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Species of the genus Phaeohelotium (Leotiomycetes: Helotiaceae) are cup fungi that grow on decaying wood, leaves, litter, and directly on soil. Northern Hemisphere species are primarily found on litter and wood, whereas in the Southern Hemisphere the genus includes a mix of saprotrophs as well as taxa that grow on soil in association with ectomycorrhizal trees. The diversity of this genus has not been fully explored in southern South America. Here we describe two species from Chile, Phaeohelotium maiusaurantium sp. nov. and Ph. pallidum sp. nov., found on soil in Patagonian Nothofagaceae-dominated forests. We present macro- and micromorphological descriptions, illustrations, and molecular phylogenetic analyses. The two new species are placed in Phaeohelotium with high support in our 15-locus phylogeny as well as phylogenetic reconstructions based on the internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene. Our ITS phylogeny places both Ph. maiusaurantium and Ph. pallidum in a well-supported subclade that includes ectomycorrhizal root tip samples from Australasia. Similar species can be separated from these new taxa based on morphological characteristics, biogeography, substrate, and sequence data. In addition, two unnamed species from Chilean Nothofagaceae forests (Phaeohelotium sp. 1 and Phaeohelotium sp. 2) are documented from scant collections and sequence data and await description until more material becomes available.

Significance A large family of membrane proteins, voltage-gated ion channels, regulate a vast array of physiological functions in essentially all life forms. How these molecules sense membrane potential and respond by creating ionic conduction is incompletely understood. The voltage sensors of these channels contain a “hydrophobic gasket,” a ring of hydrophobic amino acids near the center of the membrane, separating internal and external aqueous solutions. Although voltage-gated proton channels, HV1, resemble voltage-sensing domains of other channels, they differ fundamentally. On depolarization, HV1 conducts protons, whereas other voltage sensors open a physically distinct pore. We identify Val109, Phe150, Val177, and Val178 as the hHV1 hydrophobic gasket. Replacement with less hydrophobic amino acids accelerated channel opening and caused proton-selective leak through closed channels., The hydrophobic gasket (HG), a ring of hydrophobic amino acids in the voltage-sensing domain of most voltage-gated ion channels, forms a constriction between internal and external aqueous vestibules. Cationic Arg or Lys side chains lining the S4 helix move through this “gating pore” when the channel opens. S4 movement may occur during gating of the human voltage-gated proton channel, hHV1, but proton current flows through the same pore in open channels. Here, we replaced putative HG residues with less hydrophobic residues or acidic Asp. Substitution of individuals, pairs, or all 3 HG positions did not impair proton selectivity. Evidently, the HG does not act as a secondary selectivity filter. However, 2 unexpected functions of the HG in HV1 were discovered. Mutating HG residues independently accelerated channel opening and compromised the closed state. Mutants exhibited open–closed gating, but strikingly, at negative voltages where “normal” gating produces a nonconducting closed state, the channel leaked protons. Closed-channel proton current was smaller than open-channel current and was inhibited by 10 μM Zn2+. Extreme hyperpolarization produced a deeper closed state through a weakly voltage-dependent transition. We functionally identify the HG as Val109, Phe150, Val177, and Val178, which play a critical and exclusive role in preventing H+ influx through closed channels. Molecular dynamics simulations revealed enhanced mobility of Arg208 in mutants exhibiting H+ leak. Mutation of HG residues produces gating pore currents reminiscent of several channelopathies.

International audience; The reactive oxygen species (ROS)-producing enzyme NADPH oxidase (NOX) was first identified in the membrane of phagocytic cells. For many years, its only known role was in immune defense, where its ROS production leads to the destruction of pathogens by the immune cells. NOX from phagocytes catalyzes, via one-electron trans-membrane transfer to molecular oxygen, the production of the superoxide anion. Over the years, six human homologs of the catalytic subunit of the phagocyte NADPH oxidase were found: NOX1, NOX3, NOX4, NOX5, DUOX1, and DUOX2. Together with the NOX2/gp91$^{phox}$ component present in the phagocyte NADPH oxidase assembly itself, the homologs are now referred to as the NOX family of NADPH oxidases. NOX are complex multidomain proteins with varying requirements for assembly with combinations of other proteins for activity. The recent structural insights acquired on both prokaryotic and eukaryotic NOX open new perspectives for the understanding of the molecular mechanisms inherent to NOX regulation and ROS production (superoxide or hydrogen peroxide). This new structural information will certainly inform new investigations of human disease. As specialized ROS producers, NOX enzymes participate in numerous crucial physiological processes, including host defense, the post-translational processing of proteins, cellular signaling, regulation of gene expression, and cell differentiation. These diversities of physiological context will be discussed in this review. We also discuss NOX misregulation, which can contribute to a wide range of severe pathologies, such as atherosclerosis, hypertension, diabetic nephropathy, lung fibrosis, cancer, or neurodegenerative diseases, giving this family of membrane proteins a strong therapeutic interest.

The voltage-gated proton channel (HV1) is a voltage sensor that also conducts protons. The singular ability of protons to penetrate proteins complicates distinguishing closed and open channels. When we replaced valine with histidine at position 116 in the external vestibule of hHV1, current was potently inhibited by externally applied Zn2+ in a construct lacking the two His that bind Zn2+ in WT channels. High-affinity binding with profound effects at 10 nM Zn2+ at pHo 7 suggests additional groups contribute. We hypothesized that Asp185, which faces position 116 in our closed-state model, contributes to Zn2+ chelation. Confirming this prediction, V116H/D185N abolished Zn2+ binding. Studied in a C-terminal truncated monomeric construct, V116H channels activated rapidly. Anomalously, Zn2+ slowed activation, producing a time constant independent of both voltage and Zn2+ concentration. We hypothesized that slow turn-on of H+ current in the presence of Zn2+ reflects the rate of Zn2+ unbinding from the channel, analogous to drug-receptor dissociation reactions. This behavior in turn suggests that the affinity for Zn2+ is greater in the closed state of hHV1. Supporting this hypothesis, pulse pairs revealed a rapid component of activation whose amplitude decreased after longer intervals at negative voltages as closed channels bound Zn2+. The lower affinity of Zn2+ in open channels is consistent with the idea that structural rearrangements within the transmembrane region bring Arg205 near position 116, electrostatically expelling Zn2+. This phenomenon provides direct evidence that Asp185 opposes position 116 in closed channels and that Arg205 moves between them when the channel opens.

International audience; Small angle neutron scattering (SANS) provides a method to obtain important low-resolution information for integral membrane proteins (IMPs), challenging targets for structural determination. Specific deuteration furnishes a "stealth" carrier for the solubilized IMP. We used SANS to determine a structural envelope of SpNOX, the Streptococcus pneumoniae NADPH oxidase (NOX), a prokaryotic model system for exploring structure and function of eukaryotic NOXes. SpNOX was solubilized in the detergent lauryl maltose neopentyl glycol, which provides optimal SpNOX stability and activity. Using deuterated solvent and protein, the lauryl maltose neopentyl glycol was experimentally undetected in SANS. This affords a cost-effective SANS approach for obtaining novel structural information on IMPs. Combining SANS data with molecular modeling provided a first, to our knowledge, structural characterization of an entire NOX enzyme. It revealed a distinctly less compact structure than that predicted from the docking of homologous crystal structures of the separate transmembrane and dehydrogenase domains, consistent with a flexible linker connecting the two domains.

Bioluminescence in dinoflagellates is controlled by HV 1 proton channels. Database searches of dinoflagellate transcriptomes and genomes yielded hits with sequence features diagnostic of all confirmed HV 1, and show that HV 1 is widely distributed in the dinoflagellate phylogeny including the basal species Oxyrrhis marina. Multiple sequence alignments followed by phylogenetic analysis revealed three major subfamilies of HV 1 that do not correlate with presence of theca, autotrophy, geographic location, or bioluminescence. These data suggest that most dinoflagellates express a HV 1 which has a function separate from bioluminescence. Sequence evidence also suggests that dinoflagellates can contain more than one HV 1 gene.

Voltage-gated proton channels open appropriately in myriad physiological situations because their gating is powerfully modulated by both pHo and pHi. Cherny et al. serendipitously identify a histidine at the inner end of the S3 helix that is required for the response to pHi., We recently identified a voltage-gated proton channel gene in the snail Helisoma trivolvis, HtHV1, and determined its electrophysiological properties. Consistent with early studies of proton currents in snail neurons, HtHV1 opens rapidly, but it unexpectedly exhibits uniquely defective sensitivity to intracellular pH (pHi). The H+ conductance (gH)-V relationship in the voltage-gated proton channel (HV1) from other species shifts 40 mV when either pHi or pHo (extracellular pH) is changed by 1 unit. This property, called ΔpH-dependent gating, is crucial to the functions of HV1 in many species and in numerous human tissues. The HtHV1 channel exhibits normal pHo dependence but anomalously weak pHi dependence. In this study, we show that a single point mutation in human hHV1—changing His168 to Gln168, the corresponding residue in HtHV1—compromises the pHi dependence of gating in the human channel so that it recapitulates the HtHV1 response. This location was previously identified as a contributor to the rapid gating kinetics of HV1 in Strongylocentrotus purpuratus. His168 mutation in human HV1 accelerates activation but accounts for only a fraction of the species difference. H168Q, H168S, or H168T mutants exhibit normal pHo dependence, but changing pHi shifts the gH-V relationship on average by

Voltage-gated proton currents were first discovered in snail neurons. Thomas et al. identify a snail proton channel gene that codes for a rapidly activating proton channel that differs from other proton channels, in particular by its low sensitivity to intracellular pH., Voltage-gated proton channels, HV1, were first reported in Helix aspersa snail neurons. These H+ channels open very rapidly, two to three orders of magnitude faster than mammalian HV1. Here we identify an HV1 gene in the snail Helisoma trivolvis and verify protein level expression by Western blotting of H. trivolvis brain lysate. Expressed in mammalian cells, HtHV1 currents in most respects resemble those described in other snails, including rapid activation, 476 times faster than hHV1 (human) at pHo 7, between 50 and 90 mV. In contrast to most HV1, activation of HtHV1 is exponential, suggesting first-order kinetics. However, the large gating charge of ∼5.5 e0 suggests that HtHV1 functions as a dimer, evidently with highly cooperative gating. HtHV1 opening is exquisitely sensitive to pHo, whereas closing is nearly independent of pHo. Zn2+ and Cd2+ inhibit HtHV1 currents in the micromolar range, slowing activation, shifting the proton conductance–voltage (gH-V) relationship to more positive potentials, and lowering the maximum conductance. This is consistent with HtHV1 possessing three of the four amino acids that coordinate Zn2+ in mammalian HV1. All known HV1 exhibit ΔpH-dependent gating that results in a 40-mV shift of the gH-V relationship for a unit change in either pHo or pHi. This property is crucial for all the functions of HV1 in many species and numerous human cells. The HtHV1 channel exhibits normal or supernormal pHo dependence, but weak pHi dependence. Under favorable conditions, this might result in the HtHV1 channel conducting inward currents and perhaps mediating a proton action potential. The anomalous ΔpH-dependent gating of HtHV1 channels suggests a structural basis for this important property, which is further explored in this issue (Cherny et al. 2018. J. Gen. Physiol. https://doi.org/10.1085/jgp.201711968).

International audience; NADPH oxidases (NOX) have many biological roles, but their regulation to control production of potentially toxic ROS molecules remains unclear. A previously identified insertion sequence of 21 residues (called NIS) influences NOX activity, and its predicted flexibility makes it a good candidate for providing a dynamic switch controlling the NOX active site. We constructed NOX2 chimeras in which NIS had been deleted or exchanged with those from other NOXs (NIS1, 3 and 4). All contained functional heme and were expressed normally at the plasma membrane of differentiated PLB-985 cells. However, NOX2-ΔNIS and NOX2-NIS1 had neither NADPH-oxidase nor reductase activity and exhibited abnormal translocation of p47(phox) and p67(phox) to the phagosomal membrane. This suggested a functional role of NIS. Interestingly after activation, NOX2-NIS3 cells exhibited superoxide overproduction compared with wild-type cells. Paradoxically, the Vmax of purified unstimulated NOX2-NIS3 was only one-third of that of WT-NOX2. We therefore hypothesized that post-translational events regulate NOX2 activity and differ between NOX2-NIS3 and WT-NOX2. We demonstrated that Ser486, a phosphorylation target of ataxia telangiectasia mutated kinase (ATM kinase) located in the NIS of NOX2 (NOX2-NIS), was phosphorylated in purified cytochrome b558 after stimulation with phorbol 12-myristate-13-acetate (PMA). Moreover, ATM kinase inhibition and a NOX2 Ser486Ala mutation enhanced NOX activity whereas a Ser486Glu mutation inhibited it. Thus, the absence of Ser486 in NIS3 could explain the superoxide overproduction in the NOX2-NIS3 mutant. These results suggest that PMA-stimulated NOX2-NIS phosphorylation by ATM kinase causes a dynamic switch that deactivates NOX2 activity. We hypothesize that this downregulation is defective in NOX2-NIS3 mutant because of the absence of Ser486.

Superoxide radicals and other reactive oxygen species (ROS) are implicated in influenza A virus-induced inflammation. In thisin vitrostudy, we evaluated the effects of TG6-44, a novel quinazolin-derived myeloperoxidase-specific ROS inhibitor, on influenza A virus (A/X31) infection using THP-1 lung monocytic cells and freshly isolated peripheral blood mononuclear cells (PBMC). TG6-44 significantly decreased A/X31-induced ROS and virus-induced inflammatory mediators in THP-1 cells (IL-6, IFN-γ, MCP-1, TNF-α, MIP-1β) and in human PBMC (IL-6, IL-8, TNF-α, MCP-1). Interestingly, TG6-44-treated THP-1 cells showed a decrease in percent cells expressing viral nucleoprotein, as well as a delay in translocation of viral nucleoprotein into the nucleus. Furthermore, in influenza A virus-infected cells, TG6-44 treatment led to suppression of virus-induced cell death as evidenced by decreased caspase-3 activation, decreased proportion of Annexin V+PI+cells, and increased Bcl-2 phosphorylation. Taken together, our results demonstrate the anti-inflammatory and anti-infective effects of TG6-44.

Myeloperoxidase (MPO) is a key antimicrobial enzyme, playing a normal role in host defense, but also contributing to inflammatory conditions including neuroinflammatory diseases such as Parkinson's and Alzheimer's. We synthesized and characterized more than 50 quinazolin-4(1H)-one derivatives and showed that this class of compounds inhibits MPO with IC50 values as low as 100 nM. Representative compounds showed partially reversible inhibition that was competitive with respect to Amplex Red substrate and did not result in the accumulation of MPO Compound II. Members of this group show promise for therapeutic development for the treatment of diseases in which inflammation plays a pathogenic role.

p67phox is the paramount cytosolic regulator of the superoxide-generating Nox of phagocytes, by controlling the conformation of the catalytic component, Nox2. The initiating event of this process is a protein–protein interaction between p67phox and the part of Nox2 protruding into the cytosol, known as the dehydrogenase region. The aim of this study was to identify and characterize region(s) in Nox2 acting as binding site(s) for p67phox. For this purpose, we measured the binding of recombinant p67phox to an array of 91 overlapping synthetic pentadecapeptides covering the length of the dehydrogenase region (residues 288–570). We found that: 1) p67phox binds to a site corresponding to residues 357–383, represented by a cluster of 5 peptides (Nos. 24–28); 2) maximal binding was to peptides 24 (357–371) and 28 (369–383); 3) these shared a 369Cys-Gly-Cys371 triad, found to be responsible for binding; 4) the Cys-Gly-Cys triad was present in Nox2 of mammals, birds, and amphibians but was absent in other Nox; 5) substituting a Nox4 or Nox1 sequence for the Nox2 sequence in peptide 24 abolished binding; 6) replacing 369Cys by Arg in peptide 24 (mimicking a mutation in chronic granulomatous disease) abolished binding; 7) the same replacement in peptide 28 did not affect binding, indicating the existence of an additional binding site. Our results reveal an essential role for the Cys-Gly-Cys triad in Nox2 in binding p67phox, seconded by an additional binding region, comprising residues C terminal to Cys-Gly-Cys. The 2 regions interact with distinct partner sites in p67phox.

The examination of hair collected from crime scenes is an important and highly informative discipline relevant to many forensic investigations. However, the forensic identification of animal (non-human) hairs requires different skill sets and competencies to those required for human hair comparisons. The aim of this is paper is not only to highlight the intrinsic differences between forensic human hair comparison and forensic animal hair identification, but also discuss the utility and reliability of the two in the context of possibilities and pitfalls. It also addresses and dispels some of the more popular myths and misconceptions surrounding the microscopical examination of animal hairs. Furthermore, future directions of this discipline are explored through the proposal of recommendations for minimum standards for the morphological identification of animal hairs and the significance of the newly developed guidelines by SWGWILD is discussed.

The evolutionary origin of the autopod involved a loss of the fin-fold and associated dermal skeleton with a concomitant elaboration of the distal endoskeleton to form a wrist and digits. Developmental studies, primarily from teleosts and amniotes, suggest a model for appendage evolution in which a delay in the AER-to-fin-fold conversion fuelled endoskeletal expansion by prolonging the function of AER-mediated regulatory networks. Here, we characterize aspects of paired fin development in the paddlefishPolyodon spathula(a non-teleost actinopterygian) and catsharkScyliorhinus canicula(chondrichthyan) to explore aspects of this model in a broader phylogenetic context. Our data demonstrate that in basal gnathostomes, the autopod markerHoxA13co-localizes with the dermoskeleton componentAnd1to mark the position of the fin-fold, supporting recent work demonstrating a role forHoxA13in zebrafish fin ray development. Additionally, we show that in paddlefish, the proximal fin and fin-fold mesenchyme share a common mesodermal origin, and that components of the Shh/LIM/Gremlin/Fgf transcriptional network critical to limb bud outgrowth and patterning are expressed in the fin-fold with a profile similar to that of tetrapods. Together these data draw contrast with hypotheses of AER heterochrony and suggest that limb-specific morphologies arose through evolutionary changes in the differentiation outcome of conserved early distal patterning compartments.

A review of 92 heap leach projects from 15 countries provides a starting point for a series of experiments, at 22 °C and a vertical pressure of 2000 kPa, to examine short-term puncturing and the development of geomembrane strains that could affect longer-term performance. Underliners of gravel with some sand or those of gravel and sand caused significant puncturing and excessive strains in the geomembrane for the conditions examined. The shape of the underliner grading curve had a much greater effect on the potential for puncturing and the magnitude of the strains in the geomembrane than just the maximum particle size. Of the six granular underliners examined, the best performance was for the well graded gravelly sand with some silt which offered sufficient support to minimize the strains in the geomembrane due to the overliner while not inducing significant strains directly from the underliner. Nevertheless even in this case the maximum strain of 11% is almost double the maximum recommended in the literature for ensuring good long-term performance of the geomembrane. Consideration of composite liners with GCLs and compacted clay liners shows that the more deformable the foundation, the larger are the indentations and strains induced in the geomembrane by a given overliner. For the specific conditions examined, it is shown that there was no apparent improvement in performance for an LLDPE geomembrane versus the HDPE geomembrane tested. A 540 g/m 2 geotextile protection layer above the geomembrane was also found to be insufficient to prevent significant strains in the geomembrane due to the overliner examined.

In a real business cycle model, an agent's fear of model misspecification interacts with stochastic volatility to induce time varying worst case scenarios. These time varying worst case scenarios capture a notion of animal spirits where the probability distributions used to evaluate decision rules and price assets do not necessarily reflect the fundamental characteristics of the economy. Households entertain a pessimistic view of the world and their pessimism varies with the overall level of volatility in the economy, implying an amplification of the effects of volatility shocks. By using perturbation methods and Monte Carlo techniques we extend the class of models analyzed with robust control methods to include the sort of nonlinear production-based DSGE models that are popular in academic research and policymaking practice.

Background: Trimodality bladder preservation therapy (BPT) in muscle invasive bladder cancer (MIBC) includes a maximal transurethral resection followed by concurrent chemoradiotherapy as an alternative to radical cystectomy (RC) in appropriately selected patients, or as a treatment option in non-cystectomy candidates. Several chemotherapy regimens can be used in BPT, but little is known about current practice patterns. Objective: To describe utilization patterns of BPT and associated survival outcomes in MIBC. Methods: Data were collected from the Retrospective International Study of Cancers of the UrothelialTract (RISC), a database of 3,024 consecutive patients from 29 international academic centers from 2005 to 2013. Patients with clinical T2-T4aN0M0 urothelial cancer of the bladder were included. Results: 265 patients received BPT. Compared with the 1,447 patients who received RC, BPT patients were older, had poorer performance status, and had more comorbidities (p < 0.01 for all). Median overall survival (OS) was similar for patients treated with curative radiation doses in BPT and patients treated with RC (41 vs 46 months, p = 0.33, respectively). 45% of BPT patients received concurrent chemotherapy with radiation. The most common regimens included cisplatin alone (23%), carboplatin alone (22%), gemcitabine alone (10%), paclitaxel alone (9%), and 5-FU+mitomycin (5%). There were no significant differences in survival among chemotherapy regimens. Only 10 patients (4% of BPT patients) underwent salvage cystectomy. Conclusions: In clinical practice, BPT patients have similar survival to RC patients when treated with curative radiotherapy doses. Choice of concurrent chemotherapy regimen varied widely with no clear standard. Salvage cystectomy is rarely performed. Continued research is needed on the comparative effectiveness among BPT and RC, and among chemotherapy regimens in BPT. © 2016 - IOS Press and the authors. All rights reserved.

The fl uviolacustrine Wilkins Peak Member of the Eocene Green River Formation preserves repetitive sedimentary facies that have been interpreted as an orbitally induced climate signal. However, previous quantitative studies of cyclicity in this member have used oil-yield data derived from single locations. Here, macrostratigraphy is used to quantitatively describe the spatiotemporal patterns of three different lithofacies associations from 8 to 12 localities that span much of the basin. Macrostratigraphic time series demonstrate that there is a reciprocal basinscale relationship between carbonate-rich lacustrine facies and siliciclastic-rich alluvial facies. Spectral analyses identify statistically signifi cant periods (≥90% confi dence level) in basin-scale sedimentation that are consistent with Milankovitch-predicted orbital periodicities, with a particularly strong ~100 k.y. cycle expressed in all lithofacies associations. Numerous non-Milankovitch periods are also recognized, indicating complex depositional responses to orbital forcing, autocyclic controls on sedimentation, or harmonic artifacts. Although fl uctuations in Lake Gosiute water level did affect basin-scale patterns of sedimentation, they are not directly related to the 100 k.y. short-eccentricity cycle, as previously supposed. Instead, 100 k.y. cycles are principally recorded by the recurrence of alluvial environments, which exerted a dominant control on basin-scale patterns of sedimentation generally. Thus, the hydrologic controls on lake level that have been classically linked to short-eccentricity actually occurred at finer temporal scales (

Fogel and Hastings first hypothesized the existence of voltage-gated proton channels in 1972 in bioluminescent dinoflagellates, where they were thought to trigger the flash by activating luciferase. Proton channel genes were subsequently identified in human, mouse, and Ciona intestinalis , but their existence in dinoflagellates remained unconfirmed. We identified a candidate proton channel gene from a Karlodinium veneficum cDNA library based on homology with known proton channel genes. K. veneficum is a predatory, nonbioluminescent dinoflagellate that produces toxins responsible for fish kills worldwide. Patch clamp studies on the heterologously expressed gene confirm that it codes for a genuine voltage-gated proton channel, kH V 1: it is proton-specific and activated by depolarization, its g H – V relationship shifts with changes in external or internal pH, and mutation of the selectivity filter (which we identify as Asp 51 ) results in loss of proton-specific conduction. Indirect evidence suggests that kH V 1 is monomeric, unlike other proton channels. Furthermore, kH V 1 differs from all known proton channels in activating well negative to the Nernst potential for protons, E H . This unique voltage dependence makes the dinoflagellate proton channel ideally suited to mediate the proton influx postulated to trigger bioluminescence. In contrast to vertebrate proton channels, whose main function is acid extrusion, we propose that proton channels in dinoflagellates have fundamentally different functions of signaling and excitability.

Voltage-gated proton channels are considered completely selective for protons — no evidence exists for the permeation of ions other than H+. A study of the underlying mechanism of the high selectivity of the human voltage-gated proton channel reveals an Asp112 residue as the selectivity filter. When this residue was changed to a neutral amino acid, the mutant channel lost proton selectivity and either became anion-selective or did not conduct any ions. The ion selectivity of pumps and channels is central to their ability to perform a multitude of functions. Here we investigate the mechanism of the extraordinary selectivity of the human voltage-gated proton channel1, HV1 (also known as HVCN1). This selectivity is essential to its ability to regulate reactive oxygen species production by leukocytes2,3,4, histamine secretion by basophils5, sperm capacitation6, and airway pH7. The most selective ion channel known, HV1 shows no detectable permeability to other ions1. Opposing classes of selectivity mechanisms postulate that (1) a titratable amino acid residue in the permeation pathway imparts proton selectivity1,8,9,10,11, or (2) water molecules ‘frozen’ in a narrow pore conduct protons while excluding other ions12. Here we identify aspartate 112 as a crucial component of the selectivity filter of HV1. When a neutral amino acid replaced Asp 112, the mutant channel lost proton specificity and became anion-selective or did not conduct. Only the glutamate mutant remained proton-specific. Mutation of the nearby Asp 185 did not impair proton selectivity, indicating that Asp 112 has a unique role. Although histidine shuttles protons in other proteins, when histidine or lysine replaced Asp 112, the mutant channel was still anion-permeable. Evidently, the proton specificity of HV1 requires an acidic group at the selectivity filter.

Nox5 belongs to the calcium-regulated subfamily of NADPH oxidases (Nox). Like other calcium-regulated Noxes, Nox5 has an EF-hand-containing calcium-binding domain at its N-terminus, a transmembrane heme-containing region, and a C-terminal dehydrogenase (DH) domain that binds FAD and NADPH. While Nox1-4 require regulatory subunits, including p22phox, Nox5 activity does not depend on any subunits. We found that inactive point mutants and truncated forms of Nox5 (including the naturally expressed splice form, Nox5S) inhibit full-length Nox5, consistent with formation of a dominant negative complex. Oligomerization of full-length Nox5 was demonstrated using co-immunoprecipitation of coexpressed, differentially tagged forms of Nox5 and occurred in a manner independent of calcium ion. Several approaches were used to show that the DH domain mediates oligomerization: Nox5 could be isolated as a multimer when the calcium-binding domain and/or the N-terminal polybasic region (PBR-N) was deleted, but deletion of the DH domain eliminated oligomerization. Further, a chimera containing the transmembrane domain of Ciona intestinalis voltage sensor-containing phosphatase (CiVSP) fused to the Nox5 DH domain formed a co-immunoprecipitating complex with, and functioned as a dominant inhibitor of, full-length Nox5. Radiation inactivation of Nox5 overexpressed in HEK293 cells and endogenously expressed in human aortic smooth muscle cells indicated molecular masses of ∼350 and ∼300 kDa, respectively, consistent with a tetramer being the functionally active unit. Thus, Nox5 forms a catalytically active oligomer in the membrane that is mediated by its dehydrogenase domain. As a result of oligomerization, the short, calcium-independent splice form, Nox5S, may function as an endogenous inhibitor of calcium-stimulated ROS generation by full-length Nox5.

The Smithsonian Institution, through a grant from the Smithsonian Women's Committee, implemented a programme during the fall and winter of 2008 to teach the basics of mechanical fossil preparation, moulding and casting to individuals seeking to work as volunteer preparators in the National Museum of Natural History's exhibit preparation lab, called FossiLab. Four outside preparators were contracted to conduct the instruction; two instructors taught two five-day sessions on molding and casting and two other instructors taught two six-day sessions on preparation. The Department of Paleobiology's (Paleobiology) curatorial staff instructed the groups in paleobotanical preparation and microfossil processing and picking. All instruction took place in FossiLab during public hours. Registration for the programme was conducted via the department's web page where a detailed description of the work and the lab familiarized prospective students with the type of work they would be performing. They were also asked a series of selfevaluative questions in an attempt to filter out those who might not have the innate motor skills or temperament needed for the job. Those who were confident in their decision to volunteer were interviewed in FossiLab, and the majority of those then registered for the programme. Twenty nine people, some new and some experienced, were trained. FossiLab is now staffed each day by as many as six volunteer preparators who perform a variety of tasks for Paleobiology. The number of weekly person-hours has tripled since the training. Continued, focused training on individual projects is carried on by the Vertebrate Paleontology Preparation Laboratory staff. Eighteen hours of videotape was recorded during the training and will be edited and made available via DVD and the internet.

Voltage-gated proton channels are strongly inhibited by Zn2+, which binds to His residues. However, in a molecular model, the two externally accessible His are too far apart to coordinate Zn2+. We hypothesize that high-affinity Zn2+ binding occurs at the dimer interface between pairs of His residues from both monomers. Consistent with this idea, Zn2+ effects were weaker in monomeric channels. Mutation of His193 and His140 in various combinations and in tandem dimers revealed that channel opening was slowed by Zn2+ only when at least one His was present in each monomer, suggesting that in wild-type (WT) HV1, Zn2+ binding between His of both monomers inhibits channel opening. In addition, monomeric channels opened exponentially, and dimeric channels opened sigmoidally. Monomeric channel gating had weaker temperature dependence than dimeric channels. Finally, monomeric channels opened 6.6 times faster than dimeric channels. Together, these observations suggest that in the proton channel dimer, the two monomers are closely apposed and interact during a cooperative gating process. Zn2+ appears to slow opening by preventing movement of the monomers relative to each other that is prerequisite to opening. These data also suggest that the association of the monomers is tenuous and allows substantial freedom of movement. The data support the idea that native proton channels are dimeric. Finally, the idea that monomer–dimer interconversion occurs during activation of phagocytes appears to be ruled out.

Nitric oxide synthase (NOS) expression is regulated transcriptionally in response to cytokine induction and posttranslationally by palmitoylation and trafficking into perinuclear aggresome-like structures. We investigated the effects of multifunctional calcium/calmodulin-dependent protein kinase II protein kinase (CaMKII) on inducible NOS (iNOS) trafficking in cultured rat aortic vascular smooth muscle cells (VSMCs). Immunofluorescence and confocal microscopy demonstrated colocalization of iNOS and CaMKIIδ2with a perinuclear distribution and concentration in aggresome-like structures identified by colocalization with γ-tubulin. Furthermore, CaMKIIδ2coimmunoprecipitated with iNOS in a CaMKII activity-dependent manner. Addition of Ca2+-mobilizing stimuli expected to activate CaMKII; a purinergic agonist (UTP) or calcium ionophore (ionomycin) caused a general redistribution of iNOS from cytosolic to membrane and nuclear fractions. Similarly, adenoviral expression of a constitutively active CaMKIIδ2mutant altered iNOS localization, shifting iNOS from the cytosolic fraction. Suppression of CaMKIIδ2using an adenovirus expressing a short hairpin, small interfering RNA increased nuclear iNOS localization in resting cells but inhibited ionomycin-induced translocation of iNOS to the nucleus. Following addition of these chronic and acute CaMKII modulators, there were fewer aggresome-like structures containing iNOS. All of the treatments that chronically affected CaMKII activity or expression significantly inhibited iNOS-specific activity following cytokine induction. The results suggest that CaMKIIδ2may be an important regulator of iNOS trafficking and activity in VSMCs.

A conserved tryptophan uniquely present in the voltage-sensing domain of voltage-gated proton channels is surprisingly crucial to four channel-defining properties., Part of the “signature sequence” that defines the voltage-gated proton channel (HV1) is a tryptophan residue adjacent to the second Arg in the S4 transmembrane helix: RxWRxxR, which is perfectly conserved in all high confidence HV1 genes. Replacing Trp207 in human HV1 (hHV1) with Ala, Ser, or Phe facilitated gating, accelerating channel opening by 100-fold, and closing by 30-fold. Mutant channels opened at more negative voltages than wild-type (WT) channels, indicating that in WT channels, Trp favors a closed state. The Arrhenius activation energy, Ea, for channel opening decreased to 22 kcal/mol from 30–38 kcal/mol for WT, confirming that Trp207 establishes the major energy barrier between closed and open hHV1. Cation–π interaction between Trp207 and Arg211 evidently latches the channel closed. Trp207 mutants lost proton selectivity at pHo >8.0. Finally, gating that depends on the transmembrane pH gradient (ΔpH-dependent gating), a universal feature of HV1 that is essential to its biological functions, was compromised. In the WT hHV1, ΔpH-dependent gating is shown to saturate above pHi or pHo 8, consistent with a single pH sensor with alternating access to internal and external solutions. However, saturation occurred independently of ΔpH, indicating the existence of distinct internal and external pH sensors. In Trp207 mutants, ΔpH-dependent gating saturated at lower pHo but not at lower pHi. That Trp207 mutation selectively alters pHo sensing further supports the existence of distinct internal and external pH sensors. Analogous mutations in HV1 from the unicellular species Karlodinium veneficum and Emiliania huxleyi produced generally similar consequences. Saturation of ΔpH-dependent gating occurred at the same pHo and pHi in HV1 of all three species, suggesting that the same or similar group(s) is involved in pH sensing. Therefore, Trp enables four characteristic properties: slow channel opening, highly temperature-dependent gating kinetics, proton selectivity, and ΔpH-dependent gating.

Heat shock protein 90 (Hsp90) is a molecular chaperone that orchestrates the folding and stability of proteins that regulate cellular signaling, proliferation and inflammation. We have previously shown that Hsp90 controls the production of reactive oxygen species by modulating the activity of Noxes1-3 and 5, but not Nox4. The goal of the current study was to define the regions on Nox5 that bind Hsp90 and determine how Hsp90 regulates enzyme activity. In isolated enzyme activity assays, we found that Hsp90 inhibitors selectively decrease superoxide, but not hydrogen peroxide, production. The addition of Hsp90 alone only modestly increases Nox5 enzyme activity but in combination with the co-chaperones, Hsp70, HOP, Hsp40, and p23 it robustly stimulated superoxide, but not hydrogen peroxide, production. Proximity ligation assays reveal that Nox5 and Hsp90 interact in intact cells. In cell lysates using a co-IP approach, Hsp90 binds to Nox5 but not Nox4, and the degree of binding can be influenced by calcium-dependent stimuli. Inhibition of Hsp90 induced the degradation of full length, catalytically inactive and a C-terminal fragment (aa398-719) of Nox5. In contrast, inhibition of Hsp90 did not affect the expression levels of N-terminal fragments (aa1-550) suggesting that Hsp90 binding maintains the stability of C-terminal regions. In Co-IP assays, Hsp90 was bound only to the C-terminal region of Nox5. Further refinement using deletion analysis revealed that the region between aa490-550 mediates Hsp90 binding. Converse mapping experiments show that the C-terminal region of Nox5 bound to the M domain of Hsp90 (aa310-529). In addition to Hsp90, Nox5 bound other components of the foldosome including co-chaperones Hsp70, HOP, p23 and Hsp40. Silencing of HOP, Hsp40 and p23 reduced Nox5-dependent superoxide. In contrast, increased expression of Hsp70 decreased Nox5 activity whereas a mutant of Hsp70 failed to do so. Inhibition of Hsp90 results in the loss of higher molecular weight complexes of Nox5 and decreased interaction between monomers. Collectively these results show that the C-terminal region of Nox5 binds to the M domain of Hsp90 and that the binding of Hsp90 and select co-chaperones facilitate oligomerization and the efficient production of superoxide.

Voltage-gated proton channels, HV1, trigger bioluminescence in dinoflagellates, enable calcification in coccolithophores and play multifarious roles in human health. Because the proton concentration is minuscule, exquisite selectivity for protons over other ions is critical to HV1 function. The selectivity of the open HV1 channel requires an aspartate near an arginine in the selectivity filter (SF), a narrow region that dictates proton selectivity, but the mechanism of proton selectivity is unknown. Here we use a reduced quantum model to elucidate how the Asp–Arg SF selects protons but excludes other ions. Attached to a ring scaffold, the Asp and Arg side chains formed bidentate hydrogen bonds that occlude the pore. Introducing H3O+ protonated the SF, breaking the Asp–Arg linkage and opening the conduction pathway, whereas Na+ or Cl– was trapped by the SF residue of opposite charge, leaving the linkage intact, thus preventing permeation. An Asp–Lys SF behaved like the Asp–Arg one and was experimentally verified to be proton-selective, as predicted. Hence, interacting acidic and basic residues form favorable AspH0–H2O0–Arg+ interactions with hydronium but unfavorable Asp––X–/X+–Arg+ interactions with anions/cations. This proposed mechanism may apply to other proton-selective molecules engaged in bioenergetics, homeostasis and signaling.

Diabetes is a disorder of glucose metabolism and uncontrolled or poorly controlled glycemia has the potential to impair all organs. Cardiovascular disease, renal impairment, neuropathy, inflammatio...

The human voltage-gated proton channel (hHV1) is a transmembrane protein that is responsible for the selective permeation of protons across cell membranes in nasal mucosa, sperm, and white blood cells. hHV1 is a four-helix bundle (S1-S4) with anionic Asp112 on S1 forming a salt bridge with cationic Arg residues on helix S4 in the narrow region of the pore [Kulleperuma et al., J. Gen. Physiol. 141, 445-465 (2013)]. Mutation of Asp112 to Val abrogates channel properties. Unexpectedly, replacing Asp112 by a smaller neutral residue such as Ser turns HV1 into an anion selective channel that conducts Cl-, as does the double mutant D112V-V116S [Musset et al., Nature 480, 273-277 (2012); Morgan et al., J. Gen. Physiol. 142, 625-640 (2013)]. Although HV1 and its mutants exhibit drastic differences in ion permeation, the molecular basis of proton selectivity in WT and anion selectivity in mutants remains unexplained.As the first step towards elucidating the charge selectivity of HV1, we perform molecular dynamics simulations with umbrella sampling to compute the free energy profile for the translocation of Na+ and Cl- ions through the pore of a homology model of HV1 and its mutants. The calculations are repeated in conformational states of the channel differing in the extent of hydration of the pore and in the relative arrangements of pore residues. Results show how ion solvation and electrostatic interactions with charged side chains in the pore lumen modulate the energetics of ion permeation in HV1 and suggest a structural basis for charge selectivity.

Voltage gated proton channels (HV1) have been implicated in late stage breast cancer (Wang et al, 2012. J. Biol. Chem. 287:13877), where HV1 expression correlated with tumor size and poor prognosis. HV1 knockdown reduced cell proliferation and migration as well as matrix metalloprotease release. However, the presence of functional HV1 on cancer cell membranes has not been demonstrated and the mechanism by which they affect the function of these cells has not been elucidated.Here we show the definitive presence of functional HV1 on the membranes of MDA MB 231 cells, a highly metastatic triple negative cell line. We performed patch clamp experiments on these cells and were able to detect bona fide voltage- and pH-gated channels that were perfectly selective for protons. The membrane density of the channels in these cells was recorded as 3.5 pA/pF which is roughly 5-fold lower than the 15 pA/pF displayed in neutrophils. In order to show that HV1 expresses at a level sufficient to impact pH regulation within these cells, we acid loaded them using the ammonium prepulse technique and monitored pHi recovery utilizing SEER with SNARF-1. Inhibiting HV1 with 1 mM Zn2+ slowed recovery from an acid load by 3-fold, demonstrating that the expression of HV1 on these cells affects pH regulation in these cells.We conclude that one mechanism by which HV1 may influence the pathophysiology of breast cancer is by improving the ability of breast cancer cells to regulate their internal pH.Supported by the Bears Care and the Brian Piccolo fund.

A voltage sensor domain (VSD) is a protein module that rearranges its conformation based on the electric potential of the cell membrane. VSDs have classically been described as N-terminal modules that confer voltage sensitivity to C-terminal pore domains in ion channels. More recently, N-terminal VSDs have been shown to confer voltage response to C-terminal enzyme modules in voltage sensitive enzymes (VSE), while the isolated VSDs of voltage gated proton channels (Hv1) perform both voltage sensing and proton channel functions. So far, VSEs and Hv1s have been found only in eukaryotes. We have identified a set of prokaryotic sequences that contain a VSD homolog; however, the C-terminal domains of these sequences, which we refer to as putative prokaryotic voltage sensitive enzymes (ppVSE), are dramatically different from ion pores. As expected, predicted secondary structures of the N-terminal domain are similar to those for bona fide VSDs; however, unlike the pore domains of ion channels, which contain two transmembrane helices, predicted structures of the C-terminal domains of the ppVSEs do not contain transmembrane helices. Alignment of individual domains to the HMM of the ion channel pfam pf00520 indicates significant similarity of ppVSE N-terminal domains but no detectable similarity of ppVSE C-terminal domains to the pf00520 HMM. A phylogenetic analysis of VSDs from prokaryotic sequences indicates a distinct lineage of the ppVSE VSD. This is the first documented evidence of a prokaryotic VSD-containing protein that does not have a pore domain.

We report here that NADPH analogs such as 2'5'ADP, ATP, and 2'AMP paradoxically activate constitutive calcium/calmodulin regulated nitric oxide synthases (cNOS), including the endothelial isoform (eNOS) and the neuronal isoform (nNOS). These activators compete with NADPH by filling the binding site of the adenine moiety of NADPH, but do not occupy the entire NADPH binding domain. Effects of these analogs on cNOS's include increasing the electron transfer rate to external acceptors, as assessed by cytochrome c reductase activity in the absence of calmodulin. In addition, NO synthase activity in the presence of calmodulin (with or without added calcium) was increased by the addition of NADPH analogs. In contrast, the same NADPH analogs inhibit iNOS, the calcium insensitive inducible isoform, which lacks control elements found in constitutive isoforms. Because ATP and ADP are among the effective activators of cNOS isoforms, these effects may be physiologically relevant.

Mammalian nitric-oxide synthases are large modular enzymes that evolved from independently expressed ancestors. Calmodulin-controlled isoforms are signal generators; calmodulin activates electron transfer from NADPH through three reductase domains to an oxygenase domain. Structures of the reductase unit and its homologs show FMN and FAD in contact but too isolated from the protein surface to permit exit of reducing equivalents. To study states in which FMN/heme electron transfer is feasible, we designed and produced constructs including only oxygenase and FMN binding domains, eliminating strong internal reductase complex interactions. Constructs for all mammalian isoforms were expressed and purified as dimers. All synthesize NO with peroxide as the electron donor at rates comparable with corresponding oxygenase constructs. All bind cofactors nearly stoichiometrically and have native catalytic sites by spectroscopic criteria. Modest differences in electrochemistry versus independently expressed heme and FMN binding domains suggest interdomain interactions. These interactions can be convincingly demonstrated via calmodulin-induced shifts in high spin ferriheme EPR spectra and through mutual broadening of heme and FMNH· radical signals in inducible nitricoxide synthase constructs. Blue neutral FMN semiquinone can be readily observed; potentials of one electron couple (in inducible nitric-oxide synthase oxygenase FMN, FMN oxidized/semiquione couple =+70 mV, FMN semiquinone/hydroquinone couple =–180 mV, and heme =–180 mV) indicate that FMN is capable of serving as a one electron heme reductant. The construct will serve as the basis for future studies of the output state for NADPH derived reducing equivalents.

A single-stage polymerase-based procedure is described that allows extensive modifications of DNA. The version described here uses the QuikChange Site-Directed Mutagenesis System kit supplied by Stratagene. The original protocol is replaced by a single-stage method in which linear production of complementary strands is accomplished in separate single primer reactions. This has proved effective in introducting insertions and deletions into large gene/vector combinations without subcloning.

The oxygenase domains of nitric oxide synthases are unusual in that they contain at least three ligand binding sites; these correspond to the axial heme ligand position, the substrate binding site, and the pterin binding site. Ligands can occupy portions of a site or extend into regions of adjacent sites. Depending on the size, shape, and binding mode of ligands to these positions, cooperative and anticooperative interactions mediated conformationally and by binding domain overlap can be observed. In the present study we describe competition between arginine and imidazole at the axial heme ligand position; a second imidazole, which occupies part of the arginine site in some crystal structures, is too weak to contribute to the equilibria. All spectroscopic titrations using imidazole competition depend on displacement of the heme axial imidazole ligand, which drives the ferriheme low spin. Aminoguanidine, a partial arginine analog, has multiple binding modes. It is somewhat competitive with arginine; a ternary complex forms, but the K d for arginine increases from 1 to 15 μM in the presence of saturating aminoguanidine. Aminoguanidine competition with imidazole is very weak, amounting to approximately a factor of two increase in K d . This implies that aminoguanidine has multiple binding modes and is not well described as an arginine analog. The major binding mode occupies part of the binding site but does not extend into the imidazole axial ligand binding domain and probably corresponds to the crystal structure. The other binding mode is not significantly overlapped with the arginine site.