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Biophysical and biochemical studies of the mitochondrial pyruvate carrier (MPC) complex and the glutaminase enzyme bound to novel partners


Pyruvate is the end product of cytosolic glycolysis and has a number of possible intracellular fates, the major one being mitochondrial transport, a critical step in carbohydrate, amino acid, and lipid metabolism. Two internal mitochondrial membrane (IMM) proteins, named MPC1 and MPC2, were identified as the proteins essential for the transport of pyruvate in yeast (S. cerevisiae), Drosophila and humans. These proteins present three transmembrane helices with each subunits weighing 15 kDa. The functional macromolecular complex (MPC1 and MPC2), however, is believed to have a molecular weight of 150 kDa. Questions such as what are the role of the individual proteins (MPC 1 and MPC 2) in the complex, what is the stoichiometry between them, and what are the molecular determinants of the pyruvate transport have not been addressed so far. In order to answer these questions, the present project proposes, as a first goal, the biophysical characterization along with, within the available time, the structure determination of the MPC complex using yeast expression. Considering that MPC is a transmembrane complex, one would expect serious difficulties in the expression, purification and structural studies by X-ray crystallography. Biophysical and biochemical studies (Analytical ultra-centrifugation, isothermal titration calorimety, surface plasmonic resonance, and negative staining and/or cryo-electron microscopy) are predicted and will be carried out on the purified MPC complex, in order to determine its exact molecular weight and stoichiometry and overall architecture. On the second subject, it is already well documented that in order to sustain an inherently proliferative phenotype, cancer cells rely on aerobic glycolysis, even under normoxia, a phenomenon termed "Warburg effect". Intese use of glicose through glycolysis imposes cells the necessity of consuming higher amounts of glutamine to generate energy, shuttle carbons to the tricarboxilic cycle and other biosynthetic pathways and recycling of reducing agents, such as NADPH. The enzyme glutaminase is the first one on the glutaminolytic pathway. However, glutaminases turn out to be more complex proteins, with a distinctive combination of additional motifs and functional domains, beyond its glutaminase domain, such as ankyrin repeats and nuclear receptor binding motifs. In this context, this project has a second goal of characterizing the interaction of the glutaminases isoforms to proteins partners recently identified in our lab through yeast two-hybrid assays. Initially, we plan on to confirm the interaction in vitro by performing pull-down and gel filtration assays with the proteins expressed in heterologous system. We will then define the stoichiometry, the dissociation constants, and how these interactions influence the glutaminase activity or the function of the interacting partner, whenever the assay for the protein is available. All the interactions that render stable complexes will be subject to crystallization trials and the obtained crystals will be diffracted and have their structure solved. The proposed project once successfully completed has a potential to provide valuable insights in understanding underlying mechanisms of pyruvate shuttling across the mitochondrial membrane as well as has the potential to lead to the development of alternative and efficient therapeutic opportunities regarding the glutaminase function. (AU)

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Scientific publications (6)
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
DIAS, MARILIA M.; ADAMOSKI, DOUGLAS; DOS REIS, LARISSA M.; ASCENCAO, CAROLLINE F. R.; DE OLIVEIRA, KRISHINA R. S.; PASCHOALINI MAFRA, ANA CAROLINA; DA SILVA BASTOS, ALLINY CRISTINY; QUINTERO, MELISSA; CASSAGO, CAROLINA DE G.; FERREIRA, IGOR M.; et al. GLS2 is protumorigenic in breast cancers. Oncogene, v. 39, n. 3, p. 690-702, . (13/05668-0, 14/18061-9, 12/14298-9, 13/23510-4, 14/17820-3, 14/06512-6, 14/15968-3, 15/25832-4, 12/09452-9, 14/20673-2, 16/06625-0, 12/11577-4, 11/10127-2)
DE GUZZI CASSAGO, CAROLINA APARECIDA; DIAS, MARILIA MEIRA; PINHEIRO, MATHEUS PINTO; PASQUALI, CAMILA CRISTINA; SILVA BASTOS, ALLINY CRISTINY; ISLAM, ZEYAUL; CONSONNI, SILVIO ROBERTO; DE OLIVEIRA, JULIANA FERREIRA; GOMES, EMERSON MACHI; RODRIGUES ASCENCAO, CAROLLINE FERNANDA; et al. Glutaminase Affects the Transcriptional Activity of Peroxisome Proliferator-Activated Receptor gamma (PPAR gamma) via Direct Interaction. BIOCHEMISTRY, v. 57, n. 44, p. 6293-6307, . (14/20673-2, 11/10127-2, 17/11766-5, 16/22246-0, 15/25832-4, 10/13992-3, 14/19518-2, 11/13981-4)
FALA, ANGELA M.; OLIVEIRA, JULIANA F.; ADAMOSKI, DOUGLAS; ARICETTI, JULIANA A.; DIAS, MARILIA M.; DIAS, MARCIO V. B.; SFORCA, MAURCIO L.; LOPES-DE-OLIVEIRA, PAULO S.; ROCCO, SILVANA A.; CALDANA, CAMILA; et al. Unsaturated fatty acids as high-affinity ligands of the C-terminal Per-ARNT-Sim domain from the Hypoxia-inducible factor 3 alpha. SCIENTIFIC REPORTS, v. 5, . (12/14298-9, 10/13739-6, 14/15968-3, 14/20673-2, 14/04927-4)
QUESNAY, JOSE EDWIN NECIOSUP; POLLOCK, NAOMI L.; NAGAMPALLI, RAGHAVENDRA SASHI KRISHNA; LEE, SARAH C.; BALAKRISHNAN, VIJAYAKUMAR; DIAS, SANDRA MARTHA GOMES; MORAES, ISABEL; DAFFORN, TIM R.; AMBROSIO, ANDRE LUIS BERTELI. Insights on the Quest for the Structure-Function Relationship of the Mitochondrial Pyruvate Carrier. BIOLOGY-BASEL, v. 9, n. 11, . (14/20673-2, 17/11766-5, 19/02261-2, 13/07600-3, 14/06954-9, 18/00492-4, 17/02391-8, 15/02734-7)
KRISHNA NAGAMPALLI, RAGHAVENDRA SASHI; NECIOSUP QUESNAY, JOSE EDWIN; ADAMOSKI, DOUGLAS; ISLAM, ZEYAUL; BIRCH, JAMES; SEBINELLI, HEITOR GOBBI; BRUNO MOREIRA GIRARD, RICHARD MARCEL; RODRIGUES ASCENCAO, CAROLLINE FERNANDA; FALA, ANGELA MARIA; PAULETTI, BIANCA ALVES; et al. Human mitochondrial pyruvate carrier 2 as an autonomous membrane transporter. SCIENTIFIC REPORTS, v. 8, . (17/02391-8, 14/20673-2, 16/06034-2, 15/02734-7, 15/25832-4, 14/17820-3, 14/12663-7, 14/06954-9)
REDIS, ROXANA S.; VELA, LUZ E.; LU, WEIQIN; DE OLIVEIRA, JULIANA FERREIRA; IVAN, CRISTINA; RODRIGUEZ-AGUAYO, CRISTIAN; ADAMOSKI, DOUGLAS; PASCULLI, BARBARA; TAGUCHI, AYUMU; CHEN, YUNYUN; et al. Allele-Specific Reprogramming of Cancer Metabolism by the Long Non-coding RNA CCAT2. MOLECULAR CELL, v. 61, n. 4, p. 520-534, . (14/15968-3, 14/17820-3, 14/20673-2)

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