Oligomerization of equilibrative nucleoside transporters: a novel regulatory and functional mechanism involving PKC and PP1.

Equilibrative nucleoside transporters (ENTs) translocate nucleosides and nucleobases across plasma membranes, as well as a variety of anti-cancer, -viral, and -parasite nucleoside analogs. They are also key members of the purinome complex and regulate the protective and anti-inflammatory effects of adenosine. Despite their important role, little is known about the mechanisms involved in their regulation. We conducted membrane yeast 2-hybrid and coimmunoprecipitation studies and identified, for the first time to our knowledge, the existence of protein-protein interactions between human ENT1 and ENT2 (hENT1 and hENT2) proteins in human cells and the formation of hetero- and homo-oligomers at the plasma membrane and the submembrane region. The use of NanoLuc Binary Technology allowed us to analyze changes in the oligomeric status of hENT1 and hENT2 and how they rapidly modify the uptake profile for nucleosides and nucleobases and allow cells to respond promptly to external signals or changes in the extracellular environment. These changes in hENTs oligomerization are triggered by PKC activation and subsequent action of protein phosphatase 1.-Grañe-Boladeras, N., Williams, D., Tarmakova, Z., Stevanovic, K., Villani, L. A., Mehrabi, P., Siu, K. W. M., Pastor-Anglada, M., Coe, I. R. Oligomerization of equilibrative nucleoside transporters: a novel regulatory and functional mechanism involving PKC and PP1.

Pharmacogenomic analyzis of the responsiveness of gastrointestinal tumor cell lines to drug therapy: A transportome approach.

In this study, we have addressed the pharmacogenomic basis of the response of gastrointestinal tumors to six anticancer drugs using a panel of fifteen cell lines derived from pancreatic, stomach and biliary tract cancers. We determined the constitutive expression levels of 96 genes, whose encoded proteins contribute to drug action, and identified a major gene network that contains broad selectivity nucleoside transporter genes, as well as several genes known to be involved in cell proliferation and survival. All cell lines were exposed to 5'-DFUR, 5-FU, gemcitabine, cisplatin, doxorubicin and paclitaxel for 48h and cell response was measured using MTT assays. We correlated the cell response of the fifteen cell lines with the mRNA expression of the selected 96 genes and identified sets of 4-5 genes whose expression profiles correlated to responsiveness to each anticancer drug. These genes may be good candidates as response predictors to such therapies.

Novel nuclear hENT2 isoforms regulate cell cycle progression via controlling nucleoside transport and nuclear reservoir.

Nucleosides participate in many cellular processes and are the fundamental building blocks of nucleic acids. Nucleoside transporters translocate nucleosides across plasma membranes although the mechanism by which nucleos(t)ides are translocated into the nucleus during DNA replication is unknown. Here, we identify two novel functional splice variants of equilibrative nucleoside transporter 2 (ENT2), which are present at the nuclear envelope. Under proliferative conditions, these splice variants are up-regulated and recruit wild-type ENT2 to the nuclear envelope to translocate nucleosides into the nucleus for incorporation into DNA during replication. Reduced presence of hENT2 splice variants resulted in a dramatic decrease in cell proliferation and dysregulation of cell cycle due to a lower incorporation of nucleotides into DNA. Our findings support a novel model of nucleoside compartmentalisation at the nuclear envelope and translocation into the nucleus through hENT2 and its variants, which are essential for effective DNA synthesis and cell proliferation.

Nucleoside transporters in the purinome.

The purinome is a rich complex of proteins and cofactors that are involved in fundamental aspects of cellular homeostasis and cellular responses. The purinome is evolutionarily ancient and is made up of thousands of members. Our understanding of the mechanisms linking some parts of this complex network and the physiological relevance of the various connections is well advanced. However, our understanding of other parts of the purinome is less well developed. Our research focuses on the adenosine or nucleoside transporters (NTs), which are members of the membrane purinome. Nucleoside transporters are integral membrane proteins that are responsible for the flux of nucleosides, such as adenosine, and nucleoside analog drugs, used in a variety of anti-cancer, anti-viral and anti-parasite therapies, across cell membranes. Nucleoside transporters form the SLC28 and SLC29 families of solute carriers and the protein members of these families are widely distributed in human tissues including the central nervous system (CNS). NTs modulate purinergic signaling in the CNS primarily through their effects on modulating prevailing adenosine levels inside and outside the cell. By clearing the extracellular milieu of adenosine, NTs can terminate adenosine receptor-dependent signaling and this raises the possibility of regulatory feedback loops that tie together receptor signaling with transporter function. Despite the important role of NTs as modulators of purinergic signaling in the human body, very little is known about the nature or underlying mechanisms of regulation of either the SLC28 or SLC29 families, particularly within the context of the CNS purinome. Here we provide a brief overview of our current understanding of the regulation of members of the SLC29 family and highlight some interesting avenues for future research.