Determination of the molecular structure of the human pyruvate carrier subunit 2, MPC2

• Grant number: 18/00492-4
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: December 01, 2018
• End date: May 25, 2020
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Andre Luis Berteli Ambrosio
• Grantee: Vijayakumar Balakrishnan
• Host Institution: Instituto de Física de São Carlos (IFSC). Universidade de São Paulo (USP). São Carlos , SP, Brazil

Abstract

Pyruvate is the end product of cytosolic glycolysis and has a number of possible intracellular fates, the major one being mitochondrial transport, which is, in itself, a critical step in energy, carbohydrate, amino acid, and lipid metabolism. While pyruvate has been known to cross the mitochondrial membrane over a long period of time, it is only recently that proteins necessary for this activity have been identified. In this perspective, two internal mitochondrial membrane (IMM) proteins, named MPC1 and MPC2, were identified as the proteins essential for the transport of pyruvate. These proteins were believed to possess three transmembrane helices with each subunit weighing 15 kDa. According to the literature, the functional complex (MPC1 and MPC2) is believed to have a molecular weight of 150 kDa indicating the possibility of a decamer formation. In this regard, the brief results describing the recombinant production of human MPC subunits through a co-expression strategy that were attempted in our laboratory are as follows; the substantial complex formation between MPC1 and MPC2 was not observed and only predominantly individual subunits were purified. We demonstrated that MPC2, in contrast to MPC1 - which purifies associated to a host chaperone - can promote efficient pyruvate transport into reconstituted proteoliposomes. The derived functional requirements, kinetics and the inhibition features of MPC2 resemble those already demonstrated for pyruvate transport in mitochondrial extracts. Importantly, ectopic expression of human MPC2 in yeast lacking endogenous MPC stimulated growth in culture media lacking valine and increased consumption of oxygen, thus validating its autonomous role in yeast cells. Our results lay the initial framework for exploring the independent role of MPC2 in homeostasis and diseases related to the dysregulation of pyruvate metabolism. Now, in this proposal, we aim at the description of the molecular structure of MPC2, either by X-ray crystallography of purified MPC2 or Cryo-Electron Microscopy of MPC2 reconstituted into nanodiscs, alongside with the description of a detailed mechanism for pyruvate transport. Preliminary and successful working conditions have already been developed for both experiments, drastically increasing the chances of achieving our goals within the proposed timeframe. When completed, our research has a potential to provide valuable insights in understanding underlying the mechanisms of pyruvate shuttling across the mitochondrial membrane, besides contributing to the technological development of structural biology of membrane proteins Brazil.

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. (14/20673-2, 17/11766-5, 19/02261-2, 13/07600-3, 14/06954-9, 18/00492-4, 17/02391-8, 15/02734-7)

KARTHIK, LAKSHMINARAYANAN; VIJAYAKUMAR, BALAKRISHNAN. Screening of Anti-Cancer Properties of Beta-Sitosterol and its Derivatives against Microtubules: Molecular Modeling Approach. INTERNATIONAL JOURNAL OF PHARMACEUTICAL AND PHYTOPHARMACOLOGICAL RESEARCH, v. 10, n. 1, p. 8+, 2020. (18/00492-4)

BALAKRISHNAN, VIJAYAKUMAR; LAKSHMINARAYANAN, KARTHIK. Screening of FDA Approved Drugs Against SARS-CoV-2 Main Protease: Coronavirus Disease. INTERNATIONAL JOURNAL OF PEPTIDE RESEARCH AND THERAPEUTICS, v. 27, n. 1, p. 651-658, MAR 2021. (18/00492-4)