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The urea channel of Helicobacter pylori (HpUreI) is an ideal drug target for preventing gastric cancer but incomplete understanding of its gating mechanism has hampered development of inhibitors for the eradication of H. pylori. Here, we present the cryo-EM structures of HpUreI in closed and open conformations, both at a resolution of 2.7 Å. Our hexameric structures of this small membrane protein (~21 kDa/protomer) resolve its periplasmic loops and carboxyl terminus that close and open the channel, and define a gating mechanism that is pH dependent and requires cooperativity between protomers in the hexamer. Gating is further associated with well-resolved changes in the channel-lining residues that modify the shape and length of the urea pore. Site-specific mutations in the periplasmic domain and urea pore identified key residues important for channel function. Drugs blocking the urea pore based on our structures should lead to a new strategy for H. pylori eradication.

The Na,K-ATPase α2 subunit plays a key role in cardiac muscle contraction by regulating intracellular Ca2+, whereas α1 has a more conventional role of maintaining ion homeostasis. The β subunit differentially regulates maturation, trafficking, and activity of α-β heterodimers. It is not known whether the distinct role of α2 in the heart is related to selective assembly with a particular one of the three β isoforms. We show here by immunofluorescence and co-immunoprecipitation that α2 is preferentially expressed with β2 in T-tubules of cardiac myocytes, forming α2β2 heterodimers. We have expressed human α1β1, α2β1, α2β2, and α2β3 in Pichia pastoris, purified the complexes, and compared their functional properties. α2β2 and α2β3 differ significantly from both α2β1 and α1β1 in having a higher K0.5K+ and lower K0.5Na+ for activating Na,K-ATPase. These features are the result of a large reduction in binding affinity for extracellular K+ and shift of the E1P-E2P conformational equilibrium toward E1P. A screen of perhydro-1,4-oxazepine derivatives of digoxin identified several derivatives (e.g. cyclobutyl) with strongly increased selectivity for inhibition of α2β2 and α2β3 over α1β1 (range 22-33-fold). Molecular modeling suggests a possible basis for isoform selectivity. The preferential assembly, specific T-tubular localization, and low K+ affinity of α2β2 could allow an acute response to raised ambient K+ concentrations in physiological conditions and explain the importance of α2β2 for cardiac muscle contractility. The high sensitivity of α2β2 to digoxin derivatives explains beneficial effects of cardiac glycosides for treatment of heart failure and potential of α2β2-selective digoxin derivatives for reducing cardiotoxicity.

Septins are a family of cytoskeletal GTP-binding proteins that assemble into membrane-associated hetero-oligomers and organize scaffolds for recruitment of cytosolic proteins or stabilization of membrane proteins. Septins have been implicated in a diverse range of cancers, including gastric cancer, but the underlying mechanisms remain unclear. The hypothesis tested here is that septins contribute to cancer by stabilizing the receptor tyrosine kinase ErbB2, an important target for cancer treatment. Septins and ErbB2 were highly over-expressed in gastric cancer cells. Immunoprecipitation followed by MS analysis identified ErbB2 as a septin-interacting protein. Knockdown of septin-2 or cell exposure to forchlorfenuron (FCF), a well-established inhibitor of septin oligomerization, decreased surface and total levels of ErbB2. These treatments had no effect on epidermal growth factor receptor (EGFR), emphasizing the specificity and functionality of the septin-ErbB2 interaction. The level of ubiquitylated ErbB2 at the plasma membrane was elevated in cells treated with FCF, which was accompanied by a decrease in co-localization of ErbB2 with septins at the membrane. Cathepsin B inhibitor, but not bafilomycin or lactacystin, prevented FCF-induced decrease in total ErbB2 by increasing accumulation of ubiquitylated ErbB2 in lysosomes. Therefore, septins protect ErbB2 from ubiquitylation, endocytosis and lysosomal degradation. The FCF-induced degradation pathway is distinct from and additive with the degradation induced by inhibiting ErbB2 chaperone Hsp90. These results identify septins as novel regulators of ErbB2 expression that contribute to the remarkable stabilization of the receptor at the plasma membrane of cancer cells and may provide a basis for the development of new ErbB2-targeting anti-cancer therapies.

Septins are a family of 14 cytoskeletal proteins that dynamically form hetero-oligomers and organize membrane microdomains for protein complexes. The previously reported interactions with SNARE proteins suggested the involvement of septins in exocytosis. However, the contradictory results of up- or down-regulation of septin-5 in various cells and mouse models or septin-4 in mice suggested either an inhibitory or a stimulatory role for these septins in exocytosis. The involvement of the ubiquitously expressed septin-2 or general septin polymerization in exocytosis has not been explored to date. Here, by nano-LC with tandem MS and immunoblot analyses of the septin-2 interactome in mouse brain, we identified not only SNARE proteins but also Munc-18-1 (stabilizes assembled SNARE complexes), N-ethylmaleimide-sensitive factor (NSF) (disassembles SNARE complexes after each membrane fusion event), and the chaperones Hsc70 and synucleins (maintain functional conformation of SNARE proteins after complex disassembly). Importantly, α-soluble NSF attachment protein (SNAP), the adaptor protein that mediates NSF binding to the SNARE complex, did not interact with septin-2, indicating that septins undergo reorganization during each exocytosis cycle. Partial depletion of septin-2 by siRNA or impairment of septin dynamics by forchlorfenuron inhibited constitutive and stimulated exocytosis of secreted and transmembrane proteins in various cell types. Forchlorfenuron impaired the interaction between SNAP-25 and its chaperone Hsc70, decreasing SNAP-25 levels in cultured neuroendocrine cells, and inhibited both spontaneous and stimulated acetylcholine secretion in mouse motor neurons. The results demonstrate a stimulatory role of septin-2 and the dynamic reorganization of septin oligomers in exocytosis.

Half the world's population is chronically infected with Helicobacter pylori, causing gastritis, gastric ulcers and an increased incidence of gastric adenocarcinoma. Its proton-gated inner-membrane urea channel, HpUreI, is essential for survival in the acidic environment of the stomach. The channel is closed at neutral pH and opens at acidic pH to allow the rapid access of urea to cytoplasmic urease. Urease produces NH(3) and CO(2), neutralizing entering protons and thus buffering the periplasm to a pH of roughly 6.1 even in gastric juice at a pH below 2.0. Here we report the structure of HpUreI, revealing six protomers assembled in a hexameric ring surrounding a central bilayer plug of ordered lipids. Each protomer encloses a channel formed by a twisted bundle of six transmembrane helices. The bundle defines a previously unobserved fold comprising a two-helix hairpin motif repeated three times around the central axis of the channel, without the inverted repeat of mammalian-type urea transporters. Both the channel and the protomer interface contain residues conserved in the AmiS/UreI superfamily, suggesting the preservation of channel architecture and oligomeric state in this superfamily. Predominantly aromatic or aliphatic side chains line the entire channel and define two consecutive constriction sites in the middle of the channel. Mutation of Trp 153 in the cytoplasmic constriction site to Ala or Phe decreases the selectivity for urea in comparison with thiourea, suggesting that solute interaction with Trp 153 contributes specificity. The previously unobserved hexameric channel structure described here provides a new model for the permeation of urea and other small amide solutes in prokaryotes and archaea.

The gastric H,K-adenosine triphosphatase (ATPase) is the primary target for treatment of acid-related diseases. Proton pump inhibitors (PPIs) are weak bases composed of two moieties, a substituted pyridine with a primary pKa of about 4.0 that allows selective accumulation in the secretory canaliculus of the parietal cell, and a benzimidazole with a second pKa of about 1.0. Protonation of this benzimidazole activates these prodrugs, converting them to sulfenic acids and/or sulfenamides that react covalently with one or more cysteines accessible from the luminal surface of the ATPase. The maximal pharmacodynamic effect of PPIs as a group relies on cyclic adenosine monophosphate–driven H,K-ATPase translocation from the cytoplasm to the canalicular membrane of the parietal cell. At present, this effect can only be achieved with protein meal stimulation. Because of covalent binding, inhibitory effects last much longer than their plasma half-life. However, the short dwell-time of the drug in the blood and the requirement for acid activation impair their efficacy in acid suppression, particularly at night. All PPIs give excellent healing of peptic ulcer and produce good, but less than satisfactory, results in reflux esophagitis. PPIs combined with antibiotics eradicate Helicobacter pylori, but success has fallen to less than 80%. Longer dwell-time PPIs promise to improve acid suppression and hence clinical outcome. Potassium-competitive acid blockers (P-CABs) are another class of ATPase inhibitors, and at least one is in development. The P-CAB under development has a long duration of action even though its binding is not covalent. PPIs with a longer dwell time or P-CABs with long duration promise to address unmet clinical needs arising from an inability to inhibit nighttime acid secretion, with continued symptoms, delayed healing, and growth suppression of H. pylori reducing susceptibility to clarithromycin and amoxicillin. Thus, novel and more effective suppression of acid secretion would benefit those who suffer from acid-related morbidity, continuing esophageal damage and pain, nonsteroidal anti-inflammatory drug–induced ulcers, and nonresponders to H. pylori eradication.

Helicobacter pylori colonizes the normal human stomach by maintaining both periplasmic and cytoplasmic pH close to neutral in the presence of gastric acidity. Urease activity, urea flux through the pH-gated urea channel, UreI, and periplasmic [alpha]-carbonic anhydrase are essential for colonization. Exposure to pH 4.5 for up to 180 min activates total bacterial urease threefold. Within 30 min at pH 4.5, the urease structural subunits, UreA and UreB, and the [Ni.sup.2+] insertion protein, UreE, are recruited to UreI at the inner membrane. Formation of this complex and urease activation depend on expression of the cytoplasmic sensor histidine kinase, HP0244. Its deletion abolishes urease activation and assembly, impairs cytoplasmic and periplasmic pH homeostasis, and depolarizes the cells, with an ~7-1og loss of survival at pH 2.5, even in 10 mM urea. Associated with this assembly, UreI is able to transport N[H.sub.3], N[H.sub.4.sup.+], and C[O.sub.2], as shown by changes in cytoplasmic pH following exposure to N[H.sub.4]CI or C[O.sub.2]. To be able to colonize cells in the presence of the highly variable pH of the stomach, the organism expresses two pH-sensor histidine kinases, one, HP0165, responding to a moderate fall in periplasmic pH and the other, HP0244, responding to cytoplasmic acidification at a more acidic medium pH. Assembly of a pH-regulatory complex of active urease with UreI provides an advantage for periplasmic buffering. doi: 10.1128/JB.00848-09

Polarized distribution of plasma membrane transporters and receptors in epithelia is essential for vectorial functions of epithelia. This polarity is maintained by sorting of membrane proteins into apical or basolateral transport containers in the trans-Golgi network and/or endosomes followed by their delivery to the appropriate plasma membrane domains. Sorting depends on the recognition of sorting signals in proteins by specific sorting machinery. In the present review, we summarize experimental evidence for and against the hypothesis that N-glycans attached to the membrane proteins can act as apical sorting signals. Furthermore, we discuss the roles of N-glycans in the apical sorting event per se and their contribution to folding and quality control of glycoproteins in the endoplasmic reticulum or retention of glycoproteins in the plasma membrane. Finally, we review existing hypotheses on the mechanism of apical sorting and discuss the potential roles of the lectins, VIP36 and galectin-3, as putative apical sorting receptors. apical sorting; apical membrane retention; H-K-ATPase [beta]-subunit; lectin

Helicobacter pylori colonizes the acidic gastric environment, in contrast to all other neutralophiles, whose acid resistance and tolerance responses allow only gastric transit. This acid adaptation is dependent on regulation of gene expression in response to pH changes in the periplasm and cytoplasm. The cytoplasmic histidine kinase, HP0244, which until now was thought only to regulate flagellar gene expression via its cognate response regulator, HP0703, was found to generate a response to declining medium pH. Although not required for survival at pH 4.5, HP0244 is required for survival at pH 2.5 with 10 mM urea after 30 min. Transcriptional profiling of a HP0244 deletion mutant grown at pH 7.4 confirmed the contribution of HP0244 to [[sigma].sup.54] activation via HP0703 to coordinate flagellar biosynthesis by a pH-independent regulon that includes 14 flagellar genes. Microarray analysis of cells grown at pH 4.5 without urea revealed an additional 22 genes, including 4 acid acclimation genes (ureA, ureB, ureI, and amiE) that are positively regulated by HP0244. Additionally, 86 differentially expressed genes, including 3 acid acclimation genes (ureF, rocF [arginase], and ansB [asparaginase]), were found in cells grown at pH 2.5 with 30 mM urea. Hence, HP0244 has, in addition to the pH-independent flagellar regulon, a pH-dependent regulon, which allows adaptation to a wider range of environmental acid conditions. An acid survival study using an HP0703 mutant and an electrophoretic mobility shift assay with in vitro-phosphorylated HP0703 showed that HP0703 does not contribute to acid survival and does not bind to the promoter regions of several genes in the HP0244 pH-dependent regulon, suggesting that there is a pathway outside the HP0703 regulon which transduces the acid-responsive signal sensed by HP0244.

New models for the [E.sub.1]K and [E.sub.2]P conformations of the H, K ATPase are generated by homology modeling from the crystal structures of the [E.sub.1]2[Ca.sup.2+] and [E.sub.2]Mg[F.sub.4.sup.2-] conformations of the srCa ATPase. The [E.sub.2]P model is further modified to account for naphthyridine access to the bio-chemically defined inhibitor-binding site.

Helicobacter pylori is a gastric-dwelling pathogen responsible, with acid secretion, for peptic ulcer and a 20-fold increase in the risk of gastric cancer. Several transcriptomes have been described after short-term exposure to acidity in vitro, but there are no data identifying the effects of chronic gastric exposure on bacterial gene expression. Comparison of the in vivo to the in vitro transcriptome at pH 7.4 identified several groups of genes of known function that increased expression >2-fold, and three of these respond both to acidity in vitro and to gastric infection. Almost all known acid acclimation genes are highly up-regulated. These include ureA, ureB, and rocF and the pH-gated urea channel, urel. There is also up-regulation of two groups of motility and chemotaxis genes and for pathogenicity island genes, especially cagA, a predictor for pathogenicity. Most of these genes interact with HP0166, the response element of the pH-sensing two-component histidine kinase, HP0165/HP0166, ArsRS. Based on the pH profile of survival of urel deletion mutants in vitro and their inability to survive in gastric acidity, the habitat of the organism at the gastric surface is acidic with a pH [less than or equal to] 4.0. Hence, the pH of the habitat of H. pylori on the surface of the stomach largely determines the regulation of these specific groups of genes. acid acclimation | gastric pH | global transcriptome | microarray

The periplasmic [alpha]-carbonic anhydrase of Helicobacterpylori is essential for buffering the periplasm at acidic pH. This enzyme is an integral component of the acid acclimation response that allows this neutralophile to colonize the stomach. Transcription of the HPl186 [alpha]-carbonic anhydrase gene is upregulated in response to low environmental pH. A binding site for the HP0166 response regulator (ArsR) has been identified in the promoter region of the HPl186 gene. To investigate the mechanism that regulates the expression of HPl186 in response to low pH and the role of the HP0165-HP0166 two-component system (ArsRS) in this acidinducible regulation, Northern blot analysis was performed with RNAs isolated from two different wild-type H. pylori strains (26695 and 43504) and mutants with HP0l65 histidine kinase (ArsS) deletions, after exposure to either neutral pH or low pH (pH 4.5). ArsS-dependent upregulation of HPl186 [alpha]-carbonic anhydrase in response to low pH was found in both strains. Western blot analysis of H. pylori membrane proteins confirmed the regulatory role of ArsS in HPl186 expression in response to low pH. Analysis of the HP1186 promoter region revealed two possible transcription start points ([TSP.sub.1] and [TSP.sub.2]) located 43 and 11 bp 5' of the ATG start codon, respectively, suggesting that there are two promoters transcribing the HPl186 gene. Quantitative primer extension analysis showed that the promoter from [TSP.sub.1] (43 bp 5' of the ATG start codon) is a pH-dependent promoter and is regulated by ArsRS in combating environmental acidity, whereas the promoter from [TSP.sub.2] may be responsible for control of the basal transcription of HP1186 [alpha]-carbonic anhydrase.

About 200 genes of the gastric pathogen Helicobacter pylori increase expression at medium pHs of 6.2, 5.5, and 4.5, an increase that is abolished or much reduced by the buffering action of urease. Genes up-regulated by a low pH include the two-component system HP0165-HP0166, suggesting a role in the regulation of some of the pH-sensitive genes. To identify targets of HP0165-HP0166, the promoter regions of genes up-regulated by a low pH were grouped based on sequence similarity. Probes for promoter sequences representing each group were subjected to electrophoretic mobility shift assays (EMSA) with recombinant HP0166-[His.sub.6] or a mutated response regulator, HP0166-D52N-His6, that can specifically determine the role of phosphorylation of HP0166 in binding (including a control EMSA with inovitrophosphnrylated HP0166-[His.sub.6],). Nineteen of 45 promoter-regulatory regions were found to interact with HP0166-[His.sub.6]. Seven promoters for genes encoding s-carbonic anhydrase, omp11, fecD, lpp20, hypA, and two with unknown function (pHP1397-1396 and pHP0654-0675) were clustered in gene group A, which may respond to changes in the periplasmic pH at a constant cytoplasmic pH and showed phosphorylation-dependent binding in EMSA with HP0166-D52N-[His.sub.6]. Twelve promoters were clustered in groups B and C whose up-regulation likely also depends on a reduction of the cytoplasmic pH at a medium pH of 5.5 or 4.5. Most of the target promoters in groups B and C showed phosphorylation-dependent binding with HP0166-D52N-[His.sub.6], but promoters for ompR (pHP0166-0162), pHP0682-0681, and pHP1288-1289 showed phosphorylation-independent binding. These findings, combined with DNase I footprinting, suggest that HP0165-0166 is an acid-responsive signaling system affecting the expression of pH-sensitive genes. Regulation of these genes responds either to a decrease in the periplasmic pH alone (HP0165 dependent) or also to a decrease in the cytoplasmic pH (HP0165 independent).

Effects of an oligomeric structure on the characteristics of E(sub 1), E(sub 2) and phosphoenzyme conformations are examined by measuring binding stoichiometries of acid-stable phosphorylation (EP). Result shows that the catalytic subunits of the oligomer during turnover in intact gastric vesicles are restricted to E(sub 1):E(sub 2) configuration.

The study was conducted for the ileal apical and liver basolateral bile acid transporters catalyze the Na (super +)-dependent uptake of these amphipathic molecules in the intestine and liver. The result reveals that seven membrane segments gives a new model of the membrane domain of the protein and probably the homologues ileal transporter.

A homology modeling of the H,K-ATPase in the E2-P conformation as a means of predicting the interaction of the enzyme with two known classes of specific inhibitors is presented. All known proton pump inhibitors form a disulfide bond with cysteine 813 that is accessible from the luminal surface.