Regulation of Rho GTPases by post-translational modification in endothelial barrier function

Johanna Hendrika Majolée

Research output: ThesisResearch VU University Amsterdam, graduation VU University Amsterdam

Abstract

The inside of our blood vessels is lined with a single layer of endothelial cells, known as the endothelium. The endothelium forms a barrier with a strict degree of permeability for nutrients, waste products and cells from the blood to the tissues. Important regulators of the endothelial barrier are proteins called the Rho GTPases, of which the most researched are RhoA, RhoB, Rac1, and Cdc42. RhoA and RhoB cause the cells to contract, creating holes in the endothelial barrier that lead to vascular leakage. Rac1 and Cdc42 do the opposite: they cause the endothelial cells to expand and promote the formation of cell-cell connections. Rho GTPases can switch between an active and inactive form, allowing endothelial cells to adapt their cytoskeleton as needed. However, there are more aspects to the regulation of Rho GTPases. In this thesis, we mainly focus on the regulation of Rho GTPases by two post-translational modifications called ubiquitination and prenylation. A post-translational modification is a change made to a protein after it has been transcribed from the DNA. Ubiquitination is the attachment of a ubiquitin molecule to a protein. Ubiquitination of a protein usually leads to recognition by the proteasome, which then breaks down the protein. In chapter 2 we summarized the current knowledge on regulation of the endothelial barrier by ubiquitination. In chapter 3 we describe the role of RhoB ubiquitination in the endothelial barrier. The RhoB gene is continuously transcribed and translated into protein, but under healthy conditions the RhoB protein is also continuously broken down. By specifically removing the ubiquitin ligases Cullin-1, Cullin-2 and Cullin-3 and some adapter proteins, we found that the Cullin-3-Rbx1-KCTD10 complex is most important for RhoB ubiquitination. Next, we activated Cullin ubiquitin ligases (chapter 4). We expected that this would lead to increased RhoB degradation and an enhanced endothelial barrier. However, we soon discovered that the activation of all Cullin E3 ligases led to an imbalance in the endothelium. There was more RhoB transcription than degradation in the endothelial cells treated with CSN5i-3, leading to a high RhoB protein level and contraction of the endothelial cells. We concluded that the chemical activation of all Cullin E3 ligases is not a good strategy for reducing endothelial cell contraction. The role of a group of ubiquitin ligase adaptors called the F-box proteins in the regulation of the endothelial barrier was still unknown. In our study described in chapter 5, we found that removing FBXW7 disrupted the endothelial barrier. This was caused by a disruption of the cholesterol synthesis pathway, leading to an increase in and differential functioning of RhoB. In chapter 6, we investigated the relationship between GDI binding, activity and ubiquitination of Rac1. These three components of Rac1 regulation had not been linked in this way before. We introduced mutations in Rac1, each known to cause different activity, ubiquitination or GDI binding of Rac1. This allowed us to show that Rac1 activity and ubiquitination are positively correlated to each other. However, we did not see loss of RhoGDI binding with increased Rac1 activity as we expected, which puts the regulation of Rac1 by RhoGDI in a new light. In summary, we established the link between post-translational regulation of Rho GTPases, specifically RhoB and Rac1, and the regulation of the endothelial barrier. For some experiments, we expressed fluorescently synthesized Rho GTPases to better track them in the cells. The molecular cell biology research we performed in this thesis will help to understand the physiological regulation of Rho GTPase signaling in endothelial cells. We hope that these insights will contribute to the development or adaptation of strategies against vascular leakage.
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Vrije Universiteit Amsterdam
Supervisors/Advisors
  • Hordijk, Peter Lodewijk, Supervisor
  • Kovacevic, I., Co-supervisor
Award date12 Sep 2021
Publication statusPublished - 13 Sep 2021

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