Understanding the molecular basis of substrate specificity in nucleobase transporters – Putting the basis for the rational development of highly targeted antimicrobials
Department of Biology, School of Science, National and Kapodistrian University of Athens
Our study addresses whether regeneration could influence ageing. Many animals have the ability to fully regenerate certain organs after injury or amputation. We studied this phenomenon in crustaceans, which have the ability to fully regenerate their legs. We wondered whether regenerated legs show the same degree of senescence as non-regenerated limbs in the same animal – is the regenerated leg young or old?
To address this question we had to develop indicators of ageiAll cells membranes have embedded proteins, which function as controlled gateways that open and close transporting all essential molecules and metabolites, as well as drugs and other synthetic substances, to the cytoplasm or the extracellular space. These proteins fall into two basic categories: transporters and channels. The importance of these intra-membrane proteins is evidenced by the fact that their malfunction is linked to at least 60 genetic diseases, while their activities are associated with the cell’s ability to uptake or reject medications/antibiotics.
Since 1998, our lab has used fungal model systems (with Saccharomyces cerevisiae & Aspergillus nidulans) to study the transporters responsible for the specialized uptake of purine and pyrimidine, biochemical compounds necessary for cell life. We created a plethora of mutations in the two basic purine/pyrimidine transporters (UapA & FcyB), using both rational design and random mutagenesis and discovered a series of amino-acid residues that apparently influence the cellular expression, function and/or specialization of the transporters. Subsequently, we used modelling software to depict the three-dimensional topology of the transporters and then identified the substrate docking points (purine and pyrimidine) and the path that carries them through the “body” of the transporters. We discovered that genetic functional analysis both supports and is fully supported by a biophysical analysis of the modelling of the transporter structures.
Until recently, it was thought that transporters and channels operate in a completely different manner. Our data overturns all previous interpretations to date and proposes that while transporters do have a basic substrate docking centre, accessible either cytoplasmically or extracellularly, it also appears that independent controlled molecular gateways also exist, controlling access to the basic substrate docking centre.ng that can be measured on individual legs. We compared the expression of genes in the limbs of young versus aged animals and identified some that are differentially expressed with age. Using these genes as 'markers' of ageing, we then compared aged limbs that had regenerated to ones that had not.
We found that regenerated limbs retain the characteristics of aged appendages with respect to some markers, while displaying a youthful character with respect to others. This raises the possibility that some types of cells in the body may become rejuvenated during the lifetime of an animal, following regeneration, while others continue to age. This is the issue we want to investigate further.
Final report (in Greek)
George Diallinas, Associate Professor, Department of Biology, National and Kapodistrian University of Athens
Emmanuel Mikros, Professor, School of Pharmacy, National and Kapodistrian University of Athens
Sotiris Amillis, Researcher, Department of Biology, National and Kapodistrian University of Athens
Vassiliki Kosti, Doctoral Student, Department of Biology, National and Kapodistrian University of Athens
Aimilia Krypotou, Doctoral Student, Department of Biology, National and Kapodistrian University of Athens
Vassilis Myrianthopoulos, Post-doctoral Researcher, School of Pharmacy, National and Kapodistrian University of Athens