Fission yeast TOR complex 1 phosphorylates Psk1 through an evolutionarily conserved interaction mediated by the TOS motif.

TOR complex 1 (TORC1) is a multi-subunit protein kinase complex that controls cellular growth in response to environmental cues. The regulatory subunits of mammalian TORC1 (mTORC1) include RAPTOR (also known as RPTOR), which recruits mTORC1 substrates, such as S6K1 (also known as RPS6KB1) and 4EBP1 (EIF4EBP1), by interacting with their TOR signaling (TOS) motif. Despite the evolutionary conservation of TORC1, no TOS motif has been described in lower eukaryotes. In the present study, we show that the fission yeast S6 kinase Psk1 contains a TOS motif that interacts with Mip1, a RAPTOR ortholog. The TOS motif in Psk1 resembles those in mammals, including the conserved phenylalanine and aspartic acid residues essential for the Mip1 interaction and TORC1-dependent phosphorylation of Psk1. The binding of the TOS motif to Mip1 is dependent on Mip1 Tyr-533, whose equivalent in RAPTOR is known to interact with the TOS motif in their co-crystals. Furthermore, we utilized the mip1-Y533A mutation to screen the known TORC1 substrates in fission yeast and successfully identified Atg13 as a novel TOS-motif-containing substrate. These results strongly suggest that the TOS motif represents an evolutionarily conserved mechanism of the substrate recognition by TORC1.

Acteoside from Conandron ramondioides Reduces Microcystin-LR Cytotoxicity by Inhibiting Intracellular Uptake Mediated by OATP1B3.

The hepatotoxin microcystin-LR is a strong inhibitor of serine/threonine protein phosphatase (PP) 1 and PP2A. The onset of its cytotoxicity depends on its selective uptake via the hepatocyte uptake transporters, organic anion transporting polypeptide (OATP) 1B1 and OATP1B3. Understanding and preventing the cytotoxicity of microcystin-LR is crucial to maintain human health. This chemoprevention study demonstrates that the herbal plant extract of iwajisha (20 µg/mL) reduced microcystin-LR cytotoxicity in OATP1B3-expressing cells by approximately six times. In addition, 20 µM acteoside, which is one of the major compounds in iwajisha, reduced microcystin-LR cytotoxicity by approximately 7.4 times. Acteoside could also reduce the cytotoxicity of other compounds, such as okadaic acid and nodularin, which are both substrates of OATP1B3 and inhibitors of PP1/PP2A. To investigate the mechanism by which the cytotoxicity of microcystin-LR is attenuated by acteosides, microcystin-LR and microcystin-LR-binding proteins in cells were examined after microcystin-LR and acteosides were co-exposed. Thus, acteoside noncompetitively inhibited microcystin-LR uptake by OATP1B3-expressing cells. Furthermore, acteoside inhibited the intracellular interaction of microcystin-LR with its binding protein(s), including the 22 kDa protein. Furthermore, using immunoblot analysis, acteoside induced the phosphorylation of extracellular signal-regulated kinase (ERK), which is one of the survival signaling molecules. These results suggest that acteoside reduces microcystin-LR cytotoxicity through several mechanisms, including the inhibition of microcystin-LR uptake via OATP1B3, and decreased interaction between microcystin-LR and its binding protein(s), and that ERK signaling activation contributes to the attenuation effect of acteoside against microcystin-LR cytotoxicity.

Alteration of the neuronal and glial cell profiles in Neu1-deficient zebrafish.

Neu1 is a glycosidase that releases sialic acids from the non-reducing ends of glycoconjugates, and its enzymatic properties are conserved among vertebrates. Recently, Neu1-KO zebrafish were generated using genome editing technology, and the KO fish showed abnormal emotional behavior, such as low schooling, low aggressiveness, and excess exploratory behavior, accompanied by the downregulation of anxiety-related genes. To examine the alteration of neuronal and glial cells in Neu1-KO zebrafish, we analyzed the molecular profiles in the zebrafish brain, focusing on the midbrain and telencephalon. Using immunohistochemistry, we found that signals of Maackia amurensis (MAM) lectin that recognizes Sia α2-3 linked glycoconjugates were highly increased in Neu1-KO zebrafish brains, accompanied by an increase in Lamp1a. Neu1-KO zebrafish suppressed the gene expression of AMPA-type glutamate receptors such as gria1a, gria2a, and gria3b, and vesicular glutamate transporter 1. Additionally, Neu1-KO zebrafish induced the hyperactivation of astrocytes accompanied by an increase in Gfap and phosphorylated ERK levels, while the mRNA levels of astrocyte glutamate transporters (eaat1a, eaat1c, and eaat2) were downregulated. The mRNA levels of sypb and ho1b, which are markers of synaptic plasticity, were also suppressed by Neu1 deficiency. Abnormal activity of microglia was also revealed by IHC, and the expressions of iNOS and IL-1ß, an inflammatory cytokine, were increased in Neu1-KO zebrafish. Furthermore, drastic neuronal degeneration was detected in Neu1-KO zebrafish using Fluoro-Jade B staining. Collectively, the neuronal and glial abnormalities in Neu1-KO zebrafish may be caused by changes in the excitatory neurotransmitter glutamate and involved in the emotional abnormalities.

Enhancement of Edwardsiella piscicida infection, biofilm formation, and motility caused by N-acetylneuraminate lyase.

Sialic acid and its catabolism are involved in bacterial pathogenicity. N-acetylneuraminate lyase (NAL), which catalyzes the reversible aldol cleavage of sialic acid to form N-acetyl-D-mannosamine in the first step of sialic acid degradation, has been recently investigated to elucidate whether NAL enhances bacterial virulence; however, the role of NAL in bacterial pathogenicity remains unclear. In the present study, we demonstrated that the existence of two enzymes in Edwardsiella piscicida, referred to as dihydrodipicolinate synthase (DHDPS) and NAL, induced the cleavage/condensation activity toward sialic acids such as N-acetylneuraminic acid, N-glycolylneuraminic acid and 3-deoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid. NAL enhanced cellular infection in vitro and suppressed the survival rate in zebrafish larvae in bath-infection in vivo, whereas DHDPS did not. Furthermore, NAL strongly activated the expression of E. piscicida phenotypes such as biofilm formation and motility, whereas DHDPS did not. Besides, the gene expression level of nanK, nanE, and glmU were up-regulated in the NAL-overexpressing strain, along with an increase in the total amount of N-acetylglucosamine.

Host lactosylceramide enhances Edwardsiella tarda infection.

Edwardsiella tarda is a Gram-negative bacterium causing economic damage in aquaculture. The interaction of E. tarda with microdomains is an important step in the invasion, but the target molecules in microdomains remain undefined. Here, we found that intraperitoneal injection of E. tarda altered splenic glycosphingolipid patterns in the model host medaka (Oryzias latipes) accompanied by alteration of glycosphingolipid metabolism-related gene expressions, suggesting that glycosphingolipid levels are involved in E. tarda infection. To ascertain the significance of glycosphingolipids in the infection, fish cell lines, DIT29 cells with a high amount of lactosylceramide (LacCer) and glucosylceramide (GlcCer), and GAKS cells with a low amount of these lipids, were treated with methyl-ß-cyclodextrin to disrupt the microdomain. E. tarda infection was suppressed in DIT29 cells, but not in GAKS cells, suggesting the involvement of microdomain LacCer and GlcCer in the infection. DL-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol, an inhibitor of glycosphingolipid-synthesis, attenuated the infection in DIT29 cells, while Neu3-overexpressing GAKS cells, which accumulated LacCer, enhanced the infection. E. tarda possessed binding ability towards LacCer, but not GlcCer, and LacCer preincubation declined the infection towards fish cells, possibly due to the masking of binding sites. The present study suggests that LacCer may be a positive regulator of E. tarda invasion.

Involvement of N-acetylneuraminate cytidylyltransferase in Edwardsiella piscicida pathogenicity.

Edwardsiella piscicida is a gram-negative bacterium that causes Edwardsiellosis in cultured fish. Edwardsiellosis is accompanied by symptoms such as skin lesions, hemorrhage, and necrosis in fish organs, which leads to significant economic losses in the aquaculture industry. Recently, we found that bacterial sialoglycoconjugates may be involved in the infectivity of E. piscicida. The more infectious strains of E. piscicida contain more sialic acid in the bacterial body, and the mRNA level of putative CMP-Neu5Ac synthase (css) is upregulated compared to that in the non-pathogenic strain. However, this putative css gene is yet to be cloned, and the involvement of CSS in E. piscicida pathogenicity remains unclear. Here, we cloned and transferred the css gene from E. piscicida into the FPC498 strain. CSS promoted infection in cultured cells originating from different fish species, and enhanced the mortality of E. piscicida-infected zebrafish larvae. CSS enhanced cell attachment and motility in E. piscicida, which differs from the decreased bacterial growth observed with the sialic acid-supplemented M9 medium. Both fractions (chloroform-methanol)-soluble and -insoluble fraction) prepared from E. piscicida pellet exhibited the increment of sialo-conjugates induced by CSS. Further, lectin blotting revealed the increment of Sia α2-3- and α2-6-, but not α2-8-, -linked glycoprotein in CSS-overexpressing E. piscicida. Overall, these findings indicate the physiological significance of CSS and the role of sialylation in E. piscicida pathogenicity.

Modulation of TOR complex 2 signaling by the stress-activated MAPK pathway in fission yeast.

Sin1 is a substrate-binding subunit of target of rapamycin complex 2 (TORC2), an evolutionarily conserved protein kinase complex. In fission yeast, Sin1 has also been identified as a protein that interacts with Spc1 (also known as Sty1) in the stress-activated protein kinase (SAPK) pathway. Therefore, this study examined the relationship between TORC2 and Spc1 signaling. We found that the common docking (CD) domain of Spc1 interacts with a cluster of basic amino acid residues in Sin1. Although diminished TORC2 activity in the absence of the functional Spc1 cascade suggests positive regulation of TORC2 by Spc1, such regulation appears to be independent of the Sin1-Spc1 interaction. Hyperosmotic stress transiently inhibits TORC2, and its swift recovery is dependent on Spc1, the transcription factor Atf1, and the glycelrol-3-phosphate dehydrogenase Gpd1, whose expression is induced upon osmostress by the Spc1-Atf1 pathway. Thus, cellular adaptation to osmostress seems important for TORC2 reactivation, though Spc1 and Atf1 contribute to TORC2 activation also in the absence of osmostress. These results indicate coordinated actions of the SAPK and TORC2 pathways, both of which are essential for fission yeast cells to survive environmental stress.

Establishment and characterization of Neu1-knockout zebrafish and its abnormal clinical phenotypes.

Mammalian sialidase Neu1 is involved in various physiological functions, including cell adhesion, differentiation, cancer metastasis, and diabetes through lysosomal catabolism and desialylation of glycoproteins at the plasma membrane. Various animal models have been established to further explore the functions of vertebrate Neu1. The present study focused on zebrafish (Danio rerio) belonging to Cypriniformes as an experimental animal model with neu1 gene deficiency. The results revealed that the zebrafish Neu1 desialyzed both α2-3 and α2-6 sialic acid linkages from oligosaccharides and glycoproteins at pH 4.5, and it is highly conserved with other fish species and mammalian Neu1. Furthermore, Neu1-knockout zebrafish (Neu1-KO) was established through CRISPR/Cas9 genome editing. Neu1-KO fish exhibited slight abnormal embryogenesis with the accumulation of pleural effusion; however, no embryonic lethality was observed. Although Neu1-KO fish were able to be maintained as homozygous, they showed smaller body length and weight than the wild-type (WT) fish, and muscle atrophy and curvature of the vertebra were observed in adult Neu1-KO fish (8 months). The expression patterns of myod and myog transcription factors regulating muscle differentiation varied between Neu1-KO and WT fish embryo. Expression of lysosomal-related genes, including ctsa, lamp1a, and tfeb were up-regulated in adult Neu1-KO muscle as compared with WT. Furthermore, the expression pattern of genes involved in bone remodeling (runx2a, runx2b, and mmp9) was decreased in Neu1-KO fish. These phenotypes were quite similar to those of Neu1-KO mice and human sialidosis patients, indicating the effectiveness of the established Neu1-KO zebrafish for the study of vertebrate Neu1 sialidase.

Identification of novel fish sialidase genes responsible for KDN-cleaving activity.

2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN) is a minor component of sialic acids detected in vertebrates, such as human cancer cells, rat liver, and fish tissues. Although the enzyme activity of KDN-cleaving sialidase (KDN-sialidase) has been detected in rainbow trout, the gene responsible for its expression has not been identified in vertebrates. We evaluated sialidases in human and various fish for their KDN-cleaving activity using an artificial substrate, methylumbelliferyl-KDN (MU-KDN). Four of the human sialidases tested (NEU1, NEU2, NEU3, and NEU4) did not hydrolyze MU-KDN. Although most fish Neu1s showed negligible KDN-sialidase activity, two Neu1b sialidases from Oreochromis niloticus and Astyanax mexicanus, a paralog of Neu1, exhibited a potent KDN-sialidase activity. Further, O. niloticus and Oryzias latipes Neu3a exhibited a drastically high KDN-sialidase activity, while Danio rerio Neu3.1 showed moderate activities and other Neu3 proteins exhibited little activity. All the Neu4 sialidases tested in fish cleaved KDN and Neu5Ac from MU-KDN and MU-Neu5Ac, respectively, with equivalent potential. To our knowledge, this is the first report to identify KDN-sialidase genes in vertebrates and we believe that KDN-sialidase activity could be conserved among fish Neu4s.

Desialylation by Edwardsiella tarda is the initial step in the regulation of its invasiveness.

Edwardsiella tarda is a gram-negative bacterium causing significant economic losses to aquaculture. E. tarda possesses NanA sialidase which removes sialic acids from α2-3 sialo-glycoprotein of host cells. However, the relationship between NanA sialidase activity and E. tarda invasiveness remains poorly understood. Furthermore, the pathway of sialic acid metabolism in E. tarda remains to be elucidated. We studied sialidase activity in several E. tarda strains and found that the pathogenic strains exhibited higher sialidase activity and greater up-regulation of the NanA mRNA level than non-pathogenic strain. Pathogenic strains also showed higher rates of infection in GAKS cells, and the infection was drastically suppressed by sialidase inhibitor. Additionally, NanA gene overexpression significantly increased infection and treatment of E. tarda with free sialic acid enhanced the rate of infection in GAKS cells. Sialic acid treatment enhanced mRNA levels of two N-acetylneuraminate lyases and one N-acetylneuraminate cytidylyltransferase. E. tarda uses sialic acid as a carbon source for growth via N-acetylneuraminate lyases. The strains with high N-acetylneuraminate cytidylyltransferase level showed greater sialylation of the lipopolysaccharides and glycoproteins. Our study establishes the significance of desialylation by E. tarda sialidase in the regulation of its invasiveness.

Regulation of triglyceride metabolism in medaka (Oryzias latipes) hepatocytes by Neu3a sialidase.

Fish store triglycerides (TGs) in the liver, muscle, and adipose tissue and TGs constitute an energy source upon metabolic demand. The liver generally plays important roles in lipid metabolism. Recent studies have suggested the possibility of hepatic lipid metabolic regulation by ganglioside in mammals; however, ganglioside-mediated regulation of lipid metabolism is unclear in fish. This study aimed to clarify the role of ganglioside in fish TG metabolism, with particular reference to Neu3a, a ganglioside-specific sialidase expressed in the fish liver. Under fasting conditions, there was a decrease in hepatic TG contents, and neu3a mRNA level was significantly up-regulated in the medaka liver. To determine the role of Neu3a in hepatic lipid metabolism, Neu3a stable transfectants were generated using fish liver Hepa-T1 cells. After treating Neu3a cells with oleic acid, reduction of TG was detected in comparison with the mock cells. Furthermore, lipase activity was greater in Neu3a cells than in mock cells. To examine which ganglioside regulates these events, alterations of ganglioside composition in Neu3a cells were analyzed. Neu3a cells exhibited increased level of lactosylceramide (LacCer), a Neu3 enzymatic product originating from GM3. In addition, exposure of LacCer toward Hepa-T1 cells resulted in an increase of neutral lipase activity. The present results suggest that Neu3a up-regulation in medaka under fasting condition accelerates hepatic TG degradation for energy production via GM3 desialylation.

Elucidation of the Interleukin 12 Production Mechanism during Intracellular Bacterial Infection in Amberjack, Seriola dumerili.

Intracellular bacterial infections affect all vertebrates. Cultured fish are particularly vulnerable because no effective protection measures have been established since such infections emerged approximately 50 years ago. As in other vertebrates, the induction of cell-mediated immunity (CMI) plays an important role in protecting fish against infection. However, details of the mechanism of CMI induction in fish have not been clarified. In the present study, we focused on the production of interleukin 12 (IL-12), an important factor in CMI induction in fish. Using several different approaches, we investigated IL-12 regulation in amberjack (Seriola dumerili), the species most vulnerable to intracellular bacterial disease. The results of promoter assays and transcription factor gene expression analyses showed that the expression of interferon regulatory factor-1 (IRF-1) and activator protein-1 (AP-1) is necessary for IL-12 production. Phagocytosis of living cells (LCs) of Nocardia seriolae bacteria induced IL-12 production in neutrophils, accompanied by IRF-1 and AP-1 gene expression. Bacteria in which the exported repetitive protein (Erp)-like gene was deleted (Δerp-L) could not establish intracellular parasitism or induce IRF-1 and AP-1 expression or IL-12 production, despite being phagocytosed by neutrophils. These data suggest that IL-12 production is regulated by (i) two transcription factors, IRF-1 and AP-1, (ii) phagocytosis of LCs by neutrophils, and (iii) one or more cell components of LCs. Our results enhance the understanding of the immune response to intracellular bacterial infections in vertebrates and could facilitate the discovery of new agents to prevent intracellular bacterial disease.

Nutrient Signaling via the TORC1-Greatwall-PP2AB55δ Pathway Is Responsible for the High Initial Rates of Alcoholic Fermentation in Sake Yeast Strains of Saccharomyces cerevisiae.

Saccharomyces cerevisiae sake yeast strain Kyokai no. 7 (K7) and its relatives carry a homozygous loss-of-function mutation in the RIM15 gene, which encodes a Greatwall family protein kinase. Disruption of RIM15 in nonsake yeast strains leads to improved alcoholic fermentation, indicating that the defect in Rim15p is associated with the enhanced fermentation performance of sake yeast cells. In order to understand how Rim15p mediates fermentation control, we here focused on target-of-rapamycin protein kinase complex 1 (TORC1) and protein phosphatase 2A with the B55δ regulatory subunit (PP2AB55δ), complexes that are known to act upstream and downstream of Rim15p, respectively. Several lines of evidence, including our previous transcriptomic analysis data, suggested enhanced TORC1 signaling in sake yeast cells during sake fermentation. Fermentation tests of the TORC1-related mutants using a laboratory strain revealed that TORC1 signaling positively regulates the initial fermentation rate in a Rim15p-dependent manner. Deletion of the CDC55 gene, encoding B55δ, abolished the high fermentation performance of Rim15p-deficient laboratory yeast and sake yeast cells, indicating that PP2AB55δ mediates the fermentation control by TORC1 and Rim15p. The TORC1-Greatwall-PP2AB55δ pathway similarly affected the fermentation rate in the fission yeast Schizosaccharomyces pombe, strongly suggesting that the evolutionarily conserved pathway governs alcoholic fermentation in yeasts. It is likely that elevated PP2AB55δ activity accounts for the high fermentation performance of sake yeast cells. Heterozygous loss-of-function mutations in CDC55 found in K7-related sake strains may indicate that the Rim15p-deficient phenotypes are disadvantageous to cell survival.IMPORTANCE The biochemical processes and enzymes responsible for glycolysis and alcoholic fermentation by the yeast S. cerevisiae have long been the subject of scientific research. Nevertheless, the factors determining fermentation performance in vivo are not fully understood. As a result, the industrial breeding of yeast strains has required empirical characterization of fermentation by screening numerous mutants through laborious fermentation tests. To establish a rational and efficient breeding strategy, key regulators of alcoholic fermentation need to be identified. In the present study, we focused on how sake yeast strains of S. cerevisiae have acquired high alcoholic fermentation performance. Our findings provide a rational molecular basis to design yeast strains with optimal fermentation performance for production of alcoholic beverages and bioethanol. In addition, as the evolutionarily conserved TORC1-Greatwall-PP2AB55δ pathway plays a major role in the glycolytic control, our work may contribute to research on carbohydrate metabolism in higher eukaryotes.

Adjuvant effect of recombinant interleukin-12 in the Nocardiosis formalin-killed vaccine of the amberjack Seriola dumerili.

Nocardiosis causes serious economic damage in the fish farming of Japanese yellowtail fish. Nocardia seriolae identified as pathogenic bacterium is an intracellular-pathogen. In general, induction of cell-mediated immunity (CMI) is effective in infection defense against intracellular-pathogen. However, the conventional formalin-killed N. seriolae (FKC) vaccine induces humoral immunity. Interleukin-12 (IL-12) is Th1 type heterodimeric cytokine and induces cell differentiation in mammals. Our previous study showed that recombinant amberjack IL-12 has a role in CMI induction in vitro and could be a possible CMI inducing adjuvant. However, its adjuvant effect of fish IL-12 was not studied. In the present study, six types of amberjack recombinant IL-12 (rIL-12) were mixed and injected into amberjack with FKC. Firstly, we analyzed Th1- and Th2- related gene expression and monitored Th1/Th2 status followed by investigation of antibody titer. As a result, Th1-type immunity was induced in FKC + rIL-12 vaccinated fish. Secondly, we checked Th1/Th2 status of vaccinated fish after 10 days of N. seriolae infection using the expression of related genes. High T-bet/GATA-3 ratio was observed in FKC + rIL-12 vaccinated fish, suggesting that Th1 cells possesing antigen memory were induced against N. seriolae infection. Finally, the survival rate in challenge test showed that 88% of FKC + rIL-12 vaccinated fish was survived at 34 days after N. seriolae injection whereas PBS (control) and FKC only were exterminated. These result suggest that i) rIL-12 is viable CMI inducible adjuvant and ii) production of Th1 cells having antigen memory resulting from activation of IL-12 signaling pathway is important for defense against N. seriolae infection.

Naringenin suppresses Edwardsiella tarda infection in GAKS cells by NanA sialidase inhibition.

Edwardsiella tarda (E. tarda) is a gram-negative bacterium, which causes Edwardsiellosis in aquaculture. Previous studies indicate that E. tarda NanA sialidase plays crucial roles in infection through the desialylation of glycoproteins in fish cells. On the other hand, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, classic sialidase inhibitor, negatively regulates E. tarda infection of goldfish scale GAKS cells. Here, to development the suppression model of E. tarda infection for aquaculture application, the possibility of NanA inhibitory activities in citrus phytochemicals was evaluated as citrus extracts have widely been used as a supplement in fish diets for the improvement of meat quality. Some flavanones such as naringenin, hesperetin, hesperidin and naringin showed sialidase inhibitory activity toward recombinant NanA in vitro. Among them, naringenin showed the most potent inhibitory activity and its inhibitory pattern was non-competitive. Naringenin significantly suppressed E. tarda infection in GAKS cells at 200 and 400 µM without bactericidal effect on E. tarda. On the other hand, naringin, glycosylation form of naringenin, showed slight suppression of E. tarda infection toward GAKS cells, suggesting the glycosides on flavanone could be important for NanA inhibition. Fluorescence microscopy analysis verified that number of invading E. tarda in GAKS cells was declined by naringenin treatment. The present study exhibited the possibility of naringenin as an effective ingredient in fish diet for the inhibition of E. tarda infection.

Phosphatidic acid-mediated activation and translocation to the cell surface of sialidase NEU3, promoting signaling for cell migration.

The plasma membrane-associated sialidase NEU3 plays crucial roles in regulation of transmembrane signaling, and its aberrant up-regulation in various cancers contributes to malignancy. However, it remains uncertain how NEU3 is naturally activated and locates to plasma membranes, because of its Triton X-100 requirement for the sialidase activity in vitro and its often changing subcellular location. Among phospholipids examined, we demonstrate that phosphatidic acid (PA) elevates its sialidase activity 4 to 5 times at 50 µM in vitro at neutral pH and promotes translocation to the cell surface and cell migration through Ras-signaling in HeLa and COS-1 cells. NEU3 was found to interact selectively with PA as assessed by phospholipid array, liposome coprecipitation, and ELISA assays and to colocalize with phospholipase D (PLD) 1 in response to epidermal growth factor (EGF) or serum stimulation. Studies using tagged NEU3 fragments with point mutations identified PA- and calmodulin (CaM)-binding sites around the N terminus and confirmed its participation in translocation and catalytic activity. EGF induced PLD1 activation concomitantly with enhanced NEU3 translocation to the cell surface, as assessed by confocal microscopy. These results suggest that interactions of NEU3 with PA produced by PLD1 are important for regulation of transmembrane signaling, this aberrant acceleration probably promoting malignancy in cancers.

Glycolytic enzyme GAPDH promotes peroxide stress signaling through multistep phosphorelay to a MAPK cascade.

Phosphorelay signaling of environmental stimuli by two-component systems is prevailing in bacteria and also utilized by fungi and plants. In the fission yeast Schizosaccharomyces pombe, peroxide stress signals are transmitted from the Mak2/3 sensor kinases to the Mpr1 histidine-containing phosphotransfer (HPt) protein and finally to the Mcs4 response regulator, which activates a MAP kinase cascade. Here we show that, unexpectedly, the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) physically associates with the Mcs4 response regulator and stress-responsive MAP kinase kinase kinases (MAPKKKs). In response to H2O2 stress, Cys-152 of the Tdh1 GAPDH is transiently oxidized, which enhances the association of Tdh1 with Mcs4. Furthermore, Tdh1 is essential for the interaction between the Mpr1 HPt protein and the Mcs4 response regulator and thus for phosphorelay signaling. These results demonstrate that the glycolytic enzyme GAPDH plays an essential role in the phosphorelay signaling, where its redox-sensitive cysteine residue may provide additional input signals.

Increased sialidase activity in serum of cancer patients: Identification of sialidase and inhibitor activities in human serum.

Aberrant sialylation in glycoproteins and glycolipids is a characteristic feature of malignancy. Human sialidases, which catalyze the removal of sialic acid residues from glycoconjugates, have been implicated in cancer progression. They have been detected in a wide variety of human cells and tissues, but few studies have focused on their existence in human serum. Among the four types identified to date, we previously demonstrated that plasma membrane-associated ganglioside sialidase (NEU3) is markedly upregulated in various human cancers, including examples in the colon and prostate. Here, using a sensitive assay method, we found a significant increase of sialidase activity in the serum of patients with prostate cancer compared with that in healthy subjects having low activity, if any. Activity was apparent with gangliosides as substrates, but only to a very limited extent with 4-methylumbelliferyl sialic acid, a good synthetic substrate for sialidases other than human NEU3. The serum sialidase was also almost entirely immunoprecipitated with anti-NEU3 antibody, but not with antibodies for other sialidases. Interestingly, sera additionally contained inhibitory activity against the sialidase and also against recombinant human NEU3. The sialidase and inhibitor activities could be separated by exosome isolation and by hydrophobic column chromatography. The serum sialidase was assessed by a sandwich ELISA method using two anti-NEU3 antibodies. The results provide strong evidence that the serum sialidase is, in fact, NEU3, and this subtype may, therefore, be a potential utility for novel diagnosis of human cancers.

Utilization of paramagnetic relaxation enhancements for high-resolution NMR structure determination of a soluble loop-rich protein with sparse NOE distance restraints.

NMR structure determination of soluble proteins depends in large part on distance restraints derived from NOE. In this study, we examined the impact of paramagnetic relaxation enhancement (PRE)-derived distance restraints on protein structure determination. A high-resolution structure of the loop-rich soluble protein Sin1 could not be determined by conventional NOE-based procedures due to an insufficient number of NOE restraints. By using the 867 PRE-derived distance restraints obtained from the NOE-based structure determination procedure, a high-resolution structure of Sin1 could be successfully determined. The convergence and accuracy of the determined structure were improved by increasing the number of PRE-derived distance restraints. This study demonstrates that PRE-derived distance restraints are useful in the determination of a high-resolution structure of a soluble protein when the number of NOE constraints is insufficient.

Recombinant sialidase NanA (rNanA) cleaves α2-3 linked sialic acid of host cell surface N-linked glycoprotein to promote Edwardsiella tarda infection.

Edwardsiella tarda is one of the major pathogenic bacteria affecting both marine and freshwater fish species. Sialidase NanA expressed endogenously in E. tarda is glycosidase removing sialic acids from glycoconjugates. Recently, the relationship of NanA sialidase activity to E. tarda infection has been reported, however, the mechanism with which sialidase NanA aids the pathogenicity of E. tarda remained unclear. Here, we comprehensively determined the biochemical properties of NanA towards various substrates in vitro to provide novel insights on the potential NanA target molecule at the host cell. GAKS cell pretreated with recombinant NanA showed increased susceptibility to E. tarda infection. Moreover, sialidase inhibitor treated E. tarda showed a significantly reduced ability to infect GAKS cells. These results indicate that NanA-induced desialylation of cell surface glycoconjugates is essential for the initial step of E. tarda infection. Among the natural substrates, NanA exhibited the highest activity towards 3-sialyllactose, α2-3 linked sialic acid carrying sialoglycoconjugates. Supporting this finding, intact GAKS cell membrane exposed to recombinant NanA showed changes of glycoconjugates only in α2-3 sialo-linked glycoproteins, but not in glycolipids and α2-6 sialo-linked glycoproteins. Lectin staining of cell surface glycoprotein provided further evidence that α2-3 sialo-linkage of the N-linked glycoproteins was the most plausible target of NanA sialidase. To confirm the significance of α2-3 sialo-linkage desialylation for E. tarda infection, HeLa cells which possessed lower amount of α2-3 sialo-linkage glycoprotein were used for infection experiment along with GAKS cells. As a result, infection of HeLa cells by E. tarda was significantly reduced when compared to GAKS cells. Furthermore, E. tarda infection was significantly inhibited by mannose pretreatment suggesting that the bacterium potentially recognizes and binds to mannose or mannose containing chains following desialylation. Together, these results suggest that E. tarda may employ endogenous NanA to desialylate α2-3 glycoproteins on host cells, thus revealing one of the potential binding molecules during infection.