Structural determination of the kidney type glutaminase and the search for its bin...
| Author(s): |
Igor Monteze Ferreira
Total Authors: 1
|
| Document type: | Doctoral Thesis |
| Institution: | Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia |
| Defense date: | 11 ago. 2015 |
| Examining board members: |
Daniela Barreto Barbosa Trivella;
Paulo Sergio Lopes de Oliveira;
Rafael Victorio Carvalho Guido;
Artur Torres Cordeiro
|
| Advisor: | Andre Luis Berteli Ambrosio |
| Abstract | |
Cancer is a disease that affects millions of people each year worldwide. The identification of therapeutic targets for treatment has been the subject of extensive research. It is nowadays well established that the metabolic levels of tumor cells are adapted to support the high growth rates and proliferation characteristic of such cells; a common hallmark in that regard is the high consumption of glucose and glutamine by cancer. Glutaminases are multidomain enzymes responsible for the initial reaction for glutamine entry into the catabolic pathways. Mammals have two distinct genes for glutaminases, gls and gls2. Gls encodes for splicing isoforms KGA (Kidney-type glutaminase) and GAC (Glutaminase C) while gls2 encodes for the LGA isoforms (Liver-type glutaminase) and GAB (Glutaminase B), under different transcription initiation sites. Collectively, KGA/GAC and LGA/GAB are referred to as GLS1 and GLS2, respectively. This study focuses on the understanding of GLS2 structural and catalytic mechanisms since it has recently been reported as important for tumor growth and proliferation. Several constructs of human GLS2 were cloned for heterologous expression in bacteria and insect cells and a construct containing the glutaminase domain was crystallized and its crystal structure determined by X-ray diffraction. Structure analysis suggests key aminoacids that may be responsible for the distinct kinetic behaviours between GLS2 and GLS1. In parallel, we have established a cooperative allosteric mode of activation for GLS2, as a function of enzyme and substrate concentrations, as well as the presence of the well-known activator inorganic phosphate. The Hill coefficient calculation demonstrated that increasing the GLS2 concentration in the assays culminates in growth of cooperation among the monomers, suggesting full activation as tetramers. Analytical Gel Filtration and Electron Microscopy confirms that GLS2 never assembles into oligomers larger than tetramers - as opposed to GLS1 ¿ as a function of protein concentration and presence of phosphate. Therefore, the increasing of the cooperativity, concomitant with increasing GLS2 concentration, does not affect the number of monomers in the oligomer. Since allosteric enzymes often participate as key regulators of metabolic pathways, upon accumulation of other metabolites, we tested some of these ligands as possible GLS2 modulators and showed that, in addition to inorganic phosphate, citrate, AMP (adenosine monophosphate), glucose-6-P and fructose-6-P could possibly be biologically relevant GLS2 activators, thus allowing cross-talk between distinct metabolic pathways. Finally, by solving the crystallographic structure of the ankyrin repeats motif in the GLS2 C-terminal region, we are able to propose a molecular model for the full-length GLS2, using restraints provide by SAXS. Overall, this collection of results can be relevant for the future design of GLS2 inhibitors, thus becoming a complementary therapeutic approach for cancers that have increased expression and activity levels of GLS2. (AU) | |
| FAPESP's process: | 13/05668-0 - Understanding the mechanistic and functional details of the isozyme mammalian Liver-type glutaminase (LGA/GLS2) |
| Grantee: | Igor Monteze Ferreira |
| Support type: | Scholarships in Brazil - Doctorate (Direct) |