Correction: Constitutive Association of Tie1 and Tie2 with Endothelial Integrins is Functionally Modulated by Angiopoietin-1 and Fibronectin

<p>Correction: Constitutive Association of Tie1 and Tie2 with Endothelial Integrins is Functionally Modulated by Angiopoietin-1 and Fibronectin</p

Proposed model of Integrin Tie2 cooperative signaling in endothelial cell regulation.

(A) Based on previous studies, it is known that Ang-1 clusters Tie2 at cell-cell junctions in endothelial cells while providing pro-survival signals through the AKT and Survivin signal transduction pathway [30,46,47]. Alternatively, Tie1 may associate with integrins, including either αVß3 or α5ß1, which are preferentially localized at the cell interface with the basement membrane containing extracellular matrix components. A link between Tie1 and the extracellular matrix could explain a role for Tie1 in vessel integrity and sheer stress that has previously been described [43–45]. (B) When Tie2 is not at junctions, it is likely capable of associating with integrins. Activation of integrin-associated Tie2 via Ang-1 and fibronectin may enrich signaling through MAPK to further facilitate endothelial cell migration and proliferation.</p

Direct association between Tie1 and Tie2 and integrins using purified proteins.

(A) Purified Tie2(1–4)-Fc protein (33nM) was incubated with purified α5ß1 ectodomain protein (12.5nM) in the presence or absence of either full-length or recombinant fibronectin (5μg/mL) containing only the 9th and 10th fibronectin type-III repeats (9–10). All purified components were present at the indicated concentrations in 1mL of HBST buffer and incubated overnight with 25μL ProteinA resin. Following precipitation of Tie2(1–4)-Fc with Protein-A sepharose, proteins were electrophoresed by SDS-PAGE, transferred to nitrocellulose, and Tie2 was briefly visualized with PonceauS prior to western blotting with α5-specific antibodies. The α5ß1 ectodomain readily precipitates with Tie2, although the addition of full-length fibronectin, but not recombinant, truncated fibronectin (9–10), greatly enhances the Tie2/integrin interaction. (B) As in (A) using Tie1(1–4)-Fc protein (33nM) incubated with purified α5ß1 ectodomain protein (12.5nM) in the presence or absence of full-length fibronectin (5μg/mL). Unlike the experiments shown in (A), fibronectin does not significantly enhance the Tie1/integrin interaction. (C) Surface Plasmon Resonance depiction of purified and untagged α5ß1 binding to immobilized Tie1 (Fc tagged full extracellular domain protein). Similar experiments were completed with immobilized Tie2, however no interaction was captured. (D) 33nM purified Tie1(1–4)-Fc protein was incubated with purified α5ß1 ectodomain (12.5nM) in the presence or absence of an increasing amount of Tie2(1–4) untagged protein (either 100nM or 600nM as indicated). Following precipitation of Tie1(1–4)-Fc with Protein-A sepharose, proteins were subjected to western blotting with α5-specific antibodies. α5ß1 readily precipitates with Tie1, demonstrating that the Ig and EGF repeats are sufficient for α5ß1 recognition. Furthermore, Tie2 (1–4) effectively competes with Tie1 for integrin binding, suggesting a shared binding interface of Tie1 and Tie2 on the integrin molecule. Quantified values represent a normalized ratio of α5 to Tie1(1–4)-Fc.</p

Direct association between the Ang-1 receptor binding domain and α_Vß₃ and α₅ß₁.

<p>(A) The Ang-1 and Ang-2 receptor binding domains were expressed and purified as Fc fusion proteins, and incubated at 50nM with purified α₅ß₁ (12.5nM) in the presence or absence of the Tie2 ectodomain (33nM). Precipitating proteins were visualized with Tie2 and ß₁ -specific antibodies (α₅ migrates at the same position as Tie2 and, therefore, α₅-antibodies could not be used). The Tie2 ectodomain readily precipitates with both the Ang-1 or Ang-2 receptor binding domains, while α₅ß₁ only precipitates with Ang-1-RBD. (B) Similar to the experiment in (A) using purified ectodomain of α_Vß₃ protein incubated with either Ang-1-RBD-Fc or Ang-2-RBD-Fc. Precipitating α_Vß₃ was visualized with antibodies specific to ß₃. (C) The Ang-1 and Ang-2 receptor binding domain Fc fusion proteins (50nM) were incubated with purified α₅ß₁ (12.5nM) in the presence or absence of human fibronectin. Following precipitation of Ang-Fc proteins with Protein-A sepharose, proteins were electrophoresed by SDS-PAGE, transferred to PVDF, and briefly visualized with PonceauS prior to western blotting with α₅-specific antibodies. The α₅ß₁ ectodomain readily co-precipitates with Ang-1-RBD, but not with Ang-2-RBD. Fibronectin does not appear to significantly influence the Ang-1/integrin interaction. (D) Direct association between Ang-1-RBD (10μM) and α₅ß₁ (5μM) by size-exclusion chromatography. Indicated fractions were incubated in loading buffer with BME for 5 minutes and resolved on a 10% SDS-PAGE gel to separate proteins eluting from the column in reducible crosslinked complexes. Top panel: Purified α₅ß₁ ectodomain was chromatographed on a Superdex 200 column and elutes as a single peak at ~12 mL. Middle panel: Purified Ang-1-RBD elutes from a Superdex 200 as a single peak at ~17 mL. Bottom panel: 5μM α₅ß₁ purified protein was cross-linked to a 2 molar excess of Ang-1-RBD using DTSSP and separated on a Superdex 200 column. Under these conditions, Ang-1-RBD now elutes in a complex with α₅ß₁ at ~11.5 mL.</p

MAPK is cooperatively regulated by the Tie2/Integrin complex in response to Ang-1 and fibronectin.

Serum starved telomerase-immortalized endothelial cells (TIMEs) were plated for 15 minutes on control treated or fibronectin treated dishes with vehicle stimulation, 500 ng/mL full length Ang-1, or 500 ng/mL full length Ang-2. Cell lysates were resolved by SDS-PAGE and analyzed for p-FAK (Y397) / FAK (A) and p-ERK1/2 (T202/Y204)/ ERK (B) after treatment. (C and D) Integrin α5 protein levels were decreased using shRNA in TIME cells. (C) Ang-1 stimulated shControl cells or sh-α5 TIME cells were plated on either PBS treated or 5 μg/mL fibronectin treated plates, lysed, and probed as above for p-FAK (Y397) / FAK (C) or p-ERK1/2 (T202/Y204) / ERK1/2 (D). (E) Levels of α5 protein were monitored in control and silenced cells via western blotting. Actin was used as a loading control. Error bars represent the standard error of 3 independent experiments. * represents student t-test values of p < 0.05.</p

Fibronectin increases Tie-integrin association by sensitized emission FRET and FLIM-FRET.

<p>(A) HEK293 cells were transiently transfected with vectors for the indicated receptor fusion proteins and visualized by fluorescence microscopy. FRET images were obtained for sensitized emission calculations on a wide-field microscope and representative images are shown. PlexinA1 does not associate with Tie2 and is, therefore, used as a negative control. Alternatively, Tie1 and Tie2 readily associate on the cell membrane and, therefore, serve as a positive control. FRET values for n>3 experiments (displayed graphically in (B)), and demonstrate a robust interaction between Tie2 and α₅ß₁. Values are depicted +/- Standard Error. * indicates student t-test values of p < 0.05, ** p<0.01, *** p<0.001 (C) Fluorescence lifetime (FLIM-FRET) measurements were evaluated to corroborate the values obtained by sensitized emission using a Becker & Hickl TCSPC system. FLIM-FRET controls not shown here are available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163732#pone.0163732.s003" target="_blank">S3 Fig</a>.</p

Tie1 and Tie2 interact with the endothelial Integrins α_Vß₃ and α₅ß₁ through their extracellular domains.

<p>(A) Co-immunoprecipitation of endogenous α_V with Tie receptor in Ea.hy926 endothelial cells. Cells were serum starved and then treated with vehicle control (PBS), 500 ng/mL rhAng-1 or 500 ng/mL rhAng-2 for 30 minutes at 37 degrees Celsius prior to harvest. α_V co-precipitated under all stimulation conditions tested when lysates were incubated overnight with anti-Tie2 antibody, but not when incubated with a non-specific IgG. (B-C) Co-immunoprecipitation of α_Vß₃ and α₅ß₁ with Tie receptors from transiently transfected HEK293 cells. (B) Full-length integrins α₅ß₁ (top panel) or α_Vß₃ (bottom panel) were immunoprecipitated with anti-flag antibody, while co-precipitating HA-tagged Tie1 receptor or myc-tagged Tie2 receptor were detected by western blot using anti-HA or anti-myc antibodies, respectively. (C) Same as in (B) alterations in the transfected vectors. Here, Tie1 and Tie2 receptor fluorophore fusion constructs were used in which the intracellular tyrosine kinase domains were replaced with the GFP analogues sYFP2 and mTQ GFP variants respectively. Co-precipitating Tie1 and Tie2 fusion proteins were detected by western blot with anti-GFP antibodies indicating receptor association within the extracellular domains of the proteins (α₅ß₁- top panel and α_Vß3—bottom panel). WCL- Whole Cell Lysate. IP- Immunoprecipitation. IB: Immunoblotting.</p

Directed evolution provides insight into conformational substrate sampling by SrtA

<div><p>The Sortase family of transpeptidases are found in numerous gram-positive bacteria and involved in divergent physiological processes including anchoring of surface proteins to the cell wall as well as pili assembly. As essential proteins, sortase enzymes have been the focus of considerable interest for the development of novel anti-microbials, however, more recently their function as unique transpeptidases has been exploited for the synthesis of novel bio-conjugates. Yet, for synthetic purposes, SrtA-mediated conjugation suffers from the enzyme’s inherently poor catalytic efficiency. Therefore, to identify SrtA variants with improved catalytic efficiency, we used directed evolution to select a catalytically enhanced SrtA enzyme. An analysis of improved SrtA variants in the context of sequence conservation, NMR and x-ray crystal structures, and kinetic data suggests a novel mechanism for catalysis involving large conformational changes that delivers substrate to the active site pocket. Indeed, using DEER-EPR spectroscopy, we reveal that upon substrate binding, SrtA undergoes a large scissors-like conformational change that simultaneously translates the sort-tag substrate to the active site in addition to repositioning key catalytic residues for esterification. A better understanding of Sortase dynamics will significantly enhance future engineering and drug discovery efforts.</p></div

Supplementary Materials and Methods from Isoflavone ME-344 Disrupts Redox Homeostasis and Mitochondrial Function by Targeting Heme Oxygenase 1

Antibodies. Primers. Click Chemistry and Affinity Enrichment Mass Spectrometry. Surface Plasmon Resonance - Biacore 3000 Kinetic Determinations. Co-Immunoprecipitation. Transfections with shRNA and plasmids. Plate colony formation assay. References.</p

Supplementary Tables S11-S14 from Isoflavone ME-344 Disrupts Redox Homeostasis and Mitochondrial Function by Targeting Heme Oxygenase 1

Supplementary Table S11. All Proteins Identified. Supplementary Table S12. The Protein Groups text file from MaxQuant was processed using Perseus. Common contaminants, reversed database hits, and proteins identified by modified peptides were removed. LFQ normalized protein intensities were log2 transformed. Proteins were filtered to retain proteins identified by MS/MS (not matching between runs) in all three ME-344 pull down experiments with at least 2 peptides. Missing values in the control pull downs were imputed in Perseus with a width of 0.6 and downshift of 1.8. The difference in mean log2 protein intensities (ME344 bait - control) and fold change in abundance are provided. Supplementary Table S13. The Protein Groups text file from MaxQuant was processed using Perseus. Common contaminants, reversed database hits, and proteins identified by modified peptides were removed. LFQ normalized protein intensities were log2 transformed. Proteins were filtered to retain proteins identified by MS/MS (not matching between runs) in all three ME-344 pull down experiments with at least 2 peptides. Missing values in the control pull downs were imputed in Perseus with a width of 0.6 and downshift of 1.8. The difference in mean log2 protein intensities (ME344 bait - control) and fold change in abundance are provided. Proteins enriched with ME-344 (Difference in Log2 Protein Intensities >0). Supplementary Table S14. Proteins with at least 2 peptides identified by MS/MS in all three ME-344 pull down experiments that were not identified in the control affinity enrichment.</p