Dynamics of SLC25A51 reveal preference for oxidized NAD+ and substrate led transport.

SLC25A51 is a member of the mitochondrial carrier family (MCF) but lacks key residues that contribute to the mechanism of other nucleotide MCF transporters. Thus, how SLC25A51 transports NAD+ across the inner mitochondrial membrane remains unclear. To elucidate its mechanism, we use Molecular Dynamics simulations to reconstitute SLC25A51 homology models into lipid bilayers and to generate hypotheses to test. We observe spontaneous binding of cardiolipin phospholipids to three distinct sites on the exterior of SLC25A51's central pore and find that mutation of these sites impairs cardiolipin binding and transporter activity. We also observe that stable formation of the required matrix gate is controlled by a single salt bridge. We identify binding sites in SLC25A51 for NAD+ and show that its selectivity for NAD+ is guided by an electrostatic interaction between the charged nicotinamide ring in the ligand and a negatively charged patch in the pore. In turn, interaction of NAD+ with interior residue E132 guides the ligand to dynamically engage and weaken the salt bridge gate, representing a ligand-induced initiation of transport.

Layered mechanisms regulating the human mitochondrial NAD+ transporter SLC25A51.

SLC25A51 is the primary mitochondrial NAD+ transporter in humans and controls many local reactions by mediating the influx of oxidized NAD+. Intriguingly, SLC25A51 lacks several key features compared with other members in the mitochondrial carrier family, thus its molecular mechanism has been unclear. A deeper understanding would shed light on the control of cellular respiration, the citric acid cycle, and free NAD+ concentrations in mammalian mitochondria. This review discusses recent insights into the transport mechanism of SLC25A51, and in the process highlights a multitiered regulation that governs NAD+ transport. The aspects regulating SLC25A51 import activity can be categorized as contributions from (1) structural characteristics of the transporter itself, (2) its microenvironment, and (3) distinctive properties of the transported ligand. These unique mechanisms further evoke compelling new ideas for modulating the activity of this transporter, as well as new mechanistic models for the mitochondrial carrier family.

SLC25A51 decouples the mitochondrial NAD+/NADH ratio to control proliferation of AML cells.

SLC25A51 selectively imports oxidized NAD+ into the mitochondrial matrix and is required for sustaining cell respiration. We observed elevated expression of SLC25A51 that correlated with poorer outcomes in patients with acute myeloid leukemia (AML), and we sought to determine the role SLC25A51 may serve in this disease. We found that lowering SLC25A51 levels led to increased apoptosis and prolonged survival in orthotopic xenograft models. Metabolic flux analyses indicated that depletion of SLC25A51 shunted flux away from mitochondrial oxidative pathways, notably without increased glycolytic flux. Depletion of SLC25A51 combined with 5-azacytidine treatment limits expansion of AML cells in vivo. Together, the data indicate that AML cells upregulate SLC25A51 to decouple mitochondrial NAD+/NADH for a proliferative advantage by supporting oxidative reactions from a variety of fuels. Thus, SLC25A51 represents a critical regulator that can be exploited by cancer cells and may be a vulnerability for refractory AML.