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What We Do...

The long-term goal of our research program is to understand the structure, function, regulation and contribution to cellular life of the SLC29 family of equilibrative nucleoside transporters (ENT), specifically SLC29A1 (ENT1) and SLC29A2 (ENT2).  Over the last five years, we have shown that ENT1 and ENT2 are active members of dynamic networks of factors (receptors, enzymes, etc.) involved in cellular physiology (1) adding significantly to what was currently understood about the biology of the SLC29 family (2-4). We have established that the intracellular loop of both hENT1 and mENT1 can be phosphorylated in vitro and that the full-length protein is phosphorylated constitutively in vivo (6). We have also identified factors (motifs, glycosylation, repeated substrate translocation) that affect aspects of the ENT function and life cycle such as trafficking and internalization (7-9). We have also identified and characterized a novel, previously undescribed type of regulation of ENT1 via protein-protein interactions (PPIs), through calcium-dependent, calmodulin binding (to the large intracellular loop) activated by G-protein coupled receptor activation (10).  We have shown that ENT1 is present and plays a central role in purinergic responses in physiological contexts such as the heart (11-13) and kidney (14). We have also established that ENT2, a less well-understood ENT, contributes to regulation of cell cycle, through localization of splice variants at the nuclear membrane (5), an unexpected and highly novel finding. All of these findings have increased our understanding of the role of the SLC29 members, ENT1 and ENT2, in cellular homeostasis, in response to cellular stress and proliferative signaling, and as routes of entry of drugs used in anti-cancer, anti-viral and anti-parasitic treatments (1). Our research has also involved development of tools and techniques for biochemical and cellular analysis of these challenging membrane proteins (15-16) as well as assistance to other researchers (*17, *18) studying other aspects of ENT physiology.

Understanding the biology of the ENTs is also clinically significant since ENT1 and ENT2 are the route of entry for drugs used in many clinical settings. Our research also investigates improved chemotherapies such as nucleoside analog drug plus ultrasound microbubble treatments, a combination modality which show some promise in terms of enhanced cytotoxic effects (19). We use a variety of approaches including (but not limited to) protein biochemistry, structural biology, pharmacology, transport assays and advanced imaging to gain a comprehensive understanding of ENT1 and ENT2 life within the cell.

References

(Bold indicates our work; HQP are underlined)

1. dos Santos Rodrigues et al. 2014. Neurochem. Int. 73:229-37. doi: 10.1016/j.neuint.2014.03.014.

2. Baldwin et al. 2004. Pflugers Archive, 447:735-743.

3. Aymerich et al. 2005. Biochem. Soc. Trans. 33:216-219.

4. Gray et al. 2004. Pflugers Archive, 447:728-734.

5. Grane-Boladeras et al. 2016. Cell Mol. Life Sci. 73(23):4559-4575

6. Reyes et al. 2011a Mol Membr Biol. 2011 Sep;28(6):412-26. doi: 10.3109/09687688.2011.604861.

7. Nivillac et al. 2011 Experimental Cell Research 317(11);1567-1579.

8.  Bicket and Coe 2016a. Bioscience Reports 36(4) pii00376. doi: 10.1042/BSR20160063.

9. Zafar et al. in press. Nucleosides, Nucleotides and Nucleic Acids.

10. Bicket et al. 2016b Am J Physiol Cell Physiol. 310(10):C808-20. doi: 10.1152/ajpcell.00243.2015.

11. Marvi et al. 2010. Eur J Pharm Sci. 41(5):685-91. doi: 10.1016/j.ejps.2010.09.013.

12. Rose et al. 2010. Am J Physiol Heart Circ Physiol. 298(3):H771-7. doi: 10.1152/ajpheart.00711.2009.

13. Rose et al. 2011. Life Sci. 89(17-18):621-30. doi: 10.1016/j.lfs.2011.08.007.

14. Grenz et al 2012. J Clin Invest. 2012 Feb;122(2):693-710. doi: 10.1172/JCI60214.

15. Reyes et al. 2010 Prot. Express. Pur. 73(1):1-9.

16. Reyes et al. 2011b Biochem Cell Biol. 2011 Apr;89(2):246-55. doi: 10.1139/o10-155.

17. Kaneko et al. 2014. Biochem Biophys Res Commun. 2014 Jan 10;443(2):505-10. doi: 10.1016/j.bbrc.2013.11.132.

18. Panigrahi et al. 2015. J Virol. 89(1):626-42. doi: 10.1128/JVI.02492-14.

19. Mariglia et al. in prep.