Role of Neurotoxin Associated Proteins in the Low pH Induced Structural Changes in the Botulinum Neurotoxin Complex

Botulinum Neurotoxin (BoNT) produced by the bacterium Clostridium botulinum as a complex with NAPs causes botulism. It has been known that the NAPs protect the toxin from both extremes of pHs and proteases of the GI tract. In an attempt to emulate the physiological conditions encountered by the toxin, we examined BoNT/A, BoNT/A complex, and NAPs under different pH conditions and monitored their structural characteristics by far-UV CD and thermal denaturation analysis. BoNT/A complex showed the maximum CD signal with a mean residue weight ellipticity of ?1.8 × 10⁵° cm²/dmol at 222 nm at both acidic and neutral pHs. Thermal denaturation analysis revealed NAPs to be the most stable amongst the three protein samples examined. Interestingly and quite uniquely, at pH 2.5, there was an increase in CD signal for BoNT complex as a function of temperature, which correlated with the NAPs profile, indicating a shielding effect of NAPs on BoNT complex at low pH. Calculation of the weighted mean of the ellipticities at the T_m for thermal unfolding of toxin and NAPs at neutral and acidic pHs showed variation with that of BoNT complex, suggesting structural reorganization in BoNT complex upon the association of NAPs and BoNT. In conclusion, this study reveals the structural behavior of BoNT complex and NAPs with pH changes substantially, which could be quite relevant for BoNT survival under extreme pH conditions in vivo.     

Autoantibodies in cryptogenic fibrosing alveolitis

The pathogenesis of cryptogenic fibrosing alveolitis (CFA) involves injury, an immune/inflammatory response and fibrosis. The cause of the injury is unknown, but the identification of serum autoantibodies makes an autoimmune aetiology attractive. The core study on which this commentary is based used novel cloning and serum screening technologies in order to identify new public and private autoantibodies in sera from 12 patients with CFA. Largely negative conclusions were drawn from that study. However, we suggest that the prevalence of autoantibodies may have been underestimated, that the study was timely and that this approach is worth pursuing further.     

Binding and release of cholesterol in the Osh4 protein of yeast

Sterols have been shown experimentally to bind to the Osh4 protein (a homolog of the oxysterol binding proteins) of Saccharomyces cerevisiae within a binding tunnel, which consists of antiparallel beta-sheets that resemble a beta-barrel and three alpha-helices of the N-terminus. This and other Osh proteins are essential for intracellular transport of sterols and ultimately cell life. Molecular dynamics (MD) simulations are used to study the binding of cholesterol to Osh4 at the atomic level. The structure of the protein is stable during the course of all MD simulations and has little deviation from the experimental crystal structure. The conformational stability of cholesterol within the binding tunnel is aided in part by direct or water-mediated interactions between the 3-hydroxyl (3-OH) group of cholesterol and Trp(46), Gln(96), Tyr(97), Asn(165), and/or Gln(181) as well as dispersive interactions with Phe(42), Leu(24), Leu(39), Ile(167), and Ile(203). These residues along with other nonpolar residues in the binding tunnel and lid contribute nearly 75% to the total binding energy. The strongest and most populated interaction is between Gln(96) and 3-OH with a cholesterol/Gln(96) interaction energy of -4.5 +/- 1.0 kcal/mol. Phe(42) has a similar level of attraction to cholesterol with -4.1 +/- 0.3 kcal/mol. A MD simulation without the N-terminus lid that covers the binding tunnel resulted in similar binding conformations and binding energies when compared with simulations with the full-length protein. Steered MD was used to determine details of the mechanism used by Osh4 to release cholesterol to the cytoplasm. Phe(42), Gln(96), Asn(165), Gln(181), Pro(211), and Ile(206) are found to direct the cholesterol as it exits the binding tunnel as well as Lys(109). The mechanism of sterol release is conceptualized as a molecular ladder with the rungs being amino acids or water-mediated amino acids that interact with 3-OH.     

Stabilization of Mn(II) and Mn(III) in mononuclear complexes derived from tridentate ligands with N2O donors: Synthesis, crystal structure, superoxide dismutase activity and DNA interaction studies

A new family of tridentate ligands PhimpH (2-((2-phenyl-2-(pyridin-2-yl)hydazono)methyl)phenol), N-PhimpH (2-((2-phenyl-2-(pyridin-2-yl)hydrazono)methyl)napthalen-1-ol), Me-PhimpH (2-(1-(2-phenyl-2-(pyridine-2-yl)hydrazono)ethyl)phenol) have been synthesized and characterized. The ligands PhimpH and N-PhimpH after deprotonation react with manganese(II) and manganese(III) starting materials affording [Mn(Phimp)₂] (1), [Mn(Phimp)₂](ClO₄) (2), [Mn(N-Phimp)₂] (3), [Mn(N-Phimp)₂](ClO₄) (4). Complexes [Mn(Phimp)₂] (1) and [Mn(N-Phimp)₂] (3) convert to [Mn(Phimp)₂]⁺ (cation of 2) and [Mn(N-Phimp)₂]⁺ (cation of 4) respectively upon oxidation. Ligand Me-PhimpH stabilized only manganese(III) centre resulting [Mn(Me-Phimp)₂](ClO₄) (5). The molecular structures of [Mn(Phimp)₂], 1 and [Mn(Phimp)₂](ClO₄), 2 were determined by single crystal X-ray diffraction. X-ray crystal structures of 1 and 2 have revealed the presence of distorted octahedral MnN₄O₂ coordination sphere having meridionally spanning ligands. Electrochemical studies for the complexes showed Mn(II)/Mn(III), (E_1/2 = 0.14-0.40 V) and Mn(III)/Mn(IV), (E_1/2 = 0.80-1.06 V) couples vs. Ag/AgCl. The redox properties were exploited to examine superoxide dismutase (SOD) activity using Mn(II)/Mn(III) couple. The complexes 1, 2, 4 and 5 have been revealed to catalyze effectively the dismutation of superoxide () in xanthine-xanthine oxidase-nitro blue tetrazolium assay and IC₅₀ values were found to be 0.29, 0.39, 1.12 and 0.76 μM respectively. DNA interaction studies with complex 2 showed binding of DNA in a non-intercalative pathway. Complexes 1, 2 and 4 exhibited nuclease activity in presence of H₂O₂ and inhibition of activity was noted in presence of KI.     

Click and Cut: a click chemistry approach to developing oxidative DNA damaging agents

Metallodrugs provide important first-line treatment against various forms of human cancer. To overcome chemotherapeutic resistance and widen treatment possibilities, new agents with improved or alternative modes of action are highly sought after. Here, we present a click chemistry strategy for developing DNA damaging metallodrugs. The approach involves the development of a series of polyamine ligands where three primary, secondary or tertiary alkyne-amines were selected and ‘clicked’ using the copper-catalysed azide-alkyne cycloaddition reaction to a 1,3,5-azide mesitylene core to produce a family of compounds we call the ‘Tri-Click’ (TC) series. From the isolated library, one dominant ligand (TC1) emerged as a high-affinity copper(II) binding agent with potent DNA recognition and damaging properties. Using a range of in vitro biophysical and molecular techniques—including free radical scavengers, spin trapping antioxidants and base excision repair (BER) enzymes—the oxidative DNA damaging mechanism of copper-bound TC1 was elucidated. This activity was then compared to intracellular results obtained from peripheral blood mononuclear cells exposed to Cu(II)–TC1 where use of BER enzymes and fluorescently modified dNTPs enabled the characterisation and quantification of genomic DNA lesions produced by the complex. The approach can serve as a new avenue for the design of DNA damaging agents with unique activity profiles.     

Emergence of antibiotic-resistant extremophiles (AREs)

Excessive use of antibiotics in recent years has produced bacteria that are resistant to a wide array of antibiotics. Several genetic and non-genetic elements allow microorganisms to adapt and thrive under harsh environmental conditions such as lethal doses of antibiotics. We attempt to classify these microorganisms as antibiotic-resistant extremophiles (AREs). AREs develop strategies to gain greater resistance to antibiotics via accumulation of multiple genes or plasmids that harbor genes for multiple drug resistance (MDR). In addition to their altered expression of multiple genes, AREs also survive by producing enzymes such as penicillinase that inactivate antibiotics. It is of interest to identify the underlying molecular mechanisms by which the AREs are able to survive in the presence of wide arrays of high-dosage antibiotics. Technologically, "omics"-based approaches such as genomics have revealed a wide array of genes differentially expressed in AREs. Proteomics studies with 2DE, MALDI-TOF, and MS/MS have identified specific proteins, enzymes, and pumps that function in the adaptation mechanisms of AREs. This article discusses the molecular mechanisms by which microorganisms develop into AREs and how "omics" approaches can identify the genetic elements of these adaptation mechanisms. These objectives will assist the development of strategies and potential therapeutics to treat outbreaks of pathogenic microorganisms in the future.