Histone peptide microarray screen of chromo and Tudor domains defines new histone lysine methylation interactions

Additional file 6: Figure S4. CHD7 chromodomain histone peptide microarray. A) Representative array images of CHD7 chromodomain showing peptide binding indicated in red (right panel). The peptide tracer is shown in green (left panel). Positive antibody controls are outlined in white. B) Scatter plot of the relative binding of CHD7 chromodomain from two independent peptide arrays. All modified and unmodified H4 (1–23) peptides are shown in red. All other peptides are shown in black. C) Relative binding to the indicated histone peptides from one representative array. Data were normalized to the most intense binding and the average and standard deviation of triplicate spots is shown

Discovery of a chemical probe for the L3MBTL3 methyllysine reader domain

We describe the discovery of UNC1215, a potent and selective chemical probe for the methyl-lysine (Kme) reading function of L3MBTL3, a member of the malignant brain tumor (MBT) family of chromatin interacting transcriptional repressors. UNC1215 binds L3MBTL3 with a Kd of 120 nM, competitively displacing mono- or dimethyl-lysine containing peptides, and is greater than 50-fold selective versus other members of the MBT family while also demonstrating selectivity against more than 200 other reader domains examined. X-ray crystallography identified a novel 2:2 polyvalent mode of interaction. In cells, UNC1215 is non-toxic and binds directly to L3MBTL3 via the Kme-binding pocket of the MBT domains. UNC1215 increases the cellular mobility of GFP-L3MBTL3 fusion proteins and point mutants that disrupt the Kme binding function of GFP-L3MBTL3 phenocopy the effects of UNC1215. Finally, UNC1215 demonstrates a novel Kme-dependent interaction of L3MBTL3 with BCLAF1, a protein implicated in DNA damage repair and apoptosis

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

Ebola (EBOV) and Marburg (MARV) viruses are members of the Filoviridae family which cause outbreaks of hemorrhagic fever. The filovirus VP40 matrix protein is essential for virus assembly and budding, and its PPxY L-domain motif interacts with WW-domains of specific host proteins, such as Nedd4 and ITCH, to facilitate the late stage of virus-cell separation. To identify additional WW-domain-bearing host proteins that interact with VP40, we used an EBOV PPxY-containing peptide to screen an array of 115 mammalian WW-domain-bearing proteins. Using this unbiased approach, we identified BCL2 Associated Athanogene 3 (BAG3), a member of the BAG family of molecular chaperone proteins, as a specific VP40 PPxY interactor. Here, we demonstrate that the WW-domain of BAG3 interacts with the PPxY motif of both EBOV and MARV VP40 and, unexpectedly, inhibits budding of both eVP40 and mVP40 virus-like particles (VLPs), as well as infectious VSV-EBOV recombinants. BAG3 is a stress induced protein that regulates cellular protein homeostasis and cell survival through chaperone-mediated autophagy (CMA). Interestingly, our results show that BAG3 alters the intracellular localization of VP40 by sequestering VP40 away from the plasma membrane. As BAG3 is the first WW-domain interactor identified that negatively regulates budding of VP40 VLPs and infectious virus, we propose that the chaperone-mediated autophagy function of BAG3 represents a specific host defense strategy to counteract the function of VP40 in promoting efficient egress and spread of virus particles

Ubiquitin ligase smurf2 interacts with filovirus vp40 and promotes egress of vp40 vlps

10.3390/v13020288Viruses13228

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

siRNA knockdown of BAG3 enhances eVP40 VLP egress.

<p><b>A)</b> HEK293T cells were transfected with eVP40 plus either random (control) or BAG3-specific siRNA as indicated. Proteins were detected in cell extracts and VLPs by Western blotting. eVP40 VLPs from control cells (lane 1) was set at 1.0. <b>B)</b> Quantification of the relative budding ratio of eVP40 VLPs from four independent experiments. Statistical significance was analyzed by a student t test, *** = p<0.001.</p

SARS-CoV-2 Envelope (E) protein interacts with PDZ-domain-2 of host tight junction protein ZO1.

Newly emerged SARS-CoV-2 is the cause of an ongoing global pandemic leading to severe respiratory disease in humans. SARS-CoV-2 targets epithelial cells in the respiratory tract and lungs, which can lead to amplified chloride secretion and increased leak across epithelial barriers, contributing to severe pneumonia and consolidation of the lungs as seen in many COVID-19 patients. There is an urgent need for a better understanding of the molecular aspects that contribute to SARS-CoV-2-induced pathogenesis and for the development of approaches to mitigate these damaging pathologies. The multifunctional SARS-CoV-2 Envelope (E) protein contributes to virus assembly/egress, and as a membrane protein, also possesses viroporin channel properties that may contribute to epithelial barrier damage, pathogenesis, and disease severity. The extreme C-terminal (ECT) sequence of E also contains a putative PDZ-domain binding motif (PBM), similar to that identified in the E protein of SARS-CoV-1. Here, we screened an array of GST-PDZ domain fusion proteins using either a biotin-labeled WT or mutant ECT peptide from the SARS-CoV-2 E protein. Notably, we identified a singular specific interaction between the WT E peptide and the second PDZ domain of human Zona Occludens-1 (ZO1), one of the key regulators of TJ formation/integrity in all epithelial tissues. We used homogenous time resolve fluorescence (HTRF) as a second complementary approach to further validate this novel modular E-ZO1 interaction. We postulate that SARS-CoV-2 E interacts with ZO1 in infected epithelial cells, and this interaction may contribute, in part, to tight junction damage and epithelial barrier compromise in these cell layers leading to enhanced virus spread and severe dysfunction that leads to morbidity. Prophylactic/therapeutic intervention targeting this virus-host interaction may effectively reduce airway and/or gastrointestinal barrier damage and mitigate virus spread

VP40/BAG3 GST pulldown assay.

<p><b>A)</b> Extracts from HEK293T cells transfected with eVP40-WT or eVP40-ΔPT/PY plasmids were incubated with GSH beads conjugated with GST-BAG3WW or GST alone. Input and pulled-down proteins were detected by Western blotting using anti-eVP40 or anti-GST antisera. <b>B)</b> Extracts from HEK293T cells expressing flag-tagged mVP40-WT were incubated with GSH beads conjugated with GST-BAG3WW or GST alone. Input and pulled-down proteins were detected by Western blotting using anti-flag or anti-GST antisera.</p

BAG3 inhibits budding of eVP40 and mVP40 VLPs in a WW-domain dependent manner.

<p><b>A)</b> HEK293T cells were transfected with the indicated plasmid combinations, and proteins were detected in cell extracts and VLPs by Western blotting. <b>B)</b> Quantification of the relative budding ratios of eVP40 VLPs from three independent experiments. eVP40 VLPs from control cells was set to 1.0. Statistical significance was analyzed by a one-way ANOVA. ns: not significant, *** = p<0.001. <b>C)</b> HEK293T cells were transfected with the indicated plasmid combinations, and proteins were detected in cell extracts and VLPs by Western blotting. <b>D)</b> Quantification of the relative budding ratios of mVP40 VLPs from three independent experiments. mVP40 VLPs from control cells was set to 1.0. Statistical significance was analyzed by a one-way ANOVA. * = p<0.05, *** = p<0.001.</p

BAG3 sequesters VP40 away from the plasma membrane.

<p>HEK293T cells were mock-transfected or transfected with eVP40 <b>(A)</b> or mVP40 <b>(C)</b> plus either BAG3-WT, or BAG3-ΔN as indicated. Cytosol and plasma membrane (PM) fractions were isolated at 24 hrs post-transfection, and the indicated proteins were detected by Western blotting. β-actin served as a control protein for the cytosol fraction, whereas Na/K ATPase served as a control protein for the PM fraction. The amount of VP40 in the PM fraction in control cells (lanes 6) was set at 100% (bar graph). Quantification of the relative amount of PM-associated eVP40 <b>(B)</b> or mVP40 <b>(D)</b> from three independent experiments is shown. Statistical significance was analyzed by one-way ANOVA. ns: not significant, *** = p<0.001. <b>E)</b> HEK293T cells were transfected with eVP40 plus vector, BAG3-WT, or BAG3-ΔN as indicated. Cells were fixed at 24 hrs post-transfection, and then incubated with rabbit anti-eVP40 antiserum and mouse anti-myc antiserum (to detecting BAG3-WT and BAG3-ΔN). Cells were then stained with Alexa Fluor 488 goat anti-rabbit and 594 goat anti-mouse secondary antibodies. Microscopy was performed using a Leica SP5 FLIM inverted confocal microscope and XZY scanning. Representative images displaying eVP40 (green) and BAG3-WT (red) or BAG3-ΔN (red) localized at the PM are shown. Cell nuclei were stained with NucBlue. Scale bars = 10μm.</p