In liver and adipose cells, cytosolic citrate is a major precursor for the synthesis of fatty acids, triacylglycerols, cholesterol and low-density lipoprotein. The cytosolic citrate concentration partially depends on its direct import across the plasma membrane via the Na+-dependent citrate transporter, a member of the divalent anion/Na+ symporter (DASS) family. Mutations of the transporter gene in flies (INDY) result in reduced fat storage through calorie restriction. Knockout mice of the homologous gene are both slimmer and protected from obesity and insulin resistance. Thus, its central role in fatty acid biosynthesis makes NaCT a particularly attractive target of small-molecule therapeutic agents for obesity, diabetes and cardiovascular diseases. We have recently determined the 3.2 ? crystal structure of a bacterial INDY homolog in its inward-facing conformation. The crystal structure allows us to propose a detailed transport mechanism for the protein, including substrate specificity, ion specificity, ion- substrate coupling and conformational changes that the protein undergoes to accomplish substrate translocation across the membrane. In the current project, we will test this transport mechanism using mutagenesis and transport assays. We will further characterize the transport mechanism of the protein by determining the structure of the outward-facing conformation of the INDY protein from bacteria and mammals. Understanding of the transport mechanism of these transporters, particularly their substrate and ion specificity, will help in the design of drugs for obesity and diabetes.

Public Health Relevance

In human cells, cytosolic citrate is a major precursor for the synthesis of fatty acids, triacylglycerols, cholesterol and low-density lipoprotein, and its concentration partially depends on the direct import across the plasma membrane via the Na+-dependent citrate transporter. Mutations of the homologous gene in flies result in reduced fat storage through calorie restriction, whereas knockout mice of this gene are both slimmer and protected from obesity and insulin resistance. Understanding of the transport mechanism of these transporters, particularly their substrate and ion specificity, will help in the design of drgs for obesity and diabetes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
1R01DK099023-01
Application #
8531420
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Sechi, Salvatore
Project Start
2013-03-01
Project End
2017-02-28
Budget Start
2013-03-01
Budget End
2014-02-28
Support Year
1
Fiscal Year
2013
Total Cost
$368,663
Indirect Cost
$151,163
Name
New York University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
121911077
City
New York
State
NY
Country
United States
Zip Code
10016
Mulligan, Christopher; Fitzgerald, Gabriel A; Wang, Da-Neng et al. (2014) Functional characterization of a Na+-dependent dicarboxylate transporter from Vibrio cholerae. J Gen Physiol 143:745-59
Waight, Andrew B; Czyzewski, Bryan K; Wang, Da-Neng (2013) Ion selectivity and gating mechanisms of FNT channels. Curr Opin Struct Biol 23:499-506