Mammalian Apg12p,but not the Apg12p.Apg5p conjugate,facilitates LC3 processing

A dynamic membrane rearrangement occurs in cells during autophagy to form autophagosomes. In this dynamic process, two ubiquitin-like modifications, Apg12p-conjugation and LC3-modification, are essential for the formation of autophagosomes. Apg7p and Apg10p catalyze the conjugation of Apg12p to Apg5p. The same Apg7p and Apg3p catalyze the processing of LC3 to a membrane-bound form, LC3-II. In this paper, we investigated whether Apg12p has an influence on the second LC3-modification system. A cross-linking experiment revealed that Apg3p interacts with the endogenous Apg12p.Apg5p conjugate. However, Apg3p itself interacts with free Apg12p more preferentially than the Apg12p.Apg5p conjugate, when free Apg12p exists. When Apg12p was overexpressed, LC3 processing was significantly enhanced in the presence of Apg7p. In contrast, when the Apg12p.Apg5p conjugate itself was accumulated by the overexpression of Apg12p and Apg5p, LC3 processing was dominantly inhibited, even in the presence of Apg7p. These results indicate that both Apg12p and the Apg12p.Apg5p conjugate are regulatory factors for LC3 processing.     

Vitamin C activity in guinea pigs of 6-O-acyl-2-O-alpha-D-glucopyranosyl-L- ascorbic acids with a branched-acyl chain

A series of novel acylated ascorbic acid derivatives, 6-O-acyl-2-O-alpha-D-glucopyranosyl-L-ascorbic acids with a branched-acyl chain (6-bAcyl-AA-2G) were recently developed in our laboratory as stable and lipophilic ascorbate derivatives. In this study, the bioavailability of 6-bAcyl-AA-2G was investigated in guinea pigs. Various tissue homogenates from guinea pigs hydrolyzed 6-bAcyl-AA-2G to give ascorbic acid (AA), 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G), and 6-O-acyl AA. The releasing pattern of the three hydrolysates suggested that 6-bAcyl-AA-2G was hydrolyzed via 6-O-acyl AA to AA as a main pathway and via AA-2G to AA as a minor pathway. The former pathway seems to be of advantage, because 6-O-acyl AA, as well as AA, can have vitamin C activity. In addition, we found that a derivative with an acyl chain of C(12), 6-bDode-AA-2G, had a pronounced therapeutic effect in scorbutic guinea pigs by its repeated oral administrations. These results indicate that 6-bAcyl-AA-2G is a readily available source of AA in vivo, and may be a promising antioxidant for skin care and treatment of diseases associated with oxidative stress.     

Direct demonstration of involvement of protein kinase Calpha in the Ca2+-induced platelet aggregation

Platelets play critical roles in hemostasis and thrombosis through their aggregation following activation of integrin alphaIIbbeta3. However, the molecular mechanism of the integrin activation inside platelets remains largely unknown. Pharmacological experiments have demonstrated that protein kinase C (PKC) plays an important role in platelet aggregation. Because PKC inhibitors can have multiple substrates and given that non-PKC-phorbol ester-binding signaling molecules have been demonstrated to play important roles, the precise involvement of PKC in cellular functions requires re-evaluation. Here, we have established an assay for analyzing the Ca2+-induced aggregation of permeabilized platelets. The aggregation of platelets was inhibited by the addition of the arginine-glycine-aspartate-serine peptide, an integrin-binding peptide inhibitor of alphaIIbbeta3, suggesting that the aggregation was mediated by the integrin. The aggregation was also dependent on exogenous ATP and platelet cytosol, indicating the existence of essential cytosolic factors required for the aggregation. To examine the role of PKC in the aggregation assay, we immunodepleted PKCalpha and beta from the cytosol. The PKC-depleted cytosol lost the aggregation-supporting activity, which was recovered by the addition of purified PKCalpha. Furthermore, the addition of purified PKCalpha in the absence of cytosol did not support the aggregation, whereas the cytosol containing less PKC supported it efficiently, suggesting that additional factors besides PKC would also be required. Thus, we directly demonstrated that PKCalpha is involved in the regulation of Ca2+-induced platelet aggregation.     

NMR solution structure of lipocalin-type prostaglandin D synthase: evidence for partial overlapping of catalytic pocket and retinoic acid-binding pocket within the central cavity

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) catalyzes the isomerization of PGH(2), a common precursor of various prostanoids, to produce PGD(2), an endogenous somnogen and nociceptive modulator, in the brain. L-PGDS is a member of the lipocalin superfamily and binds lipophilic substances, such as retinoids and bile pigments, suggesting that L-PGDS is a dual functional protein acting as a PGD(2)-synthesizing enzyme and a transporter for lipophilic ligands. In this study we determined by NMR the three-dimensional structure of recombinant mouse L-PGDS with the catalytic residue Cys-65. The structure of L-PGDS exhibited the typical lipocalin fold, consisting of an eight-stranded, antiparallel beta-barrel and a long alpha-helix associated with the outer surface of the barrel. The interior of the barrel formed a hydrophobic cavity opening to the upper end of the barrel, the size of which was larger than those of other lipocalins, and the cavity contained two pockets. Molecular docking studies, based on the result of NMR titration experiments with retinoic acid and PGH(2) analog, revealed that PGH(2) almost fully occupied the hydrophilic pocket 1, in which Cys-65 was located and all-trans-retinoic acid occupied the hydrophobic pocket 2, in which amino acid residues important for retinoid binding in other lipocalins were well conserved. Mutational and kinetic studies provide the direct evidence for the PGH(2) binding mode. These results indicated that the two binding sites for PGH(2) and retinoic acid in the large cavity of L-PGDS were responsible for the broad ligand specificity of L-PGDS and the non-competitive inhibition of L-PGDS activity by retinoic acid.     

Downsloping High-Frequency Hearing Loss Due to Inner Ear Tricellular Tight Junction Disruption by a Novel ILDR1 Mutation in the Ig-Like Domain

The immunoglobulin (Ig)-like domain containing receptor 1 (ILDR1) gene encodes angulin-2/ILDR1, a recently discovered tight junction protein, which forms tricellular tight junction (tTJ) structures with tricellulin and lipolysis-stimulated lipoprotein receptor (LSR) at tricellular contacts (TCs) in the inner ear. Previously reported recessive mutations within ILDR1 have been shown to cause severe to profound nonsyndromic sensorineural hearing loss (SNHL), DFNB42. Whole-exome sequencing of a Korean multiplex family segregating partial deafness identified a novel homozygous ILDR1 variant (p.P69H) within the Ig-like domain. To address the pathogenicity of p.P69H, the angulin-2/ILDR1 p.P69H variant protein, along with the previously reported pathogenic ILDR1 mutations, was expressed in angulin-1/LSR knockdown epithelial cells. Interestingly, partial mislocalization of the p.P69H variant protein and tricellulin at TCs was observed, in contrast to a severe mislocalization and complete failure of tricellulin recruitment of the other reported ILDR1 mutations. Additionally, three-dimensional protein modeling revealed that angulin-2/ILDR1 contributed to tTJ by forming a homo-trimer structure through its Ig-like domain, and the p.P69H variant was predicted to disturb homo-trimer formation. In this study, we propose a possible role of angulin-2/ILDR1 in tTJ formation in the inner ear and a wider audiologic phenotypic spectrum of DFNB42 caused by mutations within ILDR1.     

Growth of an Escherichia coli mutant deficient in respiration

An Escherichia coli mutant deficient in genes for heme biosynthesis grew in medium of initial pH 8 containing 1% tryptone and glucose under aerobic growth conditions, and its doubling time was approximately 60 min at 37°C. The growth rate was not increased under O₂-limiting conditions. When the mutant was grown in medium of initial pH 6, growth stopped at the middle of the exponential growth phase. This could be overcome and the growth yield increased by the addition of 20 mM lysine to the growth medium. Lysine did not prevent the decrease in the medium pH as growth proceeded, making it unlikely that lysine decarboxylation stimulates growth by the alkalinization of the medium. These results indicate that respiration is not obligatory for growth under aerobic conditions, but growth without respiration at low pH requires a large amount of lysine.     

Inhibition of free radical-induced erythrocyte hemolysis by 2-O-substituted ascorbic acid derivatives

Inhibitory effects of 2-O-substituted ascorbic acid derivatives, ascorbic acid 2-glucoside (AA-2G), ascorbic acid 2-phosphate (AA-2P), and ascorbic acid 2-sulfate (AA-2S), on 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced oxidative hemolysis of sheep erythrocytes were studied and were compared with those of ascorbic acid (AA) and other antioxidants. The order of the inhibition efficiency was AA-2S> or =Trolox=uric acid> or =AA-2P> or =AA-2G=AA>glutathione. Although the reactivity of the AA derivatives against AAPH-derived peroxyl radical (ROO(*)) was much lower than that of AA, the derivatives exerted equal or more potent protective effects on AAPH-induced hemolysis and membrane protein oxidation. In addition, the AA derivatives were found to react per se with ROO(*), not via AA as an intermediate. These findings suggest that secondary reactions between the AA derivative radical and ROO(*) play a part in hemolysis inhibition. Delayed addition of the AA derivatives after AAPH-induced oxidation of erythrocytes had already proceeded showed weaker inhibition of hemolysis compared to that of AA. These results suggest that the AA derivatives per se act as biologically effective antioxidants under moderate oxidative stress and that AA-2G and AA-2P may be able to act under severe oxidative stress after enzymatic conversion to AA in vivo.     

Predicting Novel Features of Toll-Like Receptor 3 Signaling in Macrophages

The Toll-like receptor (TLR) 3 plays a critical role in mammalian innate immune response against viral attacks by recognizing double-stranded RNA (dsRNA) or its synthetic analog polyinosinic-polycytidylic acid (poly (I∶C)). This leads to the activation of MAP kinases and NF-κB which results in the induction of type I interferons and proinflammatory cytokines to combat the viral infection. To understand the complex interplay of the various intracellular signaling molecules in the regulation of NF-κB and MAP kinases, we developed a computational TLR3 model based upon perturbation-response approach. We curated literature and databases to determine the TLR3 signaling topology specifically for murine macrophages. For initial model creation, we used wildtype temporal activation profiles of MAP kinases and NF-κB and, for model testing, used TRAF6 KO and TRADD KO data. From dynamic simulations we predict i) the existence of missing intermediary steps between extracellular poly (I∶C) stimulation and intracellular TLR3 binding, and ii) the presence of a novel pathway which is essential for JNK and p38, but not NF-κB, activation. Our work shows activation dynamics of signaling molecules can be used in conjunction with perturbation-response models to decipher novel signaling features of complicated immune pathways.