , e2DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig 6 Correction of T-cell spreading by expression of nWIP and WIP. A, Intracellular fluorescence for EGFP (top row), phalloidin (middle row), and merge (bottom row) of T cells from control (Ctrl), and patients with L39P and D77G mutations transduced with lentiviruses expressing EGFP, EGFP-WIP, or EGFP-nWIP, then stimulated with immobilized anti-CD3. Bar = 10 μm. B, Pooled results represent the means ± SEMs of the percentage of spread cells from 3 controls and 2 patients (2 for each mutation). Journal of Allergy and Clinical Immunology  , e2DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Expression of EGFP-WIP and EGFP-nWIP increases WASP levels in Jurkat T cells. Intracellular WASP staining was performed in Jurkat cells expressing EGFP-nWIP, EGFP-WIP, or EGFP. The gate indicates the population of cells used in the analysis. The histogram overlay shows the levels of WASP in the gated EGFP+ populations. The bar graph shows the means ± SEMs of WASP levels from 3 independent experiments. The respective samples, their MFIs, and bars are indicated using the same color as the histograms. WASP level was calculated by subtracting the background isotype staining from the samples’ MFIs and expressing it as a percentage of WASP in EGFP+ cells. Max, Maximum. Journal of Allergy and Clinical Immunology  , e2DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Efficiency of expression of EGFP, EGFP-WIP, and EGFP-nWIP in purified primary T cells. T cells purified from 3 controls (Ctrl) and 4 patients (2 siblings each) with the mutations L39P and D77G in the WH1/EVH1 domain of WASP were transduced with lentiviruses expressing EGFP, EGFP-WIP, and EGFP-nWIP. The expression of the fusion proteins was determined 24 hours posttransduction. Shown are representative histograms from 1 control and the 2 affected siblings. Journal of Allergy and Clinical Immunology  , e2DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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