Recent studies suggest that nitrate and nitrite molecules have profound beneficial effects to human health, though they were thought to be cancerous since 1970s. To maximize their pharmaceutical potential, we need to first understand how nitrate and nitrite circulate in humans. Such circulation depends on two critical events: active accumulation of nitrate mediated by sialin transporters in salivary glands, as well as nitrate uptake and nitrite secretion by commensal bacteria in the mouth. Thus, nitrate/nitrite molecules have to cross different cellular membranes multiple times, before being used by humans. These translocation processes are mediated by a group of membrane proteins called nitrate transporters. To understand how these transporters function, we solved high-resolution crystal structures of NarK from E. coli, and further demonstrated that, surprisingly, NarK is a nitrate/nitrite exchanger. Despite the progress we made, the detailed molecular mechanisms of nitrate/nitrite translocation are still largely unknown. In this proposal, we aim to fill the knowledge gap by: 1) understand substrate selectivity and conformational flexibility using directed-mutagenesis of NarK, so to better understand the function of NarK at the molecular level; 2) obtain high-resolution structures of NarK in previously unobserved conformation, so we can reconstruct the complete transport cycle of NarK; 3) explore the structure-function relationship of human nitrate transporter sialin, so we will understand the similarities and differences among nitrate transporters from diverse species. Overall, upon completion of the proposal, we expect to expand our general understanding of the nitrate/nitrite transport, and shed light on the crucial roles that nitrate transporters (both eukaryotic and prokaryotic) play in nitrate/nitrite circulation. The knowledge gained here will facilitate potential drug development related to bacterial nitrate transporters and human sialin.

Public Health Relevance

Nitrate and nitrite translocations are fundamental steps involved in the nitrogen cycle for all life. Nitrate transporters from commensal bacteria and human sialin (highly expressed in salivary gland) are critical to regulate the homeostasis of nitrate/nitrite circulation in humans. Furthermore, sialin is closely related to various genetic diseases. To fully utilize the pharmaceutical potential of the nitrate/nitrite/nitric oxide molecules recently-proposed in the scientific community, detailed molecular understanding of their transporters is demanded. This proposal aims to decipher the molecular mechanisms of these transporters from different species, and in doing so provide thorough understanding of the nitrate/nitrite translocation processes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM126626-01
Application #
9419075
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Ainsztein, Alexandra M
Project Start
2018-01-01
Project End
2022-11-30
Budget Start
2018-01-01
Budget End
2018-11-30
Support Year
1
Fiscal Year
2018
Total Cost
Indirect Cost
Name
University of Colorado Denver
Department
Biochemistry
Type
Schools of Medicine
DUNS #
041096314
City
Aurora
State
CO
Country
United States
Zip Code
80045