NEDD4L protein role in the differentiation regulation of TGF-²-induced Treg lymphocytes

• Grant number: 17/04200-5
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: May 01, 2017
• End date: April 30, 2019
• Field of knowledge: Biological Sciences - Pharmacology - Biochemical and Molecular Pharmacology
• Principal Investigator: José Carlos Farias Alves Filho
• Grantee: Letícia Magalhães Arruda
• Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Associated research grant: 13/08216-2 - CRID - Center for Research in Inflammatory Diseases, AP.CEPID

Abstract

Regulatory T cells (Tregs) are intimately related to the homeostasis of the immune system. Unlikely T helper cells (Th) that act on adaptivity imunity Tregs act at the potential deleterious activity of Th by several suppressive activities. The main cytokine involved in Treg differentiation is TGF-², which modulate cell proliferation, recognition, differentiation, apoptosis and others1,2. TGF-² proteins bind especific membrane receptors (type I and type II) and trigger a response by Smad pathway. The phosphorylation of type II receptor by the type I receptor serves as an anchor for the Smad receptor (R-Smad), which recruit Smad2 or Smad3 proteins. Smad proteins are transcription factors (FT) imported into the nucleus, which, acting in synergy with other FTs, acts at the differentiation of CD4+ T cells into Treg by the expression of Foxp3, which is the major FT to Treg limphocytes3. In addition to the Smad pathway, TGF-² activates several other Smad-independent pathways, which vary depending on the cell2. The amplitude and duration of the TGF-² stimulus are limited by ubiquitin ligase NEDD4L, that selectively ubiquitinates Smad2 and Smad3 which will be further destroyed, changing their half-life and turn over4,5. In our laboratory, it was demonstrated that activation of lymphocytes by TGF-² activates the hexosamines pathway leading to production of UDP-GlcNAc, which is substrate for O-glycosylation of proteins by the enzyme OGT (uridine-diphospho-N-acetylglucosamine: polypeptide-²-N-acetylglicosaminyltransferase or O-GlcNactransferase) (manuscript in preparation). OGT modifies post-translationally citoplasmatic and nuclear proteins by the addition of a GlcNac group in their backbone aminoacids, more specifically in serine and threonine residues. Thus, GlcNacylation modifies, and in consequence, regulates proteins in a similar way of phosphorylation, even getting to compete with each other, once both targets the same aminoacids. In this sense, we identified that TGF-² promotes the O-GlcNacylation of NEDD4L by the activation of OGT during the differentiation of Treg cells and blocking O-GlcNacylation of NEDD4L by inhibiting OGT activity decreases their differentiation. Considering the relevance of NEDD4L in the Treg activation pathway for TGF-² and its importance in the maintenance of the cellular plasticity of the regulatory cells, this project aims to describe NEDD4L protein in the context of the differentiation of Treg lymphocytes and the rules by which UDP-GlcNac addition alters its function. (AU)