No abstract

The cascade of phosphorylation-induced conformational changes in FixJN illustrates the role of conserved residues in stabilizing the phosphoryl group in the active site, triggering the structural transition and achieving the post-phosphorylation signaling events. We propose that these phosphorylation-induced conformational changes underly the activation of response regulators in general.

The HLA-G molecule plays an important role in immune tolerance, protecting the fetus from maternal immune attack, and probably contributes to graft tolerance and tumor escape from the host immune system. HLA-G expression is tightly regulated and involves mechanisms acting in part at the transcriptional level. Nevertheless, almost all regulatory sequences that govern constitutive and inducible HLA class I gene transcription are disrupted in the HLA-G gene promoter, suggesting an unusual regulatory process. In further investigating the molecular mechanisms of HLA-G gene activation, we evaluated the influence of epigenetic mechanisms on seven HLA-G-negative cell lines that exhibit various phenotypes. Exposure of cells to histone deacetylase inhibitors, or to the demethylating agent 5-aza-2 -deoxycytidine, revealed that HLA-G gene transcription is inhibited by DNA methylation. Reversal of methylation-mediated repression may directly induce HLA-G cellsurface expression, supporting the idea that HLA-G might be activated by such a mechanism during malignancy, inflammation, and allogenic reactions.

SummaryThe`two-component' transcriptional activator FixJ controls nitrogen ®xation in Sinorhizobium meliloti. Phosphorylation of FixJ induces its dimerization, as evidenced by gel permeation chromatography and equilibrium sedimentation analysis. Phosphorylation-induced dimerization is an intrinsic property of the isolated receiver domain FixJN. Accordingly, chemical phosphorylation of both FixJ and FixJN are second-order reactions with respect to protein concentration. However, the second-order phosphorylation constant is 44-fold higher for FixJN than for FixJ. Therefore, the C-terminal transcriptional activator domain FixJC inhibits the chemical phosphorylation of the receiver domain FixJN. Conversely, FixJN has been shown previously to inhibit FixJC activity < 40-fold, re¯ecting the interaction between FixJN and FixJC. Therefore, we propose that modulation of FixJ activity involves both its dimerization and the disruption of the interface between FixJN and FixJC, resulting in the opening of the protein structure. Alanine scanning mutagenesis of FixJN indicated that the FixJ,P dimerization interface involves Val-91 and Lys-95 in helix a4. Dimerization was required for high-af®nity binding to ®xK promoter DNA.

The tissue distribution of HLA-G molecules is broader than originally reported in trophoblastic cells. On the basis of numerous studies, HLA-G is also expressed in malignant tumors and involved in tumor immune escape. The mechanisms of HLA-G gene regulation differ from those of classical HLA class I genes and involve epigenetic processes. Here, we provide additional evidence on the influence of DNA demethylation on HLA-G activation. We also analyze the 5 regulatory region of HLA-G in 2 cellular models, melanoma (FON, M8) and choriocarcinoma (JEG-3, JAR), either expressing HLA-G transcripts or not. The data strongly suggest that HLA-G is silenced as a result of CpG site hypermethylation within a 5 regulatory region encompassing 450 bp upstream of the start codon, whereas it is activated upon demethylation. This result correlates with the acetylation status of histones within this region and the putative locus control region located at -1.2 kb. cis-acting epigenetic changes and the fact that demethylating agents activate HLA-G expression at least 5 days following treatment should be taken into account in epigenetic cancer therapies.

Using high-throughput tethered particle motion single-molecule experiments, the double-stranded DNA persistence length, L p , is measured in solutions with Na + and Mg 2+ ions of various ionic strengths, I. Several theoretical equations for L p (I) are fitted to the experimental data, but no decisive theory is found which fits all the L p values for the two ion valencies. Properly extracted from the particle trajectory using simulations, L p varies from 30 to 55 nm, and is comparable to previous experimental results. For the Na + -only case, L p is an increasing concave function of I −1 , well fitted by Manning's electrostatic stretching approach, but not by classical Odjik−Skolnick−Fixman theories with or without counterion condensation. With added Mg 2+ ions, L p shows a marked decrease at low I, interpreted as an ion−ion correlation effect, with an almost linear law in I −1 , fitted by a proposed variational approach.

The Tethered Particle Motion (TPM) technique informs about conformational changes of DNA molecules, e.g. upon looping or interaction with proteins, by tracking the Brownian motion of a particle probe tethered to a surface by a single DNA molecule and detecting changes of its amplitude of movement. We discuss in this context the time resolution of TPM, which strongly depends on the particle-DNA complex relaxation time, i.e. the characteristic time it takes to explore its configuration space by diffusion. By comparing theory, simulations and experiments, we propose a calibration of TPM at the dynamical level: we analyze how the relaxation time grows with both DNA contour length (from 401 to 2080 base pairs) and particle radius (from 20 to 150 nm). Notably we demonstrate that, for a particle of radius 20 nm or less, the hydrodynamic friction induced by the particle and the surface does not significantly slow down the DNA. This enables us to determine the optimal time resolution of TPM in distinct experimental contexts which can be as short as 20 ms. arXiv:1003.0518v2 [cond-mat.soft]

Using high-throughput Tethered Particle Motion single molecule experiments, the double-stranded DNA persistence length, Lp, is measured in solutions with Na + and Mg 2+ ions of various ionic strengths, I. Several theoretical equations for Lp(I) are fitted to the experimental data, but no decisive theory is found which fits all the Lp values for the two ion valencies. Properly extracted from the particle trajectory using simulations, Lp varies from 30 nm to 55 nm, and is compared to previous experimental results. For the Na + only case, Lp is an increasing concave function of I −1 , well fitted by Manning's electrostatic stretching approach, but not by classical Odjik-Skolnick-Fixman theories with or without counter-ion condensation. With added Mg 2+ ions, Lp shows a marked decrease at low I, interpreted as an ion-ion correlation effect, with an almost linear law in I −1 , fitted by a proposed variational approach.