Pajuelo-Reguera 2017 MiP2017

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David Pajuelo-Reguera
COX4-1/COX4-2 cell knock-out/knock-in models for the study of cytochrome c oxidase regulation.

Link: MiP2017

Pajuelo-Reguera D, Cunatova K, Houstek J, Mracek T, Pecina P (2017)

Event: MiP2017

COST Action MITOEAGLE
Regulation of cytochrome c oxidase (COX), the terminal enzyme of electron transport chain, occurs through the alternating expression of subunits’ isoform pairs; a process which is controlled in developmental, tissue, or environmental manner [1]. COX4 subunit, a crucial homeostatic response switch, is optimizing respiratory chain function according to oxygen-controlled expression of its isoforms COX4-1 and COX4-2 [2,3]. However, the functional impact of the isoform switch for mammalian tissues and cells is not fully understood yet.

We created unique HEK293 cellular model of COX4-1/2 double knock-out employing novel CRISPR CAS9-10A paired nickase technology. These cells were subsequently utilized as a platform for knock-in of COX4-1 and COX4-2 variants using stable overexpression of either variant from pcDNA 3.1 vector. The ability of individual COX4 isoforms to restart de novo COX assembly was investigated by electrophoretic methods. The functional competence of either COX4-1 or COX4-2 within the enzyme complex was characterized by high-resolution respirometry.

Using this model, we further plan to asses the ability of COX4 isoforms to serve as mitochondrial energy and redox sensors, indispensable for regulation of ATP production and oxidative stress response during normoxia and hypoxia.


Bioblast editor: Kandolf G O2k-Network Lab: CZ Prague Houstek J, CZ Prague Jezek P


Labels: MiParea: Respiration, Genetic knockout;overexpression 


Organism: Human  Tissue;cell: HEK 



HRR: Oxygraph-2k 


Affiliations

Dept Bioenergetics, Inst Physiol CAS, Prague, Czech Republic. - david.reguera@fgu.cas.cz

References and support

  1. Hüttemann M, Kadenbach B, Grossman LI (2001) Mammalian subunit IV isoforms of cytochrome c oxidase. Gene 267:111–23.
  2. Hüttemann M, Lee I, Liu J, Grossman LI (2007) Transcription of mammalian cytochrome c oxidase subunit IV-2 is controlled by a novel conserved oxygen responsive element. FEBS J 274:5737–48.
  3. Fukuda R, Zhang H, Kim JW, Shimoda L, Dang CV, Semenza GL (2007) HIF-1 regulates cytochrome oxidase subunits to optimize efficiency of respiration in hypoxic cells. Cell 129:111–22.
The project is supported by Grant Agency of the Czech Republic (16-13671S).