High levels of Tryptophan reduce cell wall or membrane stress tolerance in Saccharomyces cerevisiae.

Tryptophan (Trp) is a proteinogenic aromatic amino acid; however, high levels of Trp are toxic in animals and yeast with unknown mechanisms. Previously, we suggested that aromatic aminotransferase Aro9 is important for excess Trp degradation. Besides, Schroeder and Ikui showed that aro9Δ is sensitive to membrane stress by sodium dodecyl sulfate. Therefore, Trp accumulation may reduce the cell wall or membrane (CW/M) stress tolerance through participation of cell wall integrity (CWI) pathway, which detects and responds to CW/M perturbations. In this study, we found that yeast mutants of the CWI mitogen-activated protein kinase cascade were susceptible to excess Trp. Also, the Trp degradation deficient mutant aro8Δ aro9Δ cells, in which Trp accumulation was confirmed, were sensitive to several CW/M stresses. These results indicated that accumulation of Trp is adverse for the CW/M stress resistance and may disturb appropriate signal transduction responding to the stress.

Metabolomic Analysis of Yeast and Human Cells: Latest Advances and Challenges.

Liquid chromatography-mass spectrometry (LC-MS) based nontargeted metabolomics has been applied to a wide range of biological samples and can provide information on thousands of compounds. However, reliable identification of the compounds remains a challenge affecting result interpretation. In this protocol, we describe comparable yeast cell and whole blood metabolome sample preparation for extracting similar compound groups, and we present a LC-MS method using the all ion fragmentation (AIF) approach for the purposes of increasing accuracy in metabolite annotation. Our method enables database-dependent targeted as well as nontargeted metabolomics analysis from the same data acquisition, while simultaneously improving the accuracy in metabolite identification to increase the quality of the resulting biological information.

Structural Studies of Mammalian Autophagy Lipidation Complex.

Members of the autophagy-related protein 8 (Atg8) family of ubiquitin-like proteins (ublps), including mammalian LC3 and GABARAP proteins, play crucial roles in autophagosome biogenesis, as well as selective autophagy. Upon induction of autophagy, the autophagic ublps are covalently attached to a phosphatidylethanolamine (PE) molecule of the autophagosomal membrane. This unique lipid conjugation of the autophagic ublps, which is essential for their functions, occurs in a ubiquitination-like reaction cascade consisting of the E1 enzyme ATG7, the E2 ATG3, and the E3 ATG12~ATG5-ATG16L1 complex (~denotes a covalent linkage). These enzymes are structurally unique among those of the canonical ubiquitination cascades, necessitating structural and biochemical studies of these molecules for understanding the molecular mechanisms underlying the lipidation cascade. Here, we will describe methods that were employed in our previous studies (Otomo et al., Nat Struct Mol Biol 20:59-66, 2013; Metlagel et al., Proc Natl Acad Sci U S A 110:18844-18849, 2013; Ohashi and Otomo, Biochem Biophys Res Commun 463:447-452, 2015), including the production of recombinant enzymes, in vitro enzymatic reactions, the crystallization of the E3 complexes, and the NMR-based investigations of E1-E2 and E2-E3 interactions.

Insights into autophagosome biogenesis from structural and biochemical analyses of the ATG2A-WIPI4 complex.

Autophagy is an enigmatic cellular process in which double-membrane compartments, called "autophagosomes, form de novo adjacent to the endoplasmic reticulum (ER) and package cytoplasmic contents for delivery to lysosomes. Expansion of the precursor membrane phagophore requires autophagy-related 2 (ATG2), which localizes to the PI3P-enriched ER-phagophore junction. We combined single-particle electron microscopy, chemical cross-linking coupled with mass spectrometry, and biochemical analyses to characterize human ATG2A in complex with the PI3P effector WIPI4. ATG2A is a rod-shaped protein that can bridge neighboring vesicles through interactions at each of its tips. WIPI4 binds to one of the tips, enabling the ATG2A-WIPI4 complex to tether a PI3P-containing vesicle to another PI3P-free vesicle. These data suggest that the ATG2A-WIPI4 complex mediates ER-phagophore association and/or tethers vesicles to the ER-phagophore junction, establishing the required organization for phagophore expansion via the transfer of lipid membranes from the ER and/or the vesicles to the phagophore.

Kynurenine aminotransferase activity of Aro8/Aro9 engage tryptophan degradation by producing kynurenic acid in Saccharomyces cerevisiae.

Kynurenic acid (KA) is a tryptophan (Trp) metabolite that is synthesised in a branch of kynurenine (KYN) pathway. KYN aminotransferase (KAT) catalyses deamination of KYN, yielding KA. Although KA synthesis is evolutionarily conserved from bacteria to humans, the cellular benefits of synthesising KA are unclear. In this study, we constructed a KAT-null yeast mutant defective in KA synthesis to clarify the cellular function of KA. Amino acid sequence analysis and LC/MS quantification of KA revealed that Aro8 and Aro9 are the major KATs. KA was significantly decreased in the aro8Δ aro9Δ double mutant. We found that aro8Δ aro9Δ cells did not exhibit obvious defects in growth or oxidative stress response when proper amounts of amino acids are supplied in the media. We further found that aro8Δ aro9Δ cells were sensitive to excess Trp. The Trp sensitivity was not rescued by addition of KA, suggesting that Trp sensitivity is not due to the loss of KA. In conclusion, we propose that KAT activity is required for detoxification of Trp by converting it to the less toxic KA.

Identification and characterization of the linear region of ATG3 that interacts with ATG7 in higher eukaryotes.

Transfer of GABARAP thioester from the E1 ATG7 to the E2 ATG3 requires the interaction between the N-terminal domain of ATG7 and the flexible region (FR) of ATG3. This interaction has been visualized in the yeast Atg7-Atg3 complex crystal structure, but remains to be defined in higher eukaryotes. Here, our NMR data precisely define the region of the FR of human ATG3 that interacts with ATG7 (RIA7) and demonstrate RIA7 partially overlaps with the E3-interacting region, explaining how the E1-E2 and E2-E3 interactions are mutually exclusive. Mutational analyses identify critical residues of the RIA7 for the E1 interaction and GABARAP transfer, advancing our understanding of a molecular mechanism of the autophagic conjugation cascade in higher eukaryotes.

Structural insights into E2-E3 interaction for LC3 lipidation.

The members of the LC3/Atg8 family of proteins are covalently attached to phagophore and autophagosomal membranes. At the last step of the LC3 lipidation cascade, LC3 is transferred from the E2 enzyme ATG3 to phosphatidylethanolamine (PE). This transfer is stimulated by the ATG12-ATG5-ATG16L1 E3 complex, but the mechanism is not fully understood. We recently found that ATG12 of the E3 binds to a short sequence in the flexible region (FR) of ATG3 with high affinity, and that this interaction is critical for E2-E3 complex formation. These findings, together with detailed structural analyses of this interaction, define the properties of ATG12 and provide new insights of how LC3 transfer begins with ATG3 recruitment by ATG12.

Bacterial pyruvate production from alginate, a promising carbon source from marine brown macroalgae.

Marine brown macroalgae is a promising source of material for biorefining, and alginate is one of the major components of brown algae. Despite the huge potential availability of alginate, no system has been reported for the production of valuable compounds other than ethanol from alginate, hindering its further utilization. Here we report that a bacterium, Sphingomonas sp. strain A1, produces pyruvate from alginate and secretes it into the medium. High aeration and deletion of the gene for d-lactate dehydrogenase are critical for the production of high concentrations of pyruvate. Pyruvate concentration and productivity were at their maxima (4.56 g/l and 95.0 mg/l/h, respectively) in the presence of 5% (w/v) initial alginate, whereas pyruvate produced per alginate consumed and % of theoretical yield (0.19 g/g and 18.6%, respectively) were at their maxima at 4% (w/v) initial alginate. Concentration of pyruvate decreased after it reached its maximum after cultivations for 2 or 3 days at 145 strokes per minute. Our study is the first report to demonstrate the production of other valuable compounds than ethanol from alginate, a promising marine macroalgae carbon source.

Secretion of quinolinic acid, an intermediate in the kynurenine pathway, for utilization in NAD+ biosynthesis in the yeast Saccharomyces cerevisiae.

NAD(+) is synthesized from tryptophan either via the kynurenine (de novo) pathway or via the salvage pathway by reutilizing intermediates such as nicotinic acid or nicotinamide ribose. Quinolinic acid is an intermediate in the kynurenine pathway. We have discovered that the budding yeast Saccharomyces cerevisiae secretes quinolinic acid into the medium and also utilizes extracellular quinolinic acid as a novel NAD(+) precursor. We provide evidence that extracellular quinolinic acid enters the cell via Tna1, a high-affinity nicotinic acid permease, and thereby helps to increase the intracellular concentration of NAD(+). Transcription of genes involved in the kynurenine pathway and Tna1 was increased, responding to a low intracellular NAD(+) concentration, in cells bearing mutations of these genes; this transcriptional induction was suppressed by supplementation with quinolinic acid or nicotinic acid. Our data thus shed new light on the significance of quinolinic acid, which had previously been recognized only as an intermediate in the kynurenine pathway.

Identification and characterization of a human mitochondrial NAD kinase.

NAD kinase is the sole NADP(+) biosynthetic enzyme. Despite the great significance of NADP(+), to date no mitochondrial NAD kinase has been identified in human, and the source of human mitochondrial NADP(+) remains elusive. Here we present evidence demonstrating that a human protein of unknown function, C5orf33, is a human mitochondrial NAD kinase; this protein likely represents the missing source of human mitochondrial NADP(+). The C5orf33 protein exhibits NAD kinase activity, utilizing ATP or inorganic polyphosphate, and is localized in the mitochondria of human HEK293A cells. C5orf33 mRNA is more abundant than human cytosolic NAD kinase mRNA in almost all tissues examined. We further show by database searches that some animals and protists carry C5orf33 homologues as their sole NADP(+) biosynthetic enzyme, whereas plants and fungi possess no C5orf33 homologue. These observations provide insights into eukaryotic NADP(+) biosynthesis, which has pivotal roles in cells and organelles.

Structural determinants of discrimination of NAD+ from NADH in yeast mitochondrial NADH kinase Pos5.

NAD kinase catalyzes the phosphorylation of NAD(+) to synthesize NADP(+), whereas NADH kinase catalyzes conversion of NADH to NADPH. The mitochondrial protein Pos5 of Saccharomyces cerevisiae shows much higher NADH kinase than NAD kinase activity and is therefore referred to as NADH kinase. To clarify the structural determinant underlying the high NADH kinase activity of Pos5 and its selectivity for NADH over NAD(+), we determined the tertiary structure of Pos5 complexed with NADH at a resolution of 2.0 Å. Detailed analysis, including a comparison of the tertiary structure of Pos5 with the structures of human and bacterial NAD kinases, revealed that Arg-293 of Pos5, corresponding to His-351 of human NAD kinase, confers a positive charge on the surface of NADH-binding site, whereas the corresponding His residue does not. Accordingly, conversion of the Arg-293 into a His residue reduced the ratio of NADH kinase activity to NAD kinase activity from 8.6 to 2.1. Conversely, simultaneous changes of Ala-330 and His-351 of human NAD kinase into Ser and Arg residues significantly increased the ratio of NADH kinase activity to NAD kinase activity from 0.043 to 1.39; human Ala-330 corresponds to Pos5 Ser-272, which interacts with the side chain of Arg-293. Arg-293 and Ser-272 were highly conserved in Pos5 homologs (putative NADH kinases), but not in putative NAD kinases. Thus, Arg-293 of Pos5 is a major determinant of NADH selectivity. Moreover, Ser-272 appears to assist Arg-293 in achieving the appropriate conformation.

NADPH regulates human NAD kinase, a NADP⁺-biosynthetic enzyme.

NAD kinase (NADK, EC 2.7.1.23) is the sole NADP(+)-biosynthetic enzyme that catalyzes phosphorylation of NAD(+) to yield NADP(+) using ATP as a phosphoryl donor, and thus, plays a vital role in the cell and represents a potentially powerful antimicrobial drug target. Although methods for expression and purification of human NADK have been previously established (Lerner et al. Biochem Biophys Res Commun 288:69-74, 2001), the purification procedure could be significantly improved. In this study, we improved the method for expression and purification of human NADK in Escherichia coli and obtained a purified homogeneous enzyme only through heat treatment and single column chromatography. Using the purified human NADK, we revealed a sigmoidal kinetic behavior toward ATP and the inhibitory effects of NADPH and NADH, but not of NADP(+), on the catalytic activity of the enzyme. These inhibitory effects provide insight into the regulation of intracellular NADPH synthesis. Furthermore, these attributes may provide a clue to design a novel drug against Mycobacterium tuberculosis in which this bacterial NADK is potently inhibited by NADP(+).

Roles of kinin B1 and B2 receptors in skin cancer pain produced by orthotopic melanoma inoculation in mice.

BACKGROUND: Although bradykinin is a potent algogenic peptide, the roles of this peptide and kinin receptors in cancer pain are unclear. AIMS: The present study was conducted to clarify whether kinin B(1) and B(2) receptors would be involved in pain using a mouse model of skin cancer pain. METHODS: B16-BL6 melanoma cells were inoculated into the hind paw of C57BL/6 mice. Licking, an index of spontaneous pain, allodynia and hyperalgesia were observed. Expression of kinin receptor mRNAs was analyzed with reverse transcription and polymerase chain reaction. The contents of kininogen and bradykinin-related peptides were assayed with Western blotting and enzyme immunoassay, respectively. RESULTS: Melanoma inoculation induced spontaneous licking of the melanoma-bearing paw from day 18 post-inoculation, which was inhibited by local injections of B(1) and B(2) receptor antagonists. Allodynia was briefly attenuated by B(2), but not B(1) antagonist and hyperalgesia was not inhibited by either B(1) or B(2) antagonist. Local injections of B(1) and B(2) receptor agonists increased licking behavior in melanoma-bearing, but not healthy, paw. The expression of kinin B(1), but not B(2), receptor mRNA was markedly increased in the L4/5 dorsal root ganglia on the melanoma-bearing side. Melanoma cells expressed B(1) and B(2) receptors and kininogen. The content of bradykinin and related peptides was increased in the melanoma mass as compared with healthy skin. CONCLUSIONS: Bradykinin and related peptides released from melanoma cells may cause spontaneous pain and allodynia in the melanoma-bearing paw, in which B(1) and B(2) receptors on primary afferent and melanoma cells may have different roles.