Predicting Evolutionary Site Variability from Structure in Viral Proteins: Buriedness,Packing, Flexibility,and Design

Several recent works have shown that protein structure can predict site-specific evolutionary sequence variation. In particular, sites that are buried and/or have many contacts with other sites in a structure have been shown to evolve more slowly, on average, than surface sites with few contacts. Here, we present a comprehensive study of the extent to which numerous structural properties can predict sequence variation. The quantities we considered include buriedness (as measured by relative solvent accessibility), packing density (as measured by contact number), structural flexibility (as measured by B factors, root-mean-square fluctuations, and variation in dihedral angles), and variability in designed structures. We obtained structural flexibility measures both from molecular dynamics simulations performed on nine non-homologous viral protein structures and from variation in homologous variants of those proteins, where they were available. We obtained measures of variability in designed structures from flexible-backbone design in the Rosetta software. We found that most of the structural properties correlate with site variation in the majority of structures, though the correlations are generally weak (correlation coefficients of 0.1–0.4). Moreover, we found that buriedness and packing density were better predictors of evolutionary variation than structural flexibility. Finally, variability in designed structures was a weaker predictor of evolutionary variability than buriedness or packing density, but it was comparable in its predictive power to the best structural flexibility measures. We conclude that simple measures of buriedness and packing density are better predictors of evolutionary variation than the more complicated predictors obtained from dynamic simulations, ensembles of homologous structures, or computational protein design.     

Serum cholestenoic acid as a potential marker of pulmonary cholesterol homeostasis: increased levels in patients with pulmonary alveolar proteinosis

The conversion of cholesterol into the more polar metabolites 27-hydroxycholesterol (27-OH) and cholestenoic acid by the cytochrome P450 sterol 27-hydroxylase is a cholesterol-removal mechanism used by almost all cells. Most of the cholestenoic acid present in the circulation originates from the lung, and it has been suggested that sterol 27-hydroxylase is of particular importance for cholesterol homeostasis in this organ. As an example of pulmonary cholesterol accumulation, a known disorder of surfactant homeostasis, pulmonary alveolar proteinosis (PAP), was studied. Analysis of bronchoalveolar lavage fluid from PAP patients revealed a significant accumulation of the cholesterol metabolites cholestenoic acid and 27-OH. This pattern was recapitulated in serum, with a significant increase in the levels of both cholestenoic acid (P=0.003) and 27-OH (P=0.017) in PAP patients compared with healthy controls. Analysis of PAP alveolar macrophages did not reveal a significant change in mRNA expression levels of either sterol 27-hydroxylase or the cholesterol-esterifying enzyme acyl-CoA:cholesterol acyltransferase-1. These results are consistent with the contention that substrate availability, rather than enzyme expression, is the key factor in regulating the production of cholestenoic acid by the lung and that serum cholestenoic acid may be a marker of pulmonary cholesterol homeostasis.     

The Effect of Quercetin Nanosuspension on Prostate Cancer Cell Line LNCaP via Hedgehog Signaling Pathway

Background:Prostate cancer (PCa) is the second leading cause of cancer death in American population. In this manner, novel therapeutic approaches for identification of therapeutic targets for PCa has significant clinical implications. Quercetin is a potent cancer therapeutic agent and dietary antioxidant present in fruit and vegetables.Methods:To investigate the underlying mechanism by which the PCa was regulated, nanoparticles of quercetin were administrated to cells. For in vitro experiments, human PCa cell line LNCaP were involved. Cell viability assay and quantitative RT-PCR (qRT-PCR) for hedgehog signaling pathway genes were used to determine the key signaling pathway regulated for PCa progression.Results:The cell viability gradually decreased with increased concentration of quercetin nanoparticles. At 48 h, 40 mM concentration of quercetin treatment showed near 50% of viable cells. Quercetin nanoparticles upregulates Su(Fu) mRNA expressions and downregulates gli mRNA expressions in the LNCaP cells.Conclusion:The results showed that the hedgehog signaling targeted inhibition may have important implications of PCa therapeutics. Additionally, the outcomes provided new mechanistic basis for further examination of quercetin nanoparticles to discover potential treatment strategies and new targets for PCa inhibition.Key Words: Hedgehog, Prostate cancer, Proliferation, Quercetin nanoparticles, Signaling pathway     

Drosomycin, an Innate Immunity Peptide of Drosophila melanogaster, Interacts with the Fly Voltage-gated Sodium Channel

Several peptide families, including insect antimicrobial peptides, plant protease inhibitors, and ion channel gating modifiers, as well as blockers from scorpions, bear a common CSαβ scaffold. The high structural similarity between two peptides containing this scaffold, drosomycin and a truncated scorpion β-toxin, has prompted us to examine and compare their biological effects. Drosomycin is the most expressed antimicrobial peptide in Drosophila melanogaster immune response. A truncated scorpion β-toxin is capable of binding and inducing conformational alteration of voltage-gated sodium channels. Here, we show that both peptides (i) exhibit anti-fungal activity at micromolar concentrations; (ii) enhance allosterically at nanomolar concentration the activity of LqhαIT, a scorpion alpha toxin that modulates the inactivation of the D. melanogaster voltage-gated sodium channel (DmNa_v1); and (iii) inhibit the facilitating effect of the polyether brevetoxin-2 on DmNa_v1 activation. Thus, the short CSαβ scaffold of drosomycin and the truncated scorpion toxin can maintain more than one bioactivity, and, in light of this new observation, we suggest that the biological role of peptides bearing this scaffold should be carefully examined. As for drosomycin, we discuss the intriguing possibility that it has additional functions in the fly, as implied by its tight interaction with DmNa_v1.The cysteine-stabilized αβ scaffold, CSαβ, contains an α-helix packed against a two-stranded β-sheet stabilized by three spatially conserved disulfide bonds (reviewed in Ref. 1). The CSαβ motif appears in a number of polypeptide families that can exert various biological functions such as: short chain (30–50 residues long) and long chain (60–76 residues long) scorpion toxins that affect voltage-gated ion channels, antimicrobial peptides (of insect and plants) as well as plant protease inhibitors (see Fig. 1) (2, 3).Open in a separate windowFIGURE 1.Diversity of peptides containing the CSαβ motif. Representatives from each of five major groups of peptides containing a CSαβ motif are aligned according to their conserved disulfide bridging and common structural features: two β-strands packed against an α-helix. The featured molecules are from a diverse array of organisms. Scorpion α-toxins: {"type":"entrez-protein","attrs":{"text":"P01484","term_id":"401071","term_text":"P01484"}}P01484 (Aah2 of the North African scorpion Androctonus australis hector), {"type":"entrez-protein","attrs":{"text":"AAB30413","term_id":"546246","term_text":"AAB30413"}}AAB30413 (Ts4 of the Brazilian scorpion Tityus serrulatus); Scorpion β-toxins: {"type":"entrez-protein","attrs":{"text":"P60266","term_id":"1279695545","term_text":"P60266"}}P60266 (Css4 of the Mexican scorpion Centruroides suffusus suffusus), 1BCG_A (Bj-xtrIT of the Israeli black scorpion Hottentota judaica); Scorpion potassium channel blockers: {"type":"entrez-protein","attrs":{"text":"P13487","term_id":"38503412","term_text":"P13487"}}P13487 (charybdotoxin of the Israeli yellow scorpion Leiurus quinquestriatus hebraeus), {"type":"entrez-protein","attrs":{"text":"P0C194","term_id":"93204597","term_text":"P0C194"}}P0C194 (α-KTx 6.11 of the scorpion Opisthacanthus madagascariensis of Madagascar); Insect antimicrobial peptides: {"type":"entrez-protein","attrs":{"text":"NP_523901","term_id":"17737523","term_text":"NP_523901"}}NP_523901 (drosomycin of the fruit fly Drosophila melanogaster), 1I2U_A (heliomicin of the tobacco budworm Heliothis virescens); plant γ-thionins: 1N4N (defensin of the garden petunia Petunia hybrida), {"type":"entrez-protein","attrs":{"text":"AAL85480","term_id":"28624546","term_text":"AAL85480"}}AAL85480 (defensin of peach Prunus persica), {"type":"entrez-protein","attrs":{"text":"AAM62652","term_id":"21553559","term_text":"AAM62652"}}AAM62652 (protease inhibitor II of the thale cress Arabidopsis thaliana).Analysis of the structure-function relationships of several representatives of a subclass of the long chain scorpion toxins family, the scorpion β-toxins (activators of voltage-gated sodium channels (Na_vs)⁵), elucidated their bioactive surfaces including those of the anti-insect excitatory and depressant toxins Bj-xtrIT and LqhIT2 (from Hottentota judaica and Leiurus quinquestriatus hebraeus, respectively (4–6)) and the anti-mammalian β-toxin Css4 (from Centruroides suffusus suffusus (7)). These studies highlighted a conserved pharmacophore positioned on the CSαβ protein core (7). The C-tail, loops, turns, and unstructured stretches that connect to the CSαβ protein core in long chain scorpion toxins constitute a large portion of their exteriors and bear residues that participate in bioactivity (reviewed in Ref. 8). We have recently reported that truncated scorpion β-toxins, lacking the N- and C-terminal regions of the parental peptides but maintaining the CSαβ motif (ΔΔβ-toxins), are able to interact at high affinity with Na_vs (9). Although by themselves, the ΔΔβ-toxins (ΔΔCss4 and ΔΔBj-xtrIT) were nontoxic and did not bind at the receptor sites of the parental toxins, they exhibited an unexpected ability to allosterically facilitate the activity of a scorpion α-toxin (inhibition of Na_v fast inactivation), which binds at receptor site-3 on insect Na_vs (10), and the effect of the marine polyether toxin brevetoxin-2 (PbTx-2, facilitator of Na_v activation), which binds to receptor site-5 (11). However, a short chain potassium channel blocker (charybdotoxin) with a CSαβ structural fold did not exert any of these effects (9). These results indicated that it is not only the CSαβ motif but that specific amino acids at key sites on the protein exterior that can interact with ion channels and either block voltage-gated potassium channels or induce conformational alteration of voltage-gated sodium channels. From a structural viewpoint, the ability of ΔΔBj-xtrIT and ΔΔCss4 to bind to the Na_v, as manifested in modulation of the interaction of receptor site-3 and -5 ligands, suggests that by truncation of the two β-toxins, a masked functional surface was exposed. Because the CSαβ motif appears in several protein families including antimicrobial peptides, potassium channel blockers, and sodium channel gating modifiers (Fig. 1) (2, 3), we explored the possibility that a well characterized CSαβ peptide may exert an additional function known for other peptides bearing this scaffold.For this aim, we tested the ability of a well characterized Drosophila melanogaster anti-fungal peptide drosomycin (DRS) to interact with voltage-gated sodium channels. The solution structure of DRS indicates that this 44-amino acid peptide is cross-linked by four disulfide bonds, of which three render a CSαβ structural fold (Fig. 2) (12). Sequence comparison of the truncated scorpion β-toxin ΔΔCss4 with DRS indicates moderate identity (34%) and similarity (50%), including conservation of six cysteine residues that stabilize the CSαβ motif, which is manifested by a remarkable structural similarity (Fig. 2). Moreover, Lys-3, Asp-11, Asn-12, Glu-13, Gln-21, and Gln-22 of ΔΔCss4, which are involved in the interaction with insect Na_vs, are spatially conserved in DRS (Fig. 2) (9) but not in potassium channel blockers (Fig. 1). In light of the resemblance between the truncated scorpion β-toxin and DRS, we tested whether DRS is able to interact with the D. melanogaster voltage-gated sodium channel DmNa_v1.Open in a separate windowFIGURE 2.Sequence alignment and three-dimensional structures of ΔΔCss4 and DRS. A, schematic diagrams of the Cα model structures of ΔΔCss4 and DRS covered by semitransparent molecular surfaces. The structure of DRS (right panel) is derived from the Protein Data Bank code 1MYN. The ΔΔCss4 model (left panel) is based on the NMR structure of Cn2 (Protein Data Bank code 1Cn2) and is spatially aligned with that of DRS. A was prepared using PyMOL. B, sequences were aligned according to the conserved cysteine residues, and the disulfide bonds formed between cysteine pairs are marked in solid lines. Dashes indicate gaps. Amino acid residues that were identified as part of the interacting surface of ΔΔCss4 with insect Na_vs (9) are shown in sticks according to their chemical nature (blue, positive charge; red, negative charge; green, nonpolar) and are also highlighted in the sequence alignment. Corresponding residues in DRS according to sequence and structural alignments are also shown in sticks.     

Direct interaction between the COG complex and the SM protein,Sly1, is required for Golgi SNARE pairing

The crucial roles of Sec1/Munc18 (SM)‐like proteins in membrane fusion have been evidenced in genetic and biochemical studies. SM proteins interact directly with SNAREs and contribute to SNARE pairing by a yet unclear mechanism. Here, we show that the SM protein, Sly1, interacts directly with the conserved oligomeric Golgi (COG) tethering complex. The Sly1–COG interaction is mediated by the Cog4 subunit, which also interacts with Syntaxin 5 through a different binding site. We provide evidence that disruption of Cog4–Sly1 interaction impairs pairing of SNAREs involved in intra‐Golgi transport thereby markedly attenuating Golgi‐to‐ER retrograde transport. These results highlight the mechanism by which SM proteins link tethering to SNAREpin assembly.     

Virulence Increasing of <Emphasis Type="Italic">Salmonella typhimurium</Emphasis> in Balb/c Mice After Heat-Stress Induction of Phage Shock Protein A

Salmonella typhimurium is a potentially intracellular pathogen and is responsible for thousands of reported cases of acute gastroenteritis and diarrhea each year. Although many successful physiological and genetic approaches have been taken to conclude the key virulence determinants encoded by this organism, the total number of uncharacterized reading frames observed within the S. typhimurium genome suggests that many virulence factors remain to be discovered. This study was conducted to evaluate the role of heat induced phage shock protein A (PspA), in the pathogenicity of S. typhimurium. The stress proteins detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis were identified specifically by immunoblotting with polyclonal antibody against PspA. PspA was produced in response to heat stress at 45°C and it was over-expressed at 65°C. At this temperature, the stressed bacterial cells producing PspA were more virulent (16 folds greater) to female 6–8 week-old Balb/c mice. Correspondency between decrease in LD₅₀ and increase in PspA production during heat stress and lower pathogenicity in non-producing cells that emerged during stress at 55°C represents PspA as an important virulence factor in heat stressed S. typhimurium.     

Engineering increases in sulfur amino acid contents in flax by overexpressing the yeast Met25 gene

Plant infection is accompanied by an oxidative burst that produces free radicals of various natures. The approach that we exploited in this study was to increase the antioxidative potential of flax by genetic engineering. Overexpressing the yeast Met25 gene coding for O-acetylhomoserine-O-acetylserine (OAH-OAS) sulfhydrylase in flax resulted in a significant increase in cysteine and methionine biosynthesis. This overproduction of sulfur amino acids increases the synthesis of glutathione, a tripeptide containing cysteine. The increase in glutathione content in the transgenic plant increases its antioxidative potential, and thus improves the plant's protection against Fusarium infection.     

Flow‐injection determination of vitamin A in pharmaceutical formulations using Tris(2,2′‐bipyridyl)Ru(II)–Ce(IV) chemiluminescence detection

A flow injection method with chemiluminescence detection is reported for the determination of vitamin A. The method is based on the enhancement effect of vitamin A on chemiluminescence of tris(2,2′‐bipyridyl)Ru(II)–Ce(IV) in acidic medium. The proposed procedure is used to quantitate vitamin A in the range 1.0–100 × 10^?6 mol/L with a correlation coefficient of 0.9991 (n = 9) and relative standard deviation in the range 1.2–2.3% (n = 4). The limit of detection (3 × blank) was 8.0 × 10^?8 mol/L with a sample throughput of 100/h. The effect of common excipients used in pharmaceutical formulations and some clinically important compounds was also studied. The method was applied to determine vitamin A in pharmaceutical formulations and the results obtained were in reasonable agreement with the amount quoted. The results were compared using spectrophotometric method and no significant difference was found between the results of the two methods at 95% confidence limit. Copyright © 2009 John Wiley & Sons, Ltd.     

The relationship between flexibility and EMG activity pattern of the erector spinae muscles during trunk flexion–extension

BackgroundMovements in the lumbar spine, including flexion and extension are governed by a complex neuromuscular system involving both active and passive units. Several biomechanical and clinical studies have shown the myoelectric activity reduction of the lumbar extensor muscles (flexion–relaxation phenomenon) during lumbar flexion from the upright standing posture. The relationship between flexibility and EMG activity pattern of the erector spinae during dynamic trunk flexion–extension task has not yet been completely discovered.ObjectiveThe purpose of this study was to investigate the relationship between general and lumbar spine flexibility and EMG activity pattern of the erector spinae during the trunk flexion–extension task.MethodsThirty healthy female college students were recruited in this study. General and lumbar spine flexibilities were measured by toe-touch and modified schober tests, respectively. During trunk flexion–extension, the surface electromyography (EMG) from the lumbar erector spinae muscles as well as flexion angles of the trunk, hip, lumbar spine and lumbar curvature were simultaneously recorded using a digital camera. The angle at which muscle activity diminished during flexion and initiated during extension was determined and subjected to linear regression analysis to detect the relationship between flexibility and EMG activity pattern of the erector spinae during trunk flexion–extension.ResultsDuring flexion, the erector spinae muscles in individuals with higher toe-touch scores were relaxed in larger trunk and hip angles and reactivated earlier during extension according to these angles (P < 0.001) while in individuals with higher modified schober scores this muscle group was relaxed later and reactivated sooner in accordance with lumbar angle and curvature (P < 0.05). Toe-touch test were significantly correlated with trunk and hip angles while modified schober test showed a significant correlation with lumbar angle and curvature variables.ConclusionThe findings of this study indicate that flexibility plays an important role in trunk muscular recruitment pattern and the strategy of the CNS to provide stability. The results reinforce the possible role of flexibility alterations as a contributing factor to the motor control impairments. This study also shows that flexibility changes behavior is not unique among different regions of the body.