Keeping the balance in NAD metabolism.

Research over the last few decades has extended our understanding of nicotinamide adenine dinucleotide (NAD) from a vital redox carrier to an important signalling molecule that is involved in the regulation of a multitude of fundamental cellular processes. This includes DNA repair, cell cycle regulation, gene expression and calcium signalling, in which NAD is a substrate for several families of regulatory proteins, such as sirtuins and ADP-ribosyltransferases. At the molecular level, NAD-dependent signalling events differ from hydride transfer by cleavage of the dinucleotide into an ADP-ribosyl moiety and nicotinamide. Therefore, non-redox functions of NAD require continuous biosynthesis of the dinucleotide. Maintenance of cellular NAD levels is mainly achieved by nicotinamide salvage, yet a variety of other precursors can be used to sustain cellular NAD levels via different biosynthetic routes. Biosynthesis and consumption of NAD are compartmentalised at the subcellular level, and currently little is known about the generation and role of some of these subcellular NAD pools. Impaired biosynthesis or increased NAD consumption is deleterious and associated with ageing and several pathologies. Insults to neurons lead to depletion of axonal NAD and rapid degeneration, partial rescue can be achieved pharmacologically by administration of specific NAD precursors. Restoring NAD levels by stimulating biosynthesis or through supplementation with precursors also produces beneficial therapeutic effects in several disease models. In this review, we will briefly discuss the most recent achievements and the challenges ahead in this diverse research field.

Subcellular Distribution of NAD+ between Cytosol and Mitochondria Determines the Metabolic Profile of Human Cells.

The mitochondrial NAD pool is particularly important for the maintenance of vital cellular functions. Although at least in some fungi and plants, mitochondrial NAD is imported from the cytosol by carrier proteins, in mammals, the mechanism of how this organellar pool is generated has remained obscure. A transporter mediating NAD import into mammalian mitochondria has not been identified. In contrast, human recombinant NMNAT3 localizes to the mitochondrial matrix and is able to catalyze NAD(+) biosynthesis in vitro. However, whether the endogenous NMNAT3 protein is functionally effective at generating NAD(+) in mitochondria of intact human cells still remains to be demonstrated. To modulate mitochondrial NAD(+) content, we have expressed plant and yeast mitochondrial NAD(+) carriers in human cells and observed a profound increase in mitochondrial NAD(+). None of the closest human homologs of these carriers had any detectable effect on mitochondrial NAD(+) content. Surprisingly, constitutive redistribution of NAD(+) from the cytosol to the mitochondria by stable expression of the Arabidopsis thaliana mitochondrial NAD(+) transporter NDT2 in HEK293 cells resulted in dramatic growth retardation and a metabolic shift from oxidative phosphorylation to glycolysis, despite the elevated mitochondrial NAD(+) levels. These results suggest that a mitochondrial NAD(+) transporter, similar to the known one from A. thaliana, is likely absent and could even be harmful in human cells. We provide further support for the alternative possibility, namely intramitochondrial NAD(+) synthesis, by demonstrating the presence of endogenous NMNAT3 in the mitochondria of human cells.

Immunoaffinity purification of the functional 20S proteasome from human cells via transient overexpression of specific proteasome subunits.

The proteasome is a multi-subunit proteolytic complex that plays a central role in protein degradation in all eukaryotic cells. It regulates many vital cellular processes therefore its dysfunction can lead to various pathologies including cancer and neurodegeneration. Isolation of enzymatically active proteasomes is a key step to the successful study of the proteasome regulation and functions. Here we describe a simple and efficient protocol for immunoaffinity purification of the functional 20S proteasomes from human HEK 293T cells after transient overexpression of specific proteasome subunits tagged with 3xFLAG. To construct 3xFLAG-fusion proteins, DNA sequences encoding the 20S proteasome subunits PSMB5, PSMA5, and PSMA3 were cloned into mammalian expression vector pIRES-hrGFP-1a. The corresponding recombinant proteins PSMB5-3xFLAG, PSMA5-3xFLAG, or PSMA3-3xFLAG were transiently overexpressed in human HEK 293T cells and were shown to be partially incorporated into the intact proteasome complexes. 20S proteasomes were immunoprecipitated from HEK 293T cell extracts under mild conditions using antibodies against FLAG peptide. Isolation of highly purified 20S proteasomes were confirmed by SDS-PAGE and Western blotting using antibodies against different proteasome subunits. Affinity purified 20S proteasomes were shown to possess chymotrypsin- and trypsin-like peptidase activities confirming their functionality. This simple single-step affinity method of the 20S proteasome purification can be instrumental to subsequent functional studies of proteasomes in human cells.

Two-Photon Excited Fluorescence of NADH-Alcohol Dehydrogenase Complex in a Mixture with Bacterial Enzymes.

Thorough study of composition and fluorescence properties of a commercial reagent of active equine NAD-dependent alcohol dehydrogenase expressed and purified from E. coli has been carried out. Several experimental methods: spectral- and time-resolved two-photon excited fluorescence, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, fast protein liquid chromatography, and mass spectrometry were used for analysis. The reagent under study was found to contain also a number of natural fluorophores: free NAD(P)H, NADH-alcohol dehydrogenase, NADPH-isocitrate dehydrogenase, and pyridoxal 5-phosphate-serine hydroxymethyltransferase complexes. The results obtained demonstrated the potential and limitations of popular optical methods as FLIM for separation of fluorescence signals from free and protein-bound forms of NADH, NADPH, and FAD that are essential coenzymes in redox reactions in all living cells. In particular, NADH-alcohol dehydrogenase and NADPH-isocitrate dehydrogenase complexes could not be optically separated in our experimental conditions although fast protein liquid chromatography and mass spectrometry analysis undoubtedly indicated the presence of both enzymes in the molecular sample used. Also, the results of fluorescence, fast protein liquid chromatography, and mass spectrometry analysis revealed a significant contribution of the enzyme-bound coenzyme pyridoxal 5-phosphate to the fluorescence signal that could be separated from enzyme-bound NADH by using bandpass filters, but could effectively mask contribution from enzyme-bound FAD because the fluorescence spectra of the species practically overlapped. It was shown that enzyme-bound pyridoxal 5-phosphate fluorescence can be separated from enzyme-bound NAD(P)H and FAD through analysis of short fluorescence decay times of about tens of picoseconds. However, this analysis was found to be effective only at relatively high number of peak photon counts in recorded fluorescence signals. The results obtained in this study can be used for interpretation of fluorescence signals from a mixture of enzyme-bound fluorophores and should be taken into consideration when determining the intracellular NADH/FAD ratio using FLIM.

Proteomic analysis of the 20S proteasome (PSMA3)-interacting proteins reveals a functional link between the proteasome and mRNA metabolism.

The 26S proteasome is a large multi-subunit protein complex that exerts specific degradation of proteins in the cell. The 26S proteasome consists of the 20S proteolytic particle and the 19S regulator. In order to be targeted for proteasomal degradation most of the proteins must undergo the post-translational modification of poly-ubiquitination. However, a number of proteins can also be degraded by the proteasome via a ubiquitin-independent pathway. Such degradation is exercised largely through the binding of substrate proteins to the PSMA3 (alpha 7) subunit of the 20S complex. However, a systematic analysis of proteins interacting with PSMA3 has not yet been carried out. In this report, we describe the identification of proteins associated with PSMA3 both in the cytoplasm and nucleus. A combination of two-dimensional gel electrophoresis (2D-GE) and tandem mass-spectrometry revealed a large number of PSMA3-bound proteins that are involved in various aspects of mRNA metabolism, including splicing. In vitro biochemical studies confirmed the interactions between PSMA3 and splicing factors. Moreover, we show that 20S proteasome is involved in the regulation of splicing in vitro of SMN2 (survival motor neuron 2) gene, whose product controls apoptosis of neurons.

Compartment-Specific Poly-ADP-Ribose Formation as a Biosensor for Subcellular NAD Pools.

Nicotinamide adenine dinucleotide (NAD) is vital to many cellular processes and is distributed between distinct subcellular pools in the compartmentalized eukaryotic cell. The detection and relative quantification of these individual pools is difficult because of the methods usually applied, which require cell disruption and fractionation.Here, we describe an immunochemical method to visualize and relatively quantify subcellular NAD+ pools, which relies on the NAD+-consuming activity of poly-ADP-ribose polymerase 1 (PARP1). We demonstrate that this system can be readily applied to detect changes in the mitochondrial, Golgi, endoplasmic reticulum, and peroxisomal NAD+ pools.

Polyclonal antibodies against human proteasome subunits PSMA3, PSMA5, and PSMB5.

A proteasome is a multi-subunit protein complex, which plays a central role in ubiquitin-dependent protein degradation in all eukaryotic cells. The 26S proteasome is composed of a catalytic 20S core complex and one or two 19S regulatory complexes. The 20S core complex forms a cylinder consisting of four stacked rings of seven α (PSMA1-7) or ß (PSMB1-7) subunits. Target proteins are degraded in the cavity of the 20S complex due to proteolytic activities of three ß subunits having catalytic sites located on the inner surface of the cylinder. The aim of this study was the generation of polyclonal antibodies against human proteasome subunits PSMA3, PSMA5, and PSMB5 and characterization of their experimental applications. To construct GST-fusion proteins, DNA sequences encoding PSMA3, PSMA5, and PSMB5 were cloned into prokaryotic expression vectors pGEX-5X-1 or pGEX-4T-3. Recombinant proteins GST-PSMA3, GST-PSMA5, and GST-PSMB5 were highly expressed in E. coli BL21 (DE3) cells, purified by glutathione-affinity chromatography and further used for rabbit immunization. The activity and specificity of the obtained antibody-containing sera were evaluated using Western blot analysis and immunoprecipitation. We have shown by Western blot analysis that our anti-PSMA3, anti-PSMA5, and anti-PSMB5 antibodies recognized both recombinant and endogenous proteins from different human cell lines. We have also shown that anti-PSMA3 and anti-PSMA5 sera were able to recognize and immunoprecipitate native forms of both endogenous and overexpressed FLAG-tagged proteins PSMA3 and PSMA5, respectively. Thus, the antibodies generated against PSMA3, PSMA5, and PSMB5 can be used in various experimental applications, including the evaluation of cellular levels of proteasome subunits in cell extracts and affinity purification of the endogenous and/or overexpressed proteasome subunits, which facilitates subsequent analysis of their post-translational modifications as well as protein-protein interactions in vivo.

Evolutionary relationships of Red Jungle Fowl and chicken breeds.

Published results were reassessed and original data are provided regarding the origin and relatedness of four postulated chicken breed lineages, egg-type, game, meat-type and Bantam, to each other and to the basic ancestral species of jungle fowls, Gallus gallus. A system approach was employed concerning the planning of the experiments. One element of the system approach is the choice of the breeds to be compared with G. gallus. These breeds were supposed to represent major evolutionary branches of chickens. Four experiments on genetic relationships were conducted using different estimation criteria including morphological discrete characters, body measurements, biochemical markers, and the activity of serum esterase-1. The greatest similarity was found between G. gallus and the egg-type breeds of Mediterranean roots and/or true Bantams. This fact might testify that the indicated chicken groups occupied earlier stages in the evolution from the wild progenitor to the present biodiversity of chickens in the world.