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.

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.

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.

Improved Yield for the Enzymatic Synthesis of Radiolabeled Nicotinamide Adenine Dinucleotide.

Labeled β-nicotinamide adenine dinucleotide (NAD) analogues 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 analogues, especially radiolabeled NAD isotopologues.

The mitochondrial NAD transporter SLC25A51 is a fasting-induced gene affecting SIRT3 functions.

Introduction: Nicotinamide adenine dinucleotide (NAD) is a coenzyme central to metabolism and energy production. NAD-dependent deacetylase sirtuin 3 (SIRT3) regulates the acetylation levels of mitochondrial proteins that are involved in mitochondrial homeostasis. Fasting up-regulates hepatic SIRT3 activity, which requires mitochondrial NAD.

SLC25A51 is a mammalian mitochondrial NAD transporter.

Mitochondria require nicotinamide adenine dinucleotide (NAD) to carry out the fundamental processes that fuel respiration and mediate cellular energy transduction. Mitochondrial NAD transporters have been identified in yeast and plants, but their existence in mammals remains controversial. Here we demonstrate that mammalian mitochondria can take up intact NAD, and identify SLC25A51 (also known as MCART1)-an essential mitochondrial protein of previously unknown function-as a mammalian mitochondrial NAD transporter.

Location, Location, Location: Compartmentalization of NAD Synthesis and Functions in Mammalian Cells.

The numerous biological roles of NAD are organized and coordinated via its compartmentalization within cells. The spatial and temporal partitioning of this intermediary metabolite is intrinsic to understanding the impact of NAD on cellular signaling and metabolism. We review evidence supporting the compartmentalization of steady-state NAD levels in cells, as well as how the modulation of NAD synthesis dynamically regulates signaling by controlling subcellular NAD concentrations.

Flow Cytometry Analysis of Free Intracellular NAD Using a Targeted Biosensor.

Flow cytometry approaches combined with a genetically encoded targeted fluorescent biosensor are used to determine the subcellular compartmental availability of the oxidized form of nicotinamide adenine dinucleotide (NAD ). The availability of free NAD can affect the activities of NAD -consuming enzymes such as sirtuin, PARP/ARTD, and cyclic ADPR-hydrolase family members. Many methods for measuring the NAD available to these enzymes are limited because they cannot determine free NAD as it exists in various subcellular compartments distinctly from bound NAD or NADH.

Pharmacological bypass of NAD salvage pathway protects neurons from chemotherapy-induced degeneration.

Axon degeneration, a hallmark of chemotherapy-induced peripheral neuropathy (CIPN), is thought to be caused by a loss of the essential metabolite nicotinamide adenine dinucleotide (NAD) via the prodegenerative protein SARM1. Some studies challenge this notion, however, and suggest that an aberrant increase in a direct precursor of NAD, nicotinamide mononucleotide (NMN), rather than loss of NAD, is responsible. In support of this idea, blocking NMN accumulation in neurons by expressing a bacterial NMN deamidase protected axons from degeneration.

Methods for Using a Genetically Encoded Fluorescent Biosensor to Monitor Nuclear NAD.

Free nicotinamide adenine dinucleotide (NAD) serves as substrate for NAD-consuming enzymes. As such, the local concentration of free NAD can influence enzymatic activities. Here we describe methods for using a fluorescent, genetically-encoded sensor to measure subcellular NAD concentrations.

AML suppresses hematopoiesis by releasing exosomes that contain microRNAs targeting c-MYB.

Exosomes are paracrine regulators of the tumor microenvironment and contain complex cargo. We previously reported that exosomes released from acute myeloid leukemia (AML) cells can suppress residual hematopoietic stem and progenitor cell (HSPC) function indirectly through stromal reprogramming of niche retention factors. We found that the systemic loss of hematopoietic function is also in part a consequence of AML exosome-directed microRNA (miRNA) trafficking to HSPCs.

Biosensor reveals multiple sources for mitochondrial NAD⁺.

Nicotinamide adenine dinucleotide (NAD(+)) is an essential substrate for sirtuins and poly(adenosine diphosphate-ribose) polymerases (PARPs), which are NAD(+)-consuming enzymes localized in the nucleus, cytosol, and mitochondria. Fluctuations in NAD(+) concentrations within these subcellular compartments are thought to regulate the activity of NAD(+)-consuming enzymes; however, the challenge in measuring compartmentalized NAD(+) in cells has precluded direct evidence for this type of regulation. We describe the development of a genetically encoded fluorescent biosensor for directly monitoring free NAD(+) concentrations in subcellular compartments.

miR-218 is essential to establish motor neuron fate as a downstream effector of Isl1-Lhx3.

While microRNAs have emerged as an important component of gene regulatory networks, it remains unclear how microRNAs collaborate with transcription factors in the gene networks that determines neuronal cell fate. Here we show that in the developing spinal cord, the expression of miR-218 is directly upregulated by the Isl1-Lhx3 complex, which drives motor neuron fate. Inhibition of miR-218 suppresses the generation of motor neurons in both chick neural tube and mouse embryonic stem cells, suggesting that miR-218 plays a crucial role in motor neuron differentiation.

Novel primate miRNAs coevolved with ancient target genes in germinal zone-specific expression patterns.

Major nonprimate-primate differences in cortico-genesis include the dimensions, precursor lineages, and developmental timing of the germinal zones (GZs). microRNAs (miRNAs) of laser-dissected GZ compartments and cortical plate (CP) from embryonic E80 macaque visual cortex were deep sequenced. The CP and the GZ including ventricular zone (VZ) and outer and inner subcompartments of the outer subventricular zone (OSVZ) in area 17 displayed unique miRNA profiles.

MicroRNA-134 activity in somatostatin interneurons regulates H-Ras localization by repressing the palmitoylation enzyme, DHHC9.

MicroRNA-134 (miR-134) serves as a widely accepted model for microRNA function in synaptic plasticity. In this model, synaptic activity stimulates miR-134 expression, which then regulates dendrite growth and spine formation. By using a ratiometric microRNA sensor, we found, unexpectedly, that miR-134 activity in cortical neurons was restricted to interneurons.

Comparative phosphoproteomics reveals components of host cell invasion and post-transcriptional regulation during Francisella infection.

Francisella tularensis is a facultative intracellular bacterium that causes the deadly disease tularemia. Most evidence suggests that Francisella is not well recognized by the innate immune system that normally leads to cytokine expression and cell death. In previous work, we identified new bacterial factors that were hyper-cytotoxic to macrophages.

Capturing microRNA targets using an RNA-induced silencing complex (RISC)-trap approach.

Identifying targets is critical for understanding the biological effects of microRNA (miRNA) expression. The challenge lies in characterizing the cohort of targets for a specific miRNA, especially when targets are being actively down-regulated in miRNA- RNA-induced silencing complex (RISC)-messengerRNA (mRNA) complexes. We have developed a robust and versatile strategy called RISCtrap to stabilize and purify targets from this transient interaction.

MicroRNA pathways in neural development and plasticity.

MicroRNAs contribute significantly to the development, survival, function, and plasticity of neurons. They silence expression of target genes by reducing mRNA stability and translation. Production of microRNAs is controlled via developmental and environmental cues and these small molecules, in concert with classical transcriptional regulators, amplify changes in neuronal maturation, dendrite morphogenesis, and synaptogenesis.