Study of the GAC and KGA mRNA and protein expression levels

• Grant number: 12/16881-3
• Support Opportunities: Scholarships in Brazil - Master
• Start date: March 01, 2013
• End date: December 31, 2014
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Sandra Martha Gomes Dias
• Grantee: Douglas Adamoski Meira
• Host Institution: Centro Nacional de Pesquisa em Energia e Materiais (CNPEM). Ministério da Ciência, Tecnologia e Inovação (Brasil). Campinas , SP, Brazil
• Associated research grant: 09/10875-9 - Cellular and biochemical studies of the glutaminase enzyme and its relation with cancer, AP.JP

Abstract

Tumor cells in comparison to normal ones present genetic and metabolic differences that allow them to divide at surprisingly high rates. Of special interest is the large consumption of glutamine for energy production and formation of cellular biosynthetic blocks (lipids, proteins and nucleic acids). As a first step in the metabolism of glutamine, this aminoacid is converted to glutamate within the mitochondria by the action of the enzyme glutaminase, which has three isoforms encoded by two distinct genes. The gene GLS1 encodes for the kidney-type glutaminase (KGA) and glutaminase C (GAC), products of alternative splicing, while the GLS2 encodes for the liver-type glutaminase (LGA). Our preliminary results indicate that GAC and KGA, which differ only in their C-terminal sequences, are more expressed in tumor when compared to non-transformed surrounding tissues. The GAC isoform, in particular, has elevated levels of mRNA and protein in relation to KGA in breast tumor lines SKBR3 and MDA-MB231, compared with cultured mammary epithelial cells (HMEC). These same studies indicated the protein HuR and ¾-crystallin as potential regulators of GAC`s mRNA stability, through their interaction with its mRNA`s 3'UTR, a mechanism that could explain GAC high protein levels found in transformed cells. This project aims at identifying the elements that promote mRNA stability and/or increase on translation rates of isoform GAC, in comparison to KGA, by using an array of techniques such as protein-RNA pull-down followed by protein identification by mass spectrometry, confirmation of interaction by gel-shift and UV-crosslinking, mRNA levels quantification by qPCR and RNA In Situ Hybridization (RNA FISH) (as a function of the regulating proteins knock down/overexpression), as well as the identification of interaction sequences by studies with reporter gene (luciferase).