Pecina 2017 MiP2017

From Bioblast
Pecina Petr
Mitochondrial ATP synthase disorders investigated by quantitative proteomics of CRISPR-Cas9 knockout cell lines.

Link: MiP2017

Vrbacky M, Kovalcikova J, Pecina P, Harant K, Pecinova A, Houstek J, Mracek T (2017)

Event: MiP2017


Mitochondria are cellular organelles producing the vast majority of the key energetic molecule ATP and this mitoproteome is composed of approx. 1500 proteins. Key enzyme in the mitochondrial ATP production is the multi-subunit F1Fo-ATP synthase which, together with the respiratory chain complexes, constitutes the oxidative phosphorylation (OXPHOS) system. Mutations in proteins affecting OXPHOS function comprise broad group of mitochondrial diseases and among them, disorders of ATP synthase belong to the most severe. The majority of ATP synthase disorders are caused by the mutations in the nuclear TMEM70 gene encoding mitochondrial TMEM70 transmembrane protein localized in the inner mitochondrial membrane and involved in the biogenesis of the eukaryotic ATP synthase. TMEM70 is expressed at low levels when compared with the structural subunits of ATP synthase and its molecular function is not yet known. To better understand the role of this factor, we generated TMEM70 knockout HEK293 cell lines by CRISPR-Cas9 technology. Several biochemical methods, including blue native electrophoresis, ATPase enzyme activity assay, and mitochondrial oxygen consumption revealed a defect of ATP synthase, which was fully complemented by recombinant expression of wt TMEM70. Label-free (LFQ) as well as SILAC quantitative MS analysis of knockout cell lines showed an isolated decrease of all subunits of mitochondrial ATP synthase. Co-immunoprecipitation performed as an affinity enrichment LFQ MS analysis of cellular lysates identified a few mitochondrial OXPHOS complex I proteins as interaction partners of TMEM70. Collectively, TMEM70 knockout cell lines recapitulate the pathology observed in fibroblasts from human patients and demonstrate that CRISPR-Cas9 knockouts and quantitative MS are promising tools in studying human pathologies.

β€’ Bioblast editor: Kandolf G β€’ O2k-Network Lab: CZ Prague Houstek J

Labels: MiParea: Genetic knockout;overexpression 

Organism: Human  Tissue;cell: HEK, Fibroblast 

Regulation: ATP production 

Affiliations and support

VrbackΓ½ M(1), KovalčíkovΓ‘ J(1), Pecina P(1), Harant K(2), Pecinova A(1), HouΕ‘tΔ›k J(1), MrÑček T(1)
  1. Dept Bioenergetics, Inst Physiol, Czech Academy Sciences
  2. BIOCEV & Fac Science, Charles Univ, Prague, Czech Republic. – [email protected]
This project is supported by the GACR (14-36804G) and the AZV (16-33018A).
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