Perpetuation of the hereditary sigma virus in populations of its host,Drosophila melanogaster. Geographical analysis of correlated polymorphisms

In natural populations of Drosophila melanogaster, about 10% of the individuals are infected by a virus, sigma, which is not contagious but is transmitted through gametes. These populations are also regularly polymorphic for two alleles, O and P, of a locus ref(2)P; the P allele interferes with the multiplication of the virus. Two viral Types are found in populations, differing in their sensitivity to the P allele. Many samples of flies have been collected in different parts of the world and for each of them, the P frequency has been measured and the viral Type determined. A clear geographical differentiation appears for both these traits; they present a mutual adaptation leading to relatively low frequencies of infected flies in natural populations. Most viruses are only known from highly selected laboratory strains. The observations reported in this paper give evidence of the self restraint exercised by the sigma virus at the population level; they indicate that the characteristics of wild viral clones are likely to differ from those of laboratory strains and also from one population to another.The sigma virus is comparable to other genetical elements, that can be more efficiently transmitted than a mendelian allele, such as transposable elements. The discussion illustrates some of the factors involved in the perpetuation of such elements in a population and points out the difficulty of taking them all into consideration in theoretical models dealing with their perpetuation.     

Study of the ref(2)P locus of Drosophila melanogaster

The ref(2)P gene of Drosophila melanogaster is implicated in sigma rhabdovirus multiplication. Two common alleles of ref(2)P are known, ref(2)P ⁰ which permits sigma virus multiplication and ref(2)P ^pwhich is restrictive for most sigma virus strains. This gene maps to the cytogenetic region 37E3-F3. Using Df(2L)E55 (=Df(2L)37D2-El;37F5-38A1), we have screened for lethal, semi-lethal and visible mutations following diepoxybutane (DEB) or ethyl methanesulfonate (EMS) mutagenesis. Our data confirm than DEB is mor efficient than EMS at inducing deletions. The mutations obtained in this region define 14 complementation groups. One of them, l(2)37Dh, appears to be a general enhancer of Minute and Minute-like mutations. None of the mutations were allelic to the ref(2)P locus. Loss-of-function alleles of ref(2)P (called null) were selected following DEB mutagenesis. Homozygous or hemizygous ref(2)P ^nullflies are male sterile. These flies, like homozygous or hemizygous ref(2)P ⁰flies, are fully permissive for sigma virus replication. We suggest that the ref(2)P products interact with viral products, but that this interaction is not necessary for an efficient viral cycle.     

The age and evolution of an antiviral resistance mutation in Drosophila melanogaster

What selective processes underlie the evolution of parasites and their hosts? Arms-race models propose that new host-resistance mutations or parasite counter-adaptations arise and sweep to fixation. Frequency-dependent models propose that selection favours pathogens adapted to the most common host genotypes, conferring an advantage to rare host genotypes. Distinguishing between these models is empirically difficult. The maintenance of disease-resistance polymorphisms has been studied in detail in plants, but less so in animals, and rarely in natural populations. We have made a detailed study of genetic variation in host resistance in a natural animal population, Drosophila melanogaster, and its natural pathogen, the sigma virus. We confirm previous findings that a single (albeit complex) mutation in the gene ref(2)P confers resistance against sigma and show that this mutation has increased in frequency under positive selection. Previous studies suggested that ref(2)P polymorphism reflects the progress of a very recent selective sweep, and that in Europe during the 1980s, this was followed by a sweep of a sigma virus strain able to infect flies carrying this mutation. We find that the ref(2)P resistance mutation is considerably older than the recent spread of this viral strain and suggest that—possibly because it is recessive—the initial spread of the resistance mutation was very slow.     

On the maintenance of the polymorphism at the ref(2)P locus in populations of Drosophila melanogaster

Polymorphism for two alleles of the ref(2)P locus is a very constant feature of French natural populations of Drosophila melanogaster. One of these alleles interferes with the multiplication of the hereditary sigma virus in the fly. An equilibrium, quite similar to the natural one, has been observed previously in experimental populations, whether the sigma virus is present or not. Evidence is given that one of the selection components involved in the maintenance of this equilibrium affects adult stages when flies have not suffered severe larval competition. In conditions of severe larval competition, a maternal effect seems to be involved in the differential egg-to-adult viability of heterozygotes.     

Drosophila genes which intervene in multiplication of sigma virus (author''s transl)]

Summary Distinction between Drosophila strains, differing their capacity for supporting multiplication of sigma virus, arises essentially from comparison of the incubation time after inoculation of a viral suspension. This is the most general and the most useful characteristic. By this mean five allelic differences with the reference Drosophila strain Oregon have been found. Corresponding genes, ref(1)H, ref(2)M, ref(2)P, ref(3)O and ref(3)D are located all over Drosophila chromosomes. The specific spectra of viral strains sensitive to the one or the other allele was determined for each gene.Some characteristic properties of flies in which the virus has been brought by injection or heredity were compared between heterozygotes and homozygotes for the permissive and for the non permissive allele:time of incubation as a function of the size of the inoculum,probability of initiating infection,kinetics of the virus multiplication in inoculated fly,efficiency of a viral genome brought by a spermatozoa in infecting an egg,perpetuation of the carrier state of sigma virus in germ line cells of stabilized females or males and in somatic cells.The properties concerning the perpetuation of sigma virus carrier state allow to distinguish two classes of viral functions in which the considered ref gene product can intervene: 1) functions necessary for viral genome replication and, of course, for perpetuation of carrier state, 2) other functions, (late functions — necessary for maturation - and functions necessary for cell penetration of inoculated virus).Homozygotes for each of the two alleles of a gene which acts on incubation time can show no difference in one property which is specific of a differenciated cell type only because the considered gene is not expressed in the cell type involved. Conversely genes can exist which act on such a property and which have no action on incubation time. Probably such a gene has been discovered; it intervenes in the transmission of sigma virus by stabilized males; this gene is named ref(3)V.Discussion of all the properties of flies homozygotes for each allele permits us to conclude that ref(1)H, ref(2)M, ref(2)P, ref(3)D and possibly ref(3)V genes (if this last gene intervenes directly in sigma's physiology) are involved in a function necessary for replication. No conclusive evidence has been found for ref(3)O, still it seems to intervene in a late function. Problems of functional interactions between products of the first five ref genes have been mentioned.     

Study of the ref(2)P locus of Drosophila melanogaster

The ref(2)P gene of Drosophila melanogaster is implicated in sigma rhabdovirus multiplication. Two common alleles of ref(2)P are known, ref(2)P ⁰ which permits sigma virus multiplication and ref(2)P ^pwhich is restrictive for most sigma virus strains. This gene maps to the cytogenetic region 37E3-F3. Using Df(2L)E55 (=Df(2L)37D2-El;37F5-38A1), we have screened for lethal, semi-lethal and visible mutations following diepoxybutane (DEB) or ethyl methanesulfonate (EMS) mutagenesis. Our data confirm than DEB is mor efficient than EMS at inducing deletions. The mutations obtained in this region define 14 complementation groups. One of them, l(2)37Dh, appears to be a general enhancer of Minute and Minute-like mutations. None of the mutations were allelic to the ref(2)P locus. Loss-of-function alleles of ref(2)P (called null) were selected following DEB mutagenesis. Homozygous or hemizygous ref(2)P ^nullflies are male sterile. These flies, like homozygous or hemizygous ref(2)P ⁰flies, are fully permissive for sigma virus replication. We suggest that the ref(2)P products interact with viral products, but that this interaction is not necessary for an efficient viral cycle.     

Genetic diversity of Cypripedium calceolus in Poland

In this work we assessed the genetic diversity of 32 C.?calceolus populations from Poland. Mean genetic diversity was moderate (P?=?36.4%, A?=?1.58, H _O?=?0.143, F _IS?=?0.059), and seven geographic regions did not differ significantly in their levels of polymorphism (p?>?0.05), although allele frequencies varied greatly. Only four unique alleles were found, at three sites in southern and southeastern Poland. Genetic (P, A) and genotypic diversity parameters (G, G _U) were significantly correlated with population size (p?<?0.001). In the 32 studied populations we separated 422 different multilocus genotypes; none was common to all populations. Overall population differentiation was moderate at 0.137 (p?<?0.001), but we found a significant pattern of isolation by distance for the whole dataset (r ²?=?0.65, p?<?0.001). Our chloroplast DNA (cpDNA) results suggest a single evolutionary lineage and a common origin for all Polish C.?calceolus populations. Information about the genetic health of C.?calceolus populations should be useful in developing conservation strategies.     

Dosage effects of the non permissive allele of Drosophila ref(2)P gene on sensitive strains of sigma virus (author's transl)]

Different characteristics of flies relating to sigma virus allow us to class the following drosophila genotypes according to their permissivity for the virus strains which are sensitive to the Pp allele: (formula: see text). It is concluded 1) that the two alleles Po and Pp of ref(2)P gene are active and 2) that the viral protein which interact with the product of ref(2)P is effective, or effectively transformed, without interaction with the product of ref(2)P. The delayed appearance of CO2 sensitivity symptom in flies which are issued from stabilized maternal lines, while they are immune to a superinfection non Pp sensitive virus, leads us to believe that ref(2)P is active not only on a function necessary to viral genome replication, as assumed by preceding workers, but also on a function necessary to maturation for the viral strain which was used.     

Alcohol dehydrogenase polymorphism in barrel cactus populations of Drosophila mojavensis

Starch gel electrophoresis revealed that the alcohol dehydrogenase (ADH-2) locus was polymorphic in two populations (from Agua Caliente, California and the Grand Canyon, Arizona) of cactophilic Drosophila mojavensis that utilize barrel cactus (Ferocactus acanthodes) as a host plant. Electromorphs representing products of a slow (S) and a fast (F) allele were found in adult flies. The frequency of the slow allele was 0.448 in flies from Agua Caliente and 0.659 in flies from the Grand Canyon. These frequencies were intermediate to those of the low (Baja California peninsula, Mexico) and high (Sonora, Mexico and southern Arizona) frequency Adh-2^S populations of D. mojavensis that utilize different species of host cacti.     

Reproduction in Cage Populations of a Polymorphism Regularly Observed in the Natural Populations of DROSOPHILA MELANOGASTER in France

Polymorphism for both alleles of a gene ref(2)P, which is a usual trait of French natural populations of Drosophila melanogaster , can be reproduced in experimental conditions. ref(2)P is a gene for resistance to the hereditary, noncontagious Rhabdovirus σ, responsible for CO₂ sensitivity in Drosophila melanogaster . The equilibrium frequencies observed in cages are the same as in the wild, whether σ virus is present or not. The rapid rate of return to these equilibrium frequencies indicates that strong forces, which remain to be determined, are responsible for the maintenance of this polymorphism.     

Bacteria can be selected to help beneficial plasmids spread

A recent commentary raised concerns about aspects of the model and assumptions used in a previous study which demonstrated that selection can favor chromosomal alleles that confer higher plasmid donation rates. Here, the authors of that previous study respond to the concerns raised.

In our original work [1], we demonstrated experimentally that selection can favor chromosomal alleles that confer higher plasmid donation rates, given the plasmid is beneficial and the recipient has an elevated chance of carrying the donor allele (i.e., preferential donation to kin). Our experiments demonstrated this effect via 2 mechanisms of preferential donation: biased conjugation rates and structured populations. We interpreted these results through the lens of kin selection theory (benefits via horizontal gene transfer to kin), supported by simulations and an analytical fitness function model. These results hold importance by outlining that the evolution of plasmid transfer rates (a key aspect of the antibiotic resistance crisis) is not necessarily the sole product of selection on the plasmid itself and forms part of a broader series of papers from our labs investigating the sociomicrobiology of plasmids [2–4].A new commentary raises concerns over our fitness function model, flagging issues with both the structure of the model and assumptions made in our analysis [5]. We stand by the general conclusions of our work but accept that our fitness function and stated analysis assumptions could be better formulated. Our initial fitness function is heuristic in the sense it was designed to capture general processes acting on the fitness of individuals, dependent on the plasmid and donor allele status—without explicitly modeling the myriad demographic events of dispersal, reproduction, conjugation, and death that result in selective shifts across a metapopulation of cells. Specifically, we captured the “force of infection” faced by an uninfected cell as the product of average plasmid prevalence and average donor allele prevalence in the local patch (p_jq_j; see commentary for notation details). We agree with the authors that this force of infection is better phrased as the average of the product ((1/N)∑p_ij q_ij), in part because this avoids the potential pathology under limit conditions described by the authors, but also because this approach better highlights that the particular social trait in question is an “other only” cooperative trait [6], illustrated by commentary equation [2], where transmission to self and transmission to others are separated. This separation has the important consequence of highlighting that unlike many microbial social traits where benefits accrue to a group (including self), a cooperative plasmid donor trait can only benefit other cells that lack the plasmid. Given established costs of donation (e.g., see figure S2 in our original article), this defines our “donor” behavior as an altruistic trait, which can, therefore, only be favored by selection given nonrandom interactions among individuals (e.g., [7]).Our experimental results outline 2 mechanisms of nonrandom interactions: preferential donation to kin and population structure. Each of these mechanisms will generate positive covariances between focal individual q_ij and non-self-recipient q_j donor allele states (cov(q_j, q_ij) > 0). The pathway via preferential donation to kin (order-of-magnitude differences according to our analyses and more recent measurements among lineages coexisting within natural populations [8]) will also likely generate positive covariances between donor and recipient abilities (cov(s_ij, q_ij) > 0). In contrast, to arrive at the result that selection always works against plasmid donor alleles (equation [4]), the commentary makes the assumption that both of the above covariances are zero. We suggest that the additional analyses begun by the authors are an exciting starting point to better map selection on donor alleles, under a broader array of defined assumptions on cell–cell and gene–gene structure, ideally informed by data on structures found in natural bacterial populations.     

Oxygen poisoning in Drosophila

Fruit flies live longer at the partial pressure of oxygen found in air than at either larger or smaller partial pressures. Flies exposed to 1 atm of oxygen for 8 hr every day do not recover completely in the remaining 16 hr. In general, intermittent exposures to 1 atm of oxygen are better tolerated than continuous exposure to the same average oxygen concentration per day, but exposures to higher pressures of 2–5 atm of oxygen for as little as a half hour every two days markedly shorten the life-span. Older flies consume more oxygen per minute and are more sensitive to oxygen poisoning than young flies, and the rate of dying in 6 atm of O₂, or the reciprocal of the survival time, is a linear function of the age. The oxygen pressure-time curve can be well expressed by the general empirical equation (P_OO2)² x time = 120 where P is in atmosphere and survival time in hours. The progress of oxygen poisoning appears to be linear with time rather than exponential.     

Chemogenetic evidence supporting multiple allele control of the biosynthesis of (−)-menthone and (+)-isomenthone stereoisomers in Mentha species

The essential oils of certain Mentha species and chemotypes have proportions of (?)-menthone and (+)-isomenthone which differ but show a high degree of heritability in clonal propagation. Oil from an F₂ individual (69–296), selected from numerous 4n M. longifolia (4n = 48) × M. crispa (2n = 48) hybrids for high isomenthone content, had 41.3% isomenthone; the associated but seldom observed alcohols, 1.6% isomenthol, 10.3% neoiso-menthol; and 13% of their esters; in contrast to 8% menthone with 0.1% menthol, 5.0% neo-menthol, and 1.7% esters. Self-pollination of strain 69–296 gave a 3:1 ratio of high isomenthone: high menthone. Crosses with a true breeding high menthone plant having 80% menthone and 3.2% isomenthone gave a 1:1 ratio of the parental phenotypes by GLC analyses and herbage odor. This and data from high isomenthone and high menthone crosses with tester strains lead us to postulate the involvement of a single locus having multiple alleles with true breeding menthone having the genotype P^s P^s, true breeding isomenthone P^r P^r, 69–296 P^r P^s, and high pulegone pp. The P^r allele is not completely dominant over the P^s allele in 69–296 as about 18% of the total ketone derived from pulegone is menthone. Both are dominant over the recessive allele p that largely prevents menthone development. The quantitative amounts of the two isomers are believed to be controlled by the six combinations of the three alleles in a diploid species with graded effects obtained in the more complex genotypes possible in double diploid and octoploid species. 69–296 has (?)-piperitone even though (+)-piperitone is believed to be the common isomer in Mentha.     

Control of Periplasmic Interdomain Thiol:Disulfide Exchange in the Transmembrane Oxidoreductase DsbD

The bacterial protein DsbD transfers reductant from the cytoplasm to the otherwise oxidizing environment of the periplasm. This reducing power is required for several essential pathways, including disulfide bond formation and cytochrome c maturation. DsbD includes a transmembrane domain (tmDsbD) flanked by two globular periplasmic domains (nDsbD/cDsbD); each contains a cysteine pair involved in electron transfer via a disulfide exchange cascade. The final step in the cascade involves reduction of the Cys¹⁰³-Cys¹⁰⁹ disulfide of nDsbD by Cys⁴⁶¹ of cDsbD. Here we show that a complex between the globular periplasmic domains is trapped in vivo only when both are linked by tmDsbD. We have found previously (Mavridou, D. A., Stevens, J. M., Ferguson, S. J., & Redfield, C. (2007) J. Mol. Biol. 370 ,643 -658) that the attacking cysteine (Cys⁴⁶¹) in isolated cDsbD has a high pK_a value (10.5) that makes this thiol relatively unreactive toward the target disulfide in nDsbD. Here we show using NMR that active-site pK_a values change significantly when cDsbD forms a complex with nDsbD. This modulation of pK_a values is critical for the specificity and function of cDsbD. Uncomplexed cDsbD is a poor nucleophile, allowing it to avoid nonspecific reoxidation; however, in complex with nDsbD, the nucleophilicity of cDsbD increases permitting reductant transfer. The observation of significant changes in active-site pK_a values upon complex formation has wider implications for understanding reactivity in thiol:disulfide oxidoreductases.DsbD is a unique protein that transfers reductant across the cytoplasmic membrane to the periplasm in many Gram-negative bacteria (1, 2). Provision of reductant to the periplasm is required because this compartment is otherwise considered to be an oxidizing environment (2). DsbD includes three domains, each containing a pair of cysteine residues that perform a series of disulfide exchange reactions (Fig. 1A). In the first step, the transmembrane domain (tmDsbD) accepts electrons from thioredoxin in the cytoplasm; these are then transferred to the periplasmic C-terminal domain (cDsbD) and finally to the N-terminal domain (nDsbD), which is also located in the periplasm (3-5). nDsbD acts as a junction point for several pathways that require reductant, including the general disulfide isomerase system and the pathway that is thought to reduce the cysteine thiols of apocytochromes in the cytochrome c biogenesis pathway (6). In Gram-positive bacteria, CcdA, an integral membrane protein, and ResA, which has a thioredoxin fold, provide the reductant required for cytochrome c maturation (7).Open in a separate windowFIGURE 1.Schematic representation of DsbD. A, proposed pathway of electron flow from thioredoxin (TrxA) in the cytoplasm, via the three domains of DsbD, to the cytochrome c maturation (Ccm) and disulfide bond isomerization pathways in the periplasm is shown. The crystal structure of nDsbD is from Protein Data Bank code 1L6P (8), cDsbD from Protein Data Bank code 1UC7 (11), and the nDsbD-cDsbD complex from Protein Data Bank code 1VRS (12). The cyan boxes indicate the thrombin cleavage sites introduced into full-length DsbD to allow detection of the nDsbD-cDsbD complex following its formation in vivo. The cysteine residues are shown in yellow. B, schematic representation of the active site of cDsbD in the covalent complex with nDsbD (12). Some active-site residues of cDsbD are indicated in stick representation and the inter-domain disulfide (Cys⁴⁶¹-SS-Cys¹⁰⁹) is shown in yellow.Structural studies have sought to explain how DsbD functions and interacts with its various partners. The structures of the two soluble periplasmic domains have been determined (Fig. 1A, left). nDsbD has an immunoglobulin-like structure (8, 9) and is the only known thiol:disulfide oxidoreductase with this fold. cDsbD has the more typical thioredoxin fold found in many oxidoreductases; this has the characteristic active-site CXXC motif (10, 11). A covalent complex between single-cysteine variants of each of these two domains was produced in vitro and its x-ray structure solved (12), revealing the interface between the two domains (Fig. 1A, right). Although this mixed disulfide is accepted as a physiological intermediate in the function of DsbD, an in vivo complex between the two soluble domains has not been reported previously (3). Further complexes between nDsbD and its other physiological partners have also been trapped and their structures examined (9, 13). Interestingly, all of the interaction partners of nDsbD are thioredoxin-like proteins; similarities in their folds are congruous with common interaction interfaces (14). However, only cDsbD will reduce nDsbD, whereas nDsbD will reduce several partners. This raises questions about how the direction of reductant flow is maintained and controlled within the series of disulfide-exchange reactions.As part of our structural and mechanistic characterization of DsbD and its domains in solution, we have previously measured by NMR the pK_a values of the active-site cysteine pair, Cys⁴⁶¹ and Cys⁴⁶⁴, of cDsbD (numbered according to the full-length Escherichia coli DsbD sequence) (15). An unusually high pK_a value of 10.5 was measured for the N-terminal cysteine of the CXXC motif, Cys⁴⁶¹, and the pK_a value of the second cysteine, Cys⁴⁶⁴, was significantly higher than the maximum pH value that was studied (pH 12.2). The pK_a value of 10.5 is the highest reported for the N-terminal cysteine of the CXXC motif in a thioredoxin fold. The striking consequence of the elevated pK_a value is that the active-site cysteine of cDsbD, Cys⁴⁶¹, is not strongly nucleophilic, raising critical questions about how this cysteine reacts with the disulfide in nDsbD. It was demonstrated using site-directed mutagenesis that the negatively charged side chains of Asp⁴⁵⁵ and Glu⁴⁶⁸, which are located close to the CXXC motif (Fig. 1B), are responsible for the unusually high pK_a value of Cys⁴⁶¹; mutation of one or both of these residues to Asn and Gln, respectively, resulted in decreases in the pK_a value of Cys⁴⁶¹ from 10.5 to 9.9 (E468Q), to 9.3 (D455N), and to 8.6 (D455N/E468Q). The pK_a values for Asp⁴⁵⁵ were found to be 5.9 and 6.6 in oxidized and reduced cDsbD; these values are significantly higher than the value of ∼4 for an unperturbed aspartic acid. We postulated that the properties of the amino acid side chains in the immediate environment of the cysteines in cDsbD would change upon complex formation with nDsbD, changing the reactivity of the cysteines and explaining how the reaction between the two domains is initiated (15). Specifically, we proposed that an increase in the pK_a value of Asp⁴⁵⁵ upon complex formation would lead to a decrease in the pK_a value of Cys⁴⁶¹, thereby making it a better nucleophile. Stirnimann et al. (10) previously presented pK_a calculations suggesting an increase in the Asp⁴⁵⁵ pK_a value upon complex formation.The aim of this work has been to determine the molecular basis of the control of the reactivity of the active-site cysteine residues in cDsbD, using NMR to compare the active-site properties of cDsbD alone and in its physiological complex with nDsbD. We demonstrate that the pK_a value of Asp⁴⁵⁵ is elevated by at least 1.1 pH units when cDsbD forms a complex with nDsbD. This modulation of the pK_a value is critical for the specificity and function of cDsbD. These in vitro studies are complemented by in vivo studies on complex formation, in which we have trapped the nDsbD-cDsbD complex for the first time. The results of our experiments explain how the intramolecular disulfide cascade within the soluble domains of DsbD functions, and demonstrate the importance of the transmembrane domain in controlling and facilitating complex formation between the soluble domains.     

Evolution of the proportions of two sigma viral types in experimental populations of Drosophila melanogaster in the absence of the allele that is restrictive of viral multiplication

A minority of flies in natural populations of Drosophila melanogaster are endemically infected by a rhabdovirus, sigma. The virus is vertically transmitted through male and female gametes. Two alleles of a fly locus, the ref(2)P locus, are present as a polymorphism in all populations: O permissive, and P restrictive for viral multiplication and transmission. Two viral types are known, Type I, which is very sensitive to the P allele, and Type II, which is more resistant. Previous observations have shown that, in presence of the P allele, viral Type II is selected for, in both natural and experimental populations. The aim of the present study was to determine whether, in the absence of P, Type I is selected for, or whether the two types are equivalent. For this purpose, experimental populations deprived of the P allele and differing in the initial proportions of the two viral types were established. After several generations, and despite a possible bias toward Type I, the frequencies of Type I and Type II clones differed in the various populations, depending on their initial values. These findings do not rule out selective advantage of viral Type I in the absence of P, but suggest that, if any, this advantage is in no way comparable to that displayed by viral Type II in the presence of P.     

Several polymorphisms of KCNQ1 gene are associated with plasma lipid levels in general Chinese populations

Background

Potassium voltage-gated channel, KQT-like subfamily, member 1 (KCNQ1) is thought to be an important candidate gene of diabetes. Several single nucleotide polymorphisms (SNPs) in a 40-kb linkage disequilibrium (LD) block in its intron 15 have been identified to be associated with diabetes in East Asian populations in recent genome-wide association studies. The aim of this study was to investigate whether KCNQ1 polymorphisms influence the levels of the metabolic phenotypes in general Chinese populations.

Methodology/Principal Findings

We investigated the associations of two SNPs (rs2237892 and rs2237895) in the aforementioned 40-kb LD block, a missense variant rs12720449 (P448R) in exon 10, and a synonymous variant rs1057128 (S546S) in exon 13 with metabolic phenotypes in a Uyghur population (n = 478) and replicated these associations in a Han population (n = 2,485). We found that rs2237892-T allele was significantly associated with decreased triglyceride levels (p_combined = 0.001). The minor G allele of the rs12720449, with sharp difference of the allelic frequency between European and East Asian populations (0.2% versus 14%, respectively), was associated with a lower triglyceride levels than G allele in Uyghur subjects (p = 0.004), in Han subjects (p = 0.052), and in subjects of meta-analysis (p_combined = 0.001). Moreover, the minor A allele of the rs1057128 was also associated with decreased triglyceride levels in meta-analysis (p_combined = 0.010).

Conclusions

To the best of our knowledge, this is the first report associating a missense mutation of KCNQ1, rs12720449, with triglyceride levels. Rs2237892, representing the 40-kb LD block, is also associated with triglyceride levels in Han population. Further studies are required to replicate these findings in other East Asian populations.     

Disaccharide 1-phosphate polymers of some representatives of the Bacillus subtilis group

Disaccharide 1-phosphate polymers as well as teichoic acids of various structures have been found in the cell walls of the representatives of the Bacillus subtilis group, namely Bacillus subtilis subsp. spizizenii VKM B-720 and VKM B-916, B. subtilis VKM B-517, and Bacillus vallismortis VKM B-2653^T. Disaccharide 1-phosphate polymers are composed of repeating units of the following structure: -P-4)-β-D-GlcpNAc-(1→6)-α-D-Galp-(1-, the N-acetylglucosamine residues are partially acetylated at positions O3 and O6 (VKM B-720 and VKM B-916); -P-4)-β-D-Glcp-(1→6)-α-D-GlcpNAc-(1-, the glucopyranose residues are partially acetylated at positions O2 or O3 (VKM B-517); -P-6)-α-D-GlcpNH ₃ ⁺ /α-D-GlcpNAc-(1→2)-α-D-Glcp-(1-, the N-acetylglucosamine residues are partially deacetylated (VKM B-2653^T). The structures of the two last disaccharide 1-phosphate polymers have not been reported so far for Gram-positive bacteria. The teichoic acids in the studied strains are O-D-alanyl-1,5-poly(ribitol phosphates) substituted with β-D-glucopyranose (VKM B-517, VKM B-720, VKM B-916) or 2-acetamido-2-deoxy-β-D-glucopyranose (VKM B-2653^T). The structures of the phosphate-containing polymers have been studied by chemical methods and by NMR spectroscopy.     

Life and Death of Sensory Hair Cells Expressing Constitutively Active TRPML3

The varitint-waddler mutation A419P renders TRPML3 constitutively active, resulting in cationic overload, particularly in sustained influx of Ca²⁺. TRPML3 is expressed by inner ear sensory hair cells, and we were intrigued by the fact that hair cells are able to cope with expressing the TRPML3(A419P) isoform for weeks before they ultimately die. We hypothesized that the survival of varitint-waddler hair cells is linked to their ability to deal with Ca²⁺ loads due to the abundance of plasma membrane calcium ATPases (PMCAs). Here, we show that PMCA2 significantly reduced [Ca²⁺]_i increase and apoptosis in HEK293 cells expressing TRPML3(A419P). The deaf-waddler isoform of PMCA2, operating at 30% efficacy, showed a significantly decreased ability to rescue the Ca²⁺ loading of cells expressing TRPML3(A419P). When we combined mice heterozygous for the varitint-waddler mutant allele with mice heterozygous for the deaf-waddler mutant allele, we found severe hair bundle defects as well as increased hair cell loss compared with mice heterozygous for each mutant allele alone. Furthermore, 3-week-old double mutant mice lacked auditory brainstem responses, which were present in their respective littermates containing single mutant alleles. Likewise, heterozygous double mutant mice exhibited severe circling behavior, which was not observed in mice heterozygous for TRPML3(A419P) or PMCA2(G283S) alone. Our results provide a molecular rationale for the delayed hair cell loss in varitint-waddler mice. They also show that hair cells are able to survive for weeks with sustained Ca²⁺ loading, which implies that Ca²⁺ loading is an unlikely primary cause of hair cell death in ototoxic stress situations.Varitint-waddler (Va) mice express a mutant isoform (A419P) of the transient receptor potential channel TRPML3 (murine gene symbol, Mcoln3) that results in profound hearing loss, vestibular defects (circling behavior, imbalance, head bobbing, waddling), pigmentation deficiencies, sterility, and perinatal lethality in homozygous animals (1). A second Mcoln3 variant (Va^J) that arose in the Va background carries two mutations (I362T and A419P) and shows a phenotype with reduced severity, particularly in heterozygous animals (1). The A419P mutation in Va and Va^J mice is located in transmembrane-spanning domain 5(TM5)³ of TRPML3, where it leads to a constitutively open channel, resulting in highly elevated [Ca²⁺]_i (2-5). In contrast to the effect of the A419P mutation on TRPML3 channel activity, the single I362T mutation does not appear to affect [Ca²⁺]_i (3, 5). When combined with the A419P mutation, as found in Va^J mice, the constitutive activity of this mutant TRPML3 isoform is comparable with that of A419P alone (2-5).Here, we show that HEK293 cells expressing TRPML3-(A419P) or TRPML3(I362T/A419P) undergo rapid apoptosis. This apoptosis is suppressed by coexpression of plasma membrane calcium ATPase type 2 (PMCA2). In varitint-waddler mice, sensory hair cells survive for weeks after birth (6), which raised the question of whether this survival could be the result of the hair cells'' ability to deal with normally transient and localized Ca²⁺ influx, a feature that is centered around the high levels of mobile Ca²⁺ buffers and PMCA isoforms found in sensory hair cells (7-10). We decided to test this hypothesis in vivo by utilizing deaf-waddler mice that carry a mutation (G283S) in the Atp2b2 gene encoding mutant PMCA2. Mice homozygous for PMCA2(G283S) (Atp2b2^dfw/dfw) are deaf and have poor balance (11). Compared with Atp2b2 knock-out mice, deaf-waddler mice display a milder phenotype because PMCA2(G283S) retains 30% of its biological activity compared with the wild-type isoform (12). We found that sensory hair cell loss, hearing loss, and vestibular dysfunction were aggravated in mice carrying varitint-waddler and deaf-waddler alleles compared with animals carrying the single mutant alleles. Our results reveal that the Ca²⁺-buffering and Ca²⁺ extrusion abilities of hair cells are powerful enough to prevent cell death for weeks, even in the presence of constitutively active TRPML3(A419P), which is able to induce rapid apoptosis in other cells.     

Novel Protective Mechanism against Irreversible Hyperoxidation of Peroxiredoxin: N��-TERMINAL ACETYLATION OF HUMAN PEROXIREDOXIN II*

Peroxiredoxins (Prxs) are a group of peroxidases containing a cysteine thiol at their catalytic site. During peroxidase catalysis, the catalytic cysteine, referred to as the peroxidatic cysteine (C_P), cycles between thiol (C_P-SH) and disulfide (–S–S–) states via a sulfenic (C_P-SOH) intermediate. Hyperoxidation of the C_P thiol to its sulfinic (C_P-SO₂H) derivative has been shown to be reversible, but its sulfonic (C_P-SO₃H) derivative is irreversible. Our comparative study of hyperoxidation and regeneration of Prx I and Prx II in HeLa cells revealed that Prx II is more susceptible than Prx I to hyperoxidation and that the majority of the hyperoxidized Prx II formation is reversible. However, the hyperoxidized Prx I showed much less reversibility because of the formation of its irreversible sulfonic derivative, as verified with C_P-SO₃H-specific antiserum. In an attempt to identify the multiple hyperoxidized spots of the Prx I on two-dimensional PAGE analysis, an N-acetylated Prx I was identified as part of the total Prx I using anti-acetylated Lys antibody. Using peptidyl-Asp metalloendopeptidase (EC 3.4.24.33) peptide fingerprints, we found that N^α-terminal acetylation (N^α-Ac) occurred exclusively on Prx II after demethionylation. N^α-Ac of Prx II blocks Prx II from irreversible hyperoxidation without altering its affinity for hydrogen peroxide. A comparative study of non-N^α-acetylated and N^α-terminal acetylated Prx II revealed that N^α-Ac of Prx II induces a significant shift in the circular dichroism spectrum and elevation of T_m from 59.6 to 70.9 °C. These findings suggest that the structural maintenance of Prx II by N^α-Ac may be responsible for preventing its hyperoxidation to form C_P-SO₃H.Peroxiredoxins (Prxs)⁴ are a family of peroxidases that possess a conserved cysteine residue at the catalytic site for the reduction of peroxide/peroxynitrite. Using thiol-based reducing equivalents, like thioredoxin, Prxs catalyze the reduction of hydrogen peroxide, alkylhydroperoxides, and peroxynitrite to water, corresponding alcohols, and nitrite, respectively (1–8). Based on the number and location of conserved cysteine residue(s) directly involved in peroxide reduction, the six isotypes of mammalian Prx can be grouped into three distinct subgroups as follows: 2-Cys Prx, atypical 2-Cys Prx, and 1-Cys Prx, (1–2, 5). Human Prx I (hPrx I) and Prx II (hPrx II) are members of the 2-Cys Prx subgroup and thus contain two conserved cysteine residues that are directly involved in peroxidase activity. Cys⁵² for hPrx I and Cys⁵¹ for hPrx II are designated the peroxidatic cysteines (C_P). These residues attack the O–O bond of the peroxide (ROOH) substrate to form the product (ROH) and the sulfenic derivative C_P-SOH. This sulfenic derivative then forms a disulfide bond with the other conserved cysteine residue, which is referred to as the resolving cysteine (C_R; Cys¹⁷³ in hPrx I and Cys¹⁷² in hPrx II). In the case of 2-Cys Prxs, the disulfide partners, C_P and C_R, reside within different subunits; therefore, the disulfide bond established between C_P and C_R (C_P-S–S-C_R) is intermolecular. The reduced thioredoxin molecule is responsible for reducing the C_P-S–S-C_R disulfide bond to generate sulfhydryls (1–3, 5, 9).The C_P of eukaryotic 2-Cys Prxs is vulnerable to hyperoxidation, which results in the loss of its peroxidase activity. This feature is referred to as the “floodgate” mechanism, by which Prxs function as a redox sensor for the regulation of cell signaling (10–11). Hyperoxidation of C_P does not occur when the disulfide bond (C_P-S–S-C_R) is formed. However, the thiol (C_P-SH) can be hyperoxidized via the sulfenic (C_P-SOH) derivative intermediate in the absence of C_P-S-S-C_R formation during catalysis (12). Two different hyperoxidation products of C_P, the reversible sulfinic (C_P-SO₂H) derivative and the irreversible sulfonic (C_P-SO₃H) derivative, have been identified. The irreversible C_P-SO₃H was reported in Tsa1p, a yeast 2-Cys Prx, based on in vivo and in vitro regeneration assay results, and a stronger reactivity to an anti-Tsa1p-SO₃H antibody, which exhibits high specificity toward Tsa1p-C_P-SO₃H relative to Tsa1p-C_P-SO₂H (13). Both forms of hyperoxidized Prxs, C_P-SO₂H and C_P-SO₃H, are superimposed on the acidic migrated spot instead of the Prx-SH spot on a two-dimensional polyacrylamide gel because of the introduction of one negative charge by hyperoxidation (12–16). The protein sulfinic acid reductase, sulfiredoxin, is responsible for reversing 2-Cys Prx-SO₂H to Prx-SH in the presence of ATP and thiol-reducing equivalents like thioredoxin or glutathione (17–24). Until now, an intracellular enzymatic regeneration system for Prx-SO₃H has not been reported.Because mammalian Prx I and Prx II have been studied independently in a number of different organisms and cultured cells, the comparative biochemical data supporting their distinctive functional identities is still very limited. Recombinant Prx I (rPrx I) showed a 2.6-fold higher specific activity as a peroxidase than the recombinant Prx II (rPrx II) without any obvious catalytic or mechanistic differences (25, 26). Recent competition kinetics studies of hPrx II revealed a rate constant of 1.3 × 10⁷ m^–1 s^–1, which is fast enough to favor an intracellular hydrogen peroxide target even in competition with catalase or glutathione peroxidase (27, 28). The kinetic parameters of the competition assay for hPrx I are still not available. Mammalian Prx I interacts with and regulates a broad spectrum of proteins, such as the Src homology domain 3 of c-Abl (29), the Myc box II (MBII) domain of c-Myc (30), the macrophage migration inhibitory factor (MIF, 31), the androgen receptor (32), and the apoptosis signal-regulating kinase-1 (ASK-1) (33). The suggested roles of Prx I in interactions with these molecules are those of a tumor repressor, a survival enhancer, and a growth regulator. Although these suggested functions are controversial (34), all of them can be attributed to the peroxide-scavenging capacity of Prx I (at least in part), except for the enhancement of androgen receptor transactivation (32). Prx II interacts with platelet-derived growth factor receptor and functions as a negative regulator for platelet-derived growth factor signaling (35). Prx II also binds to phospholipase D1 (PLD1) and functions as a hydrogen peroxide-stimulated PLD1 signal terminator (36). Both of these suggested Prx II roles are attributable to the peroxidase activity of Prx II. The major phenotypes of Prx I knock-out mice involve the development of a variety of age-related cancers, hemolytic anemia (37), and dramatic shifts in subcellular reactive oxygen species localization (38). Prx II knock-out mice exhibit splenomegaly and a lack of tumor development in any cell type or tissue (39). Until now, the protein molecule that interacts with Prx I and Prx II has not been characterized, and there is no indication of a heterodimer between Prx I and Prx II. Despite their similar peroxide-scavenging capacities, it is reasonable to conclude that the Prx I and Prx II are unable to compensate for each other in terms of physiological roles. There are several examples of tissue- or cell type-specific expression patterns, such as exclusive Prx I expression in astrocytes and Leydig cells and Prx II expression in neurons and Sertoli cells (40, 41); however, Prx I and Prx II are coexpressed in the majority of mammalian cells and tissues, suggesting distinguished biochemical characteristics of their cellular regulatory mechanisms. Recently, the unique presence of Cys⁸³ in hPrx I, which contributes to the stability of the dimer-dimer interface and suppresses local unfolding, has been claimed to be prone to overoxidation of Prx I (42). The contribution of the dimer-decamer interconversion to the regulation of Prx I activity has also been reported (43).In this study, we confirmed that hPrx II was more susceptible to hyperoxidation as well as more prone to regeneration than hPrx I in HeLa cells. We also found that the difficulty in regenerating hPrx I was caused by irreversible sulfonic (C_P-SO₃H) hyperoxidation. Using AspN (EC 3.4.24.33) peptide fingerprints, we identified the N^α-terminal acetylation exclusively on hPrx II. In addition, we provide evidence for the structural maintenance offered by N^α-terminal acetylation of hPrx II, which possibly contributes to preventing irreversible overoxidation of C_P-SO₃H.