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The cytoplasmic enzyme, NAD⁺ kinase (ATP: NAD⁺ 2-phosphotransferase, [E.C. 2.7.1.23}) has been characterized and purified from yeast. A continuous fluorescence assay was developed. A purification procedure was developed utilizing NAD⁺-Agarose affinity column chromatography.

The levels of total nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), and their redox states were determined as the function of growth in S. cerevisiae. Cells growing in a medium containing 0.8% glucose exhibit two phases of exponential growth, utilizing glucose and ethanol, respectively. The NAD pool is 50% reduced during both stages of growth while the NADP pool is 67% reduced in glucose growth and 48% reduced in ethanol growth. The NAD/NADP ratio is constant during growth on glucose and a two-fold increase in the NAD/NADP ratio occurs upon exhaustion of glucose. The increased ratio is maintained during growth on ethanol. This alteration in the regulation of the relative levels of NAD and NADP may be due to a change in the regulation of NAD kinase and/or NADP phosphatase activities. These changes may be related to the redox state of the NADP pool.

A protein exhibiting NADH oxidase and NADH peroxidase activity has been isolated from Lactobacillus casei in high purity. Evidence obtained through gel filtration, electrophoresis, and electrofocusing indicates that the two activities are characteristic of a single tetrameric protein with an approximate molecular weight of 240,000.

Nicotinamide adenine dinucleotide (NAD+) is a cofactor in hydride transfer reactions and consumed substrate of several classes of glycohydrolyitc enzymes, including sirtuins. NAD+, its biosynthetic intermediates, breakdown products, and related nucleotides (the NAD metabolome) is altered in many metabolic disorders, such as aging and obesity. Supplementation with the novel NAD+ precursor, nicotinamide riboside (NR), ameliorates these alterations and opposes systemic metabolic dysfunctions in rodent models. Based on the hypothesis that perturbations of the NAD metabolome are both a symptom and cause of metabolic disease, accurate assessment of the abundance of these metabolites is expected to provide insight into the biology of diseases and the mechanism of action of NR in promoting metabolic health. Current quantitative methods, such as HPLC, lack specificity and sensitivity to detect distinct alterations to the NAD metabolome. In this thesis, I developed novel sensitive, accurate, robust liquid chromatography mass spectrometry methodologies to quantify the NAD metabolome and applied these methods to determine the effects of disease states and NR supplementation on NAD+ metabolism. My investigations indicate that NR robustly increases the NAD metabolome, especially NAD+ in a manner kinetically different than any other NAD+ precursor. I provide the first evidence of effective NAD+ supplementation from NR in a healthy, 52 year old human male, suggesting the metabolic promoting qualities of NR uncovered in rodent studies are translatable to humans. During my investigation of NR supplementation, my work establishes an unexpected robust, dramatic increase in deamino–NAD+, NAAD, directly from NR, which I argue could serve as an accessible biomarker for efficacious NAD+ supplementation and the effect of disease upon the NAD metabolome. Lastly, I further establish NR as a general therapeutic against metabolic disorder by detailing its ability to oppose aspects of chronic alcoholism and diabetes mellitus.

Thesis (doctoral)--Rijksuniversiteit te Groningen. / Description based on print version record.

Poly(ADP-ribosyl)ation is a reversible post-translational modification of histones and nuclear proteins rapidly stimulated by DNA damage. Its homeostasis is a dynamic process regulated by the synthesizing enzymes poly(ADP-ribose) polymerases (PARPs) and the degrading enzyme poly(ADP-ribose) glycohydrolase (PARG). PARP-1, the first-discovered and major PARP, has been the focus of many studies aimed at clarifying the biological function of poly(ADP-ribose) (PAR). This abundant nuclear enzyme plays key roles in a variety of cellular processes, including the regulation of chromatin structure, transcription and genomic integrity. Its multifunctionality has made it an attractive and potential target for therapy, as evidenced by the numerous PARP-1 inhibitors currently undergoing clinical trials. The transient nature of PAR, explained by the close coordination between PARP-1 and PARG, has also highlighted the potential of targeting PARG for diseases of inappropriate cell death. A number of obstacles, however, have prevented PARG from being studied as extensively as PARP-1. The extreme sensitivity of PARG to proteases and its insolubility at high concentrations have limited structure-activity relationship analyses and structural studies of PARG, and the unavailability of high-throughput activity assays has stalled the discovery and development of specific and cell permeable PARG inhibitors, subsequently slowing down the validation of PARG as a therapeutic target. The work presented in this dissertation describes in detail strategies devised to overcome these difficulties. First, a novel colorimetric high-throughput assay for PARG was evaluated and its sensitivity and precision were compared to a widely-used radiolabelling assay. Second, several expression and purification systems were constructed in order to obtain high quantities of soluble human PARG protein adequate for in vitrostructural studies. The efficacy of these strategies was demonstrated in structure-activity analyses of PARG which led to the identification of a regulatory segment far removed linearly from the catalytic site of PARG. This region, necessary for catalytic activity, corresponds with a recently identified mitochondrial targeting sequence (MTS) and was thus named the ‘regulatory segment/MTS’ (REG/MTS). Finally, based on structural data obtained, secondary structure predictions were made to provide insight into the molecular composition of the different domains of PARG, whose structures still remain to be determined.

The F420-dependent NADP reductase of Methanobrevibacter smithii has been partially purified employing a combination of affinity chromatography with Blue Sepharose (Cl-6B) and molecular sieve chromatography with Sephacryl S-200, The enzyme, which requires reduced F420 as an electron donor, has been purified over 145 fold with a recovery of 6%. A molecular weight of 120,00 for the native enzyme was determined by Sephacryl S-200 chromatography. A subunit molecular weight of 28,200 was determined by SDS-PAGE, indicating that the native enzyme is a tetramer. The optimal temperature for enzymatic activity was found to be 45°C, with a pH optimum of 7.5. The NADP reductase had an apparent Km of 42 uM for reduced F420, and an apparent Km of 4l uM for NADP. The enzyme was stable in 0.05 M sodium phosphate buffer (plus 10 mM cysteine) at pH 7.0, when gassed with nitrogen or hydrogen and stored at 4°C.

O balanço redox em mitocôndrias de mamíferos é realizado pelo transportador de aspartato-glutamato (AGC), o qual é o principal mecanismo para o movimento de equivalentes redutores na forma de NADH. A citrulinemia do tipo II (CTLN2) é uma doença autossômica recessiva de início tardio, causada por mutações no gene SLC25A13 que codifica a citrina. A citrina é uma isoforma do transportador AGC e catalisa o transporte de glutamato citosólico através da troca com o aspartato mitocondrial, o qual será utilizado no ciclo da ureia. A CTLN2 promove uma deficiência no ciclo da ureia e consequente hiperamonemia. A deficiência da citrina promove um aumento da razão NADH/NAD+ citosólica. O aumento dessa razão inibe a glicólise e a gliconeogênese. O desenvolvimento de modelo in vitro da CTLN2 é importante para estudos do mecanismo da doença e de novas terapias. A expressão heteróloga de proteínas entre diferentes reinos tem sido utilizado como uma forma de corrigir algumas doenças mitocondriais. Estudos bioquímicos e moleculares em nosso laboratório demonstraram a presença de um transportador mitocondrial de nicotinamida adenina dinucleotídeo (Ndt1) em Aspergillus fumigatus. Ndt1 realiza o transporte de NAD+ citosólico para a matriz mitocondrial, sendo dessa forma uma proteína importante para manter o balanço redox em A. fumigatus. Assim, o objetivo deste trabalho foi obter uma linhagem de células de mamífero HEK293 com knockdown para o gene SLC25A13, ou seja, um modelo in vitro de CTLN2 e a expressão heteróloga da proteína Ndt1 como uma forma de recuperação do metabolismo. As células com knockdown para o gene SLC25A13 apresentaram um aumento da razão NADH/NAD+ citosólico, redução da glicólise, redução da concentração da ureia e aumento da concentração de amônia. A expressão de Ndt1 foi capaz de reduzir a razão NADH/NAD+ citosólico e recuperou a atividade glicolítica. Entretanto, a expressão de Ndt1 não foi capaz de aumentar a concentração de ureia e reduzir a concentração de amônia causadas pela CTLN2. Dessa forma, nossos resultados sugerem que a expressão da proteína Ndt1 em células de mamíferos recupera o metabolismo mitocondrial e atividade glicolítica das células com CTLN2, mas não melhora o ciclo da ureia e o aumento da concentração de amônia. / Redox balance in mammalian mitochondria is performed by the aspartate-glutamate carrier (AGC), which is the main mechanism for the movement of reducing equivalents in the form of NADH.Type II citrullinemia (CTLN2) is an adult-onset autosomal recessive disease caused by mutations in SLC25A13 gene, and that coding citrin. Citrin is an isoform of AGC and catalyzes the transport of cytosolic glutamate through exchange with mitochondrial aspartate. It will be used in the urea cycle. CTLN2 causes urea cycle deficiency and hyperammonemia. Citrin deficiency cause an increase in the cytosolic NADH/NAD+ ratio. The increase in this ratio inhibits glycolysis and gluconeogenesis. The development an in vitro CTLN2 model is important for new studies about the disease mechanism and new therapies. Heterologous expression of proteins from different organisms has been used to recover some mitochondrial diseases. Biochemical and molecular studies in our laboratory demonstrated the presence of a mitochondrial nicotinamide adenine dinucleotide transporter (Ndt1) in Aspergillus fumigatus. Ndt1 protein performs cytosolic NAD+ transport to the mitochondria matrix, thus being an important protein to keep the redox balance in A. fumigatus. Thus, the aim of this work was to obtain a line of HEK293 mammalian cells with knockdown for the SLC25A13 gene, an in vitro CTLN2 model and the heterologous expression of the Ndt1 protein as a form of metabolism recovery. Cells with citrin knockdown showed an increase of cytosolic NADH/NAD+ ratio, reduction of glycolysis, reduction of urea concentration, and increase of ammonia concentration. Expression of Ndt1 protein was able to reduce cytosolic NADH/NAD+ ratio and recovered the glycolytic activity. However, Ndt1 protein was not able to increase the urea concentration and reduce of ammonia concentration caused by CTLN2. Thus, our results suggest that expression of Ndt1 protein in mammal cells recovers the mitochondrial metabolism and glycolytic activity in CTLN2 cells but does not improve urea cycle and reduce ammonia concentration.

O A. fumigatus é um fungo saprofítico e tornou-se um dos principais agente patogênico oportunista em pacientes imunossuprimidos. Estudos prévios em nosso laboratório foi demonstrado que em mitocôndrias de P. brasiliensis e de A. fumigatus, o NAD+ era capaz de induzir a formação de potencial de membrana mitocondrial, o qual podia ser dissipado por FCCP, sugerindo a presença de um transportador de NAD+/NADH, conforme havia sido descrito em S. cerevisiae. Através de ferramentas de bioinformática, foi identificado no Aspergillus Gene Database, uma sequência com 32% de identidade com o gene ndt1p de S. cerevisiae. A sequência de cDNA, contendo 1.194 pb foi obtida usando PCR-Overlaping e clonada em vetor pGEM®-T Easy. Em seguida, a sequência foi subclonada em vetor de expressão pET28-a(+) e expressa em E. coli BL21(DE3). A proteína recombinante foi purificada a partir dos corpos de inclusão e sua identidade confirmada por espectrometria de massas e por Western Blotting usando anticorpo anti-His-tag. A proteína recombinante foi utilizada para produção de anticorpo policlonal anti-Ndt1 em coelho. Para expressão em levedura, o cDNA do gene ndt1 de A. fumigatus foi subclonado em vetor pYES2 e as leveduras S. cerevisiae ?ndt1?ndt2 foram transformadas. Foi realizada a curva de crescimento e indução da expressão da proteína recombinante Ndt1, a presença da proteína foi detectada utilizando anticorpo policlonal anti-Ndt1 após 16 horas de expressão. Nesse período foi verificado que as leveduras estavam em fase de crescimento exponencial. A cepa duplo mutante apresenta uma taxa de crescimento menor quando comparada com a cepa expressando a proteína recombinante quando crescidas em meio fermentável. As mitocôndrias isoladas de ambas as cepas foram submetidas à medida do potencial de membrana onde apresentavam acoplamento entre a oxidação de substratos e a fosforilação oxidativa. Além disso, ficou evidenciado que na cepa expressando a proteína recombinante, NAD+ induziu a formação de um potencial de membrana maior que na cepa controle. O transporte de NAD+ foi realizado e demonstrou que a cepa expressando a proteína Ndt1 tinha um aumento na fluorescência de NADH, mostrando que NAD+ foi capaz de entrar na matriz mitocondrial e posteriormente ser reduzido a NADH por enzimas da matriz mitocondrial. A determinação da produção de espécies reativas de oxigênio foi realizada utilizando as sondas fluorescentes CM-H2DCFDA e MitoSox Red em esferoplastos da levedura S. cerevisiae. Ambos experimentos não houve diferença significativa entre a cepa expressando a proteína Ndt1 e a cepa controle. As proteínas carboniladas foram determinadas utilizando anticorpo anti-DNP, após a reação com dinitrofenilhidrazona e não há diferença significativa entre as cepas. Finalmente, para confirmação da localização celular da proteína Ndt1, os esferoplastos de S. cerevisiae foram submetidos à microscopia confocal, onde ficou evidenciado a co-localização da proteína Ndt1 com as mitocôndrias na cepa de S. cerevisiae transformada com a construção pYES/ndt1, o mesmo perfil não foi observado na cepa ?ndt1?ndt2. / The A. fumigatus is a saprophytic fungus and is a major opportunistic pathogen in immunosuppressed patients. Previous studies in our lab has showed that mitochondrias of the P. brasiliensis and A. fumigatus, NAD+ is able to induce the formation of mitochondrial membrane potential, which could be dissipated by FCCP, suggesting the presence of a NAD+/NADH carrier as described in S. cerevisiae. Using bioinformatics tools, it was identified in Aspergillus Gene database a sequence containing 32% of identity with the gene ndt1p of S. cerevisiae. A fragment of cDNA was obtained from the ndt1p mRNA sequence, containing 1194 bp by using PCR-overlaping and cloned in pGEM®-T Easy vector. Then, the sequence was subcloned in pET28-a(+) vector and expressed in E. coli BL21 (DE3). The recombinant protein was purified from inclusion bodies and the identity confirmed by mass spectrometry and by Western Blotting using anti-His-tag antibody. The recombinant protein was used to produce polyclonal antibody anti-Ndt1 in rabbit. For expression in yeast, the ndt1 cDNA of A. fumigatus was subcloned into pYES2 vector and the yeast S. cerevisiae ?ndt1?ndt2 were transformed. Growth curve and induction of the recombinant protein expression Ndt1 was performed and the protein was detected using a polyclonal anti-Ndt1 antibody after 16 hours of expression. During this period it was found that the yeast were in exponential growth phase. The double mutant strain shows a slower growth rate compared to the strain expressing the recombinant protein growth rate when grown in fermentable medium. The isolated mitochondria from both strains were subjected to measurement of the membrane potential which showed coupling between substrate oxidation and oxidative phosphorylation. Furthermore, these measurements evidenced that the strain expressing the recombinant protein NAD+ induced the formation of a membrane potential larger than the control strain. The transport NAD+ was evaluated and showed that the strain expressing the protein Ndt1 has an increase in NADH fluorescence, indicating that NAD+ was able to enter into the mitochondrial matrix and then reduced to NADH by enzymes from the matrix. The determination of the production of reactive oxygen species was performed using the fluorescent probe CM-H2DCFDA and MitoSox Red in spheroplasts of the yeast S. cerevisiae. Both experiments showed no significant difference between the strain expressing Ndt1 protein and strain control. The carbonylated proteins levels were checked using anti-DNP, after the reaction with dinitrophenylhydrazone and there is no significant difference between the strains. Finally, to confirm the cellular localization of the protein Ndt1, spheroplasts of S. cerevisiae were subjected to confocal microscopy, which evidenced the co-location of Ndt1 protein with mitochondria in S. cerevisiae strain transformed with the construction pYES/ndt1. This same profile was not observed in strain ?ndt1?ndt2.