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Direct analysis of Holliday junction resolving enzyme in a DNA origami nanostructure

Nucleic Acids Res. 2014; 42 (11): 7421-7428.

Suzuki Y, Endo M, Cañas C, Ayora S, Alonso JC, Sugiyama H, Takeyasu K.

Nucleic Acids Res. 2014; 42 (11): 7421-7428Holliday junction (HJ) resolution is a fundamental step for completion of homologous recombination. HJ resolving enzymes (resolvases) distort the junction structure upon binding and prior cleavage, raising the possibility that the reactivity of the enzyme can be affected by a particular geometry and topology at the junction.

Here, we employed a DNA origami nano-scaffold in which each arm of a HJ was tethered through the base-pair hybridization, allowing us to make the junction core either flexible or inflexible by adjusting the length of the DNA arms. Both flexible and inflexible junctions bound to Bacillus subtilis RecU HJ resolvase, while only the flexible junction was efficiently resolved into two duplexes by this enzyme.

This result indicates the importance of the structural malleability of the junction core for the reaction to proceed. Moreover, cleavage preferences of RecU-mediated reaction were addressed by analyzing morphology of the reaction products.

Direct measurement of the dielectric polarization properties of DNA

Proc Natl Acad Sci USA. 2014; pii: 201405702.

Cuervo A, Dans PD, Carrascosa JL, Orozco M, Gomila G, Fumagalli L.

Proc Natl Acad Sci USA. 2014; pii: 201405702The electric polarizability of DNA, represented by the dielectric constant, is a key intrinsic property that modulates DNA interaction with effector proteins. Surprisingly, it has so far remained unknown owing to the lack of experimental tools able to access it.

Here, we experimentally resolved it by detecting the ultraweak polarization forces of DNA inside single T7 bacteriophages particles using electrostatic force microscopy. In contrast to the common assumption of low-polarizable behavior like proteins (εr ∼ 2–4), we found that the DNA dielectric constant is ∼8, considerably higher than the value of ∼3 found for capsid proteins. State-of-the-art molecular dynamic simulations confirm the experimental findings, which result in sensibly decreased DNA interaction free energy than normally predicted by Poisson–Boltzmann methods.

Our findings reveal a property at the basis of DNA structure and functions that is needed for realistic theoretical descriptions, and illustrate the synergetic power of scanning probe microscopy and theoretical computation techniques.

Kinetic and phenotypic analysis of CD8+ T cell responses after priming with alphavirus replicons and homologous or heterologous booster immunizations

J Virol. 2014; pii: JVI.02223-14.

Knudsen ML, Ljungberg K, Kakoulidou M, Kostic L, Hallengärd D, García-Arriaza J, Merits A, Esteban M, Liljeström P.

J Virol. 2014; pii: JVI.02223-14Alphavirus replicons are potent inducers of CD8+ T cell responses and thus constitute an attractive vaccine vector platform for developing novel vaccines. However, the kinetics and memory phenotype of CD8+ T cell responses induced by alphavirus replicons are not well characterized. Furthermore, little is known how priming with alphavirus replicons affects booster immune responses induced by other vaccine modalities. We demonstrate that a single immunization with an alphavirus replicon, administered as viral particles or naked DNA, induced an antigen-specific CD8+ T cell response that had a sharp peak, followed by a rapid contraction. Administering a homologous boost before contraction had occurred did not further increase the response. In contrast, boosting after contraction when CD8+ T cells had obtained a memory phenotype (based on CD127/CD62L expression), resulted in maintenance of CD8+ T cells with a high recall capacity (based on CD27/CD43 expression). Increasing the dose of replicon particles promoted T effector memory (Tem) and inhibited T central memory (Tcm) development. Moreover, infection with a replicating alphavirus induced a similar distribution of CD8+ T cells as the replicon vector. Lastly, the distribution of T cell subpopulations induced by a DNA-launched alphavirus replicon could be altered by heterologous boosts. For instance, boosting with a poxvirus vector (MVA) favored expansion of the Tem compartment. In summary, we have characterized the antigen-specific CD8+ T cell response induced by alphavirus replicon vectors and demonstrated how it can be altered by homologous and heterologous boost immunizations.

Importance Alphavirus replicons are promising vaccine candidates against a number of diseases and are by themselves developed as vaccines against for example chikungunya virus infection. Replicons are also considered to be used for priming followed by booster immunization using different vaccine modalities. In order to rationally design prime-boost immunization schedules with these vectors, characterization of the magnitude and phenotype of CD8+ T cell responses induced by alphavirus replicons is needed. Here, we demonstrate how factors such as timing and dose affect the phenotype of the memory T cell populations induced by immunization with alphavirus replicons. These findings are important for designing future clinical trials with alphaviruses, as they can be used to tailor vaccination regimens in order to induce a CD8+ T cell response that is optimal for control and/or clearance of a specific pathogen.

Crystal structure of the lytic CHAPK domain of the endolysin LysK from Staphylococcus aureus bacteriophage K

Virol J. 2014; 11 (1) :133.

Sanz-Gaitero M, Keary R, Garcia-Doval C, Coffey A, van Raaij MJ.

Virol J. 2014; 11 (1) :133Background: Bacteriophages encode endolysins to lyse their host cell and allow escape of their progeny. Endolysins are also active against Gram-positive bacteria when applied from the outside and are thus attractive anti-bacterial agents. LysK, an endolysin from staphylococcal phage K, contains an N-terminal cysteine-histidine dependent amido-hydrolase/peptidase domain (CHAPK), a central amidase domain and a C-terminal SH3b cell wall-binding domain. CHAPK cleaves bacterial peptidoglycan between the tetra-peptide stem and the penta-glycine bridge.

Methods: The CHAPK domain of LysK was crystallized and high-resolution diffraction data was collected both from a native protein crystal and a methylmercury chloride derivatized crystal. The anomalous signal contained in the derivative data allowed the location of heavy atom sites and phase determination. The resulting structures were completed, refined and analyzed. The presence of calcium and zinc ions in the structure was confirmed by X-ray fluorescence emission spectroscopy. Zymogram analysis was performed on the enzyme and selected site-directed mutants.

Results: The structure of CHAPK revealed a papain-like topology with a hydrophobic cleft, where the catalytic triad is located. Ordered buffer molecules present in this groove may mimic the peptidoglycan substrate. When compared to previously solved CHAP domains, CHAPK contains an additional lobe in its N-terminal domain, with a structural calcium ion, coordinated by residues Asp45, Asp47, Tyr49, His51 and Asp56. The presence of a zinc ion in the active site was also apparent, coordinated by the catalytic residue Cys54 and a possible substrate analogue. Site-directed mutagenesis was used to demonstrate that residues involved in calcium binding and of the proposed active site were important for enzyme activity.

Conclusions: The high-resolution structure of the CHAPK domain of LysK was determined, suggesting the location of the active site, the substrate-binding groove and revealing the presence of a structurally important calcium ion. A zinc ion was found more loosely bound. Based on the structure, we propose a possible reaction mechanism. Future studies will be aimed at co-crystallizing CHAPK with substrate analogues and elucidating its role in the complete LysK protein. This, in turn, may lead to the design of site-directed mutants with altered activity or substrate specificity.

Anticancer activities of pterostilbene-isothiocyanate conjugate in breast cancer cells: involvement of PPARγ

PLoS One. 2014; 9 (8): e104592.

Nikhil K, Sharan S, Singh AK, Chakraborty A, Roy P.

PLoS One. 2014; 9 (8): e104592Trans-3,5-dimethoxy-4′-hydroxystilbene (PTER), a natural dimethylated analog of resveratrol, preferentially induces certain cancer cells to undergo apoptosis and could thus have a role in cancer chemoprevention. Peroxisome proliferator-activated receptor γ (PPARγ), a member of the nuclear receptor superfamily, is a ligand-dependent transcription factor whose activation results in growth arrest and/or apoptosis in a variety of cancer cells.

Here we investigated the potential of PTER-isothiocyanate (ITC) conjugate, a novel class of hybrid compound (PTER-ITC) synthesized by appending an ITC moiety to the PTER backbone, to induce apoptotic cell death in hormone-dependent (MCF-7) and -independent (MDA-MB-231) breast cancer cell lines and to elucidate PPARγ involvement in PTER-ITC action. Our results showed that when pre-treated with PPARγ antagonists or PPARγ siRNA, both breast cancer cell lines suppressed PTER-ITC-induced apoptosis, as determined by annexin V/propidium iodide staining and cleaved caspase-9 expression. Furthermore, PTER-ITC significantly increased PPARγ mRNA and protein levels in a dose-dependent manner and modulated expression of PPARγ-related genes in both breast cancer cell lines. This increase in PPARγ activity was prevented by a PPARγ-specific inhibitor, in support of our hypothesis that PTER-ITC can act as a PPARγ activator. PTER-ITC-mediated upregulation of PPARγ was counteracted by co-incubation with p38 MAPK or JNK inhibitors, suggesting involvement of these pathways in PTER-ITC action. Molecular docking analysis further suggested that PTER-ITC interacted with 5 polar and 8 non-polar residues within the PPARγ ligand-binding pocket, which are reported to be critical for its activity.

Collectively, our observations suggest potential applications for PTER-ITC in breast cancer prevention and treatment through modulation of the PPARγ activation pathway.