Interference of para-aminobenzoic acid with Coenzyme Q metabolism in mammals : molecular causes, physiological consequences and biosynthesis of CoQ analogues. – pABACoQ

Mammalian cell culture, HPLC-ECD quantification of coenzyme Q and structural analogues. Oxygen consumption measurements on intact and permeabilized cells and spectrophotometric assays for enzymatic activities.

We have confirmed that N-DMQ is synthesized in many murines and human cell lines when the cells are cultured in the presence of pABA. We have also showed that the cellular coenzyme Q content depends on the serum used for culturing the cells and a serum appropriate to our applications has been selected. Studies concerning the functionality of N-DMQ in the respiratory chain are in progress.

During the course of the next year, we will focus on experiments which aim at assessing a pro or antioxydant function of N-DMQ and new coenzyme Q analogues in cultured cells. Within 6 months, we will initiate the experiment with mice to verify whether N-DMQ forms also in whole organisms. Finally, we will continue to establish the molecular causes of the formation of N-DMQ in cells exposed to pABA.

Impact of Chemical Analogs of 4-Hydroxybenzoic Acid on Coenzyme Q Biosynthesis: From Inhibition to Bypass of Coenzyme Q Deficiency.
Pierrel F.
Front Physiol. (2017) 8:436
DOI: 10.3389/fphys.2017.00436

The COQ2 genotype predicts the severity of coenzyme Q10 deficiency
Desbats M. A., Morbidoni V., Silic-Benussi M., Doimo M., Ciminale V., Cassina M., Sacconi S., Hirano M., Basso G., Pierrel F., Navas P., Salviati L., Trevisson E.
Hum Mol Genet. (2016) 25:4256-4265
DOI: 10.1093/hmg/ddw257

Ubiquinone or Coenzyme Q (CoQ) is a redox active lipid essential to electron transport in eukaryotic mitochondrial respiratory chain. CoQ is an important membrane antioxidant but also participates to the formation of deleterious reactive oxygen species (ROS) under certain physiological conditions. CoQ is a substituted benzoquinone coupled to a long polyprenyl chain. The susbtituents of the benzoquinone determine the redox potential of CoQ and are added step by step by CoQ biosynthetic enzymes on the precursor, 4-hydroxybenzoic acid (4-HB). CoQ is synthesized in mitochondria of all eukaryotic cells and mutations in five genes essential for CoQ biosynthesis have been identified to cause severe pathologies. Despite the crucial role of CoQ, some aspects of its biosynthesis are still obscure and very little is known concerning the availability and biosynthesis of 4-HB.
We have reported in 2010 that 4-aminobenzoic acid (pABA), the 4-amino analogue of 4-HB, competes with 4-HB as a precursor of CoQ in the yeast S. cerevisiae. We have shown that the C4-amino (NH2) group originating from pABA is converted into a C4-hydroxyl (OH) group at an undefined step of CoQ biosynthesis in yeast. Based on these results, we have tested whether pABA is also a precursor of CoQ in murine cell cultures. Our results show that murine cell cultures can not synthesize CoQ from pABA. In place, pABA decreases CoQ cellular level and causes the accumulation of an unprecedented amino-containing CoQ biosynthetic analogue (4-amino-6-demethoxyquinone, N-DMQ) in a dose dependent manner. This result shows that pABA enters the CoQ biosynthetic pathway in murine cells in place of 4-HB. However, contrary to yeast, the C4-amine is not converted into a C4-hydroxyl, thus causing a strong depletion of CoQ and the accumulation of N-DMQ in murine cells exposed to pABA.
Since CoQ is essential to normal cellular function, it is important to evaluate the consequences on the cellular physiology of the interference of pABA with CoQ metabolism. We propose to investigate the functionality of N-DMQ in the mitochondrial respiratory chain and its propensity to form and to detoxify ROS. In addition, we want to identify the molecular reasons for the formation of N-DMQ. We will also address whether pABA interferes with CoQ metabolism in human cell cultures and in live mammals like mice. These fundamental researches are important because pABA is widely used as a food supplement and could therefore interfere with CoQ biosynthesis in humans. Our research will indicate whether pABA intake may represent a risk for humans by perturbing CoQ biosynthesis.
In addition, we propose to induce the biosynthesis of CoQ analogues with various benzoquinone substitutents by adding different 4-HB analogues to murine cells cultures. These CoQ analogues are expected to have different redox potentials from CoQ and should therefore possess different properties in electron transport, in ROS production and in antioxidant activity. Our experiments will evaluate in particular whether changing the substituents of the benzoquinone of CoQ may improve its antioxidant activity. Positive results will be validated by synthesizing the corresponding analogues of MitoQ, a CoQ based antioxidant which is presently tested on patients with pathologies implicating mitochondrial oxidative damage. The study of the CoQ analogues will also provide further details about the function of CoQ as an electron transporter, antioxidant and prooxidant. This knowledge in combination with the use of various 4-HB analogues could be a new approach in treatment of human CoQ deficiencies, i.e. the use of a suitable precursor may bypass some genetic mutations in CoQ biosynthesis.