y-p000  (Echelon Biosciences)

Journal: PLoS ONE

Article Title: Unconventional Secretion of Tissue Transglutaminase Involves Phospholipid-Dependent Delivery into Recycling Endosomes

doi: 10.1371/journal.pone.0019414

Figure Lengend Snippet: (A) Binding of tTG to recycling endosomes. The amounts of purified 125 I-tTG bound at 4°C to recycling endosomes immunoisolated from NIH3T3 cells lacking tTG were determined in a gamma counter with all measurements performed in triplicates. Shown are means ± SEM for three independent experiments. (B) The role of phosphoinositides in the tTG interaction with recycling endosomes. Binding of 50 nM purified tTG to recycling endosomes was tested in the presence of 100 µM free phosphoinositides. Bound tTG and endosomal Rab11A/Rab11B and transferrin receptor (TfR) were detected by immuno-blotting. The relative amounts of vesicle-bound tTG were quantified and compared to that in the sample without phosphoinositides in four independent experiments. Bars depict means ± SEM, *p<0.05. (C) The putative phospholipid-binding sequence and site within the fourth domain of tTG. (D) Comparative analysis of tTG binding to phosphoinositides. 125 I-tTG binding to synthetic liposomes (PolyPIPosomes™) containing indicated phiosphoinositides was determined in a gamma counter with all measurements performed in triplicates. Shown are means ± SEM for three independent experiments. (E) Association of tTG with phosphoinositides in cells. tTG was immunoprecipitated from extracts of WI-38 fibroblasts and the immune complexes were probed by immunoblotting for PI(3)P or PI(4)P. (F) The phospholipid-binding site in tTG is required for membrane targeting and externalization of the protein. K598A,K600A,R601A,K602A mutations (m-plbs) were generated within the phospholipid-binding site of tTG. NIH3T3 fibroblasts transfected with wild type tTG (wt) or its mutant deficient in phosphoinositide binding (m-plbs) were left untreated or treated with 10 mM neomycin for 24 h before induction of these proteins for 4 h. The contents of wt and m-plbs tTG in the recycling endosomes and cytosol were defined by immunoblotting ( left panels ). Cell surface and total levels of wt and m-plbs tTG were defined by surface biotinylation, isolation of surface protein fractions, and immunoblotting ( right panels ). The relative contents of wt and m-plbs tTG in the recycling endosomes and on the cell surface were quantified and compared to those for untreated cells expressing wt protein. Shown are representative of three independent experiments. Bars depict means ± SEM, **p<0.005. See also . (G) A proposed mechanism of unconventional tTG secretion. The de novo synthesized intracellular (cytoplasmic) tTG ( hexagons ) does not follow the classical, ER/Golgi-dependent secretion pathway, but is exported via a four-step unconventional secretion process mediated by recycling endosomes ( dotted arrows ). Solid arrows mark the major intracellular recycling route through the PNRC utilized by β1 integrins. See further comments in the text.

Article Snippet: Membrane lipid strips (#P6002); PIP strips (#P6001), PolyPIPosomes (#YP000; #YP003; #YP004; #YP005; #YP034; #YP035; #YP045; #YP039), and purified synthetic phosphoinositides were from Echelon Biosciences.

Techniques: Binding Assay, Purification, Sequencing, Immunoprecipitation, Western Blot, Generated, Transfection, Mutagenesis, Isolation, Expressing, Synthesized

Journal: Scientific Reports

Article Title: A novel bi-domain plant defensin MtDef5 with potent broad-spectrum antifungal activity binds to multiple phospholipids and forms oligomers

doi: 10.1038/s41598-017-16508-w

Figure Lengend Snippet: MtDef5 binds to multiple phospholipids but with higher affinity for PIP. ( A ) PIP strip showing the binding of MtDef5 to PI3P, PI4P and PI5P and also to PI4,5P 2 , PI3,5P 2 , PA and PS. ( B ) PIP array displays the relative binding of MtDef5 to phospholipids. ( C ) PolyPIPosome binding assay shows that MtDef5 binds with higher affinity to PI3P and PI4P as compared to PI5P. The full length gel is presented in Supplementary Figure .

Article Snippet: PolyPIPosome binding assays were performed in 200 µL of binding buffer (20 mM HEPES pH 7.4, 120 mM NaCl, 1 mM EGTA, 1 mM MgCl 2 , 1 mg/mL BSA, 0.2 mM CaCl 2 , 5 mM KCl) containing 10 µL each of control PolyPIPosome, PI PolyPIPosome, PI3P PolyPIPosome, PI4P PolyPIPosomes and PI5P PolyPIPosome (Echelon Biosciences).

Techniques: Stripping Membranes, Binding Assay

Journal: Scientific Reports

Article Title: A novel bi-domain plant defensin MtDef5 with potent broad-spectrum antifungal activity binds to multiple phospholipids and forms oligomers

doi: 10.1038/s41598-017-16508-w

Figure Lengend Snippet: γ-core motif sequences of MtDef5 are critical for membrane permeabilization, oligomerization and antifungal activity of MtDef5, but not for PIP binding. ( A ) Sequence of MtDef5 and its variants. Amino acid residues of the two γ-core motifs (highlighted) were mutated to alanine ( A ). ( B ) Quantitative assessment of the in vitro antifungal activity of MtDef5 and its variants against F . graminearum conidia after 20 h of treatment. Values are means of three replications. Error bars indicate standard deviations. ( C ) Quantitative measurement of fluorescence emitted by hyphae treated with 0.75 µM MtDef5 or its variants plus 0.5 µM of SYTOX Green. Values are means of three replications. Error bars indicate standard deviations. ( D ) Interaction of MtDef5 and it variants to phospholipids. PIP strip shows that MtDef5 and it variants strongly bind to PI3P, PI4P and PI5P and also to PI4,5P 2 , PI3,5P 2 , PA and PS. ( E ) PolyPIPosome binding assays of MtDef5 and its γ-core motif variants. It shows that MtDef5 binds to PI3P and PI4P with higher affinity than PI5P. It also binds to PI. The full length gels are presented in Supplementary Figure . ( F ) Oligomerization of MtDef5 γ-core motif variants in presence of PI(3)P. MtDef5_V1 variant containing the H36A, R37A, H93A and R94A substitutions loses its ability to oligomerize in presence of PI3P. The full length gel is presented in Supplementary Figure .

Article Snippet: PolyPIPosome binding assays were performed in 200 µL of binding buffer (20 mM HEPES pH 7.4, 120 mM NaCl, 1 mM EGTA, 1 mM MgCl 2 , 1 mg/mL BSA, 0.2 mM CaCl 2 , 5 mM KCl) containing 10 µL each of control PolyPIPosome, PI PolyPIPosome, PI3P PolyPIPosome, PI4P PolyPIPosomes and PI5P PolyPIPosome (Echelon Biosciences).

Techniques: Activity Assay, Binding Assay, Sequencing, In Vitro, Fluorescence, Stripping Membranes, Variant Assay