CEEPC/IPM/CMSC - Abstrakt prezentace

(CEEPC/IPM/CMSC 2022 - ThP-05)
Complexome profiling of mitochondrial OXPHOS knockout models

Marek Vrbacký 1 *, Kristýna Čunátová 1, Thomas Stehrer 2, Aleksandra Marković 1, Petr Pecina 1, Tomáš Mráček 1

  1. Institute of Physiology, CAS, Prague
  2. Institute of Molecular Genetics, CAS, Prague

Abstrakt

Proteins are key biomolecules that mostly exert their function thru interaction with other macromolecules. Several techniques to elucidate the protein-protein interactions (PPIs) are available and they frequently employ the mass spectrometry detection. One such technique is complexome profiling that quantifies the proteins separated by the native, non-denaturing, electrophoresis. Correlative analysis of migration patterns is used to infer the multiprotein assemblies. Here we used the blue native polyacrylamide gel electrophoresis (BN-PAGE) followed by 96 well plate-based processing to analyze the HEK293 cellular CRISPR/Cas9 knockout models of mitochondrial oxidative phosphorylation (OXPHOS) defects. Isolated mitochondria of cytochrome c oxidase COX4I1+4I2 or COX6B1 knockouts allowed us to study interdependency between the mitochondrial Complex I and Complex IV (COX). We observed accumulation of Complex I assembly intermediates, indicating that its biogenesis, rather than stability, was affected. Complexome profiling was also used to characterize the knockout disease model of inner mitochondrial membrane protein TMEM70, an assembly factor of ATP synthase. We observed an aberrant ATP synthase biogenesis with accumulation of sub-assemblies and also characterized two novel ATP synthase subunit c interacting proteins (TMEM242, c15orf61). Recently proposed standards "The Minimum Information About a Complexome profiling Experiment" (MIACE) published within a CEDAR (ComplexomE profiling DAta Resource) project (PMID: 33722514) were followed. The technique of complexome profiling is a reproducible method that provides deeper understanding of cellular disease models.

* Korespondující autor: vrbacky@fgu.cas.cz

Poděkování:

This project is supported by the GACR (20-25768S) and the AZV (NU21-07-00550).


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Partneři

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