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: Molecular Cell

Article Title: Initiation of Quality Control during Poly(A) Translation Requires Site-Specific Ribosome Ubiquitination

doi: 10.1016/j.molcel.2016.11.039

Figure Lengend Snippet:

Article Snippet: GST-UBE1 (human) , Boston Biochem , Cat. #E-306.

Techniques: Recombinant, Protease Inhibitor, Methylation, Ubiquitin Proteomics, Expressing, Plasmid Preparation, Sequencing, Negative Control, Software

Journal: Journal of Biomedical Science

Article Title: Localization, traffic and function of Rab34 in adipocyte lipid and endocrine functions

doi: 10.1186/s12929-023-00990-8

Figure Lengend Snippet: UBA1 conveys Rab34 action on FABP5 stability to regulate lipid metabolism. A Transfected 3T3-L1 cells with the indicated plasmids (GFP-Rab34 or FABP5-c-Myc) were treated with MG132 (10 μmol/L, 12 h) and lysed under denaturing conditions. c-Myc-tagged FABP5 was purified by anti-c-Myc immunoprecipitation and ubiquitinated FABP5 was detected by Western Blot. An expression vector coding for hemagglutinin (HA)-tagged ubiquitin (HA-Ubiquitin) was employed for cotransfection of cells expressing FABP5-c-Myc, alone or in combination with GFP-Rab34. The graph shows the ratio of Ubiquitinated-FABP5-c-Myc immunosignal to Ponceau S immunosignal. Data are referred to values in non-transfected cells (100%) and expressed as mean ± SEM (n = 3 biological replicates). **P < 0.01; ***P < 0.001. B Co-immunoprecipitation analysis in cells expressing GFP-Rab34 and vectors coding for LD-associated proteins related to ubiquitination/deubiquitination processes: UBA1-c-Myc (top panel), UCHL3-c-Myc (middle panel), or ISG15-c-Myc (bottom panel). In each experimental setting, proteins were purified by anti-c-Myc immunoprecipitation and detected by Western Blot using anti-c-Myc or anti-GFP antibodies. C Representative immunoblots and quantification of FABP5 levels in 3T3-L1 cells transfected with GFP-Rab34 (+ , 0.8 µg/µL), UBA1-c-Myc (+ , 0.8 µg/µL; + + , 1.6 µg/µL) or both expression vectors in the absence or presence of MG132 (10 µmol/L, 12 h). Data represent the ratio of FABP5 immunosignal to β-actin immunosignal and referred to values in non-transfected cells (100%). Data are expressed as mean ± SEM (n = 3 biological replicates). *P < 0.05; **P < 0.01; ***P < 0.001 vs. non-transfected cells. $$ P < 0.01; $$$ P < 0.001 vs. their respective condition treated with MG132. # P < 0.05; ## P < 0.01. D, E Rescue experiments of FABP5 in 3T3-L1 cells expressing GFP-Rab34 and UBA1 siRNA (siUBA1), alone or in combination. At the end of the experiments, cells were processed for immunoblotting studies ( D ) (see also Fig. S4) and for measurement of TGs (lipogenesis) and glycerol content (lipolysis) ( E ). Data are referred to values in control cells (100%; Scr), and expressed as mean ± SEM (n = 3 biological replicates). *P < 0.05; **P < 0.01; ***P < 0.001

Article Snippet: Plasmid coding for E1 Ubiquitin-Activating Enzyme 1 (UBA1) (pCMV3-UBA1-c-Myc) was purchased from SiNo Biological (Düsseldorfer, Eschborn, Germany).

Techniques: Transfection, Purification, Immunoprecipitation, Western Blot, Expressing, Plasmid Preparation, Cotransfection

Journal: Autophagy

Article Title: ALDH1A1 and HLTF modulate the activity of lysosomal autophagy inhibitors in cancer cells

doi: 10.1080/15548627.2017.1377377

Figure Lengend Snippet: Expression levels of ALDH1A1 and HLTF predict sensitivity to HCQ in cancer cell lines. (A) MTT (72 h) in colon and lung cancer cells. (B) Differentially expressed genes in HCQ-S (HT29) and HCQ-R (HCT15) colon cancer cells. (C) HCQ IC50 and Hill Slope for 33 human cancer cell lines. Blue dots indicate sensitive cell lines (<16 µM IC50); green indicates intermediate resistant cell lines (IC50 > 16 µM, Slope > −2.1); red indicates resistant cell lines (IC50 > 16, slope < − 2.1) (D) Protein expression detected by western blot of the 2 most upregulated (ALDH1A1, LYZ) and the 2 most downregulated (ABCB1, HLTF) genes in HCQ-sensitive (Sen), HCQ-intermediate resistant (Int Res), and HCQ-resistant (Res) cells. ANOVA indicates no single gene predicts sensitivity or resistance. (E) CART analysis of expression level of 4 genes (ALDH1A1, LYZ, HLTF, ABCB1) identifies a 2-gene signature that is sufficient to predict all sensitive cell lines. Exp.: expression as detected by fluorescence intensity of the band/ control.

Article Snippet: ALDH1A1 transient overexpression was performed with pCMV6-AC ALDH1A1 (OriGene, RC200723).

Techniques: Expressing, Western Blot, Fluorescence

Journal: Autophagy

Article Title: ALDH1A1 and HLTF modulate the activity of lysosomal autophagy inhibitors in cancer cells

doi: 10.1080/15548627.2017.1377377

Figure Lengend Snippet: ALDH1A1 levels control entry and activity of chloroquine derivatives in cancer cells. (A-B) Aldeflour assay shows (A) CQ derivatives produce no impairment of ALDH1 enzyme function, (B) siRNA against ALDH1A1 impairs enzymatic function. (C) DC341-C3 strucutre. (D) Fluorescence microscopy of DC340-Cy3 (red fluorescence) in A375 cells treated with nontargeting siRNA (NT) or siALDH1A1; or vehicle and DEAB for 24 h. Mean +/− SD from multiple experiments. (E) DC340-Cy3 fluorescence following SiNT or SiALDH1A1 in HT29 in the presence or absence of verapamil. (F) CD340-Cy3 in A375P cells transiently transfected with control or ALDH1A1-expressing vector. (G) Immunoblotting against autophagy markers in HCT15 cells transfected with control or ADLH1A1-expressing vector +- HCQ. Mean +/− SD for band quantification from multiple experiments shown. (H) LysoSensor fluorescence (green) of HCT15 and HT29 cells transfected with control or ALDH1A1-expressing vector, or siNT or siALDHA1, respectively. (I-K) 72-h MTT mean +/− SD. (I) Knockdown of ALDH1A1 promotes resistance to HCQ. (J) DEAB co-treatment promotes resistance to HCQ. (K) Overexpression of ALDH1A1 promotes HCQ-sensitivity. *p < 0.05.

Article Snippet: ALDH1A1 transient overexpression was performed with pCMV6-AC ALDH1A1 (OriGene, RC200723).

Techniques: Activity Assay, Fluorescence, Microscopy, Transfection, Expressing, Plasmid Preparation, Western Blot, Over Expression

Journal: Autophagy

Article Title: ALDH1A1 and HLTF modulate the activity of lysosomal autophagy inhibitors in cancer cells

doi: 10.1080/15548627.2017.1377377

Figure Lengend Snippet: Modulation of HCQ efficacy by ALDH1A1 and HLTF. (A) ROS levels in HCT15 and HT29 cells with overexpression or knockdown of ALHD1A1. (B) HCT15 cells transiently transfected with Control or ALDH1A1-expressing vector with or without HCQ (20 µM) for 12 h. (C) HT29 cells transfected with siNT (Non-Target) or siALDH1A1 with or without HCQ (20 µM) for 12 h. (D) Immunoblotting in lysates from the indicated cells treated with retinoic acid (RA 5 µM) +/− EZH2 inhibitor EPZ005687 2 µM (24 h). (E) Immunoblotting of lysates from HCT 15 cells transfected with control or ALDH1A1-expressing vector +/- EPZ005687 (2 µM), 24 h. (F) Immunoblotting from lysates from HCT15 cells treated with HCQ (20 µM) +/− Tiron (10 µM). (G) Immunoblotting of HCT15 cells transiently transfected with control or ALDH1A1-expressing vector +/− HCQ (20 µM) for 12 h. (H) ALDH1A1 facilitates entry of HCQ into the cell, where it accumulates in the lysosome. Lysosomal impairment leads to autophagy inhibition and the generation of ROS. ROS-associated DNA damage produces single-strand breaks with stalled replication forks. If HLTF is expressed, recruitment of the low fidelity DNA polymerase POLH allows translesion synthesis to occur promoting resistance to therapy. In the absence of HLTF-associated TLS, stalled replication forks collapse into double-strand breaks triggering cell death. Both RA and ROS can regulate KDM5D-PRC2-driven degradation of DNMT1 and upregulation of HLTF.

Article Snippet: ALDH1A1 transient overexpression was performed with pCMV6-AC ALDH1A1 (OriGene, RC200723).

Techniques: Over Expression, Transfection, Expressing, Plasmid Preparation, Western Blot, Inhibition, Translesion Synthesis

Journal: Autophagy

Article Title: ALDH1A1 and HLTF modulate the activity of lysosomal autophagy inhibitors in cancer cells

doi: 10.1080/15548627.2017.1377377

Figure Lengend Snippet: Validation set and TCGA analysis of 2-gene profile. (A) HCQ IC50 and Hill Slope for 16 human cancer cell lines. Blue dots indicate sensitive cell lines (<16 µM IC50); green indicates intermediate resistant cell lines (IC50 > 16 µM, Slope > −2.1); red indicates resistant cell lines (IC50 > 16, slope < −2.1). (B) Protein expression of ALDH1A1 and HLTF, and percentage of HCQ-sensitive cell lines in the validation set based on the CART analysis in Fig. 1. (C) Percentage of stage IV cancer patients in The Cancer Genome Atlas (TCGA) with the indicated malignancies that had the indicated profile of ALDH1A1 and HLTF expression predicted by RNA sequencing. Blue, HCQ-S profile; red, HCQ-R profile; white, unknown. HNSCC, head and neck squamous cell carcinoma. (D) Distribution of 4 ALDH1A1 HLTF RNA-Seq profiles in stage I-IV melanoma tumors from the TCGA. WT, wild type; Mu, mutant.

Article Snippet: ALDH1A1 transient overexpression was performed with pCMV6-AC ALDH1A1 (OriGene, RC200723).

Techniques: Expressing, RNA Sequencing Assay, Mutagenesis

Journal: Cell Death Discovery

Article Title: TRAIL DR5-CTSB crosstalk participates in breast cancer autophagy initiated by SAHA

doi: 10.1038/cddiscovery.2017.52

Figure Lengend Snippet: The effects of SAHA and TRAIL DR5 on the expressions of autophagy-related genes in cancer cells. RNA was isolated from MDA-MB-231 or MCF-7 cells using high pure RNA isolation kit following manufacturer’s instructions. To obtain the first-strand cDNA, transcriptor first strand cDNA synthesis kit was used and cDNA was as a template in real-time PCR reactions with power SYBR green PCR master mix. Exprofile human autophagy Gene qPCR array kit was employed to describe related mRNA expression. Relative changes of gene expression in the array were calculated using the 2 −ΔΔCt (threshold cycle) method. ( a – c ). MDA-MB-231 cells, ( d – f ). MCF-7 cells, ( g ). Related data analysis. Relative quantitative values represent mean±S.E.M.; * represents statistical significance of P <0.05 comparing with Basal, ** represents statistical significance of P <0.01 comparing with Basal.

Article Snippet: Exprofile human autophagy gene qPCR array kit was obtained from Genecopoeia (Rockville, MD.

Techniques: Isolation, cDNA Synthesis, Real-time Polymerase Chain Reaction, SYBR Green Assay, Expressing, Gene Expression