Double Point Mutations Research Articles

Molecular Characterization of Fluoroquinolone Resistance of Methicillin –Resistant Clinical Staphylococcus aureus Isolates from Rawalpindi, Pakistan

We have characterized 10 multidrug-resistant Staphylococcus aureus clinical isolates from Pakistan, with respect to their fluoroquinolone resistance, mutations in gyrA/B, and grlA/B genes, and the presence and expression of the efflux pump genes norA, norB, norC, and mdeA. All the isolates were highly resistant to ciprofloxacin, enrofloxacin, levofloxacin, and norfloxacin and possessed one or more efflux pump genes. The efflux pumps, along with the single and double point mutations S84L, S84L and G106D observed in gyrA; and S80Y, S81P, E84G, and S80F and E84G in grlA genes; enhanced fluoroquinolone resistance in these isolates. A single deletion of nucleotide “A” upstream of the putative Fur-binding box and the presence of two novel mutations, G291D in isolate 30, 32, 35, and 41 and V371I in isolate 10 within the norA coding region, were also observed. An in silico structural analysis revealed an increase in NorAG291D stability with a predicted DDG of 0.48 kcal/mol, but a decrease in NorAV371I stability by -1.12 kcal/mol. Moreover, an increased transcriptional expression of the norA, norB, norC, and mdeA genes and relatively higher fluoroquinolone resistance in isolates with a G291D mutation, compared to that of the isolate with a V371I mutation, suggests a possible role of these mutations in fluoroquinolone resistance. The data provide new insights into the mechanism of fluoroquinolone resistance and may lead to an enhanced understanding of multidrug resistance observed in MRSA isolates and the development of effective mitigation strategies.

Abstract 2448: Towards structure-based drug design of 3-phosphoglycerate dehydrogenase inhibitors

Abstract Background The NAD+-requiring enzyme 3-phosphoglycerate dehydrogenase (PHGDH) diverts glycolytic flux into serine production and folate metabolism by catalyzing the oxidization of 3-phosphoglycerate to phosphohydroxypyruvate. The PHGDH gene is located on chromosome 1(1p12), a region frequently amplified in melanoma and certain breast cancer forms. PHGDH knockdown in cells with amplified PHGDH or overexpressing PHGDH at the protein level resulted in cell growth inhibition. In addition ectopic overexpression of PHGDH in a non-tumorigenic cell line induced morphological changes characteristic of transformation. Therefore, inhibitors of PHGDH may be therapeutically valuable. No PHGDH inhibitors have been reported to date, our aim is to develop PHGDH inhibitors targeting the cofactor (and substrate) binding site. Methods Full length PHGDH and the catalytic subunit (sPHGDH) were expressed in, and isolated from E. coli. PHGDH was found to co-purify with its cofactor, making it necessary to abolish the cofactor-protein interaction to investigate inhibitor binding at the co-factor site. To accomplish this, site-directed sPHGDH mutants were constructed and purified. To characterize the mutants, their enzymatic activity and ability to bind to NADH (assessed by thermal stability and isothermal titration calorimetry (ITC)) were measured, and their structures were characterized by circular dichroism (CD) spectroscopy and on-going x-ray crystallographic studies. Results sPHGDH retained 60% of the enzymatic activity of the full length protein whereas introduction of single or double point mutations around the cofactor or substrate binding site resulted in complete abolition of enzymatic activity. The binding of NADH (1mM) to PHGDH and sPHGDH resulted in increases in melting temperature (ΔTm) of 8.4 ± 0.2 °C and 9.3 ± 0.5 °C respectively. Mutation of the cofactor binding site resulted in reduced ability to bind NADH (ΔTm = 0.8 ± 0.2 °C, 2.3 ± 0.1 °C) whereas mutation at the substrate binding site, directly adjacent to the cofactor binding site, had no significant effect on the ability to bind NADH. Congruent findings were obtained via ITC determining a binding affinity (Kd) of 0.66 μM for wt sPHGDH and a reduced binding affinity of 3-30 fold for the mutated proteins. CD measurements showed that the proteins mutated at the co-factor binding site had undergone minor changes in the secondary structure compared to the wt sPHGDH whereas mutation at the substrate binding site had no effect on the secondary structure elements. Conclusion Although the mutations carried out were single or double point mutations only, cofactor binding could be reduced substantially, giving rise to markedly lower catalytic activity. These investigations illustrate a promising way of making proteins with large, tight binding ligands accessible to structure-based drug-design. Citation Format: Judith E. Unterlass, Nabila Aljufri, Sophie Bex, Celine Cano, Martin E M Noble, Nicola J. Curtin. Towards structure-based drug design of 3-phosphoglycerate dehydrogenase inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2448. doi:10.1158/1538-7445.AM2015-2448

Abstract 2438: Identification of the mitochondrial binding site on the amino-terminal end of hexokinase II

Abstract Hexokinase II is responsible for the first step in the glycolysis pathway. Hexokinase II adds a phosphate onto the glucose molecule so it can proceed down the pathway to give the cancer cell energy for its continuous growth. Tumor cells overexpress the hexokinase II enzyme. In fact, it is the overexpression of the hexokinase II enzyme that makes the diagnosis of cancer possible when imaged by positron emission tomography (PET). Hexokinase II binds to the voltage-dependent anion channel (VDAC) on the mitochondrial membrane and when it is attached to the mitochondrial membrane it is blocking a major pathway for cell death. Thus, hexokinase II is responsible for two characteristics of cancer cells, rapid tumor growth and difficulty to kill. Therefore the overall goal of our laboratory is to identify novel compounds with the ability to displace or translocate hexokinase II from the mitochondrial membrane to the cytoplasm. To attain our goal, we propose to use the crystal structure of hexokinase II to create a molecular model of the enzyme. However the amino acids responsible for binding to VDAC are not known. Therefore, we made a series of truncations and point mutations to identify the binding site to VDAC. Truncations of the first 10 and 20 amino acids indicated that important amino acids for binding were located within the first 10 amino acids. We made a series of single, double and triple point mutations within the first 10 amino acids. Immunofluorescence and immunoblot data has confirmed that mutating the fifth amino acid from histidine to proline completely abolished binding to the mitochondrial membrane. To prepare hexokinase II for crystallization, we have cloned the enzyme downstream of a thrombin cleavage site and His-tag. We were able to purify hexokinase II using a cobalt column, then eluting hexokinase II using thrombin to cleave at the thrombin cleavage site. Purified hexokinase II is isolated after removal of the thrombin using Protein A beads. After the crystal structure of hexokinase II is obtained, we will use the University of Florida Structure-based Molecular Docking Core facility to identify compounds predicted to fit into the VDAC binding site. The compounds will be characterized to validate a mechanism-of-action of translocating hexokinase II from the mitochondrial membrane to the cytoplasm, blocking glycolysis and decreasing ATP synthesis. The positive compounds will be analyzed to determine the best strategy for moving our studies closer to clinical application in cancer patients. Citation Format: Nadezda Bryan, Michelle M. Hwang, Kevin P. Raisch. Identification of the mitochondrial binding site on the amino-terminal end of hexokinase II. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2438. doi:10.1158/1538-7445.AM2015-2438

Kv1.3 contains an alternative C-terminal ER exit motif and is recruited into COPII vesicles by Sec24a.

BackgroundPotassium channels play a fundamental role in resetting the resting membrane potential of excitable cells. Determining the intracellular trafficking and localization mechanisms of potassium channels provides a platform to fully characterize their maturation and functionality. Previous investigations have discovered residues or motifs that exist in their primary structure, which directly promote anterograde trafficking of nascent potassium channels. Recently, a non-conical di-acidic motif (E483/484) has been discovered in the C-terminus of the mammalian homologue of the Shaker voltage-gated potassium channel subfamily member 3 (Kv1.3), and was shown to disrupt the anterograde trafficking of Kv1.3.ResultsWe have further investigated the intracellular trafficking requirements of Kv1.3 both in vivo and in vitro. First, three alternative C-terminal acidic residues, E443, E445, E447 were probed for their involvement within the early secretory pathway of Kv1.3. Single point (E443A, E445A, and E447A) and double point (E443A-E445A, E445A-E447A) mutations exhibited no significant changes in their endoplasmic reticulum (ER) retention. The triple point mutant E443A-E445A-E447A displayed a modest ER retention while deletion of the C-terminus showed dramatic ER retention. Second, we demonstrate in vivo the requirement for the Sec24a isoform to confer anterograde trafficking using a siRNA knockdown assay. Third, we show in vitro the association of recombinantly expressed Kv1.3 and Sec24a proteins.ConclusionThese results expand upon previous studies aimed at deciphering the Kv1.3 secretory trafficking mechanisms and further show in vitro evidence of the association between Kv1.3 and the COPII cargo adaptor subunit isoform Sec24a.Electronic supplementary materialThe online version of this article (doi:10.1186/s12858-015-0045-6) contains supplementary material, which is available to authorized users.

Three Novel Mutations of CHD7 Gene in Two Turkish Patients with Charge Syndrome; A Double Point Mutation and an Insertion

The CHARGE (coloboma, heart defects, atresia, retardation, genital, ear) syndrome is a genetic disease characterized by ocular coloboma, choanal atresia or stenosis and semicircular canal abnormalities. Most of the patients clinically diagnosed with CHARGE syndrome have mutations in chromodomain helicase DNA-binding protein 7 (CHD7) gene. The CHD7 gene is located on chromosome 8q12.1, and up to now, there are more than 500 pathogenic mutations identified in the literature. We report two patients diagnosed with CHARGE syndrome with two novel mutations in the CHD7 gene: the first patient has double consecutive novel mutations in three adjacent codons, and the other has a novel insertion.

Genetic Diversity of the Hepatitis B Virus Strains in Cuba: Absence of West-African Genotypes despite the Transatlantic Slave Trade.

Cuba is an HBsAg low-prevalence country with a high coverage of anti-hepatitis B vaccine. Its population is essentially the result of the population mix of Spanish descendants and former African slaves. Information about genetic characteristics of hepatitis B virus (HBV) strains circulating in the country is scarce. The HBV genotypes/subgenotypes, serotypes, mixed infections, and S gene mutations of 172 Cuban HBsAg and HBV-DNA positive patients were determined by direct sequencing and phylogenetic analysis. Phylogenetic analysis of HBV S gene sequences showed a predominance of genotype A (92.4%), subgenotype A2 (84.9%) and A1 (7.6%). Genotype D (7.0%) and subgenotype C1 (0.6%) were also detected but typical (sub)genotypes of contemporary West-Africa (E, A3) were conspicuously absent. All genotype A, D, and C strains exhibited sequence characteristics of the adw2, ayw2, and adrq serotypes, respectively. Thirty-three (19.1%) patients showed single, double, or multiple point mutations inside the Major Hydrophilic domain associated with vaccine escape; eighteen (10.5%) patients had mutations in the T-cell epitope (amino acids 28-51), and there were another 111 point mutations downstream of the S gene. One patient had an HBV A1/A2 mixed infection. This first genetic study of Cuban HBV viruses revealed only strains that were interspersed with strains from particularly Europe, America, and Asia. The absence of genotype E supports previous hypotheses about an only recent introduction of this genotype into the general population in Africa. The presence of well-known vaccine escape (3.5%) and viral resistance mutants (2.9%) warrants strain surveillance to guide vaccination and treatment strategies.

Identification of essential residues for binding and activation in the human 5-HT7(a) serotonin receptor by molecular modeling and site-directed mutagenesis.

The human 5-HT7 receptor is expressed in both the central nervous system and peripheral tissues and is a potential drug target in behavioral and psychiatric disorders. We examined molecular determinants of ligand binding and G protein activation by the human 5-HT7(a) receptor. The role of several key residues in the 7th transmembrane domain (TMD) and helix 8 were elucidated combining in silico and experimental mutagenesis. Several single and two double point mutations of the 5-HT7(a) wild type receptor were made (W7.33V, E7.35T, E7.35R, E7.35D, E7.35A, R7.36V, Y7.43A, Y7.43F, Y7.43T, R8.52D, D8.53K; E7.35T-R7.36V, R8.52D-D8.53K), and their effects upon ligand binding were assessed by radioligand binding using a potent agonist (5-CT) and a potent antagonist (SB269970). In addition, the ability of the mutated 5-HT7(a) receptors to activate G protein after 5-HT-stimulation was determined through activation of adenylyl cyclase. In silico investigation on mutated receptors substantiated the predicted importance of TM7 and showed critical roles of residues E7.35, W7.33, R7.36 and Y7.43 in agonist and antagonist binding and conformational changes of receptor structure affecting adenylyl cyclase activation. Experimental data showed that mutants E7.35T and E7.35R were incapable of ligand binding and adenylyl cyclase activation, consistent with a requirement for a negatively charged residue at this position. The mutant R8.52D was unable to activate adenylyl cyclase, despite unaffected ligand binding, consistent with the R8.52 residue playing an important role in the receptor-G protein interface. The mutants Y7.43A and Y7.43T displayed reduced agonist binding and AC agonist potency, not seen in Y7.43F, consistent with a requirement for an aromatic residue at this position. Knowledge of the molecular interactions important in h5-HT7 receptor ligand binding and G protein activation will aid the design of selective h5-HT7 receptor ligands for potential pharmacological use.

The Proapoptotic F-box Protein Fbxl7 Regulates Mitochondrial Function by Mediating the Ubiquitylation and Proteasomal Degradation of Survivin

Fbxl7, a component of the Skp1·Cul1·F-box protein type ubiquitin E3 ligase, regulates mitotic cell cycle progression. Here we demonstrate that overexpression of Fbxl7 in lung epithelia decreases the protein abundance of survivin, a member of the inhibitor of apoptosis family. Fbxl7 mediates polyubiquitylation and proteasomal degradation of survivin by interacting with Glu-126 within its carboxyl-terminal α helix. Furthermore, both Lys-90 and Lys-91 within survivin serve as ubiquitin acceptor sites. Ectopically expressed Fbxl7 impairs mitochondrial function, whereas depletion of Fbxl7 protects mitochondria from actions of carbonyl cyanide m-chlorophenylhydrazone, an inhibitor of oxidative phosphorylation. Compared with wild-type survivin, cellular expression of a survivin mutant protein deficient in its ability to interact with Fbxl7 (E126A) and a ubiquitylation-resistant double point mutant (KK90RR/KK91RR) rescued mitochondria to a larger extent from damage induced by overexpression of Fbxl7. Therefore, these data suggest that the Skp1·Cul1·F-box protein complex subunit Fbxl7 modulates mitochondrial function by controlling the cellular abundance of survivin. The results raise opportunities for F-box protein targeting to preserve mitochondrial function.

Design of Nitrilases with Superior Activity and Enantioselectivity towards Sterically Hindered Nitrile by Protein Engineering

AbstractThe enantioselective hydrolysis of ortho‐chloromandelonitrile with nitrilase is one of the most attractive approaches to prepare (R)‐ortho‐chloromandelic acid. To date, efforts to develop this nitrilase‐mediated process were plagued by either insufficient eep (enantiomeric excess of product) or low activity due to the steric hindrance from the ortho‐substituted substrate. To improve the nitrilase potential for producing (R)‐ortho‐chloromandelic acid, an enhancement of both activity and enantioselectivity towards sterically hindered nitriles would be highly desirable. Molecular docking of the (R)‐ortho‐chloromandelonitrile into the active site of wild‐type 2A6 nitrilase (nitA) allowed the identification of proximal nitA active site residues. Several residues (52, 132, 189 and 190) were selected as targets for single and double point mutation to improve nitA activity and enantioselectivity towards ortho‐chloromandelonitrile. Targeted mutagenesis yielded several nitA variants with superior activity and enantioselectivity. The best mutant T132A/F189T exhibited a 4.37‐fold higher specific activity (7.39 U/mg) towards ortho‐chloromandelonitrile than the wild‐type nitA. More importantly, the enantioselectivity (E) was improved from 17.34 to >200, resulting in a highly enantiopure product. Molecular docking experiments further support the enhanced activity and enantioselectivity shown experimentally and the structural effects of this amino acid substitution on the active site of nitA are provided. The amino acids at sites 189 and 132 determine the activity and enantioselectivity towards ortho‐chloromandelonitrile. With mutant T132A/F189T as a catalyst, a maximum of 450 mM of (R)‐ortho‐chloromandelic acid was produced with a 90% conversion and >99% eep within 3 h. This is the first time that a high productivity of (R)‐ortho‐chloromandelic acid of up to 671.76 g L−1 d−1 using a nitrilase‐mediated approach is reported. The engineered T132A/F189T variant represents a promising and competitive biocatalyst for practical application in synthesizing (R)‐ortho‐chloromandelic acid.magnified image

Two conserved amino acids of juxtaposed domains of a ribosomal maturation protein CgtA sustain its optimal GTPase activity

CgtA is a highly conserved ribosome binding protein involved in ribosome biogenesis and associated with stringent response. It is a 55 KDa GTPase protein consisting of GTPase, Obg and C-terminal domains. The function of the latter two domains was not clear and despite the importance, the mode of action of CgtA is still largely unknown. Knocking out of CgtA gene is lethal and mutations lead to growth, sporulation and developmental defects in bacteria. It was found that a growth defect and pinhole size colony morphology of Bacillus subtilis was associated with a Gly92Asp point mutation on the Obg domain of its CgtA protein, instead of its GTPase domain. CgtA is an important and essential protein of the deadly diarrhea causing bacteria Vibrio cholerae and in order to investigate the mode of action of the V. cholerae CgtA we have utilized this information. We measured the GTPase activity of V. cholerae CgtA (CgtAvc) protein in the presence of purified ribosome. Our results showed 5-fold increased GTP hydrolysis activity compared to its intrinsic activity. Then we explored the GTPase activity of the mutated CgtAvc (Gly98Asp) located at the Obg domain, which reduced the GTP hydrolysis rate to half. The double point mutations (Gly98Asp, and Tyr194Gly) encompassing another conserved residue, Tyr194, located at the diagonally opposite position in the GTPase domain largely restored (about 82%) the reduced GTPase activity, revealing a fine-tuned inter-domain movement readily associated with the GTPase activity of CgtA and thus maintaining the proper functioning of the CgtA protein.

Spectroscopic Characterization of Staphylococcal Nuclease Mutants with Tryptophan at Internal Sites**Supported by the National Key Basic Research Program of China under Grant Nos 2013CB921904, 2013CB328701-2013CB328706, and the National Natural Science Foundation of China under Grant Nos 11074016, 11121091, 61177020, 11134001 and 10828407.

Tryptophan (Trp) is an intrinsic fluorescent probe for detecting the site-specified dynamics inside/outside protein. It is found that the Trp can easily be inserted in desired sites of protein, which affects the integrity of the overall structure. To evaluate this effect, we design thirteen double point mutants of staphylococcal nuclease, each of which has a single Trp residue planted at an internal site. The studies on Trp fluorescence, ANS-binding fluorescence, far- and near-UV CD spectra, and enzymatic activity are carried out. It is found that the mutation at the hydrophobic core of protein generates molten globular state conformation, which is a loose structure compared to their original compactness in wild type (WT). Its enzyme activity and surface hydrophobicity are also affected. The studies show that by proper site designing and external binding, Trp mutagenesis is a suitable method for carrying out the study on site specified dynamics of proteins.

Theoretical investigation on the glycan-binding specificity of Agrocybe cylindracea galectin using molecular modeling and molecular dynamics simulation studies.

Galectins are β-galactoside binding proteins which have the ability to serve as potent antitumor, cancer biomarker, and induce tumor cell apoptosis. Agrocybe cylindracea galectin (ACG) is a fungal galectin which specifically recognizes α(2,3)-linked sialyllactose at the cell surface that plays extensive roles in the biological recognition processes. To investigate the change in glycan-binding specificity upon mutations, single point and double point site-directed in silico mutations are performed at the binding pocket of ACG. Molecular dynamics (MD) simulation studies are carried out for the wild-type (ACG) and single point (ACG1) and double point (ACG2) mutated ACGs to investigate the dynamics of substituted mutants and their interactions with the receptor sialyllactose. Plausible binding modes are proposed for galectin-sialylglycan complexes based on the analysis of hydrogen bonding interactions, total pair-wise interaction energy between the interacting binding site residues and sialyllactose and binding free energy of the complexes using molecular mechanics-Poisson-Boltzmann surface area. Our result shows that high contribution to the binding in different modes is due to the direct and water-mediated hydrogen bonds. The binding specificity of double point mutant Y59R/N140Q of ACG2 is found to be high, and it has 26 direct and water-mediated hydrogen bonds with a relatively low-binding free energy of -47.52 ± 5.2 kcal/mol. We also observe that the substituted mutant Arg59 is crucial for glycan-binding and for the preference of α(2,3)-linked sialyllactose at the binding pocket of ACG2 galectin. When compared with the wild-type and single point mutant, the double point mutant exhibits enhanced affinity towards α(2,3)-linked sialyllactose, which can be effectively used as a model for biological cell marker in cancer therapeutics.

Rational and efficient site-directed mutagenesis of adenylation domain alters relative yields of luminmide derivatives in vivo.

Cloning and engineering of natural product biosynthetic pathways followed by heterologous expression in a tractable host is a widely used approach for expression and genetic modification of microbial secondary metabolites. Herein, we employed ccdB counterselection combined with oligonucleotide-mediated recombineering to efficiently create point mutations in a complex nonribosomal peptide synthetase (NRPS) from Photorabdus luminescens directing the biosynthesis of luminmides. After in depth analysis of the luminmide production profile from the native NRPS, single and double point mutations were rationally constructed within the adenylation (A) domain from NRPS module 3 which turned out to have a broad substrate tolerance. Expression of mutated versions of the 15.6 kb NRPS gene plu3263 in E. coli led to alterations in luminmide production profiles and allowed to direct the biosynthesis towards certain derivatives. These results demonstrate the suitability of counterselection recombineering for site-directed mutagenesis of complex expression constructs, e.g., genes encoding NRPS biosynthetic pathways in multi-copy plasmids.

The structure of C290A:C393A Aurora A provides structural insights into kinase regulation.

Aurora A is a Ser/Thr protein kinase that functions in cell-cycle regulation and is implicated in cancer development. During mitosis, Aurora A is activated by autophosphorylation on its activation loop at Thr288. The Aurora A catalytic domain (amino acids 122-403) expressed in Escherichia coli autophosphorylates on two activation-loop threonine residues (Thr288 and Thr287), whereas a C290A,C393A double point mutant of the Aurora A catalytic domain autophosphorylates only on Thr288. The structure of the complex of this mutant with ADP and magnesium was determined to 2.1 Å resolution using molecular replacement. This is an improvement on the existing 2.75 Å resolution structure of the equivalent wild-type complex. The structure confirms that single phosphorylation of the activation loop on Thr288 is insufficient to stabilize a `fully active' conformation of the activation loop in the absence of binding to TPX2.

Insights into the binding specificity of wild type and mutated wheat germ agglutinin towards Neu5Acα(2‐3)Gal: a study by in silico mutations and molecular dynamics simulations

Wheat germ agglutinin (WGA) is a plant lectin, which specifically recognizes the sugars NeuNAc and GlcNAc. Mutated WGA with enhanced binding specificity can be used as biomarkers for cancer. In silico mutations are performed at the active site of WGA to enhance the binding specificity towards sialylglycans, and molecular dynamics simulations of 20 ns are carried out for wild type and mutated WGAs (WGA1, WGA2, and WGA3) in complex with sialylgalactose to examine the change in binding specificity. MD simulations reveal the change in binding specificity of wild type and mutated WGAs towards sialylgalactose and bound conformational flexibility of sialylgalactose. The mutated polar amino acid residues Asn114 (S114N), Lys118 (G118K), and Arg118 (G118R) make direct and water mediated hydrogen bonds and hydrophobic interactions with sialylgalactose. An analysis of possible hydrogen bonds, hydrophobic interactions, total pair wise interaction energy between active site residues and sialylgalactose and MM-PBSA free energy calculation reveals the plausible binding modes and the role of water in stabilizing different binding modes. An interesting observation is that the binding specificity of mutated WGAs (cyborg lectin) towards sialylgalactose is found to be higher in double point mutation (WGA3). One of the substituted residues Arg118 plays a crucial role in sugar binding. Based on the interactions and energy calculations, it is concluded that the order of binding specificity of WGAs towards sialylgalactose is WGA3 > WGA1 > WGA2 > WGA. On comparing with the wild type, double point mutated WGA (WGA3) exhibits increased specificity towards sialylgalactose, and thus, it can be effectively used in targeted drug delivery and as biological cell marker in cancer therapeutics.

A new restriction endonuclease-based method for highly-specific detection of DNA targets from methicillin-resistant Staphylococcus aureus.

PCR multiplexing has proven to be challenging, and thus has provided limited means for pathogen genotyping. We developed a new approach for analysis of PCR amplicons based on restriction endonuclease digestion. The first stage of the restriction enzyme assay is hybridization of a target DNA to immobilized complementary oligonucleotide probes that carry a molecular marker, horseradish peroxidase (HRP). At the second stage, a target-specific restriction enzyme is added, cleaving the target-probe duplex at the corresponding restriction site and releasing the HRP marker into solution, where it is quantified colorimetrically. The assay was tested for detection of the methicillin-resistant Staphylococcus aureus (MRSA) pathogen, using the mecA gene as a target. Calibration curves indicated that the limit of detection for both target oligonucleotide and PCR amplicon was approximately 1 nM. Sequences of target oligonucleotides were altered to demonstrate that (i) any mutation of the restriction site reduced the signal to zero; (ii) double and triple point mutations of sequences flanking the restriction site reduced restriction to 50–80% of the positive control; and (iii) a minimum of a 16-bp target-probe dsDNA hybrid was required for significant cleavage. Further experiments showed that the assay could detect the mecA amplicon from an unpurified PCR mixture with detection limits similar to those with standard fluorescence-based qPCR. Furthermore, addition of a large excess of heterologous genomic DNA did not affect amplicon detection. Specificity of the assay is very high because it involves two biorecognition steps. The proposed assay is low-cost and can be completed in less than 1 hour. Thus, we have demonstrated an efficient new approach for pathogen detection and amplicon genotyping in conjunction with various end-point and qPCR applications. The restriction enzyme assay may also be used for parallel analysis of multiple different amplicons from the same unpurified mixture in broad-range PCR applications.

Oligomerization of the Nrdp1 E3 Ubiquitin Ligase Is Necessary for Efficient Autoubiquitination but Not ErbB3 Ubiquitination

Overexpression of the ErbB3 receptor tyrosine kinase protein in breast and other cancers contributes to tumor malignancy and therapeutic resistance. The RBCC/TRIM family RING finger E3 ubiquitin ligase Nrdp1 mediates the ubiquitination of ErbB3 in normal mammary epithelial cells to facilitate receptor degradation and suppress steady-state receptor levels. Post-transcriptional loss of Nrdp1 in patient breast tumors allows ErbB3 overexpression and receptor contribution to tumor progression, and elevated lability through autoubiquitination contributes to the observed loss of Nrdp1 in tumors relative to normal tissue. To begin to understand the mechanisms underlying Nrdp1 protein self-regulation through lability, we investigated the structural determinants required for efficient autoubiquitination and ErbB3 ubiquitination. Using mutagenesis, chemical cross-linking, size exclusion chromatography, and native polyacrylamide gel electrophoresis, we demonstrate that Nrdp1 self-associates into a stable oligomeric complex in cells. Deletion of its coiled-coil domain abrogates oligomerization but does not affect Nrdp1-mediated ErbB3 ubiquitination or degradation. On the other hand, the presence of the coiled-coil domain is necessary for efficient Nrdp1 autoubiquitination via a trans mechanism, indicating that Nrdp1 ubiquitination of its various targets is functionally separable. Finally, a GFP fusion of the coiled-coil domain stabilizes Nrdp1 and potentiates ErbB3 ubiquitination and degradation. These observations point to a model whereby the coiled-coil domain plays a key role in regulating Nrdp1 lability by promoting its assembly into an oligomeric complex, and raise the possibility that inhibition of ligase oligomerization via its coiled-coil domain could be of therapeutic benefit to breast cancer patients by restoring Nrdp1 protein.

Characterization of an enhanced antigenic change in the pandemic 2009 H1N1 influenza virus haemagglutinin

Murine hybridomas producing neutralizing mAbs specific to the pandemic influenza virus A/California/07/2009 haemagglutinin (HA) were isolated. These antibodies recognized at least two different but overlapping new epitopes that were conserved in the HA of most Spanish pandemic isolates. However, one of these isolates (A/Extremadura/RR6530/2010) lacked reactivity with the mAbs and carried two unique mutations in the HA head (S88Y and K136N) that were required simultaneously to eliminate reactivity with the murine antibodies. This unusual requirement directly illustrates the phenomenon of enhanced antigenic change proposed previously for the accumulation of simultaneous amino acid substitutions at antigenic sites of the influenza A virus HA during virus evolution (Shih et al., Proc Natl Acad Sci USA, 104 , 6283-6288, 2007). The changes found in the A/Extremadura/RR6530/2010 HA were not found in escape mutants selected in vitro with one of the mAbs, which contained instead nearby single amino acid changes in the HA head. Thus, either single or double point mutations may similarly alter epitopes of the new antigenic site identified in this work in the 2009 H1N1 pandemic virus HA. Moreover, this site is relevant for the human antibody response, as shown by competition of mAbs and human post-infection sera for virus binding. The results are discussed in the context of the HA antigenic structure and challenges posed for identification of sequence changes with possible antigenic impact during virus surveillance.

A novel mechanism of regulating breast cancer cell migration via palmitoylation-dependent alterations in the lipid raft affiliation of CD44

IntroductionMost breast cancer-related deaths result from metastasis, a process involving dynamic regulation of tumour cell adhesion and migration. The adhesion protein CD44, a key regulator of cell migration, is enriched in cholesterol-enriched membrane microdomains termed lipid rafts. We recently reported that raft affiliation of CD44 negatively regulates interactions with its migratory binding partner ezrin. Since raft affiliation is regulated by post-translational modifications including palmitoylation, we sought to establish the contribution of CD44 palmitoylation and lipid raft affiliation to cell migration.MethodsRecovery of CD44 and its binding partners from raft versus non-raft membrane microdomains was profiled in non-migrating and migrating breast cancer cell lines. Site-directed mutagenesis was used to introduce single or double point mutations into both CD44 palmitoylation sites (Cys286 and Cys295), whereupon the implications for lipid raft recovery, phenotype, ezrin co-precipitation and migratory behaviour was assessed. Finally CD44 palmitoylation status and lipid raft affiliation was assessed in primary cultures from a small panel of breast cancer patients.ResultsCD44 raft affiliation was increased during migration of non-invasive breast cell lines, but decreased during migration of highly-invasive breast cells. The latter was paralleled by increased CD44 recovery in non-raft fractions, and exclusive non-raft recovery of its binding partners. Point mutation of CD44 palmitoylation sites reduced CD44 raft affiliation in invasive MDA-MB-231 cells, increased CD44-ezrin co-precipitation and accordingly enhanced cell migration. Expression of palmitoylation-impaired (raft-excluded) CD44 mutants in non-invasive MCF-10a cells was sufficient to reversibly induce the phenotypic appearance of epithelial-to-mesenchymal transition and to increase cell motility. Interestingly, cell migration was associated with temporal reductions in CD44 palmitoylation in wild-type breast cells. Finally, the relevance of these findings is underscored by the fact that levels of palmitoylated CD44 were lower in primary cultures from invasive ductal carcinomas relative to non-tumour tissue, while CD44 co-localisation with a lipid raft marker was less in invasive ductal carcinoma relative to ductal carcinoma in situ cultures.ConclusionOur results support a novel mechanism whereby CD44 palmitoylation and consequent lipid raft affiliation inversely regulate breast cancer cell migration, and may act as a new therapeutic target in breast cancer metastasis.

Role of Intrinsic Helicity Within N-Terminal Flanking Sequences on Huntingtin Aggregation Mechanisms

Huntington's disease (HD) is associated with CAG repeats within exon 1 that lead to polyglutamine (polyQ) expansions in the protein huntingtin (htt). Aberrant splicing of htt transcripts generates N-terminal fragments in a polyQ length dependent manner that span exon 1 of htt. These N-terminal fragments can be trafficked into the nucleus and form neuronal nuclear inclusions. Within exon 1 of htt the polyQ region is flanked by a N-terminal 17-residue amphipathic stretch (N17) and a C-terminal proline-rich stretch. Modulation of these flanking sequences has profound effects on HD progression. Specifically, post-translational modifications and single / double point mutations within N17 can modulate the intrinsic helicity of N17, as well as aggregation in vitro and HD phenotypes in animal models. However, the role of helicity within N17 on the modulation of polyQ aggregation remains unresolved. Here, we focus on understanding the synergy between the intrinsic helicity within N17 and polyQ length on htt aggregation mechanisms. Through the use of atomistic Monte Carlo simulations utilizing the ABSINTH implicit solvation model, we examine the monomeric properties of N17 permutants with varying degrees of intrinsic helicity as a function of polyQ length. Results suggest that the degree of coupling between N17 constructs and polyQ is dependent on the intrinsic helicity within N17 constructs. A comparative analysis of N17 constructs in cis with a range of polyQ lengths will be combined with biophysical experiments to understand how monomeric properties of N17 modulate the solubility and mechanism of polyQ aggregation.