Binding thermodynamics of peptide ligands to YAP WW domains.

<p>The variability in the experimental values was estimated to be about 1% for the number of sites, 5% for the binding enthalpy and 10% for the dissociation constant. n.b.: no binding detected.</p><p>¹Data obtained using a binding model for one set of sites with n = 2.</p><p>²Data obtained using a binding model for two different sets of sites with n = 1 for each set of sites. (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113828#sec002" target="_blank">methods</a> for details).</p><p>³Number of binding sites fixed to 1 in fitting procedure.</p><p>Binding thermodynamics of peptide ligands to YAP WW domains.</p

A) Modular organization of the two isoforms of human YAP transcriptional regulator.

<p>Both forms are comprised of one TEAD transcription factor-binding domain (TB), one (YAP-WW1) or two (YAP-WW1 and YAP-WW2) WW domains, one trans-activation domain (TAD) and one PDZ binding motif. The symbol P indicates the proposed serine phosphorylation sites. <b>B) Sequence alignment of the individual WW domains of human YAP1 and YAP2 isoforms</b>. Identical residues are shown on a black background and conservative changes on a gray background. Black arrows indicate those residues constituting the xP and xY pockets at the binding site. Red numbers indicate their position in the sequence of the full protein. <b>C) Modular organization of the human LATS1 and LATS2 kinases</b> containing an ubiquitin-associated domain (UBA), a C-terminal protein kinase domain (KIN-D) and one or two PY sequences. <b>D) Modular architecture of human PATCHED homologue 1</b> including a Sterol-sensing domain (SSD) and two PY sequences.</p

YAP interacts with PTCH1 Through its WW Domains.

<p><b>A)</b> Intact PPxY motifs in PTCH1 are required for binding to YAP. Two PPxY sequences in PTCH1 were mutated to PPxA. Wild type (WT) or single (PY1* or PY2*) or double (PY1*&2*) mutants of PTCH1 in Flag-tag vector were transiently co-transfected with YAP2 (in pcDNA4/HisMax) into HEK293 cells. Cell lysates were immunoprecipitated with Flag antibodies, resolved on SDS-PAGE and immunoblotted with YAP antibody or Flag antibody. <b>B)</b> Intact WW domains in YAP are required for binding to PTCH1. Two WW domains in YAP were mutated to render them inactive in terms of ligand binding. Wild type (WT) or double (1&2 WW*) mutant of YAP2 in pcDNA4/HisMax vector were co-transfected with Flag-PTCH1 or Flag vector alone. Cells were lysed and analyzed as in A.</p

Differential Scanning Calorimetry thermal denaturation profiles of YAP1-WW1 (A) and YAP1-WW2 (B) domains.

<p>Symbols correspond to experimental data for the temperature dependency of the partial molar heat capacity at different pH values (black circles for pH 7.0, green squares for pH 5.0, orange triangles for pH 4.0 and red rhombi for pH 3.0). Solid lines correspond to the global fitting to the two-states model. The heat capacities functions for the folded and unfolded states [C_p,N(T) and C_p,U(T)] resulting from the analysis are shown as short dashed lines. The dotted lines show the C_p,N(T) function calculated according to the molecular weight [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113828#pone.0113828.ref028" target="_blank">28</a>] and the C_p,U(T) function estimated from the contributions of the amino acid composition [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113828#pone.0113828.ref041" target="_blank">41</a>]. <b>C) DSC profile for the YAP1-WW1-WW2 tandem</b>. Shown are the DSC profiles for YAP-WW1 (dark blue circles), for YAP-WW2 (light blue circles) and YAP-WW1-WW2 tandem (black circles) at pH 7.0. The dashed line corresponds to the addition of the heat capacity profiles of the individual WW domains and the continuous black line represents the addition of the DSC experiments of the individual WW domains plus the contribution to the heat capacity for the linker sequence in the tandem [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113828#pone.0113828.ref041" target="_blank">41</a>]. <b>D) DSC profile for the YAP1-WW1-WW2 tandem at different pH values.</b> Symbols correspond to experimental data for the temperature dependency of the partial molar heat capacity of the tandem at different pH values (black circles for pH 7.0, orange triangles for pH 4.0 and red rhombi for pH 3.0). The continuous lines correspond to the curves resulting from the addition of the heat capacity profiles for the individual WW domains and linker sequences under each condition.</p

Peptide ligands derived from YAP functional targets.

<p>¹Numbers indicate the position of each ligand in the context of the full-length protein sequence.</p><p>Peptide ligands derived from YAP functional targets.</p

Yes-Associated Protein (YAP) Modulates Oncogenic Features and Radiation Sensitivity in Endometrial Cancer

<div><p>Background</p><p>Yes-associated protein (YAP) is a transcriptional co-activator and regulates cell proliferation and apoptosis. We investigated the clinical and biological significance of YAP in endometrial cancer (EMCA).</p><p>Methods</p><p>YAP expression in 150 primary tumor tissues from patients with EMCA was evaluated by immunohistochemistry and its association with clinicopathological data was assessed. The biological functions of YAP were determined in EMCA cell lines through knockdown/overexpression of YAP. The role of YAP in modulating radiation sensitivity was also investigated in EMCA cells.</p><p>Results</p><p>Increased nuclear YAP expression was significantly associated with higher grade, stage, lympho-vascular space invasion, postoperative recurrence/metastasis and overall survival in estrogen mediated EMCA, called type 1 cancer (p = 0.019,  = 0.028,  = 0.0008,  = 0.046 and  = 0.015, respectively). In multivariate analysis, nuclear YAP expression was confirmed as an independent prognostic factor for overall survival in type 1 EMCA. YAP knockdown by siRNA resulted in a significant decrease in cell proliferation (p<0.05), anchorage-dependent growth (p = 0.015) and migration/invasion (p<0.05), and a significant increase in the number of cells in G0/G1 phase (p = 0.002). Conversely, YAP overexpression promoted cell proliferation. Clonogenic assay demonstrated enhanced radiosensitivity by approximately 36% in YAP inhibited cells.</p><p>Conclusions</p><p>Since YAP functions as a transcriptional co-activator, its differential localization in the nucleus of cancer cells and subsequent impact on cell proliferation could have important consequences with respect to its role as an oncogene in EMCA. Nuclear YAP expression could be useful as a prognostic indicator or therapeutic target and predict radiation sensitivity in patients with EMCA.</p></div

Clonogenic assay in HEC-1-B cells after radiation exposure.

<p>Knockdown of YAP expression by siRNA reduced clonogenic survival in HEC-1-B cells, resulting in an increase in radiation sensitivity with a dose enhancement factor at 10% survival (DEF 0.1) of 1.36. Results are shown in means ±standard deviations (bars) in triplicate experiments. Similar trends were obtained in other three independent experiments.</p

The Kaplan-Meier curve for overall survival rate of patients with type 1 EMCA (n = 120) according to the nuclear expression of YAP.

<p>Increased nuclear immunoreactivity of YAP was significantly associated with worse overall survival (P = 0.015, log-rank test).</p

Correction: Chaperone-Mediated Autophagy Protein BAG3 Negatively Regulates Ebola and Marburg VP40-Mediated Egress

<p>Correction: Chaperone-Mediated Autophagy Protein BAG3 Negatively Regulates Ebola and Marburg VP40-Mediated Egress</p

Yes-Associated Protein (YAP) Modulates Oncogenic Features and Radiation Sensitivity in Endometrial Cancer - Figure 2

<p>(A) Expression of YAP and phospho-YAP (Ser127) in three EMCA cell lines by western blotting. (B) Immunofluorescent cytochemical staining of endogenous YAP using anti-YAP antibody (YAP: green, DAPI: blue).</p