Cyclic lipopeptide antibiotics bind to the N-terminal domain of the prokaryotic Hsp90 to inhibit the chaperone activity

Chemical arrays were employed to screen ligands for HtpG, the prokaryotic homologue of Hsp (heat-shock protein) 90. We found that colistins and the closely related polymyxin B interact physically with HtpG. They bind to the N-terminal domain of HtpG specifically without affecting its ATPase activity. The interaction caused inhibition of chaperone function of HtpG that suppresses thermal aggregation of substrate proteins. Further studies were performed with one of these cyclic lipopeptide antibiotics, colistin sulfate salt. It inhibited the chaperone function of the N-terminal domain of HtpG. However, it inhibited neither the chaperone function of the middle domain of HtpG nor that of other molecular chaperones such as DnaK, the prokaryotic homologue of Hsp70, and small Hsp. The addition of colistin sulfate salt increased surface hydrophobicity of the N-terminal domain of HtpG and induced oligomerization of HtpG and its N-terminal domain. These structural changes are discussed in relation to the inhibition of the chaperone function.     

Glycosylphosphatidylinositol anchor-dependent stimulation pathway required for generation of baculovirus-derived recombinant scrapie prion protein

The pathogenic isoform (PrP(Sc)) of the host-encoded cellular prion protein (PrP(C)) is considered to be an infectious agent of transmissible spongiform encephalopathy (TSE). The detailed mechanism by which the PrP(Sc) seed catalyzes the structural conversion of endogenous PrP(C) into nascent PrP(Sc) in vivo still remains unclear. Recent studies reveal that bacterially derived recombinant PrP (recPrP) can be used as a substrate for the in vitro generation of protease-resistant recPrP (recPrP(res)) by protein-misfolding cyclic amplification (PMCA). These findings imply that PrP modifications with a glycosylphosphatidylinositol (GPI) anchor and asparagine (N)-linked glycosylation are not necessary for the amplification and generation of recPrP(Sc) by PMCA. However, the biological properties of PrP(Sc) obtained by in vivo transmission of recPrP(res) are unique or different from those of PrP(Sc) used as the seed, indicating that the mechanisms mediated by these posttranslational modifications possibly participate in reproductive propagation of PrP(Sc). In the present study, using baculovirus-derived recombinant PrP (Bac-PrP), we demonstrated that Bac-PrP is useful as a PrP(C) substrate for amplification of the mouse scrapie prion strain Chandler, and PrP(Sc) that accumulated in mice inoculated with Bac-PrP(res) had biochemical and pathological properties very similar to those of the PrP(Sc) seed. Since Bac-PrP modified with a GPI anchor and brain homogenate of Prnp knockout mice were both required to generate Bac-PrP(res), the interaction of GPI-anchored PrP with factors in brain homogenates is essential for reproductive propagation of PrP(Sc). Therefore, the Bac-PMCA technique appears to be extremely beneficial for the comprehensive understanding of the GPI anchor-mediated stimulation pathway.     

A comparison of hydration effect on body fluid and temperature regulation between Malaysian and Japanese males exercising at mild dehydration in humid heat

Background

This study investigated the effect of hydration differences on body fluid and temperature regulation between tropical and temperate indigenes exercising in the heat.

Methods

Ten Japanese and ten Malaysian males with matched physical characteristics (height, body weight, and peak oxygen consumption) participated in this study. Participants performed exercise for 60 min at 55% peak oxygen uptake followed by a 30-min recovery at 32°C and 70% relative air humidity with hydration (4 times each, 3 mL per kg body weight, 37°C) or without hydration. Rectal temperature, skin temperature, heart rate, skin blood flow, and blood pressure were measured continuously. The percentage of body weight loss and total sweat loss were calculated from body weight measurements. The percentage change in plasma volume was estimated from hemoglobin concentration and hematocrit.

Results

Malaysian participants had a significantly lower rectal temperature, a smaller reduction in plasma volume, and a lower heart rate in the hydrated condition than in the non-hydrated condition at the end of exercise (P <0.05), whereas Japanese participants showed no difference between the two hydration conditions. Hydration induced a greater total sweat loss in both groups (P <0.05), and the percentage of body weight loss in hydrated Malaysians was significantly less than in hydrated Japanese (P <0.05). A significant interaction between groups and hydration conditions was observed for the percentage of mean cutaneous vascular conductance during exercise relative to baseline (P <0.05).

Conclusions

The smaller reduction in plasma volume and percentage body weight loss in hydrated Malaysians indicated an advantage in body fluid regulation. This may enable Malaysians to reserve more blood for circulation and heat dissipation and thereby maintain lower rectal temperatures in a hydrated condition.     

Torque Generation of Enterococcus hirae V-ATPase

V-ATPase (V_oV₁) converts the chemical free energy of ATP into an ion-motive force across the cell membrane via mechanical rotation. This energy conversion requires proper interactions between the rotor and stator in V_oV₁ for tight coupling among chemical reaction, torque generation, and ion transport. We developed an Escherichia coli expression system for Enterococcus hirae V_oV₁ (EhV_oV₁) and established a single-molecule rotation assay to measure the torque generated. Recombinant and native EhV_oV₁ exhibited almost identical dependence of ATP hydrolysis activity on sodium ion and ATP concentrations, indicating their functional equivalence. In a single-molecule rotation assay with a low load probe at high ATP concentration, EhV_oV₁ only showed the “clear” state without apparent backward steps, whereas EhV₁ showed two states, “clear” and “unclear.” Furthermore, EhV_oV₁ showed slower rotation than EhV₁ without the three distinct pauses separated by 120° that were observed in EhV₁. When using a large probe, EhV_oV₁ showed faster rotation than EhV₁, and the torque of EhV_oV₁ estimated from the continuous rotation was nearly double that of EhV₁. On the other hand, stepping torque of EhV₁ in the clear state was comparable with that of EhV_oV₁. These results indicate that rotor-stator interactions of the V_o moiety and/or sodium ion transport limit the rotation driven by the V₁ moiety, and the rotor-stator interactions in EhV_oV₁ are stabilized by two peripheral stalks to generate a larger torque than that of isolated EhV₁. However, the torque value was substantially lower than that of other rotary ATPases, implying the low energy conversion efficiency of EhV_oV₁.     

Essential role of ferrous iron in cyanide-resistant respiration in Hansenula anomala

Antimycin A-dependent induction of cyanide-resistant respiration in Hansenula anomala was completely blocked by o-phenanthroline, alpha,alpha'-dipyridyl, or 8-hydroxyquinoline. Pulse-labeling of the cells with [35S]methionine in the presence of both antimycin A and o-phenanthroline indicated that the 36-kDa protein previously reported to be involved in cyanide-resistant respiration [(1989) J. Biochem. 105, 864-866] was formed in mitochondria even under these conditions. The addition of Fe2+, but not Fe3+, ions to these cells in the presence of cycloheximide resulted in the rapid expression of cyanide-resistant respiration activity. These results suggest that in the presence of both antimycin A and o-phenanthroline an inactive form of the 36-kDa protein was formed and Fe2+ ions converted it to the active form. It is also likely that Fe2+ ions are involved in the reaction mechanism of cyanide-resistant respiration.     

V domain of human SLAM (CDw150) is essential for its function as a measles virus receptor

Human signaling lymphocytic activation molecule (SLAM; also known as CDw150) has been shown to be a cellular receptor for measles virus (MV). Chinese hamster ovary cells transfected with a mouse SLAM cDNA were not susceptible to MV and the vesicular stomatitis virus pseudotype bearing MV envelope proteins alone, indicating that mouse SLAM cannot act as an MV receptor. To determine the functional domain of the receptor, we tested the abilities of several chimeric SLAM proteins to function as MV receptors. The ectodomain of SLAM comprises the two immunoglobulin superfamily domains (V and C2). Various chimeric transmembrane proteins possessing the V domain of human SLAM were able to act as MV receptors, whereas a chimera consisting of human SLAM containing the mouse V domain instead of the human V domain no longer acted as a receptor. To examine the interaction between SLAM and MV envelope proteins, recombinant soluble forms of SLAM were produced. The soluble molecules possessing the V domain of human SLAM were shown to bind to cells expressing the MV hemagglutinin (H) protein but not to cells expressing the MV fusion protein or irrelevant envelope proteins. These results indicate that the V domain of human SLAM is necessary and sufficient to interact with the MV H protein and allow MV entry.     

The Aspergillus nidulans genes chsA and chsD encode chitin synthases which have redundant functions in conidia formation

 We previously isolated three chitin synthase genes (chsA, chsB, and chsC) from Aspergillus nidulans. In the present work, we describe the isolation and characterization of another chitin synthase gene, named chsD, from A. nidulans. Its deduced amino acid sequence shows 56.7% and 55.9% amino acid identity, respectively, with Cal1 of Saccharomyces cerevisiae and Chs3 of Candida albicans. Disruption of chsD caused no defect in cell growth or morphology during the asexual cycle and caused no decrease in chitin content in hyphae. However, double disruption of chsA and chsD caused a remarkable decrease in the efficiency of conidia formation, while double disruption of chsC and chsD caused no defect. Thus it appears that chsA and chsD serve redundant functions in conidia formation.     

Hydroxylations of substituted naphthalenes by Escherichia coli expressing aromatic dihydroxylating dioxygenase genes from polycyclic aromatic hydrocarbon-utilizing marine bacteria

Bioconversion experiments of various mono- or di-substituted naphthalenes such as dimethylnaphthalenes were carried out using the cells of Escherichia coli that expressed aromatic dihydroxylating dioxygenase genes (phnA1A2A3A4 and phdABCD) from polycyclic aromatic hydrocarbon-utilizing marine bacteria, Nocardioides sp. KP7 and Cycloclasticus sp. A5, respectively. We found that the former dioxygenase PhnA1A2A3A4 had broad substrate preference for these compounds and often was able to hydroxylate their methyl groups. Specifically, 1,4-dimethylnaphthalene was predominantly bioconverted into 1,4-dihydroxymethylnaphthalene.