Depletion of hsp90beta induces multiple defects in B cell receptor signaling

Hsp90 participates in many distinct aspects of cellular functions and accomplishes these roles by interacting with multiple client proteins. To gain insight into the interactions between Hsp90 and its clients, here we have reduced the protein level of Hsp90 in avian cells by gene targeting in an attempt to elicit the otherwise undetectable (because of the vast amount of cellular Hsp90) Hsp90-interacting proteins. Hsp90beta-deficient cells can grow, albeit more slowly than wild-type cells. B cell antigen receptor signaling is multiply impaired in these mutant cells; in particular, the amount of immunoglobulin M heavy chain protein is markedly reduced. Furthermore, serum activation does not promote ERK phosphorylation in Hsp90beta-deficient cells. These multifaceted depressive effects seem to be provoked independently of each other and possibly recapitulate the proteome-wide in vivo functions of Hsp90. Reintroduction of the Hsp90beta gene efficiently restores all of the defects. Unexpectedly, however, introducing the Hsp90alpha gene is also effective in restoration; thus, these defects might be caused by a reduction in the total expression of Hsp90 rather than by loss of Hsp90beta-specific function.     

Molecular interaction between fukutin and POMGnT1 in the glycosylation pathway of alpha-dystroglycan

The recent identification of mutations in genes encoding demonstrated or putative glycosyltransferases has revealed a novel mechanism for congenital muscular dystrophy. Hypoglycosylated alpha-dystroglycan (alpha-DG) is commonly seen in Fukuyama-type congenital muscular dystrophy (FCMD), muscle-eye-brain disease (MEB), Walker-Warburg syndrome (WWS), and Large(myd) mice. POMGnT1 and POMTs, the gene products responsible for MEB and WWS, respectively, synthesize unique O-mannose sugar chains on alpha-DG. The function of fukutin, the gene product responsible for FCMD, remains undetermined. Here we show that fukutin co-localizes with POMGnT1 in the Golgi apparatus. Direct interaction between fukutin and POMGnT1 was confirmed by co-immunoprecipitation and two-hybrid analyses. The transmembrane region of fukutin mediates its localization to the Golgi and participates in the interaction with POMGnT1. Y371C, a missense mutation found in FCMD, retains fukutin in the ER and also redirects POMGnT1 to the ER. Finally, we demonstrate reduced POMGnT1 enzymatic activity in transgenic knock-in mice carrying the retrotransposal insertion in the fukutin gene, the prevalent mutation in FCMD. From these findings, we propose that fukutin forms a complex with POMGnT1 and may modulate its enzymatic activity.     

Participation of proteasome-associating complex PC500 in starfish oocyte maturation as revealed by monoclonal antibodies

We previously reported that immature starfish oocytes contain a novel 530-kDa proteasome-associating complex PC500 [previously named PC530; E. Tanaka, M. Takagi Sawada, C. Morinaga, H. Yokosawa, H. Sawada, Isolation and characterization of a novel 530-kDa protein complex (PC 530) capable of associating with the 20S proteasome from star fish oocytes, Arch. Biochem. Biophys. 374 (2000) 181-188]. In the present study, in order to obtain an insight into the biological function of this complex, we investigated the effects of anti-PC500 monoclonal antibodies on oocyte maturation of the starfish Asterina pectinifera. A monoclonal antibody 7C5 strongly inhibited germinal vesicle breakdown (GVBD) in a concentration-dependent manner. In contrast to the inhibitory effect of the 7C5 antibody on GVBD, no inhibition of egg cleavage was observed in a 7C5-antibody-microinjected single blastomere in a 2-cell stage embryo. These results indicate that PC500 plays a key role in starfish oocyte maturation in a meiosis-specific manner.     

Sperm-egg interaction is mediated by a sperm-associated body in quail

The present study describes the holes in the inner vitelline membrane of fertile eggs of the quail Coturnix japonica, which remain after the spermatozoa pass through. It was shown that the light-microscopically observable 'holes' correspond mostly to electron-microscopically defined 'disks', and, to a lesser extent (about 5%), real holes. Immunofluorescent staining of the vitelline membranes with an antiquail ZPC antiserum was used to discriminate the holes from the disks light-microscopically. Over 96% of holes were accompanied by calcium-coated sperm-associated bodies, indicating a close relationship between the two. There was no preferential localization of the disks, holes or sperm-associated bodies in the vitelline membrane around the egg. The sperm-associated bodies bound with the spermatozoa at the posterior end of sperm flagella. Incubation of the inner vitelline membranes, isolated from the largest follicles, with spermatozoa resulted in production only of the disks, whereas the holes (about 9%) were produced when the sperm-associated bodies were added to the system. It was suggested that the sperm-associated bodies assist fertile spermatozoa in binding to the inner vitelline membrane, making holes in the membrane and passing through them in fertile eggs.     

A novel ATP/ADP hydrolysis activity of hyperthermostable group II chaperonin in the presence of cobalt or manganese ion

A novel ATPase activity that was strongly activated in the presence of either cobalt or manganese ion was discovered in the chaperonin from hyperthermophilic Pyrococcus furiosus (Pfu-cpn). Surprisingly, a significant ADPase activity was also detected under the same conditions. A more extensive search revealed similar nucleotide hydrolysis activities in other thermostable chaperonins. Chaperonin activity, i.e., thermal stabilization and refolding of malate dehydrogenase from the guanidine-hydrochloride unfolded state were also detected for Pfu-cpn under the same conditions. We propose that the novel cobalt/manganese-dependent ATP/ADPase activity may be a common trait of various thermostable chaperonins.     

High-throughput determination of free D-aspartic acid in mammals by enzyme immunoassay using specific monoclonal antibody

A method for rapid determination of free d-aspartic acid (d-Asp) in mammals has been established using a highly specific mouse monoclonal antibody against d-Asp for the first time. An anti-d-Asp monoclonal antibody was obtained by the immunization of bovine-serum-albumin-conjugated d-Asp to BALB/c mice. The obtained antibody has a high specificity toward d-Asp but shows a slight cross-reactivity to all other d- and l- amino acids including l-Asp. The calibration range of the competitive enzyme linked immunosorbent assay (ELISA) is 0.016-16 μmol/mL d-Asp in rat serum samples. The precisions of this method were evaluated by inter-plate and intraplate assays, and the relative standard deviation values were 4.8% and 4.5%, respectively. The values of d-Asp determined by the present ELISA have a good correlation to those determined by high-performance liquid chromatography with the correlation coefficient of 0.963. Using this ELISA, the time course of d-Asp in the rat serum after intravenous administration was successfully demonstrated. The present method provides a simple and high-throughput determination of d-Asp in mammals, and is a useful tool for clarifying the physiological roles and diagnostic values of this d-amino acid.     

Why does avian influenza A virus hemagglutinin bind to avian receptor stronger than to human receptor? Ab initio fragment molecular orbital studies

Influenza A viruses attach to alpha-sialosides on the target cell surface by their hemagglutinins, which strictly recognize the difference in sialic acid-galactose linkage. Why does avian virus H3 subtype bind to avian receptor Neu5Ac(alpha2-3)Gal stronger than to human receptor Neu5Ac(alpha2-6)Gal? Why does avian H3 mutated Gln226 to Leu preferentially bind to human receptor? In this paper, we theoretically answer the questions by molecular mechanics and ab initio fragment molecular orbital (FMO) calculations. The binding energy between avian H3 and avian receptor is 8.2kcal/mol larger than that of the avian H3-human receptor complex estimated at the FMO-HF/STO-3G level, which is a reason that avian H3 binds to avian receptor stronger than to human receptor. Avian Leu226 H3 clashes to Gal unit on the avian receptor to quite decrease its binding affinity. In contrast, Gal unit on the human receptor forms intermolecular hydrophobic interaction with avian Leu226 H3 to afford moderate binding affinity.     

Identification of novel citrullinated autoantigens of synovium in rheumatoid arthritis using a proteomic approach

Recently, autoantibodies to some citrullinated autoantigens have been reported to be specific for rheumatoid arthritis (RA). However, an entire profile of and autoimmunity of the citrullinated proteins have been poorly understood. To understand the profile, we examined citrullinated autoantigens by a proteomic approach and further investigated the significance of citrullination in antigenicity of one of the autoantigens. Specifically, we detected citrullinated autoantigens in synovial tissue of a patient with RA by two-dimensional electrophoresis and Western blotting by using pooled sera from five patients with RA and anti-citrulline antibodies. After identifying the detected autoantigens by mass spectrometry, we investigated the contribution of citrullination to autoantigenicity by using a recombinant protein with or without citrullination on one of the identified novel citrullinated autoantigens. As a result, we found 51 citrullinated protein spots. Thirty (58.8%) of these spots were autoantigenic. We identified 13 out of the 30 detected citrullinated autoantigenic proteins. They contained three fibrinogen derivatives and several novel citrullinated autoantigens (for example, asporin and F-actin capping protein alpha-1 subunit [CapZalpha-1]). We further analyzed the contribution of citrullination to autoantigenicity in one of the detected citrullinated autoantigens, CapZalpha-1. As a result, frequencies of autoantibodies to non-citrullinated CapZalpha-1 were 36.7% in the RA group tested, 10.7% in the osteoarthritis (OA) group, and 6.5% in healthy donors. On the other hand, those to citrullinated CapZalpha-1 were 53.3% in the RA group, 7.1% in the OA group, and 6.5% in the healthy donors. This shows that autoantigenicity of citrullinated or non-citrullinated CapZalpha-1 is relevant to RA. The antibody titers to the citrullinated CapZalpha-1 were significantly higher than those to the non-citrullinated CapZalpha-1 in 36.7% of patients; however, the other patients showed almost equal antibody titers to both citrullinated and non-citrullinated CapZalpha-1. Therefore, the autoantibodies would target citrulline-related and/or citrulline-unrelated epitope(s) of CapZalpha-1. In conclusion, we report a profile of citrullinated autoantigens for the first time. Even though citrullination is closely related to autoantigenicity, citrullination would not always produce autoantigenicity in RA. Citrullinated and non-citrullinated autoantigens/autoepitopes would have different pathological roles in RA.     

Lycopene inhibits Helicobacter pylori-induced ATM/ATR-dependent DNA damage response in gastric epithelial AGS cells

Oxidative stress linked to DNA damage is involved in the pathogenesis of Helicobacter pylori-associated gastric diseases. The DNA damage response (DDR) coordinates cell-cycle transitions, DNA repair, and apoptosis through the activation of ataxia-telangiectasia-mutated (ATM) and ATM and Rad3-related (ATR) and their target proteins. However, neither H. pylori-induced DDR nor the effects of antioxidants on the DNA damage have been established. This study aimed to investigate the detailed process of H. pylori-induced DNA damage and to examine whether lycopene, a natural antioxidant, inhibits DNA damage and cellular response of gastric epithelial AGS cells infected with H. pylori. AGS cells were cultured with H. pylori in Korean isolates and treated with or without lycopene. Cell viability, DNA damage indices, levels of 8-OH-dG, and reactive oxygen species (ROS) as well as cell-cycle distributions were determined. The activation of ATM, ATR, Chk1, and Chk2; histone H2AX focus formation; activation and induction of p53; and levels of Bax and Bcl-2 and poly(ADP-ribose) polymerase-1 (PARP-1) were assessed. The results showed that H. pylori induced apoptosis in AGS cells with increased Bax and decreased Bcl-2 expression as well as PARP-1 cleavage. Culture with H. pylori led to increases in intracellular ROS, 8-OH-dG, double-strand DNA breaks (DSBs), and DNA fragmentation. H. pylori induced activation of the ATM/Chk2 and ATR/Chk1 pathways, phosphorylation of H2AX and p53, and a delay in the progression of the cells entering the S phase. Lycopene inhibited H. pylori-induced increases in ROS, apoptosis, alterations in cell-cycle distribution, DSBs, and ATM- and ATR-mediated DDR in AGS cells. In conclusion, lycopene may be beneficial for treatment of H. pylori-induced gastric diseases associated with oxidative DNA damage.