Kovalcikova 2018 Thesis
|Kovalčíková J (2018) Functional characterisation of new components of mitochondrial proteome. PhD Thesis p104.|
Abstract: It has been estimated that the mammalian mitochondrial proteome consists of ~1500 distinct proteins and approximately one quarter of them is still not fully characterized.
One of these proteins is TMEM70, protein involved in the biogenesis of the eukaryotic F1Fo-ATP synthase. TMEM70 mutations cause isolated deficiency of ATP synthase often resulting in a fatal neonatal mitochondrial encephalocardiomyopathies in patients. To understand the molecular mechanism of TMEM70 action, we generated constitutive Tmem70 knockout mice, which led to embryonic lethal phenotype with disturbed ATP synthase biogenesis. Subsequently generated inducible Tmem70 mouse knockout was lethal by the week 8 post induction. It exhibited primarily impaired liver function, which contrasts with the predominantly cardiologic phenotype at disease onset in humans. Liver mitochondria revealed formation of labile ATP synthase subcomplexes lacking subunit c. Thus, in case of TMEM70 deficiency c-oligomer was not incorporated into ATP synthase, which led to critical impairment of mitochondrial energy provision, analogous to TMEM70 dysfunction in humans. In TMEM70 deficient models, the ATP synthase deficiency reached the ‘threshold’ for its pathologic presentation, which we quantified at 30 %. We observed compensatory increases in the content of most OXPHOS complexes but unexpectedly also of ANT and PiC, components of ATP synthasome, which should associate with ATP synthase.
We also studied ATP synthase subunit DAPIT (coded by Usmg5 gene). We generated DAPIT deficient rats, which were fully viable but had lower body weight, pronounced decrease of fat tissue and right ventricular hypertrophy. We observed normal levels of assembled ATP synthase, however, it was predominantly present in the monomeric form, pointing at the role of DAPIT in formation of ATP synthase dimers. ATP synthase function was reduced by ~10 % in both liver and heart. Its higher sensitivity to inhibitor oligomycin than to aurovertin indicated that DAPIT shields oligomycin binding site at Fo moiety.
In conclusion, we generated unique models of mitochondrial proteins deficiency and characterised TMEM70 and DAPIT function.
Labels: MiParea: Respiration, mtDNA;mt-genetics, Genetic knockout;overexpression
Organism: Mouse, Rat Tissue;cell: Heart, Liver Preparation: Homogenate Enzyme: Complex V;ATP synthase
Coupling state: LEAK, OXPHOS, ET Pathway: N, NS HRR: Oxygraph-2k