CERT and intracellular trafficking of ceramide

The transport and sorting of lipids from the sites of their synthesis to their appropriate destinations are fundamental for membrane biogenesis. In the synthesis of sphingolipids in mammalian cells, ceramide is newly produced at the endoplasmic reticulum (ER), and transported from the ER to the trans Golgi regions, where it is converted to sphingomyelin. CERT has been identified as a key factor for the ER-to-Golgi trafficking of ceramide. CERT contains several functional domains including (i) a START domain capable of catalyzing inter-membrane transfer of ceramide, (ii) a pleckstrin homology domain, which serves to target the Golgi apparatus by recognizing phosphatidylinositol 4-monophosphate, and (iii) a short peptide motif named FFAT motif which interacts with the ER-resident membrane protein VAP. CERT is preferentially distributed to the Golgi region in cells, and Golgi-targeted CERT appears to retain the activity to interact with VAP. On the basis of these results, it has been proposed that CERT extracts ceramide from the ER and carries it to the Golgi apparatus in a non-vesicular manner and that a particularly efficient cycle of CERT movement for trafficking of ceramide may proceed at membrane contact sites between the ER and the Golgi apparatus.     

Coexpression of alpha1,2 galactosyltransferase and UDP-galactose transporter efficiently galactosylates N- and O-glycans in Saccharomyces cerevisiae

We have studied in vivo neo-galactosylation in Saccharomyces cerevisiae and analyzed the critical factors involved in this system. Two heterologous genes, gma12(+) encoding alpha1, 2-galactosyltransferase (alpha1,2 GalT) from Schizosaccharomyces pombe and UGT2 encoding UDP- galactose (UDP-Gal) transporter from human, were functionally expressed to examine the intracellular conditions required for galactosylation. Detection by fluorescence labeled alpha-galactose specific lectin revealed that 50% of the cells incorporated galactose to cell surface mannoproteins only when the gma12(+) and hUGT2 genes were coexpressed in galactose media. Integration of both genes in the Delta mnn1 background cells increased galactosylation to 80% of the cells. Correlation between cell surface galactosylation and UDP-galactose transport activity indicated that an exogenous supply of UDP-Gal transporter rather than alpha1,2 GalT played a key role for efficient galactosylation in S.cerevisiae. In addition, this heterologous system enabled us to study the in vivo function of S. pombe alpha1,2 GalT to prove that it transfers galactose to both N - and O -linked oligosaccharides. Structural analysis indicated that this enzyme transfers galactose to O -mannosyl residue attached to polypeptides and produces Galalpha1,2-Man1-O-Ser/Thr structure. Thus, we have successfully generated a system for efficient galactose incorporation which is originally absent in S. cerevisiae, suggesting further possibilities for in vivo glycan remodeling toward therapeutically useful galactose containing heterologous proteins in S. cerevisiae.      

Efficacy comparison of adjuvants in PcrV vaccine against Pseudomonas aeruginosa pneumonia

Vaccination against the type III secretion system of P. aeruginosa is a potential prophylactic strategy for reducing the incidence and improving the poor prognosis of P. aeruginosa pneumonia. In this study, the efficacies of three different adjuvants, Freund's adjuvant (FA), aluminum hydroxide (alum) and CpG oligodeoxynucleotide (ODN), were examined from the viewpoint of inducing PcrV‐specific immunity against virulent P. aeruginosa. Mice that had been immunized intraperitoneally with recombinant PcrV formulated with one of the above adjuvants were challenged intratracheally with a lethal dose of P. aeruginosa. The PcrV–FA immunized group attained a survival rate of 91%, whereas the survival rates of the PcrV–alum and PcrV–CpG groups were 73% and 64%, respectively. In terms of hypothermia recovery after bacterial instillation, PcrV–alum was the most protective, followed by PcrV–FA and PcrV–CpG. The lung edema index was lower in the PcrV–CpG vaccination group than in the other groups. PcrV–alum immunization was associated with the greatest decrease in myeloperoxidase in infected lungs, and also decreased the number of lung bacteria to a similar number as in the PcrV–FA group. There was less neutrophil recruitment in the lungs of mice vaccinated with PcrV–alum or PcrV–CpG than in those of mice vaccinated with PcrV–FA or PcrV alone. Overall, in terms of mouse survival the PcrV–CpG vaccine, which could be a relatively safe next‐generation vaccine, showed a comparable effect to the PcrV–alum vaccine.     

Hybridization-dependent fluorescence of oligodeoxynucleotides incorporating new pyrene-modified adenosine residues

We report the synthesis and properties of oligonucleotides incorporating N(6)-[N-(pyren-1-ylmethyl)carbamoyl]-deoxyadenosine (dA(pymcm)). We designed the ODN which incorporated two consecutive dA(pymcm) residues. It was revealed that on hybridization with the target DNA and RNA oligomers, the fluorescence spectra of ODNs having two consecutive dA(pymcm) molecules near the 5'-terminal position can change from the pyrene monomer emission to the excimer, depending on the chain length of the target DNA and RNA. These results indicated that dA(pymcm)-modified ODNs can be used as interesting hybridization sensors that are sensitive to the size of the target strand.     

Characterization of the {alpha}-helix region in domain 3 of the haemolytic lectin CEL-III: implications for self-oligomerization and haemolytic processes

CEL-III is a haemolytic lectin, which has two beta-trefoil domains (domains 1 and 2) and a beta-sheet-rich domain (domain 3). In domain 3 (residues 284-432), there is a hydrophobic region containing two alpha-helices (H8 and H9, residues 317-357) and a loop between them, in which alternate hydrophobic residues, especially Val residues, are present. To elucidate the role of the alpha-helix region in the haemolytic process, peptides corresponding to different parts of this region were synthesized and characterized. The peptides containing the sequence that corresponded to the loop and second alpha-helix (H9) showed the strongest antibacterial activity for Staphylococcus aureus and Bacillus subtilis through a marked permeabilization of the bacterial cell membrane. The recombinant glutathione S-transferase (GST)-fusion proteins containing domain 3 or the alpha-helix region peptide formed self-oligomers, whereas mutations in the alternate Val residues in the alpha-helix region lead to decreased oligomerization ability of the fusion proteins. These results suggest that the alpha-helix region, particularly its alternate Val residues are important for oligomerization of CEL-III in target cell membranes, which is also required for a subsequent haemolytic action.     

CERT-mediated trafficking of ceramide

The transport and sorting of lipids from the sites of their synthesis to their appropriate destinations are fundamental for membrane biogenesis. In the synthesis of sphingolipids in mammalian cells, ceramide is newly produced at the endoplasmic reticulum (ER), and transported from the ER to the trans Golgi regions, where it is converted to sphingomyelin. CERT mediates the ER-to-Golgi trafficking of ceramide. CERT contains several functional domains and motifs including i) a START domain capable of catalyzing inter-membrane transfer of ceramide, ii) a pleckstrin homology domain, which serves to target the Golgi apparatus, iii) a FFAT motif which interacts with the ER-resident membrane protein VAP, and iv) a serine-repeat motif, of which hyperphosphorylation down-regulates CERT activity. It has been suggested that CERT extracts ceramide from the ER and carries it to the Golgi apparatus in a non-vesicular manner and that efficient CERT-mediated trafficking of ceramide occurs at membrane contact sites between the ER and the Golgi apparatus.     

The NADPH thioredoxin reductase C functions as an electron donor to 2-Cys peroxiredoxin in a thermophilic cyanobacterium Thermosynechococcus elongatus BP-1

An NADPH thioredoxin reductase C was co-purified with a 2-Cys peroxiredoxin by the combination of anion exchange chromatography and electroelution from gel slices after native PAGE from a thermophilic cyanobacterium Thermosynechococcus elongatus as an NAD(P)H oxidase complex induced by oxidative stress. The result provided a strong evidence that the NADPH thioredoxin reductase C interacts with the 2-Cys peroxiredoxin in vivo. An in vitro reconstitution assay with purified recombinant proteins revealed that both proteins were essential for an NADPH-dependent reduction of H₂O₂. These results suggest that the reductase transfers the reducing power from NADPH to the peroxiredoxin, which reduces peroxides in the cyanobacterium under oxidative stress. In contrast with other NADPH thioredoxin reductases, the NADPH thioredoxin reductase C contains a thioredoxin-like domain in addition to an NADPH thioredoxin reductase domain in the same polypeptide. Each domain contains a conserved CXYC motif. A point mutation at the CXYC motif in the NADPH thioredoxin reductase domain resulted in loss of the NADPH oxidation activity, while a mutation at the CXYC motif in the thioredoxin-like domain did not affect the electron transfer, indicating that this motif is not essential in the electron transport from NADPH to the 2-Cys peroxiredoxin.     

A gain-of-function screen identifies wdb and lkb1 as lifespan-extending genes in Drosophila

The insulin/insulin-like growth factor (IGF) and the target of rapamycin (TOR) signaling pathways are known to regulate lifespan in diverse organisms. However, only a limited number of genes involved in these pathways have been examined regarding their effects on lifespan. Through a gain-of-function screen in Drosophila, we found that overexpression of the wdb gene encoding a regulatory subunit of PP2A, and overexpression of the lkb1 gene encoding a serine/threonine kinase, reduced organ size and extended lifespan. Overexpression of wdb also reduced the level of phosphorylated AKT, while overexpression of lkb1 increased the level of phosphorylated AMPK and decreased the level of phosphorylated S6K. Taken together, our results suggest that wdb- and lkb1-dependent lifespan extension is mediated by downregulation of S6K, a downstream component of the insulin/IGF and TOR signaling pathways.     

iNKT cells in microbial immunity: recognition of microbial glycolipids

Natural killer T cells expressing an invariant T cell antigen receptor (iNKT cells) are cells of the innate immune system. After recognizing glycolipid antigens presented by CD1d molecules on antigen presenting cells (APCs), iNKT cells rapidly produce large quantities of cytokines, thereby stimulating many types of cells. Recent studies have described several mechanisms of iNKT cell activation and the contribution of these cells to antimicrobial responses. iNKT cells can be activated by endogenous antigens and/or inflammatory cytokines from APCs. However, iNKT cells also recognize certain microbial glycolipids by their invariant T cell antigen receptor (TCR), and they contribute to pathogen clearance in certain microbial infections. These findings indicate that the iNKT TCR is useful for detecting certain microbial pathogens. Moreover, recent studies suggest that iNKT cell glycolipid antigens may be useful in antimicrobial therapy and vaccines.