Roles of coenzyme F420-reducing hydrogenases and hydrogen- and F420-dependent methylenetetrahydromethanopterin dehydrogenases in reduction of F420 and production of hydrogen during methanogenesis

Reduced coenzyme F420 (F420H2) is an essential intermediate in methanogenesis from CO2. During methanogenesis from H2 and CO2, F420H2 is provided by the action of F420-reducing hydrogenases. However, an alternative pathway has been proposed, where H2-dependent methylenetetrahydromethanopterin dehydrogenase (Hmd) and F420H2-dependent methylenetetrahydromethanopterin dehydrogenase (Mtd) together reduce F420 with H2. Here we report the construction of mutants of Methanococcus maripaludis that are defective in each putative pathway. Their analysis demonstrates that either pathway supports growth on H2 and CO2. Furthermore, we show that during growth on formate instead of H2, where F420H2 is a direct product of formate oxidation, H2 production occurs. H2 presumably arises from the oxidation of F420H2, and the analysis of the mutants during growth on formate suggests that this too can occur by either pathway. We designate the alternative pathway for the interconversion of H2 and F420H2 the Hmd-Mtd cycle.     

Carrier-mediated transport systems of tetraethylammonium in rat renal brush-border and basolateral membrane vesicles

Transport of [3H]tetraethylammonium, an organic cation, has been studied in brush-border and basolateral membrane vesicles isolated from rat kidney cortex. Some characteristics of carrier-mediated transport for tetraethylammonium were demonstrated in brush-border and basolateral membrane vesicles; the uptake was saturable, was stimulated by the countertransport effect, and showed discontinuity in an Arrhenius plot. In brush-border membrane vesicles, the presence of an H+ gradient ( [H+]i greater than [H+]o) induced a marked stimulation of tetraethylammonium uptake against its concentration gradient (overshoot phenomenon), and this concentrative uptake was completely inhibited by HgCl2. In contrast, the uptake of tetraethylammonium by basolateral membrane vesicles was unaffected by an H+ gradient. Tetraethylammonium uptake by basolateral membrane vesicles was significantly stimulated by a valinomycin-induced inside-negative membrane potential, while no effect of membrane potential was observed in brush-border membrane vesicles. These results suggest that tetraethylammonium transport across brush-border membranes is driven by an H+ gradient via an electroneutral H+-tetraethylammonium antiport system, and that tetraethylammonium is transported across basolateral membranes via a carrier-mediated system and this process is stimulated by an inside-negative membrane potential.     

Characteristics of tetraethylammonium transport in rabbit renal plasma-membrane vesicles.

Transport of [14C]tetraethylammonium (TEA), an organic cation, was studied in brush-border (BBMV) and basolateral (BLMV) membrane vesicles isolated from rabbit kidney cortex. In BBMV, the presence of an outwardly directed H+ gradient induced a marked stimulation of TEA uptake against its concentration gradient (overshoot phenomenon), whereas a valinomycin-induced inside-negative potential had no effect on TEA uptake. In BLMV, TEA uptake was significantly stimulated by the presence of an outwardly directed H+ gradient and by an inside-negative potential, but the effect of H+ gradient was absent when the vesicles were chemically 'voltage clamped'. In BBMV, internal H+ stimulated TEA uptake in a non-competitive manner by binding at a site with apparent pKa of 6.87. External H+ inhibited TEA uptake through a direct interaction with the putative H+/organic-cation exchanger at a site with apparent pKa of 6.78. Changing external pH while maintaining the pH gradient constant produced a result similar to that obtained by changing external pH alone. Increasing external H+ showed a mixed-type inhibition of TEA uptake. These results suggest that in the rabbit TEA transport across the basolateral membranes is driven by an inside-negative potential and that transport across the brush-border membrane is driven by a H+ gradient via an electroneutral H+/TEA antiport system.     

Role of histone pairs H2A,H2B and H3,H4 in the self-assembly of nucleosome core particles

The role of the histone pairs H2A,H2B and H3,H4 in the kinetics of core particle formation was investigated by using N-(1-pyrene)maleimide-labeled histone H3. The excimer emission intensity of a DNA-core histone complex prepared by direct mixing of DNA and histones in 0.2 m-NaCl is reduced by half when H2A,H2B is omitted. Fluorescence quenching studies and lifetime measurements indicate that the emission differences are probably due to static quenching. In a correctly folded nucleosome or a DNA-(H3,H4) complex, the two pyrene rings are buried and are held very close. DNA-(H3,H4) can interact with additional copies of H3,H4, but only when two dimers of H2A,H2B are correctly bound is there a specific twofold increase in excimer emission.The kinetics of the reaction of H3,H4 with DNA in 0.2 m-NaCl were followed by measuring the increase in 460 nm fluorescence. The apparent rate constant of the dominant kinetic component is ~ 2 × 10^?1 s^?1. If histones H2A,H2B are added immediately after the preparation of the DNA-(H3,H4) complex, an increase in excimer fluorescence is observed, with an apparent rate constant of ~ 6 × 10^?3 s^?1. However, if histones H2A,H2B are added one hour after DNA-(H3,H4) complex formation, there is no increase in excimer fluorescence. These results suggest that an intermediate involving the H3,H4 tetramer is formed first in nucleosome assembly. In the presence of H2A,H2B, this intermediate evolves to the final folded nucleosome, but in the absence of H2A,H2B it rearranges to an unmaturable dead-end complex. Additional experiments show that a very fast transfer of histone pairs (probably H2A,H2B) can take place between partially reconstituted nucleosomes.     

Histamine modulation of eosinophil migration.

Preincubation of eosinophils with 10(-5) M or higher concentrations of histamine inhibited the eosinophil chemotactic response to endotoxin-activated serum whether by using the nucleopore filter assay and counting the cells migrating through the filter, or by using the Zigmond-Hirsch assay and counting the cells at each 10-mum interval. When the H2-receptor sites on the eosinophils were blocked by metiamide, the inhibitory capacity of histamine was prevented. Preincubation of eosinophils with 10(-6) M histamine increased the number of responding eosinophils to endotoxin-activated serum and this enhancement was blocked by an H1-receptor antagonist. Isoproteronol and aminophylline inhibited eosinophil movement and increasing concentrations of dibutryl cyclic AMP inhibited eosinophil migration. Concentrations of histamine that consistently resulted in inhibition of eosinophil movement stimulated an increase in cyclic AMP that was prevented by blocking the H2-receptor but not the H1-receptor. Thus, histamine-dependent inhibition of the eosinophil chemotactic response to other agents is mediated through the H2-receptor and is associated with an increase in the intracellular level of cyclic AMP whereas histamine dependent enhancement of eosinophil migration to other agents appears to be mediated through the H1-receptor. Eosinophils behave as a heterogeneous population as assessed by the ability of histamine to augment or inhibit cell migration. This may reflect differences in H1 to H2 receptor density or cell responsiveness to receptor stimulation. The chemoattractant activity of histamine itself is not influenced by H1 or H2 receptor antagonists, thus it is possible that an eosinophil has a third type of histamine receptor.     

Mitotic phosphorylation of histone H3 threonine 80

The onset and regulation of mitosis is dependent on phosphorylation of a wide array of proteins. Among the proteins that are phosphorylated during mitosis is histone H3, which is heavily phosphorylated on its N-terminal tail. In addition, large-scale mass spectrometry screens have revealed that histone H3 phosphorylation can occur at multiple sites within its globular domain, yet detailed analyses of the functions of these phosphorylations are lacking. Here, we explore one such histone H3 phosphorylation site, threonine 80 (H3T80), which is located on the nucleosome surface. Phosphorylated H3T80 (H3T80ph) is enriched in metazoan cells undergoing mitosis. Unlike H3S10 and H3S28, H3T80 is not phosphorylated by the Aurora B kinase. Further, mutations of T80 to either glutamic acid, a phosphomimetic, or to alanine, an unmodifiable residue, result in an increase in cells in prophase and an increase in anaphase/telophase bridges, respectively. SILAC-coupled mass spectrometry shows that phosphorylated H3T80 (H3T80ph) preferentially interacts with histones H2A and H4 relative to non-phosphorylated H3T80, and this result is supported by increased binding of H3T80ph to histone octamers in vitro. These findings support a model where H3T80ph, protruding from the nucleosome surface, promotes interactions between adjacent nucleosomes to promote chromatin compaction during mitosis in metazoan cells.     

Transport of H+ in mitochondria induced by the uptake of sulfite, sulfate and thiosulfate

The addition of sulphite to rat-liver mitochondria (RLM) causes an uptake of H+ that is unaffected by NEM and butylmalonate. The uptake of H+ induced by sulphate or thiosulphate is abolished by NEM and butylmalonate in freshly isolated RLM, whereas it is inhibited only by butylmalonate in sulphite-pretreated mitochondria. The data suggest that sulphite is cotransported with H+, whereas the movement of H+ associated to the uptake of sulphate or thiosulphate by RLM is mediated by either phosphate or sulphite.     

Mammalian growth-associated H1 histone kinase: a homolog of cdc2+/CDC28 protein kinases controlling mitotic entry in yeast and frog cells.

Mammalian growth-associated H1 histone kinase, an enzyme whose activity is sharply elevated at mitosis, is similar to cdc2+ protein kinase from Schizosaccharomyces pombe and CDC28 protein kinase from Saccharomyces cerevisiae with respect to immunoreactivity, molecular size, and specificity for phosphorylation sites in H1 histone. Phosphorylation of specific growth-associated sites in H1 histone is catalyzed by yeast cdc2+/CDC28 kinase, as shown by the in vitro thermal lability of this activity in extracts prepared from temperature-sensitive mutants. In addition, highly purified Xenopus maturation-promoting factor catalyzes phosphorylation of the same sites in H1 as do the mammalian and yeast kinases. The data indicate that growth-associated H1 kinase is encoded by a mammalian homolog of cdc2+/CDC28 protein kinase, which controls entry into mitosis in yeast and frog cells. Since H1 histone is known to be an in vivo substrate of the mammalian kinase, this suggests that phosphorylation of H1 histone or an H1 histone counterpart is an important component of the mechanism for entry of cells into mitosis.     

Inhibition of Influenza H7 Hemagglutinin-Mediated Entry

The recent outbreak of H7N9 influenza in China is of high concern to public health. H7 hemagglutinin (HA) plays a critical role in influenza entry and thus HA presents an attractive target for antivirals. Previous studies have suggested that the small molecule tert-butyl hydroquinone (TBHQ) inhibits the entry of influenza H3 HA by binding to the stem loop of HA and stabilizing the neutral pH conformation of HA, thereby disrupting the membrane fusion step. Based on amino acid sequence, structure and immunogenicity, H7 is a related Group 2 HA. In this work we show, using a pseudovirus entry assay, that TBHQ inhibits H7 HA-mediated entry, as well as H3 HA-mediated entry, with an IC50∼6 µM. Using NMR, we show that TBHQ binds to the H7 stem loop region. STD NMR experiments indicate that the aromatic ring of TBHQ makes extensive contact with the H7 HA surface. Limited proteolysis experiments indicate that TBHQ inhibits influenza entry by stabilizing the H7 HA neutral pH conformation. Together, this work suggests that the stem loop region of H7 HA is an attractive target for therapeutic intervention and that TBHQ, which is a widely used food preservative, is a promising lead compound.     

Structure of the histone chaperone ASF1 bound to the histone H3 C-terminal helix and functional insights

Asf1 is a histone chaperone that favors histone H3/H4 assembly and disassembly. We solved the structure of the conserved domain of human ASF1A in complex with the C-terminal helix of histone H3 using nuclear magnetic resonance spectroscopy. This structure is fully compatible with an association of ASF1 with the heterodimeric form of histones H3/H4. In our model, ASF1 substitutes for the second H3/H4 heterodimer that is normally found in heterotetrameric H3/H4 complexes. This result constitutes an essential step in the fundamental understanding of the mechanisms of nucleosome assembly by histone chaperones. Point mutations that perturb the Asf1/histone interface were designed from the structure. The decreased binding affinity of the Asf1-H3/H4 complex correlates with decreased levels of H3-K56 acetylation and phenotypic defects in vivo.     

A novel mode of TRPML3 regulation by extracytosolic pH absent in the varitint-waddler phenotype

TRPML3 belongs to the TRPML subfamily of the transient receptor potential (TRP) channels. The A419P mutation in TRPML3 causes the varitint-waddler phenotype as a result of gain-of-function mutation (GOF). Regulation of the channels and the mechanism by which the A419P mutation leads to GOF are not known. We report here that TRPML3 is a Ca(2+)-permeable channel with a unique form of regulation by extracytosolic (luminal) H(+) (H(+)(e-cyto)). Regulation by H(+)(e-cyto) is mediated by a string of three histidines (H252, H273, H283) in the large extracytosolic loop between transmembrane domains (TMD) 1 and 2. Each of the histidines has a unique role, whereby H252 and H273 retard access of H(+)(e-cyto) to the inhibitory H283. Notably, the H283A mutation has the same phenotype as A419P and locks the channel in an open state, whereas the H283R mutation inactivates the channel. Accordingly, A419P eliminates regulation of TRPML3 by H(+)(e-cyto), and confers full activation to TRPML3(H283R). Activation of TRPML3 and regulation by H(+)(e-cyto) are altered by both the alpha-helix-destabilizing A419G and the alpha-helix-favouring A419M and A419K. These findings suggest that regulation of TRPML3 by H(+)(e-cyto) is due to an effect of the large extracytosolic loop on the orientation of fifth TMD and thus pore opening and show that the GOF of TRPML3(A419P) is due to disruption of this communication.     

Properties of the apo-form of the NADP(H)-binding domain III of proton-pumping Escherichia coli transhydrogenase: implications for the reaction mechanism of the intact enzyme

Proton-translocating nicotinamide nucleotide transhydrogenases contain an NAD(H)-binding domain (dI), an NADP(H)-binding domain (dIII) and a membrane domain (dII) with the proton channel. Separately expressed and isolated dIII contains tightly bound NADP(H), predominantly in the oxidized form, possibly representing a so-called "occluded" intermediary state of the reaction cycle of the intact enzyme. Despite a K(d) in the micromolar to nanomolar range, this NADP(H) exchanges significantly with the bulk medium. Dissociated NADP(+) is thus accessible to added enzymes, such as NADP-isocitrate dehydrogenase, and can be reduced to NADPH. In the present investigation, dissociated NADP(H) was digested with alkaline phosphatase, removing the 2'-phosphate and generating NAD(H). Surprisingly, in the presence of dI, the resulting NADP(H)-free dIII catalyzed a rapid reduction of 3-acetylpyridine-NAD(+) by NADH, indicating that 3-acetylpyridine-NAD(+) and/or NADH interacts unspecifically with the NADP(H)-binding site. The corresponding reaction in the intact enzyme is not associated with proton pumping. It is concluded that there is a 2'-phosphate-binding region in dIII that controls tight binding of NADP(H) to dIII, which is not a required for fast hydride transfer. It is likely that this region is the Lys424-Arg425-Ser426 sequence and loops D and E. Further, in the intact enzyme, it is proposed that the same region/loops may be involved in the regulation of NADP(H) binding by an electrochemical proton gradent.     

Proton gradient-dependent transport of glycine in rabbit renal brush-border membrane vesicles

This study describes evidence for the existence of a H+/glycine symport system in rabbit renal brush-border membrane vesicles. An inward proton gradient stimulates glycine transport across the brush-border membrane, and this H+-driven glycine uptake is attenuated by the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone. It is a positive rheogenic process, i.e. the H+-dependent glycine uptake is further enhanced by an intravesicular negative potential. Glycine uptake is stimulated to a lesser degree by an inward Na+ gradient. H+-dependent glycine uptake is inhibited by sarcosine (69%), an analog amino acid, imino acids (proline 81%, hydroxy proline 67%), and beta-alanine (31%), but not by neutral (L-leucine) or basic (L-lysine) amino acids. The results demonstrate that H+ glycine co-transport system in rabbit renal brush-border membrane vesicles is a carrier-mediated electrogenic process and that transport is shared by imino acids and partially by beta-alanine.