Деякі проблеми використання тимчасово зайнятих земель

<div><p>Glucocorticoid induced-leucine zipper (GILZ) has been shown to be induced in cells by different stimuli such as glucocorticoids, IL-10 or deprivation of IL-2. GILZ has anti-inflammatory properties and may be involved in signalling modulating apoptosis. Herein we demonstrate that wildtype <em>Yersinia enterocolitica</em> which carry the pYV plasmid upregulated GILZ mRNA levels and protein expression in epithelial cells. Infection of HeLa cells with different <em>Yersinia</em> mutant strains revealed that the protease activity of YopT, which cleaves the membrane-bound form of Rho GTPases was sufficient to induce GILZ expression. Similarly, <em>Clostridium difficile</em> toxin B, another bacterial inhibitor of Rho GTPases induced GILZ expression. YopT and toxin B both increased transcriptional activity of the GILZ promoter in HeLa cells. GILZ expression could not be linked to the inactivation of an individual Rho GTPase by these toxins. However, forced expression of RhoA and RhoB decreased basal <em>GILZ</em> promoter activity. Furthermore, MAPK activation proved necessary for profound GILZ induction by toxin B. Promoter studies and gel shift analyses defined binding of upstream stimulatory factor (USF) 1 and 2 to a canonical c-Myc binding site (E-box) in the <em>GILZ</em> promoter as a crucial step of its trans-activation. In addition we could show that USF-1 and USF-2 are essential for basal as well as toxin B induced GILZ expression. These findings define a novel way of <em>GILZ</em> promoter trans-activation mediated by bacterial toxins and differentiate it from those mediated by dexamethasone or deprivation of IL-2.</p> </div

Plk1 phosphorylates YY1 at threonine 39 <i>in vivo</i>.

<p>(<b>A</b>) Western blot analysis of cold <i>in vitro</i> kinase assay reactions using HeLa whole cell extracts (WCE) as the source for kinase activity and bacterially expressed GST-YY1 bound to glutathione beads, as substrate. WCEs were prepared from HeLa cells, asynchronously growing or double-thymidine blocked and released for eight hours (T/T 8 h). Plk1 inhibitor, Cyclapolin 9, was added to the kinase reactions of T/T 8 h extracts at the indicated concentrations. Reactions were separated on SDS-PAGE, transferred to nitrocellulose membrane, and probed with anti-pT39, then anti-YY1 antibody. WCEs were also analyzed on a separate Western blot (right panel), using anti-Plk1 and anti-Cyclin B1 antibodies. Anti-GAPDH was used as a loading control. (<b>B</b>) Flag-YY1 was immunoprecipitated from HeLa-Flag-YY1 cells, synchronized by double-thymidine block and released for eight hours. Cyclapolin 9 (or DMSO, for the negative control for the inhibitor) was added to the cells four hours prior to cell collection. Flag-YY1 was also immunoprecipitated from cell extracts collected two hours after release as a negative control. The resulting Western blot was probed with anti-pT39 and anti-YY1 antibodies. (<b>C</b>) Co-immunoprecipitation of Plk1 with YY1 from WCEs prepared from HeLa cells released for eight hours after double thymidine block. YY1 was immunoprecipitated using an antibody specific for the last 20 amino acids of the YY1 (C-20). IgG was used as a control for the specificity of the immunoprecipitation. The Western blot was probed with anti-Plk1 and then anti-YY1 antibodies.</p

YY1 phosphorylation at T39 is rapidly dephosphorylated upon entry into mitosis.

<p>Stable HeLa-Flag-YY1 cells were synchronized by double-thymidine block and then released. Cells were collected for WCE preparation at the indicated time points. (<b>A</b>) Western blot analysis of HeLa WCEs and immunoprecipitated Flag-YY1 at the indicated time points. WCEs were probed with anti-Plk1, anti-Cyclin B1, and anti-YY1 antibodies. Immunoprecipitated Flag-YY1 was probed with anti-pT39 and anti-YY1 antibodies. (<b>B</b>) <i>In vitro</i> kinase assay using the same WCEs tested in (A) at the indicated time points with GST-YY1 attached to beads, in the presence of phosphatase inhibitors. Reactions were separated on SDS-PAGE, transferred to nitrocellulose membrane, and probed with anti-pT39, then anti-YY1 antibody.</p

Plk1 phosphorylates YY1 at threonine 39 <i>in vitro</i>.

<p>(<b>A</b>) Diagram displaying the different domains of the YY1 protein. Amino acid residues 2–62 are shown; serine and threonine residues in amino acids 2–62 are indicated by arrows. The predicted phosphorylation site at threonine 39 is indicated with a star. (<b>B</b>) Coomassie blue staining and phosphorimager exposure of the SDS-PAGE gel analysis of the radioactive <i>in vitro</i> kinase assay reactions. Kinase reactions include Plk1 only (no substrate lane), Plk1 with GST-YY1 wild type (WT) or mutant (T39A). (<b>C</b>) Amino acid sequence alignment of the N-terminal domain of the YY1 protein from different species, as indicated.</p

Plk1 phosphorylates YY1 in the N-terminal activation domain <i>in vitro</i>.

<p>(<b>A</b>) Coomassie blue staining (left) and phosphorimager exposure (right) of SDS-PAGE gel analysis of the radioactive <i>in vitro</i> kinase assay reactions using purified Plk1 and a panel of GST-tagged YY1 deletion mutants. The specific YY1 deletions are indicated above the lanes. Equal amounts of purified Plk1 were added to all reactions. (<b>B</b>) Diagram of the deletion mutants of YY1 used in the kinase assay in (A). Evidence of phosphorylation shown in (A) is indicated by the (+) sign, whereas the absence of evidence of phosphorylation is indicated by a (--) sign. The region identified as the site for phosphorylation by Plk1 is indicated (amino acid residues 2–62).</p

Threonine 39 phosphorylation on YY1 peaks at G2/M transition.

<p>Stable HeLa-Flag-YY1 cells were synchronized by double-thymidine block and then released. (<b>A</b>) Analysis of the cell-cycle progression of HeLa cells released after double-thymidine block using fluorescence-activated cell sorting. Cells were stained with propidium iodide and analyzed based on their DNA content. An asynchronous population of cells was used as a control. (<b>B</b>) Whole cell extracts were prepared from HeLa-Flag-YY1 cells collected at the indicated times after release from double-thymidine block. Total WCE were analyzed on a Western blot after SDS-PAGE separation, and probed with anti-Plk1, anti-Cyclin B1, and anti-YY1 antibodies. Flag-YY1 was immunoprecipitated from the extracts of each time point, and then analyzed on a Western blot using anti-pT39 and anti-YY1 antibodies.</p

Anti-p-T39 antibody specifically recognizes YY1 phosphorylation at threonine 39.

<p>(<b>A</b>) Western blot analysis of the cold <i>in vitro</i> kinase assay reactions using purified Plk1 and purified YY1, after SDS-PAGE. The blot was probed with anti-YY1, anti-Plk1 and anti-pT39 antibodies. (<b>B</b>) Western blot analysis of the cold <i>in vitro</i> kinase assay reactions after SDS-PAGE, using purified Plk1 and purified GST-YY1 wild type (WT) or mutant (T39A). The blot was probed with anti-YY1, anti-Plk1 and anti-pT39 antibodies.</p

Plk1 phosphorylates YY1 <i>in vitro</i>.

<p>(<b>A</b>) Coomassie blue staining of SDS-PAGE gel showing the purification of bacterially expressed non-tagged YY1 under denaturing conditions. An arrow indicates the position of overexpressed YY1. Lysates: sample from total bacterial lysates; Depleted lysates: sample from bacterial lysates after passing through the Ni-NTA column. E1, 2, and 3 are samples from elutions at pH 5.9; E4, 5, and 6 are samples from elutions at pH 4.5; the last lane shows a sample of the Ni-NTA beads after the last elution step. (<b>B</b>) Coomassie blue staining (left panel) and Western blot analysis (right panel) of purified YY1 after renaturation, and separation by SDS-PAGE. In the Western blot, YY1 was probed with anti-YY1 (H-10) antibody. (<b>C</b>) EMSA testing the DNA binding activity of purified and renatured YY1 side by side with HeLa whole cell extract. A 22 bp oligonucleotide encompassing the YY1 binding site in histone H3.2 coding region was used as the radioactively labeled probe. The Western blot shows equal amounts of purified YY1 and YY1 protein in HeLa WCEs. (<b>D</b>) Coomassie blue staining and phosphorimager exposure of SDS-PAGE gel after radioactive <i>in vitro</i> kinase assay, using purified Plk1 and YY1. The gel shows three reactions: Plk1 only, YY1 only, and Plk1 plus YY1; as indicated.</p

The NAD⁺ binding site in full-length SIRT2.

<p><b>(A)</b> Overview of the SIRT2/NAD⁺ complex. The NAD⁺ (yellow) and catalytic residue H150 (green) are represented by sticks. The CT is highlighted in pink. The sites A, B and C of SIRT2, which are involved in NAD⁺ binding, are indicated by blue circles. <b>(B)</b> Close-up view of the binding site of nicotinamide and nicotinamide ribose. <b>(C)</b> Close-up view of the binding site of adenine. Hydrogen bonds are highlighted by dashed lines. <b>(D)</b> Superposition of two crystal structures of Sir2-Af2/NAD⁺ (PDB ID: 1S7G [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139095#pone.0139095.ref016" target="_blank">16</a>]) with the SIRT2/NAD⁺ complex (in green). The chain A (NAD⁺ in non-productive conformation) and chain B (NAD⁺ in productive conformation) of 1S7G are colored in blue and salmon, respectively. The NAD⁺ cofactors are represented by sticks.</p

Structural models of the lowest interaction energy docking structure of SIRT2 in complex with cCDK2 in the largest cluster.

<p>The structures of Models 1–6 were initially generated by homology modeling, while Model 7 was generated by <i>ab initio</i> modeling (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139095#sec002" target="_blank">Materials and Methods</a> for details). The cartoon representations of CDK2, cyclin A, and SIRT2 are colored in blue, orange, and green, respectively. The CT of SIRT2 is colored in pink.</p