NADPH oxidases (NOX) are transmembrane proteins, widely spread in eukaryotes and prokaryotes, that produce reactive oxygen species (ROS). Eukaryotes use the ROS products for innate immune defense and ...signaling in critical (patho)physiological processes. Despite the recent structures of human NOX isoforms, the activation of electron transfer remains incompletely understood. SpNOX, a homolog from
, can serves as a robust model for exploring electron transfers in the NOX family thanks to its constitutive activity. Crystal structures of SpNOX full-length and dehydrogenase (DH) domain constructs are revealed here. The isolated DH domain acts as a flavin reductase, and both constructs use either NADPH or NADH as substrate. Our findings suggest that hydride transfer from NAD(P)H to FAD is the rate-limiting step in electron transfer. We identify significance of F397 in nicotinamide access to flavin isoalloxazine and confirm flavin binding contributions from both DH and Transmembrane (TM) domains. Comparison with related enzymes suggests that distal access to heme may influence the final electron acceptor, while the relative position of DH and TM does not necessarily correlate with activity, contrary to previous suggestions. It rather suggests requirement of an internal rearrangement, within the DH domain, to switch from a resting to an active state. Thus, SpNOX appears to be a good model of active NOX2, which allows us to propose an explanation for NOX2's requirement for activation.
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 paddlefish Polyodon spathula (a non-teleost actinopterygian) and catshark Scyliorhinus canicula (chondrichthyan) to explore aspects of this model in a broader phylogenetic context. Our data demonstrate that in basal gnathostomes, the autopod marker HoxA13 co-localizes with the dermoskeleton component And1 to mark the position of the fin-fold, supporting recent work demonstrating a role for HoxA13 in 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.
Voltage-gated proton channel in a dinoflagellate Smith, Susan M. E; Morgan, Deri; Musset, Boris ...
Proceedings of the National Academy of Sciences - PNAS,
11/2011, Letnik:
108, Številka:
44
Journal Article
Recenzirano
Odprti dostop
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, kHV1: it is proton-specific and activated by depolarization, its gH–V relationship shifts with changes in external or internal pH, and mutation of the selectivity filter (which we identify as Asp51) results in loss of proton-specific conduction. Indirect evidence suggests that kHV1 is monomeric, unlike other proton channels. Furthermore, kHV1 differs from all known proton channels in activating well negative to the Nernst potential for protons, EH. 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.
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.
Voltage-gated proton channels, H
1, were first reported in
snail neurons. These H
channels open very rapidly, two to three orders of magnitude faster than mammalian H
1. Here we identify an H
1 gene ...in the snail
and verify protein level expression by Western blotting of
brain lysate. Expressed in mammalian cells, HtH
1 currents in most respects resemble those described in other snails, including rapid activation, 476 times faster than hH
1 (human) at pH
7, between 50 and 90 mV. In contrast to most H
1, activation of HtH
1 is exponential, suggesting first-order kinetics. However, the large gating charge of ∼5.5
suggests that HtH
1 functions as a dimer, evidently with highly cooperative gating. HtH
1 opening is exquisitely sensitive to pH
, whereas closing is nearly independent of pH
Zn
and Cd
inhibit HtH
1 currents in the micromolar range, slowing activation, shifting the proton conductance-voltage (
-
) relationship to more positive potentials, and lowering the maximum conductance. This is consistent with HtH
1 possessing three of the four amino acids that coordinate Zn
in mammalian H
1. All known H
1 exhibit ΔpH-dependent gating that results in a 40-mV shift of the
-
relationship for a unit change in either pH
or pH
This property is crucial for all the functions of H
1 in many species and numerous human cells. The HtH
1 channel exhibits normal or supernormal pH
dependence, but weak pH
dependence. Under favorable conditions, this might result in the HtH
1 channel conducting inward currents and perhaps mediating a proton action potential. The anomalous ΔpH-dependent gating of HtH
1 channels suggests a structural basis for this important property, which is further explored in this issue (Cherny et al. 2018.
https://doi.org/10.1085/jgp.201711968).
NADPH oxidases (NOX) are transmembrane proteins, widely spread in eukaryotes and prokaryotes, that produce reactive oxygen species (ROS). Eukaryotes use the ROS products for innate immune defense and ...signaling in critical (patho)physiological processes. Despite the recent structures of human NOX isoforms, the activation of electron transfer remains incompletely understood. SpNOX, a homolog from Streptococcus pneumoniae , can serves as a robust model for exploring electron transfers in the NOX family thanks to its constitutive activity. Crystal structures of SpNOX full-length and dehydrogenase (DH) domain constructs are revealed here. The isolated DH domain acts as a flavin reductase, and both constructs use either NADPH or NADH as substrate. Our findings suggest that hydride transfer from NAD(P)H to FAD is the rate-limiting step in electron transfer. We identify significance of F397 in nicotinamide access to flavin isoalloxazine and confirm flavin binding contributions from both DH and Transmembrane (TM) domains. Comparison with related enzymes suggests that distal access to heme may influence the final electron acceptor, while the relative position of DH and TM does not necessarily correlate with activity, contrary to previous suggestions. It rather suggests requirement of an internal rearrangement, within the DH domain, to switch from a resting to an active state. Thus, SpNOX appears to be a good model of active NOX2, which allows us to propose an explanation for NOX2’s requirement for activation.
Does SpNox directly produce hydrogen peroxide? Scott, James; Notice, Brittany; Gonzalez, Victoria ...
The FASEB journal,
April 2019, 2019-04-00, Letnik:
33, Številka:
S1
Journal Article
Recenzirano
NADPH oxidases (NOXs) are a family of transmembrane enzymes that generate reactive oxygen species (ROS), which in humans acts both as a cytotoxic agent in autoimmunity and as a signaling molecule in ...many physiological processes. Human NOXes can produce different ROS species; for example, NOX2 isoform generates superoxide whereas NOX4 isoform produces mainly hydrogen peroxide. The ROS species produced affects downstream signaling, and misregulation produces disease states. A recently discovered prokaryotic NOX, SpNOX, provides an ideal model system to investigate controls on ROS product. SpNOX was previously shown to produce superoxide. Because superoxide spontaneously dismutes at a fast rate, assays that detect hydrogen peroxide cannot be used to assess direct hydrogen peroxide formation. Instead, as in the case of xanthine oxidase, we estimate total electron flux and also superoxide production; we assume that any difference between these values reflects direct hydrogen peroxide production by the enzyme. We estimate electron flux using three assays: i/ monitoring NADPH consumption using absorbance (340 nm); ii/ monitoring NADPH consumption using fluorescence (ex 345/em 454); iii/ monitoring cytochrome c reduction by the FAD of a dehydrogenase‐domain only construct of SpNOX. Superoxide generation from full‐length SpNOX is estimated by the superoxide‐dismutase‐inhibitable portion of cytochrome c reduction, monitored by absorbance at 550 nm. Here we present our findings regarding relative production of superoxide and hydrogen peroxide by SpNOX, as a first step toward manipulating ROS product in this model system.
This is from the Experimental Biology 2019 Meeting. There is no full text article associated with this published in The FASEB Journal.
p67(phox) 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 p67(phox) 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 p67(phox). For this purpose, we measured the binding of recombinant p67(phox) to an array of 91 overlapping synthetic pentadecapeptides covering the length of the dehydrogenase region (residues 288-570). We found that: 1) p67(phox) 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 (369)Cys-Gly-Cys(371) 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 (369)Cys 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 p67(phox), seconded by an additional binding region, comprising residues C terminal to Cys-Gly-Cys. The 2 regions interact with distinct partner sites in p67(phox).
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 <20 mV/unit. Thus, His168 is critical to pHi sensing in hHV1. His168, located at the inner end of the pore on the S3 transmembrane helix, is the first residue identified in HV1 that significantly impairs pH sensing when mutated. Because pHo dependence remains intact, the selective erosion of pHi dependence supports the idea that there are distinct internal and external pH sensors. Although His168 may itself be a pHi sensor, the converse mutation, Q229H, does not normalize the pHi sensitivity of the HtHV1 channel. We hypothesize that the imidazole group of His168 interacts with nearby Phe165 or other parts of hHV1 to transduce pHi into shifts of voltage-dependent gating.
Part of the "signature sequence" that defines the voltage-gated proton channel (...) is a tryptophan residue adjacent to the second Arg in the S4 transmembrane helix: RxWRxxR, which is perfectly ...conserved in all high confidence ... genes. Replacing ... in human ... (...) 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 ... establishes the major energy barrier between closed and open ... Cation-p interaction between ... and ... evidently latches the channel closed. ... mutants lost proton selectivity at ... >8.0. Finally, gating that depends on the transmembrane pH gradient (...pH-dependent gating), a universal feature of ... that is essential to its biological functions, was compromised. In the WT ..., ...-dependent gating is shown to saturate above ... or ... 8, consistent with a single pH sensor with alternating access to internal and external solutions. However, saturation occurred independently of ..., indicating the existence of distinct internal and external pH sensors. In ... mutants, ...-dependent gating saturated at lower ... but not at lower ... That ... mutation selectively alters ... sensing further supports the existence of distinct internal and external pH sensors. Analogous mutations in ... from the unicellular species Karlodinium veneficum and Emiliania huxleyi produced generally similar consequences. Saturation of ...-dependent gating occurred at the same ... and ...in ... 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 ...-dependent gating. (ProQuest: ... denotes formulae/symbols omitted.)
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