Proton transfer drives protein radical formation in Helicobacter pylori catalase but not in Penicillium vitale catalase.

Heme catalases prevent cells from oxidative damage by decomposing hydrogen peroxide into water and molecular oxygen. Here we investigate the factors that give rise to an undesirable side reaction competing with normal catalase activity, the migration of a radical from the heme active site to the protein in the principal reaction intermediate compound I (Cpd I). Recently, it has been proposed that this electron transfer reaction takes place in Cpd I of Helicobacter pylori catalase (HPC), but not in Cpd I of Penicillium vitale catalase (PVC), where the oxidation equivalent remains located on the heme active site. Unraveling the factors determining the different radical locations could help engineer enzymes with enhanced catalase activity for detection or removal of hydrogen peroxide. Using quantum mechanics/molecular mechanics metadynamics simulations, we show that radical migration in HPC is facilitated by the large driving force (-0.65 eV) of the subsequent proton transfer from a histidine residue to the ferryl oxygen atom of reduced Cpd I. The corresponding free energy in PVC is significantly smaller (-0.19 eV) and, as we argue, not sufficiently high to support radical migration. Our results suggest that the energetics of oxoferryl protonation is a key factor regulating radical migration in catalases and possibly also in hydroperoxidases.

Genome-wide association identifies SKIV2L and MYRIP as protective factors for age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly in the developed world. We conducted a genome-wide association study in a series of families enriched for AMD and completed a meta-analysis of this new data with results from reanalysis of an existing study of a late-stage case-control cohort. We tested the top findings for replication in 1896 cases and 1866 controls and identified two novel genetic protective factors for AMD. In addition to the complement factor H (CFH) (P=2.3 × 10⁻64) and age-related maculopathy susceptibility 2 (ARMS2) (P=1.2 × 10⁻6°) loci, we observed a protective effect at rs429608, an intronic SNP in SKIV2L (P=5.3 × 10⁻¹5), a gene near the complement component 2 (C2)/complement factor B (BF) locus, that indicates the protective effect may be mediated by variants other than the C2/BF variants previously studied. Haplotype analysis at this locus identified three protective haplotypes defined by the rs429608 protective allele. We also identified a new potentially protective effect at rs2679798 in MYRIP (P=2.9 × 10⁻4), a gene involved in retinal pigment epithelium melanosome trafficking. Interestingly, MYRIP was initially identified in the family-based scan and was confirmed in the case-control set. From these efforts, we report the identification of two novel protective factors for AMD and confirm the previously known associations at CFH, ARMS2 and C3.

Polymorphisms in C2, CFB and C3 are associated with progression to advanced age related macular degeneration associated with visual loss.

BACKGROUND: Age related macular degeneration (AMD) is a leading cause of blindness. AMD is a complex disorder caused by genetic and environmental factors in which single nucleotide polymorphisms (SNPs) in the genes CFH and LOC387715/HTRA1/ARMS2 have prognostic importance for progression to advanced AMD (with visual loss). CFH may also have a pharmacogenetic role by affecting treatment response to widely used nutritional supplements. This paper examines other AMD susceptibility genes to determine if these genotypes influenced disease progression and treatment response. METHODS: Three cohorts, totalling 3137 individuals, were genotyped for SNPs in 13 genes previously published to be associated with advanced AMD (other than CFH and LOC387715/ARMS2/HTRA1). Those genes found associated were then evaluated for their involvement in disease progression. Interactions between the genes and with AREDS (Age-Related Eye Disease Study) nutritional supplements were investigated. RESULTS: Positive independent associations were noted in SNPs in the genes C2 (p = 0.0001, odds ratio (OR) 0.35, 95% confidence interval (CI) 0.2 to 0.6), CFB (p = 0.0001, OR 0.35, 95% CI 0.2 to 0.6), C3 (p = 0.0001, OR 3.91, 95% CI 1.94 to 7.88), APOE (epsilon4, p = 0.01, OR 0.50, 95% CI 0.29 to 0.86) and VEGFA (p = 0.01, OR 2.23, 95% CI 1.06 to 4.68). C2/CFB and C3 were independently related to progression from early/intermediate to advanced AMD with OR 0.32 (95% CI 0.14 to 0.73) and 3.32 (95% CI 1.46 to 7.59), respectively. Gene-gene and pharmacogenetic interactions were not observed. No preferential associations were observed with geographic atrophy or choroidal neovascularisation. CONCLUSION: This study provides insights into the genetic pathogenesis of AMD. Five genes have now been shown to be independently involved in progression from intermediate disease (before vision loss has occurred) to advanced disease in which blindness is frequent.

The interaction of cytochrome c and the heme domain of cytochrome P-450BM-3 with the reductase domain of cytochrome P-450BM-3.

Cytochrome P-450BM-3 from Bacillus megaterium is a soluble, catalytically self-sufficient fatty acid mono-oxygenase that resembles the Class II P-450 systems of the eukaryotic endoplasmic reticulum. Its single polypeptide chain contains both a P-450 heme domain and an NADPH:P-450 reductase domain, each of which bears significant structural and functional homology with its microsomal counterparts. We report here that cytochrome c, which can accept NADPH-derived electrons from the reductase domain of P-450-BM-3, did not inhibit myristate hydroxylation catalyzed by P-450BM-3 or by two reductase domain mutant enzymes (W574Y, W574F) which have diminished hydroxylase activity relative to wild-type enzyme but retain cytochrome c reductase activity levels comparable to wild-type enzyme. Because reduced cytochrome c generated independently of the reductase domain of P-450BM-3 did not support myristate hydroxylation, it seems likely that cytochrome c binds to a site on the reductase domain which does not overlap the site of the heme domain interaction. We also found that myristate did not inhibit P-450BM-3-mediated cytochrome c reduction. Since neither substrate inhibited the conversion of the other, we conclude that the rate-limiting steps for both myristate hydroxylation and cytochrome c reduction by P-450BM-3 do not involve electron transfer through the reductase domain.

A simple protocol for identification of helical and mobile residues in membrane proteins.

A simple molecular dynamics (MD) simulations is protocol shown to predict whether a residue is in a structured alpha-helical segment or in a mobile loop or terminal segment of a membrane protein. The results are verified by comparisons with experimental NMR data. The protocol consists of performing several independent MD simulations on a polypeptide sequence of interest in a dielectric continuum with a relative permittivity epsilon = 2. The time histories of the individual angles between NH bond vectors at time 0 and time t later are calculated, and then Gaussian smoothing of typically 50 ps is applied. The smoothed data are subtracted from the original data to yield the short time fluctuations of the NH bond vectors, and then the rms deviations of the angles are calculated and compared to experimental NMR results. Pf1 coat protein and the c subunit of the E. coli F1F0 ATP synthase are used as examples of membrane proteins. The calculated NH bond rms fluctuations are in qualitative agreement with experimental NMR data in showing that each of these proteins has a mobile segment connecting two helices, as well as mobile N and C-terminal regions. This MD simulations protocol can demonstrate the presence of both the amphipathic and hydrophobic helices while hydropathy plots are able to detect only the hydrophobic helices present in membrane proteins.

Novel mutation of the initiation codon of PAX9 causes oligodontia.

Tooth development is under strict genetic control. Oligodontia is defined as the congenital absence of 6 or more permanent teeth, excluding the third molar. The occurrence of non-syndromic oligodontia is poorly understood, but in recent years several cases have been described where a single gene mutation is associated with oligodontia. Several studies have shown that MSX1 and PAX9 play a role in early tooth development. We screened one family with non-syndromic oligodontia for mutations in MSX1 and PAX9. The pedigree showed an autosomal-dominant pattern of inheritance. Direct sequencing and restriction enzyme analysis revealed a novel heterozygous A to G transition mutation in the AUG initiation codon of PAX9 in exon 1 in the affected members of the family. This is the first mutation found in the initiation codon of PAX9, and we suggest that it causes haploinsufficiency.

Genetic and phenotypic heterogeneity in pattern dystrophy.

BACKGROUND: The pattern dystrophies (PD) represent a clinically heterogeneous family of inherited macular diseases frequently caused by mutations in the peripherin/RDS gene. Most previous studies have detailed the clinical findings in single families, making it difficult to derive data from which progression and visual outcome can be generalised. METHODS: Families were ascertained and clinically evaluated including angiography and electrophysiology where appropriate. RESULTS: In each of the six families with autosomal dominant PD, a mutation in the peripherin/RDS gene was identified, including a novel Cys250Phe variant. These data suggest that the condition is characterised by the accumulation of yellow to grey subretinal flecks, followed by pigmentary change accompanied by patches of chorioretinal atrophy. Subsequently, 50% (16/32) of individuals with PD developed poor central vision because of chorioretinal geographic atrophy or subretinal neovascularisation. The risk of these complications appears to increase with age. CONCLUSION: PD should not necessarily be considered a benign condition. Instead, patients should be counselled that there is a significant chance of losing central vision in their later years. Some elderly patients with probands showing PD may be misdiagnosed with age related macular degeneration owing to the phenotypic similarities between these conditions in the advanced state.

Molecular genetic heterogeneity in autosomal dominant drusen.

OBJECTIVE: Autosomal dominant drusen is of particular interest because of its phenotypic similarity to age related macular degeneration. Currently, mutation R345W of EFEMP1 and, in a single pedigree, linkage to chromosome 6q14 have been causally related to the disease. We proposed to investigate and quantify the roles of EFEMP1 and the 6q14 locus in dominant drusen patients from the UK and USA. DESIGN: Molecular genetic analysis. PARTICIPANTS: Ten unrelated families and 17 young drusen patients. MAIN OUTCOME MEASURES: Exons 1 and 2 of EFEMP1 were characterised by 5' rapid amplification of cDNA ends and direct sequencing. Exons 1-12 of EFEMP1 were then investigated for mutation by direct sequencing. A HpaII restriction digest test was constructed to detect the EFEMP1 R345W mutation. Marker loci spanning the two dominant drusen linked loci were used to generate haplotype data. RESULTS: Only seven of the 10 families (70%) and one of the 17 sporadic patients (6%) had the R345W mutation. The HpaII restriction digest test was found to be a reliable and quick method for detecting this. No other exonic or splice site mutation was identified. Of the three families without EFEMP1 mutation, two were linked to the 2p16 region. CONCLUSIONS: EFEMP1 R345W accounts for only a proportion of the dominant drusen phenotype. Importantly, other families linked to chromosome 2p16 raise the possibility of EFEMP1 promoter sequence mutation or a second dominant drusen gene at this locus. Preliminary haplotype data suggest that the disease gene at the 6q14 locus is responsible for only a minority of dominant drusen cases.

Molecular dynamics simulation of a synthetic four-alpha-helix bundle that binds the anesthetic halothane.

The structural features of binding sites for volatile anesthetics are examined by performing a molecular dynamics simulation study of the synthetic four-alpha-helix bundles (Aalpha2)2, which are formed by association of two 62-residue di-alpha-helical peptides. The peptide bundle (Aalpha2)2 was designed by Johansson et al. [Biochemistry 37 (1998) 1421-1429] and was shown experimentally to have a high affinity for the binding of the anesthetic halothane (CF3CBrCIH) in a hydrophobic cavity. Since (Aalpha2)2 can exhibit either the anti or syn topologies, the two distinct bundles are simulated both in the presence and in the absence of halothane. Nanosecond length molecular dynamics trajectories were generated for each system at room temperature (T = 298 K). The structural and dynamic effects of the inclusion of halothane are compared, illustrating that the structures are stable over the course of the simulation; that the (Aalpha2)2 bundles have suitable pockets that can accommodate halothane; that the halothane remains in the designed hydrophobic cavity in close proximity to the Trp residues with a preferred orientation; and that the dimensions of the peptide are perturbed by the inclusion of an anesthetic molecule.

Molecular dynamics simulation of four-alpha-helix bundles that bind the anesthetic halothane.

The mutation of a single leucine residue (L38) to methionine (M) is known experimentally to significantly increase the affinity of the synthetic four-alpha-helix bundle (Aalpha(2))(2) for the anesthetic halothane. We present a molecular dynamics study of the mutant (Aalpha(2)-L38M)(2) peptide, which consists of a dimer of 62-residue U-shaped di-alpha-helical monomers assembled in an anti topology. A comparison between the simulation results and those obtained for the native (Aalpha(2))(2) peptide indicates that the overall secondary structure of the bundle is not affected by the mutation, but that the side chains within the monomers are better packed in the mutant structure. Unlike the native peptide, binding of a single halothane molecule to the hydrophobic core of (Aalpha(2)-L38M)(2) deforms the helical nature of one monomer in a region close to the mutation site. Increased exposure of the cysteine side chain to the hydrophobic core in the mutant structure leads to the enhancement of the attractive interaction between halothane and this specific residue. Since the mutated residues are located outside the hydrophobic core the observed increased affinity for halothane appears to be an indirect effect of the mutation.

Simulation of the HIV-1 Vpu transmembrane domain as a pentameric bundle.

The transmembrane domain of oligomeric protein Vpu encoded by HIV-1 has been studied by means of a molecular dynamics simulation. A pentameric bundle of unconstrained helices (residues 6-28 of Vpu) with a water filled pore was initially assembled in a membrane mimetic octane/water system. This system was simulated, using the CHARMm19 and OPLS united atom force fields with no constraints at a temperature of 300 K and a pressure of 1 atm. For these forcefields and the initial conditions tested, the oligomeric bundle expelled most of the pore water molecules. The resulting bundle and residual waters adopt a conical structural motif with some resemblance to a potassium channel.

Molecular dynamics study of the LS3 voltage-gated ion channel.

Molecular dynamics calculations have been carried out on a model of the LS3 synthetic ion channel in a membrane mimetic environment. In the absence of an external electrostatic field, the LS3 channel, which consists of a bundle of six alpha-helices with sequence Ac-(LSSLLSL)3-CONH2, exhibits large structural fluctuations. However, in the presence of the field, the bundle adopts a well defined coiled-coil structure with an inner pore of water. The observed structural changes induced by the applied field are consistent with the proposed gating mechanism of the ion channel.

Two possible conducting states of the influenza A virus M2 ion channel.

Molecular dynamics simulations have been performed on protonated four-helix bundles based on the 25-residue Duff-Ashley transmembrane sequence of the M2 channel of the influenza A virus. Well-equilibrated tetrameric channels, with one, two and four of the H37 residues protonated, were investigated. The protonated peptide bundles were immersed in the octane portion of a phase-separated water/octane system, which provided a membrane-mimetic environment. The simulations suggest that there could be two conducting states of the M2 channel corresponding to tetramers containing one or two protonated histidines. The more open structure of the doubly protonated state suggests it would have the higher conductance.

The M2 channel of influenza A virus: a molecular dynamics study.

Molecular dynamics simulations have been performed on a tetramer of the 25-residue (SSDPLVVAASIIGILHLILWILDRL) synthetic peptide [1] which contains the transmembrane domain of the influenza A virus M2 coat protein. The peptide bundle was initially assembled as a parallel alpha-helix bundle in the octane portion of a phase separated water/octane system, which provided a membrane-mimetic environment. A 4-ns dynamics trajectory identified a left-handed coiled coil state of the neutral bundle, with a water filled funnel-like structural motif at the N-terminus involving the long hydrophobic sequence. The neck of the funnel begins at V27 and terminates at H37, which blocks the channel. The C-terminus is held together by inter-helix hydrogen bonds and contains water below H37. Solvation of the S23 and D24 residues, located at the rim of the funnel, appears to be important for stability of the structure. The calculated average tilt of the helices in the neutral bundle is 27 +/- 5 degrees, which agrees well with recent NMR data.

Sensitivities in older eyes with good acuity: cross-sectional norms.

We measured several indices of foveal visual function for a large group of people aged 60 and older. The data reported in this paper are from individuals who had good acuity in each eye and met a number of other criteria for good ocular health. For each index, we described the rate of cross-sectional change with age using linear regression statistics. We found age-related change for eyes having 20/20 or better acuity to exist for several different indices. Sensitivity mediated by the blue-sensitive cones decreased with age, as expected. However, the rate of decrease was faster for females than for males. At least part of the difference was associated with different rates of lenticular change. Absolute sensitivity at long wavelengths also decreased with age, but at the same rate for each sex. Rayleigh color matches changed with age in a manner consistent with underlying age-related decreases of effective foveal cone photopigment density. However, not all indices showed age-dependent changes. For instance, the time constant describing the rate of photopic dark adaptation did not appear to change with age.

Sensitivities in older eyes with good acuity: eyes whose fellow eye has exudative AMD.

We compared several indices of foveal visual function between two groups of people aged 60 and older. One group was comprised of individuals who had good acuity in one eye, but had a history of exudative aging macular degeneration (AMD) in the other eye. We measured visual function in these individuals' good eyes only. The second group was a normative group; it was comprised of individuals who had good acuity in each eye. None of the eyes which we tested from either group had funduscopic evidence of macular pathology other than macular drusen and/or hypopigmentation. We found that eyes whose fellow eye had suffered from exudative AMD themselves suffered compromised foveal function, even when they retained 20/20 or better acuity. Losses of sensitivity mediated by blue-sensitive cones tended to be greater for 1 degree than for 3 degrees diameter test stimuli. Absolute sensitivity losses at long test wavelengths were probably due to several factors, including decreased effective cone photopigment density. Slow rates of recovery during dark adaptation were associated with the presence of many macular drusen and/or macular hypopigmentation. Eyes whose fellow eye had suffered from exudative AMD had more macular drusen and hypopigmentation than eyes whose fellow eye had not suffered from exudative AMD.

Relations between fundus appearance and function. Eyes whose fellow eye has exudative age-related macular degeneration.

Foveal visual function was compared with fundus appearance for 41 eyes that had good acuity but whose fellow eye had exudative age-related macular degeneration (AMD). The visual functions tested were among those reported to be compromised by AMD. They included: (1) dark adaptation, (2) absolute sensitivity, (3) S cone-mediated sensitivity, and (4) color matching. The fundus features used to evaluate the risk of developing exudative AMD included: (1) drusen confluence, (2) drusen size, and (3) focal hyperpigmentation. For the group of eyes defined by the presence of one or more high-risk fundus characteristics, all visual functions were compromised significantly. In particular, all 21 eyes with abnormally slow rates of dark adaptation had high-risk fundi, and all 16 eyes with abnormal color matching (ie, a small effect of test area on the color match or rejection of all potential color matches) had high-risk fundi. Conversely, 30 of the 32 eyes with high-risk fundi had abnormally slow rates of dark adaptation or abnormal color matching. In addition, reduced acuity in the fellow exudative eye was associated significantly with a high-risk fundus in the nonexudative eye.

Visual function and the subsequent development of exudative age-related macular degeneration.

The eyes of 47 subjects with exudative age-related macular degeneration in the fellow eye were tested with a battery of visual function tests at baseline and followed for at least 18 mo. Fundus photographs also were obtained at baseline. These photographs were used to verify the absence of exudative lesions in the 47 eyes tested. Functional and funduscopic baseline data each were compared against outcome data obtained typically at 18 mo. The baseline data were analyzed for their ability to distinguish eyes that had developed detectable exudative age-related macular degeneration from eyes that had not. Eyes with relatively slow foveal dark adaptation rates despite low foveal quantum absorption capabilities (as inferred from the effects of test area on the Rayleigh color match) were especially likely to develop subretinal neovascularization. The resulting sensitivity/specificity and odds ratios were comparable to those of the most effective funduscopic risk indicators. Low S (blue) cone-mediated sensitivity also was associated with an exudative outcome.

Diet-related macular anomalies in monkeys.

Fundus color photographs and retinal fluorescein angiograms were obtained from 48 nonhuman primates of three macaque species. Yellow pigmentation of the macula was present in monkeys fed a standard laboratory diet containing xanthophylls but was absent in animals maintained on semipurified or liquid formula diets with no xanthophyll content. Plasma levels of xanthophylls ranged from 0.5 to 2.4 microliters/ml in monkeys receiving the standard diet but were undetectable in animals raised on semipurified or liquid formula diets. Fluorescein angiograms revealed foveal areas of hyperfluorescence in almost all monkeys; however, the degree of hyperfluorescence was significantly greater in monkeys maintained on the semipurified or liquid formula diets.

Indirect glutamate neurotoxicity.

Indirect glutamate toxicity can be demonstrated by exposing dissociated rat hippocampal cultures to the media of the same culture transiently exposed (1 min) to glutamate (0.5 mM). The toxicity was maximum when the media was collected 5 min after the glutamate exposure. While the primary glutamate toxicity was attenuated by ionotropic glutamate receptor antagonists, the transferred, indirect toxicity was unaffected by the same antagonists. The changes in nuclear morphology indicated chromatin condensation and nuclear fragmentation in both primary and transferred toxicity. The stain for DNA damage by TUNEL method also revealed cells staining positive in both primary and transferred glutamate toxicity. These observations demonstrate that glutamate-induced neurotoxicity can be propagated to the uninjured cells by an unknown toxin released into the extracellular space. This neurotoxin induced both apoptosis and necrosis in cultured rat hippocampal cells.