Structural Characterization of the VDAC2-Bak complex from mitochondrial outer membrane by Single Particle Cryo-Electron Microscopy

• Grant number: 16/14350-1
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: September 01, 2016
• End date: October 31, 2018
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Andre Luis Berteli Ambrosio
• Grantee: Antônio Carlos Borges
• Host Institution: Centro Nacional de Pesquisa em Energia e Materiais (CNPEM). Ministério da Ciência, Tecnologia e Inovação (Brasil). Campinas , SP, Brazil

Abstract

Structural Characterization of the VDAC2-Bak complex from mitochondrial outer membrane by Single Particle Cryo-Electron Microscopy.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.