Biology subjects

Cambronne, X. A.

Publications and source records attributed to Cambronne, X. A..

3 recordsLinked to original sources

bioRxiv2022

Improved Yield for the Enzymatic Synthesis of Radiolabeled Nicotinamide Adenine Dinucleotide

Labeled {beta}-nicotinamide adenine dinucleotide (NAD) analogs have been critical for uncovering new biochemical connections and quantitating enzymatic activity. They function as tracers for enzymology, flux analyses, and in situ measurements. Nevertheless, there is limited availability of specific types of analogs, especially radiolabeled NAD analogs. Here, we describe an improved enzymatic synthesis reaction for 32P-NAD+ with a yield of 98% {+/-} 1%, using lowered concentrations of reactants and standard equipment. This represents the highest reported yield for the enzymatic synthesis of NAD+ to date. With the high yield we were able to directly use the reaction product to generate derivatives, such as 32P-NADP. The high-yield enzymatic synthesis is versatile for a broad variety of labels and NAD derivatives. Its advantages include lowered concentrations of reactants, providing sufficient amounts of product for downstream applications, and minimizing intermediate purification steps.

biochemistry↗

bioRxiv2022

SLC25A51 impacts drug sensitivity in AML cells by sustaining mitochondrial oxidative flux

SLC25A51 imports oxidized NAD+ into the mitochondrial matrix and is required for sustaining oxidative metabolism in human mitochondria. We observed that higher expression of SLC25A51 correlated with poorer survival in Acute Myeloid Leukemia (AML) patient data. Given AMLs dependency on oxidative cell metabolism, we sought to determine the role SLC25A51 may serve in this disease. We found that depleting SLC25A51 in AML cells led to increased apoptosis, as well as prolonged survival in a xenograft model. Metabolic flux analyses indicated that depletion of SLC25A51 shunted flux away from oxidative pathways and promoted glutamine utilization for reductive carboxylation to support aspartate production. Consequently, SLC25A51 loss sensitized AML cells to glutamine deprivation and glutaminase inhibitor CB-839. Together, the work highlights connections between SLC25A51 and oxidative mitochondrial flux in AML. We identified a rationale for targeting SLC25A51 in myeloid cancers with potential for a therapeutic window, especially when coupled with glutaminase inhibition. Statement of significanceThis investigation describes an approach to directly modulate the tricarboxylic acid cycle as a potential vulnerability in oxidative tumors. Using AML models, the work is an inaugural look into SLC25A51s role supporting oxidative mitochondrial metabolism and identifies SLC25A51 levels as a potential marker for stratification of AML.

cancer biology↗

bioRxiv2022

Dynamics of Mitochondrial NAD+ Import Reveal Preference for Oxidized Ligand and Substrate Led Transport

SLC25A51 is a member of the mitochondrial carrier family (MCF) but lacks key residues that have been attributed 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 used Molecular Dynamic simulations to study reconstituted SLC25A51 homology models in lipid bilayers. We observed spontaneous binding of cardiolipin phospholipids to three distinct sites on the exterior of SLC25A51s central pore and found that mutation of these sites impaired transporter activity. We also observed that stable formation of the required matrix gate was controlled by a single salt bridge. Using simulation data and in-cell activity assays we identified binding sites in SLC25A51 for NAD+ and showed that its binding was guided by an electrostatic interaction between NAD+ and a negatively charged patch in the pore. In turn, interaction of NAD+ with interior residue E132 guided the ligand to dynamically engage and weaken the salt bridge gate, representing a ligand-induced initiation of transport. SignificanceNAD+ is an intermediary metabolite whose multiple functions are entwined with respiration, catabolism, and stress responses in cells. Previous sensor measurements had indicated that its continuous biosynthesis was required to sustain mitochondrial matrix levels in respiring cells, and SLC25A51 was identified as the required importer of NAD+ across the inner mitochondrial membrane. However, SLC25A51 has little homology to other nucleotide carriers at its substrate binding site. By combining modeling approaches and experimental assays, this work provides mechanistic insight into how human SLC25A51 recognizes its ligand, how the transporter can be regulated by its lipid environment, and an observation of ligand-induced gate opening. This represents the first description of the ligand binding site for an NAD+ mitochondrial carrier.

biochemistry↗