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Goncalves Pinho, L.

Publications and source records attributed to Goncalves Pinho, L..

2 recordsLinked to original sources

Parthenolide boosts megakaryocyte maturation and restores platelet responses in Wiskott Aldrich syndrome

Wiskott-Aldrich syndrome (WAS) is an inborn error of immunity with a broad disease spectrum, classified into class I (late onset) or II (early onset) variants. Thrombocytopenia and small platelets are the most consistent findings among patients, difficult to treat and related to development of autoimmunity. To identify new treatment options for thrombocytopenia in WAS, we developed a FACS-based screening for drug repurposing. We identified parthenolide as a lead small molecule that increased WASp abundance in cells with residual WASp expression. Using the megakaryocytic MEG-01 cells, gene edited to express WAS class I and II genetic variants, parthenolide induced megakaryocyte maturation as evident by upregulation of CD61, increased cell size and complexity, increased phosphorylation of ERK1/2, and higher DNA ploidy. We generated a new mouse model harboring a WAS class I missense variant WASp-R88C, corresponding to human WASp-R86C, with reduced expression of WASp. WASp-R88C mice had lower numbers of platelets compared to WT mice. Bone marrow-derived WASp-R88C and WASp-KO megakaryocytes, differentiated in the presence of parthenolide derivative, DMAPT, showed increased ploidy and upregulation of the maturation markers CD61, CD41 and CD42d, resulting in improved platelet production in vitro. Treatment with parthenolide derivative, DMAPT, led to increased platelet numbers in vivo in WASp-R88C mice and dampened the hyperactivation of WAS patient platelets by reducing thrombin-induced CD62P exposure after activation. The identification of parthenolide offers a promising therapeutic approach for WAS patients who are unresponsive or unsuitable for definitive therapies.

cell biology↗

A systematic approach to understanding how patient variants affect the activity of Wiskott-Aldrich syndrome protein

Wiskott-Aldrich syndrome (WAS) and X-linked neutropenia (XLN) are caused by genetic variants in the WAS gene. How WAS variants lead to clinical disease remains unsolved in many cases. We expressed human WASp using a spider silk inspired solubility tag (NT*-tag) and inserted patients variants. Native mass spectrometry and pyrene actin assays showed that five variants (L270P, F271S, S272P, I290T, I294T) predicted to cause XLN led to open protein conformation and high actin polymerization rate in the absence of the WASp activator, Cdc42. One previously reported XLN variant (R268W), two loss-of-function WAS variants (A236G, D485N), and one variant of unknown significance (R431W) behaved similarly to wildtype WASp in terms of structural conformation and actin polymerization. Patient CD4+ T cells were used for analysis of WASp expression and phosphorylation, actin polymerization, anti-CD3 induced proliferation capacity, and upregulation of high affinity LFA-1, distinguishing loss-of-function and gain-of-function variants from benign WAS variants. This systematic approach reveals how WAS genetic variants cause severe human disease and stratify variants to guide clinical decision for definitive therapy. Key PointsO_LIGain-of-function WASp variant has extended protein conformation probed by native mass spectrometry and raised pyrene actin polymerization. C_LIO_LIFunctional analysis of patients CD4+ T cells classifies WASp variants as loss-of-function, reduced-function, gain-of-function, and benign. C_LI

immunology↗