Macrothrombocytopenia of Takenouchi-Kosaki syndrome is ameliorated by CDC42 specific- and lipidation inhibitors in MEG-01 cells

Etsuko Daimon,Natsuko Yamazaki,Nobuhiko Okamoto,Yukinao Shibukawa,Suganya Thanasegaran

doi:10.1038/s41598-021-97478-y

Etsuko Daimon, Natsuko Yamazaki + Show 3 more

Open Access

https://doi.org/10.1038/s41598-021-97478-y

Copy DOI

Journal: Scientific Reports	Publication Date: Sep 9, 2021
Citations: 3	License type: open-access

Affiliation: Osaka Women's and Children's Hospital

Abstract

Macrothrombocytopenia is a common pathology of missense mutations in genes regulating actin dynamics. Takenouchi-Kosaki syndrome (TKS) harboring the c.191A > G, Tyr64Cys (Y64C) variant in Cdc42 exhibits a variety of clinical manifestations, including immunological and hematological anomalies. In the present study, we investigated the functional abnormalities of the Y64C mutant in HEK293 cells and elucidated the mechanism of macrothrombocytopenia, one of the symptoms of TKS patients, by monitoring the production of platelet-like particles (PLP) using MEG-01 cells. We found that the Y64C mutant was concentrated at the membrane compartment due to impaired binding to Rho-GDI and more active than the wild-type. The Y64C mutant also had lower association with its effectors Pak1/2 and N-WASP. Y64C mutant-expressing MEG-01 cells demonstrated short cytoplasmic protrusions with aberrant F-actin and microtubules, and reduced PLP production. This suggested that the Y64C mutant facilitates its activity and membrane localization, resulting in impaired F-actin dynamics for proplatelet extension, which is necessary for platelet production. Furthermore, such dysfunction was ameliorated by either suppression of Cdc42 activity or prenylation using chemical inhibitors. Our study may lead to pharmacological treatments for TKS patients.

Highlights

Cdc[42] belongs to the Ras superfamily of small GTPase proteins and is implicated in a variety of biological activities to regulate proteins interacting with the actin cytoskeleton such as p21-activated kinase (PAK) and Wiskott-Aldrich syndrome protein (WASP)[1,2,3]
SDS-PAGE followed by silver staining and peptide mass fingerprinting revealed that Y64C did not associate with Rho GDP-dissociation inhibitor (Rho-guanine nucleotide dissociation inhibitors (GDI)) (Fig. 1c and Supplemental Fig. 1b for full blots)
Thrombocytopenia is a common pathology of missense mutations in the switch II region of Cdc[42], which mediates its binding to effectors and regulators[6,13,14]

Summary

Introduction

Cdc[42] belongs to the Ras superfamily of small GTPase proteins and is implicated in a variety of biological activities to regulate proteins interacting with the actin cytoskeleton such as p21-activated kinase (PAK) and Wiskott-Aldrich syndrome protein (WASP)[1,2,3]. The expression of Y64C in neurons impairs their morphology and migration both in vitro and in vivo in a mouse m odel[16] These defects are Scientific Reports | (2021) 11:17990. Palazzo et al reported that Cdc[42] regulates proplatelet formation through N-WASP23. These studies suggested the importance of Cdc[42] in MK maturation and thrombopoiesis. The dysfunction in proplatelet formation processes, including cytoplasmic extension and PLP production, was ameliorated by either suppression of Cdc[42] activity or cellular localization using chemical inhibitors, which eventually led to the improvement of PLP production by Y64C to the same extent as wild-type Cdc[42] (WT)

Methods

Results

Conclusion