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Biophysical and biochemical studies of transmembrane mitochondrial proteins and their implication on the tumor metabolism adaptation process

Abstract

This proposal is divided in two main parts, as determined by the protein subjects: (1) it aims at the crystallization and determination of the crystal structure of the human mitochondrial pyruvate carrier; (2) it aims at the structural characterization of the human VDAC2-Bak complex from mitochondrial outer membrane by single particle cryo-electron microscopy. (1) The active transport of glycolytic pyruvate across the human inner mitochondrial membrane (IMM) is thought to involve two mitochondrial pyruvate carrier subunits, MPC1 and MPC2, assembled as a 150 kDa oligomeric heterotypic structure. We have recently demonstrated that homodimeric human MPC2 can promote efficient pyruvate transport into reconstituted proteoliposomes. Importantly, the derived functional requirements and kinetic features resemble those already demonstrated for pyruvate transport in mitochondrial extracts. Our results lay the initial framework for exploring the independent role of MPC2 in homeostasis and diseases related to the dysregulation of pyruvate metabolism. However, to establish the molecular basis of pyruvate transport, the determination of the atomic structure of human MPC2 becomes of major importance. In this context, we propose to continue quest on the pursuit of the crystal structure of MPC2. The experience acquired will bring benefits back to the future endeavors of the in the field of structural biology of membrane proteins. (2) Also known as mitochondrial porins, VDACs (voltage-dependent anion-selective channel) are the most abundant integral membrane proteins present in the mitochondrial outer membrane and considered a new target for anti-cancer drugs. Bak (Bcl-2-homologous antagonist / killer) belongs to the pro-apoptotic members of the Bcl-2 protein family and is constitutively integrated into the mitochondrial outer membrane. In healthy cells, VDAC2 and Bak are together in a complex where Bak appears inactivated or repressed. The association and dissociation mechanisms of VDAC2-Bak complex is closely related to the mitochondrial apoptotic pathway. Studies have shown that interference in this complex induces melanoma cell death, a type of cancer with high resistance to the traditional chemotherapies, and that the VDAC2 homozygous deficient is lethal to mouse embryos. Although there is a reasonable understanding of Bak regulatory mechanism, little is known about the functions of the VDAC2-Bak complex. This project aims to establish bases for the structural investigation of the complex formed by VDAC2 and Bak and obtain structural information by Cryo-Electron Microscopy (Cryo-EM) method and Single Particle Analysis. For this, human cell lines will be established for the production of recombinant VDAC2 and Bak, aiming a subsequent target-complex purification. After purification, the conditions of investigation for the complex by scanning electron microscopy will be initially set by negative staining technique and then, by preparing amorphous ice (Cryo-EM). The obtaining of images of the pure complex allows the processing by the technique of Single Particle Analysis. Obtaining structural complex model will contribute to elucidate how Bak interacts structurally with VDAC2, paving the way for future studies to use the complex as a therapeutic target. (AU)

Scientific publications
(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)
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 NOV 2020. Web of Science Citations: 0.
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; HONORATO, RODRIGO; PAULETTI, BIANCA ALVES; INDOLFO, NATHALIA DE CARVALHO; RIBEIRO FILHO, HELDER VERAS; LOPES DE OLIVEIRA, PAULO SERGIO; MIGLIORINI FIGUEIRA, ANA CAROLINA; PAES LEME, ADRIANA FRANCO; BERTELI AMBROSIO, ANDRE LUIS; GOMES DIAS, SANDRA MARTHA. Glutaminase Affects the Transcriptional Activity of Peroxisome Proliferator-Activated Receptor gamma (PPAR gamma) via Direct Interaction. BIOCHEMISTRY, v. 57, n. 44, p. 6293-6307, NOV 6 2018. Web of Science Citations: 1.

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