A mass spectrometric approach to the study of DNA-binding proteins: interaction of human TRF2 with telomeric DNA

Human telomeric repeat binding factor 2 (hTRF2) is a protein that plays an important role in capping human telomeres to protect them from DNA damage repair systems. The ineffectiveness of hTRF2 may be linked to aging and cancer. We report the use of PLIMSTEX (protein-ligand interactions by mass spectrometry, titration, and H/D exchange) and selective acetylation of lysine residues to study the interaction of the DNA-binding domain and double-stranded telomeric DNA (repeats of TTAGGG). By increasing the resolution of PLIMSTEX to the peptide level, we localized the changes in deuterium uptake of hTRF2 as a function of varying amounts of a model oligodeoxynucleotide. From these experiments, we determined the affinity constant for binding to DNA, which is within a factor of 3 of the previously reported value. Amide H/D exchange revealed portions of the protein that have contacts with the phosphate backbone of DNA, whereas acetylation disclosed the decrease in solvent accessibility of regions containing Lys 447 and 488, which must be involved in interactions with the DNA major and minor grooves. These complementary approaches of amide H/D exchange and selective side chain modification can be employed effectively to pinpoint and quantify protein-ligand, in particular protein-DNA, interactions.     

Adhesion and viability of waterborne pathogens on p-DADMAC coatings

The attachment of waterborne pathogens onto surfaces can be increased by coating the surfaces with positive charge-enhancing polymers. In this paper, the increased efficacy of polydiallyldimethylammonium chloride (p-DADMAC) coatings on glass was evaluated in a parallel plate flow chamber with the use of waterborne pathogens (Raoultella terrigena, Escherichia coli, and Brevundimonas diminuta). p-DADMAC coatings strongly compensated the highly negative charges on the glass surface and even yielded a positively charged surface when applied from a 500 ppm solution. Whereas none of the strains adhered from water to glass due to electrostatic repulsion, R. terrigena and E. coli readily adhered in high numbers to p-DADMAC coated glass slides applied from 1, 100, or 500 ppm aqueous solutions. B. diminuta only adhered to a positively charged p-DADMAC coating applied from a 500 ppm solution. In addition, all p-DADMAC coatings indicated strong contact killing with the bacterial species used in this study by live/dead staining techniques. In summary, this paper demonstrates the potential of p-DADMAC coatings to strongly enhance bacterial adhesion. Moreover, once adhered, bacterial viability can be reduced by the positively charged ammonium groups in the coating.     

Modest loss of peripheral axons, muscle atrophy and formation of brain inclusions in mice with targeted deletion of gigaxonin exon 1

Mutations in the gigaxonin gene are responsible for giant axonal neuropathy (GAN), a progressive neurodegenerative disorder associated with abnormal accumulations of Intermediate Filaments (IFs). Gigaxonin is the substrate-specific adaptor for a new Cul3-E3-ubiquitin ligase family that promotes the proteasome dependent degradation of its partners MAP1B, MAP8 and tubulin cofactor B. Here, we report the generation of a mouse model with targeted deletion of Gan exon 1 (Gan(Deltaexon1;Deltaexon1)). Analyses of the Gan(Deltaexon1;Deltaexon1) mice revealed increased levels of various IFs proteins in the nervous system and the presence of IFs inclusion bodies in the brain. Despite deficiency of full length gigaxonin, the Gan(Deltaexon1;Deltaexon1) mice do not develop overt neurological phenotypes and giant axons reminiscent of the human GAN disease. Nonetheless, at 6 months of age the Gan(Deltaexon1;Deltaexon1) mice exhibit a modest hind limb muscle atrophy, a 10% decrease of muscle innervation and a 27% axonal loss in the L5 ventral roots. This new mouse model should provide a useful tool to test potential therapeutic approaches for GAN disease.     

Mechanism of antiviral drug resistance of vaccinia virus: identification of residues in the viral DNA polymerase conferring differential resistance to antipoxvirus drugs

The acyclic nucleoside phosphonate (ANP) family of drugs shows promise as therapeutics for treating poxvirus infections. However, it has been questioned whether the utility of these compounds could be compromised through the intentional genetic modification of viral sequences by bioterrorists or the selection of drug resistance viruses during the course of antiviral therapy. To address these concerns, vaccinia virus (strain Lederle) was passaged 40 times in medium containing an escalating dose of (S)-1-[3-hydroxy-2-(phosphonomethoxypropyl)-2,6-diaminopurine [(S)-HPMPDAP], which selected for mutant viruses exhibiting a ~15-fold-increased resistance to the drug. (S)-HPMPDAP-resistant viruses were generated because this compound was shown to be one of the most highly selective and effective ANPs for the treatment of poxvirus infections. DNA sequence analysis revealed that these viruses encoded mutations in the E9L (DNA polymerase) gene, and marker rescue studies showed that the phenotype was produced by a combination of two (A684V and S851Y) substitution mutations. The effects of these mutations on drug resistance were tested against various ANPs, both separately and collectively, and compared with E9L A314T and A684V mutations previously isolated using selection for resistance to cidofovir, i.e., (S)-1-[3-hydroxy-2-(phosphonomethoxypropyl)cytosine]. These studies demonstrated a complex pattern of resistance, although as a general rule, the double-mutant viruses exhibited greater resistance to the deoxyadenosine than to deoxycytidine nucleotide analogs. The S851Y mutant virus exhibited a low level of resistance to dCMP analogues but high-level resistance to dAMP analogues and to 6-[3-hydroxy-2-(phosphonomethoxy)propoxy]-2,4-diaminopyrimidine, which is considered to mimic the purine ring system. Notably, (S)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]-3-deazaadenine retained marked activity against most of these mutant viruses. In vitro studies showed that the A684V mutation partially suppressed a virus growth defect and mutator phenotype created by the S851Y mutation, but all of the mutant viruses still exhibited a variable degree of reduced virulence in a mouse intranasal challenge model. Infections caused by these drug-resistant viruses in mice were still treatable with higher concentrations of the ANPs. These studies have identified a novel mechanism for the development of mutator DNA polymerases and provide further evidence that antipoxviral therapeutic strategies would not readily be undermined by selection for resistance to ANP drugs.     

Tomato ethylene receptor-CTR interactions: visualization of NEVER-RIPE interactions with multiple CTRs at the endoplasmic reticulum

In the model plant Arabidopsis, members of a family of two-componentsystem His kinase-like ethylene receptors have direct protein–proteininteractions with a single downstream Ser/Thr kinase CTR1. Thesecomponents of the ethylene signalling network found in Arabidopsisare conserved in the climacteric fruit tomato, but both theethylene receptors and CTR1-like proteins (LeCTRs) in tomatoare encoded by multigene families. Here, using a yeast two-hybridinteraction assay, it is shown that the tomato receptors LeETR1,LeETR2, and NEVER-RIPE (NR) can interact with multiple LeCTRs.In vivo protein localization studies with fluorescent taggedproteins revealed that the ethylene receptor NR was targetedto the endoplasmic reticulum (ER) when transiently expressedin onion epidermal cells, whereas the four LeCTR proteins werefound in the cytoplasm and nucleus. When co-expressed with NR,three LeCTRs (1, 3, and 4), but not LeCTR2, also adopted thesame ER localization pattern in an NR receptor-dependent mannerbut not in the absence of NR. The receptor–CTR interactionswere confirmed by biomolecular fluorescence complementation(BiFC) showing that NR could form a protein complex with LeCTR1,3, and 4. This suggested that ethylene receptors recruit theseLeCTR proteins to the ER membrane through direct protein–proteininteraction. The receptor–CTR interactions and localizationobserved in the study reinforce the idea that ethylene receptorstransmit the signal to the downstream CTRs and show that a singlereceptor can interact with multiple CTR proteins. It remainsunclear whether the different LeCTRs are functionally redundantor have unique roles in ethylene signalling. Key words: BiFC, endoplasmic reticulum, Ser/Thr kinase, tomato ethylene signalling, two-component system His kinase     

Male-specific protein (MSP): a new gene linked to sexual behavior and aggressiveness of tilapia males

MSP is a male-specific protein initially identified in the serum of sexually active Sarotherodon galilaeus males, and is shown herein to be present in the serum of sexually mature males, but not females, of three other tilapia species. Cloning of the MSP cDNA and analysis of its predicted amino-acid sequence revealed that it is an outlier lipocalin that contains a signal peptide in its N-terminal region. The abundance of highly homologous sequences found in fish and the monophyletic relationship to tetrapod Alpha-1-acid glycoprotein (AGP) places it as a clade XII lipocalin. MSP was shown to undergo major N-glycosylation, characteristic of many lipocalins. The expression pattern of MSP, as determined at both the RNA and protein levels, points to the liver, head kidney and testis as production tissues, and resembles a pattern typical of some hormones. We found that MSP is secreted in urine and seminal fluids, and is present in the skin mucus of socially dominant males. Moreover, we discovered a positive correlation between MSP levels in the serum and the dominance and aggressive behavior displayed by socially dominant males. Based on these data, we suggest that MSP is a novel male-specific lipocalin that may function in intra and inter-sex communication.     

Resonance Raman spectroscopy can detect structural changes in haemozoin (malaria pigment) following incubation with chloroquine in infected erythrocytes

Resonance Raman spectroscopy was applied to monitor the effects of chloroquine (CQ) treatment on cultures of Plasmodium falciparum trophozoites. A number of bands assigned to A(1g) and B(1g) modes characteristic of the haemozoin aggregate are reduced in intensity in the CQ-treated cells, however, no bands from the CQ are observed. The intensity changes are attributed to intermolecular drug binding of the CQ in a sandwich type complex between ferriprotoporphyrin IX (FePPIX) dimer units. It is postulated that the CQ binds via pi-pi interactions between adjacent and orientated porphyrins thereby disrupting the haemozoin aggregate and reducing excitonic interactions between adjacent haems. The results show the potential of Raman microscopy as a screening tool for FePPIX:drug interactions in live cells.