Journal: bioRxiv

Article Title: A Degenerate PCNA-Interacting Peptide (DPIP) box targets RNF168 to replicating DNA to limit 53BP1 signaling

doi: 10.1101/2021.03.17.435897

Figure Lengend Snippet: (A) Domain organization of RNF168 indicating key functional domains and a degenerate PIP-like sequence residing in a disordered C-terminal region. The protein disorder profile was generated using the Protein Disorder Prediction (PrDOS) tool at http://prdos.hgc.jp/cgi-bin/top.cgi . (B) Replicate plates of H1299 cells were co-infected with adenovirus vectors encoding HA-PCNA and RNF168 WT or RNF168 ΔDPIP. 24 h post infection, some cultures were treated with HU (2mM, 2 h), camptothecin (CPT, 100 nM, 2 h), ATM inhibitor KU55933 100 nM, 2 h), or the WEE1 inhibitor MK1775 (10 μM, 2h). Chromatin extracts from the treated and untreated (control) cells were normalized for protein content and immunoprecipitated with anti-HA antibodies. Anti-HA immunoprecipitates were resolved on SDS-PAGE, transferred to nitrocellulose, and analyzed by immunoblotting with the indicated antibodies. (C) Replicate plates of RNF8 -/- U2OS cells were transiently infected with adenovirus vectors encoding FLAG-RNF168 WT, FLAG-RNF168 ΔDPIP, or FLAG-RNF168 ΔMIU2, or with a control ‘empty’ adenoviral vector. 48 h post-infection chromatin extracts were prepared, normalized for protein content and immunoprecipitated with anti-FLAG antibodies. Anti-FLAG immunoprecipitates were resolved on SDS-PAGE, transferred to nitrocellulose, and analyzed by immunoblotting with the indicated antibodies. (D) Isothermal titration calorimetry (left panel) was conducted by titrating synthetic peptide (p21, GRKRR QTSMTDFY HSKRRLIFS-amide where underlined residues denote PIP box; syringe, 150 - 300 μM) into a solution of PCNA (cell; 30 μM) in TBS. Control experiments using peptide injected into buffer alone showed minimal heats with no evidence of titration. Analysis of the isotherm yielded Kd = 76.3 nM and stoichiometry = 0.93 (Microcal Origin software). Replicate plates of H1299 cells were infected with adenovirus vectors encoding FLAG-RNF168 WT and HA-PCNA (or an ‘empty’ adenovirus vector for control). 36 h post-infection, chromatin extracts were prepared, normalized for protein content and immunoprecipitated with anti-FLAG antibody in the presence of different concentrations of peptides corresponding to the p21 or Polη PIP boxes. Anti-FLAG immunoprecipitates were resolved on SDS-PAGE, transferred to nitrocellulose, and analyzed by immunoblotting with the indicated antibodies (right panel) . (E) Replicate plates of H1299 cells were co-infected with adenovirus vectors encoding HA-PCNA and FLAG-RNF168 WT or HA-PCNA and RNF168 ΔDPIP. Some cells received an ‘empty’ adenovirus vector instead of HA-PCNA (for control). 36 h post-infection, some plates were treated with 2 mM HU for 2 h. Chromatin extracts were prepared, normalized for protein content and immunoprecipitated with anti-HA antibody in the presence or absence of the p21 PIP box peptide (1 mM). Anti-HA immune complexes were resolved on SDS-PAGE, transferred to nitrocellulose, and analyzed by immunoblotting with the indicated antibodies. (F) Replicate plates of H1299 cells were infected with adenovirus vectors encoding wild-type PCNA (HA-PCNA WT), ubiquitylation-resistant PCNA (HA-PCNA K164R), or a PCNA-ubiquitin fusion (HA-PCNA-Ub) in combination with FLAG-RNF168 WT adenovirus. Some cells received an ‘empty’ adenovirus vector instead of HA-PCNA viruses(for control). 36 h post-infection, chromatin extracts were prepared, normalized for protein content and immunoprecipitated with anti-HA antibody. Anti-HA immune complexes were resolved on SDS-PAGE, transferred to nitrocellulose, and analyzed by immunoblotting with the indicated antibodies. (G) H1299 cells were transiently co-transfected with expression constructs encoding HA-PCNA and FLAG-RNF168 WT or FLAG-RNF168 ΔDPIP. 48 h post-transfection cells were fixed, then stained with anti-HA and anti-FLAG antibodies prior to analysis by immunofluorescence confocal microscopy. The photographs are of representative cells co-expressing HA-PCNA and WT or ΔDPIP forms of FLAG-RNF168. The bar chart shows enumeration of cells containing HA-PCNA-co-localizing FLAG-RNF168 foci. Each data point represents the mean of results from 3 separate experiments and the error-bars represent the standard deviation. (H) RNF168 -/- U2OS cells were infected with adenoviral vectors encoding FLAG-RNF168 WT or FLAG-RNF168 ΔDPIP. 24 h post-infection cells were fixed and subject to proximity ligation assays to compare proximities of WT and ΔDPIP RNF168 with endogenous PCNA. The photographs are of representative DAPI-stained nuclei from each experimental condition. The bar chart showing enumeration of cells containing PCNA / FLAG-RNF168 PLA foci. Each data point represents the mean of results from 3 separate experiments and the error-bars represent the standard deviation. Ordinary one-way ANOVA demonstrated statistically significant difference between RNF168 WT and PIP (p = 0.0049).

Article Snippet: Ubiquitylation assays were performed in 25 mL reactions in which the components were added in the following order: dd H2O, 1x Energy regeneration solution (#B-10 R&D systems), 75 mM ubiquitin (#U-100H R&D systems) 1.6 mM FLAG-PCNA substrate (expressed and purified in bacteria), 0.1 mM E1 Ubiquitin Activating Enzyme (#E-304 R&D systems), 0.2 mM E2 conjugase (UbcH5c, #E2-627 R&D systems, or RAD6 #E2-613 R&D Systems), 0.2 mM E3 ligase (recombinant bacterial RAD18-RAD6 complex or RNF168 both purified in-house).

Techniques: Functional Assay, Sequencing, Generated, Infection, Control, Immunoprecipitation, SDS Page, Western Blot, Plasmid Preparation, Isothermal Titration Calorimetry, Injection, Titration, Software, Ubiquitin Proteomics, Transfection, Expressing, Construct, Staining, Immunofluorescence, Confocal Microscopy, Standard Deviation, Ligation

Journal: bioRxiv

Article Title: A Degenerate PCNA-Interacting Peptide (DPIP) box targets RNF168 to replicating DNA to limit 53BP1 signaling

doi: 10.1101/2021.03.17.435897

Figure Lengend Snippet: (A) Replicate plates of RNF8 +/+ and RNF8 -/- U2OS cells were infected with adenovirus vectors encoding RNF168 WT, RNF168 ΔDPIP, RNF168 super-PIP, and RNF168 Δ121-157, or with an empty adenovirus vector as control. After 23 h, one of the empty vector control plates was irradiated with UVC (60 J/m 2 ). All plates of cells were collected 24 h post-infection and fractionated to give chromatin and soluble extracts. Cell extracts were normalized for protein content, resolved by SDS-PAGE and transferred to nitrocellulose membranes prior to immunoblotting with the indicated antibodies. (B) Replicate cultures of U2OS cells were infected with adenovirus vectors encoding RNF168 WT, RNF168 ΔDPIP, RNF168 super-PIP, and RNF168 Δ121-157, or with an empty adenovirus vector as control. After 23 h cells were labelled with EdU, extracted with nonionic detergent to remove unbound MCM, fixed, and stained with anti-MCM2 (a marker for the MCM2-7 complex), PI (total DNA), and for EdU incorporation (active DNA synthesis). Cell cycle phases are defined by DNA content (PI-A) and DNA synthesis (Edu-A) in the upper plots. Nuclei containing loaded MCM2 in G1 and S phase are represented by blue and orange dots respectively. G1/G2/M phase cells negative for chromatin-loaded MCM2 are shown in grey. (C) Replicate plates of RAD18 +/+ and RAD18 -/- H1299 cells were infected with adenovirus vectors encoding different RNF168 variants (RNF168 WT, RNF168 ΔDPIP, RNF168 super-PIP, and RNF168 Δ121-157) in combination with RAD18 adenovirus, or with an empty adenovirus vector as control. After 22 h, two cultures were irradiated with UVC (60 J/m 2 ). All plates of cells were collected 24 h post-infection and fractionated to give chromatin and soluble extracts. Cell extracts were normalized for protein content, resolved by SDS-PAGE and transferred to nitrocellulose membranes for immunoblotting with the indicated antibodies. (D) Replicate plates of RAD18 +/+ and RAD18 -/- H1299 cells were sequentially transfected with HLTF-directed siRNA or with non-targeting control siRNA (siCon), then with CMV-FLAG RNF168 WT (or with an empty vector for control). 48 h post-transfection, some cultures were conditionally irradiated with UVC (20 J/m 2 ). After 2 h chromatin fractions were prepared and analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. (E) Purified PCNA substrate was incubated in vitro with recombinant RNF168 and recombinant UBCH5 individually or in combination, with recombinant RAD18-RAD6 complex, or with a combination of RAD18-RAD6 complex and RNF168 in the presence of E1, ubiquitin and an ATP-regenerating system. Reactions were terminated after 15 min or 30 min and products were separated on SDS-PAGE, transferred to nitrocellulose and analyzed by immunoblotting with the indicated antibodies.

Article Snippet: Ubiquitylation assays were performed in 25 mL reactions in which the components were added in the following order: dd H2O, 1x Energy regeneration solution (#B-10 R&D systems), 75 mM ubiquitin (#U-100H R&D systems) 1.6 mM FLAG-PCNA substrate (expressed and purified in bacteria), 0.1 mM E1 Ubiquitin Activating Enzyme (#E-304 R&D systems), 0.2 mM E2 conjugase (UbcH5c, #E2-627 R&D systems, or RAD6 #E2-613 R&D Systems), 0.2 mM E3 ligase (recombinant bacterial RAD18-RAD6 complex or RNF168 both purified in-house).

Techniques: Infection, Plasmid Preparation, Control, Irradiation, SDS Page, Western Blot, Staining, Marker, DNA Synthesis, Transfection, Purification, Incubation, In Vitro, Recombinant, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: A Degenerate PCNA-Interacting Peptide (DPIP) box targets RNF168 to replicating DNA to limit 53BP1 signaling

doi: 10.1101/2021.03.17.435897

Figure Lengend Snippet: RNF168 and RAD18 both ubiquitinate histone H2A in the vicinity of DSB to promote 53BP1 signaling and NHEJ (left panel) and also ubiquitinate PCNA to promote TLS (right panel) . RAD18 additionally acts as a molecular chaperone for the RAD51D recombinase and promotes HR independently of its ubiquitin ligase activity (middle) . See ‘Discussion’ for details.

Article Snippet: Ubiquitylation assays were performed in 25 mL reactions in which the components were added in the following order: dd H2O, 1x Energy regeneration solution (#B-10 R&D systems), 75 mM ubiquitin (#U-100H R&D systems) 1.6 mM FLAG-PCNA substrate (expressed and purified in bacteria), 0.1 mM E1 Ubiquitin Activating Enzyme (#E-304 R&D systems), 0.2 mM E2 conjugase (UbcH5c, #E2-627 R&D systems, or RAD6 #E2-613 R&D Systems), 0.2 mM E3 ligase (recombinant bacterial RAD18-RAD6 complex or RNF168 both purified in-house).

Techniques: Ubiquitin Proteomics, Activity Assay

Journal: iScience

Article Title: Evolutionary conserved regulation of TFEB stability by the E3 ubiquitin ligase WWP2 modulates response to stress in vivo

doi: 10.1016/j.isci.2025.111838

Figure Lengend Snippet:

Article Snippet: recombinant human 6His-Ube1 , RD System , Cat# E-305.

Techniques: Virus, Recombinant, Ubiquitin Proteomics, Luciferase, Mutagenesis, Reverse Transcription, SYBR Green Assay, Plasmid Preparation, Software, Lysis

Journal: Journal of Assisted Reproduction and Genetics

Article Title: Identification and characterization of RSIY-11, a novel seminal peptide derived from semenogelin-1, which acts as a neutral endopeptidase inhibitor modulating sperm motility

doi: 10.1007/s10815-019-01524-8

Figure Lengend Snippet: Determination of the effect of pre-incubation of RSIY-11 with NEP on inhibition. Fifty microliters of recombinant human NEP enzyme (0.1 μg/ml) and 50 μl of RSIY-11 (8.2 μM) were incubated for 0, 3, or 24 h at 37 °C prior to the addition of 50 μl of fluorogenic NEP substrate to determine enzymatic activity, and then, activity was measured every minute over 15 min. A control was performed by incubating NEP for the indicated time without RSIY-11, which was used as the 100% for each time point. Assays were performed in triplicate and averaged. Enzymatic activity was expressed as a percentage of the activity of the control. Error bars represent standard deviation. Asterisks (*) = statistically different from the control, *p value < 0.05; **p value < 0.01. NS no significant difference

Article Snippet: Michaelis-Menten Constant A fluorometric assay was performed using recombinant human NEP enzyme at 0.1 μg/ml (R&D Systems, Minneapolis, USA) and fluorogenic NEP peptide substrate ((7-methoxycoumarin-4-yl)-acetyl-R-P-P-G-F-S-A-F-K-(2, 4-dinitrophenyl)-OH, R&D Systems).

Techniques: Incubation, Inhibition, Recombinant, Activity Assay, Standard Deviation

Journal: Cellular and Molecular Life Sciences

Article Title: Snake venom phospholipase A 2 s exhibit strong virucidal activity against SARS-CoV-2 and inhibit the viral spike glycoprotein interaction with ACE2

doi: 10.1007/s00018-021-03985-6

Figure Lengend Snippet: Molecular models for HDP-2P (gold) complexes with ACE2 (red), and RBD (turquoise) obtained by protein docking. A Structures of HDP-2P complexed with ACE2 (left panel) and S-protein RBD (right panel) determined using Frodock. The central (control) panel shows re-docking of ACE2 to RBD, which results in a structure that resembles published X-ray structures; B A Rosetta-optimized binding interface between HDP-2P and RBD intersects the ACE2-RBD binding surface, which could prevent RBD from interacting with ACE2; C A different Rosetta-optimized binding interface between HDP-2P and ACE2 where HDP-2P docks away from the ACE2-RBD binding surface, implying no direct interplay between viral protein and PLA 2

Article Snippet: Recombinant human ACE2 (Cusabio, Houston, TX, USA) was immobilized on 3D carboxymethyl dextran hydrogel-coated sensor slides.

Techniques: Control, Binding Assay

Journal: Cellular and Molecular Life Sciences

Article Title: Snake venom phospholipase A 2 s exhibit strong virucidal activity against SARS-CoV-2 and inhibit the viral spike glycoprotein interaction with ACE2

doi: 10.1007/s00018-021-03985-6

Figure Lengend Snippet: PLA 2 s exhibit antiviral activity against viruses containing mutations and interfere with different stages of the SARS-CoV-2 replication cycle. a CPE inhibition assay. Vero E6 cells were infected with SARS-CoV-2 strain PMVL-19 or PMVL-20 at 100 TCID 50 in the presence of different concentrations of HDP-2 for 72 h. CPE inhibition was then measured by colorimetric assay with MTT. b Time-of-drug-addition assay. Vero E6 cells were infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.01, and virus yield in the infected cell supernatants was quantified by qRT-PCR 18 h after infection. c 293T/ACE2 were infected with different pseudo-SARS-CoV-2-GFP either in the presence of vehicle (PBS, Control) or HDP-2 (10 µg/ml). Representative fluorescent microscopy images of 293T/ACE2 cells infected with the pseudo-SARS-CoV-2 and treated with HDP-2. HDP-2 treatment led to a decrease in the entry of pseudoviruses, which was manifested in a decrease in the number of GFP-positive cells compared to the control. Scale bars, 100 µm. d Infectivity of pseudo-SARS-CoV-2 particles on 293T/ACE2 cells was quantified by measuring GFP fluorescence. Wuhan, B.1.1.7 and B.1.351 are the Wuhan reference strain, the lineage B.1.1.7 (United Kingdom) and the lineage B.1.351 (South Africa), respectively. Significant difference was determined using a Student’s t test: *p ˂0.05; **p ˂0.01; ***p ˂0.001. All results are shown as mean ± SD of n = 3 or 5 biologically independent samples. RFU relative fluorescence units

Article Snippet: Recombinant human ACE2 (Cusabio, Houston, TX, USA) was immobilized on 3D carboxymethyl dextran hydrogel-coated sensor slides.

Techniques: Activity Assay, Inhibition, Infection, Colorimetric Assay, Virus, Quantitative RT-PCR, Control, Microscopy, Fluorescence

Journal: Cellular and Molecular Life Sciences

Article Title: Snake venom phospholipase A 2 s exhibit strong virucidal activity against SARS-CoV-2 and inhibit the viral spike glycoprotein interaction with ACE2

doi: 10.1007/s00018-021-03985-6

Figure Lengend Snippet: HDP-2P reduces the binding of an anti-ACE2 antibody and RBD of glycoprotein S to ACE2 receptor at 293T/ACE2 cells. a , b Inhibition of anti-ACE2 antibody binding to 293T/ACE2 cells by HDP-2P. 293T/ACE2 cells were incubated with (100 µg/ml) or without (control) HDP-2P for 30 min and stained using human phycoerythrin (PE) conjugated anti-mouse ACE2 antibody. MFI mean fluorescence intensity. c , d Cells were incubated with PBS (control) or HDP-2P. The RBD protein fused with human Fc was then added for 1 h. After washing, the binding of RBD was detected using a DyLight 650-conjugated secondary anti-human Fc antibody. e Representative RBD binding profile in control and HDP-2P-treated cells. The cell populations binding high (RBD hi ) and low (RBD lo ) amount of RBD are shown. The percentage of positive cells was determined using flow cytometry analysis. Significant difference was determined using a Student’s t test: *p ˂0.05, ** p ˂0.01. Results are mean ± SD and are representative of at least three independent determinations

Article Snippet: Recombinant human ACE2 (Cusabio, Houston, TX, USA) was immobilized on 3D carboxymethyl dextran hydrogel-coated sensor slides.

Techniques: Binding Assay, Inhibition, Incubation, Control, Staining, Fluorescence, Flow Cytometry