Role of Exostosin and Rab-Gtpase proteins in cell homeostasis

Abstract

Exostosin is a family of type II transmembrane glycosyltransferase enzymes, containing five members: EXT1, EXT2, EXTL1, EXTL2, EXTL3. These proteins are linked with heparan sulfate chains polymerization on heparan sulfate proteoglycans, but recent advances demonstrated that some of these members exhibit novel functions. Rab GTPases family is composed of at least 60 members of membrane-bound proteins that regulate many steps of membrane trafficking. The aim of this study is to analyse potential interplay between EXT and Rab family members in Notch signaling. Rab GTPases were cloned in an expression vector and transfected in human cells to observe their potential effect on NOTCH1 expression. Cell lines silenced for each EXT member were created and their characteristics assessed. EXT1 and EXT2 protein production in Yarrowia lipolytica was also considered to perform in vitro analyzes using purified proteins. As a result, a functional library of Rab GTPases in expression vector was created, verified, and is ready to be used for transfection experiments. Stable knockdown cell lines were successfully created for EXT genes, a reduction of NOTCH1 expression was observed when EXT2 or EXTL3 were silenced, probably due to modification of the trafficking mechanism and/or genetic co-regulation. Protein production did not show significant results. These results suggest a new model for increased NOTCH1 trafficking: overexpression of EXT2 and Rab10 with simultaneous silencing of EXT1. Rab GTPases were cloned in an expression vector and transfected in human cells to observe their potential effect on NOTCH1 expression. Cell lines silenced for each EXT member were created and their characteristics assessed. EXT1 and EXT2 protein production in Yarrowia lipolytica was also considered to perform in vitro analyzes using purified proteins. As a result, a functional library of Rab GTPases in expression vector was created, verified, and is ready to be used for transfection experiments. Stable knockdown cell lines were successfully created for EXT genes, a reduction of NOTCH1 expression was observed when EXT2 or EXTL3 were silenced, probably due to modification of the trafficking mechanism and/or genetic co-regulation. Protein production did not show significant results. These results suggest a new model for increased NOTCH1 trafficking: overexpression of EXT2 and Rab10 with simultaneous silencing of EXT1.