Journal: Science Advances

Article Title: Type II kinase inhibitors that target Parkinson’s disease–associated LRRK2

doi: 10.1126/sciadv.adt2050

Figure Lengend Snippet: ( A ) Schematic domain structure of LRRK2. The three constructs used in this study are indicated: full-length LRRK2, LRRK2 RCKW , and LRRK2 KW . ( B and C ) Close-up of the inhibitor binding pocket from cryo–electron microscopy (cryo-EM) maps and models of LRRK2 RCKW bound to the type I inhibitor MLi-2 [Protein Data Bank (PDB): 8TXZ] (B) and type II inhibitor GZD-824 (PDB: 8TZE) (C). Key residues and features are labelled. Both structures are shown in the same view, aligned through the C-lobe of the kinase. Dark orange, C-lobe; light orange, N-lobe; black, DYG motif; gray, G-loop; green, activation loop. ( D ) Scheme depicting our hybrid design strategy to develop potent type II inhibitors targeting LRRK2.

Article Snippet: For analysis of inhibitor activity against wild-type and G2019S mutant LRRK2, 293T cells were transfected with 500 ng of GFP-Rab8a and either 1000 ng of GFP-11 tagged wild-type LRRK2 (pcDNA5-FRT-TO-GFP11-LRRK2, RRID: Addgene_231174) or GFP-11 tagged G2019S LRRK2 (pcDNA5-FRT-TO-GFP11-LRRK2-G2019S, RRID: Addgene_231175).

Techniques: Construct, Binding Assay, Cryo-Electron Microscopy, Cryo-EM Sample Prep, Activation Assay

Journal: Science Advances

Article Title: Type II kinase inhibitors that target Parkinson’s disease–associated LRRK2

doi: 10.1126/sciadv.adt2050

Figure Lengend Snippet: ( A ) The co-crystal structure of RN129 ( 28 ) with CLK3 highlighting the type II binding mode and interactions between the protein and inhibitor (PDB: 9EZ3). ( B ) Ribbon diagram of the atomic model of LRRK2 RCKW :RN277:E11 DARPin complex (PDB: 9DMI) built into the cryo-EM map. ( C and D ) Close-ups of the active sites of the cryo-EM structures of LRRK2 RCKW :RN277 (C) and LRRK2 RCKW :GZD824 (PDB: 8TZE) (D). ( E ) Superposition of the atomic model of LRRK2 RCKW :RN277:E11 DARPin complex (in lighter shades) and our previously published structure of a LRRK2 RCKW :MLi-2:E11 DARPin complex (PDB: 8TXZ) (in darker shades). Only the kinase domains, which were aligned on their C-lobes, are shown. Major features of the kinase, including those that are indicators of type I and type II inhibitor binding, are shown.

Article Snippet: For analysis of inhibitor activity against wild-type and G2019S mutant LRRK2, 293T cells were transfected with 500 ng of GFP-Rab8a and either 1000 ng of GFP-11 tagged wild-type LRRK2 (pcDNA5-FRT-TO-GFP11-LRRK2, RRID: Addgene_231174) or GFP-11 tagged G2019S LRRK2 (pcDNA5-FRT-TO-GFP11-LRRK2-G2019S, RRID: Addgene_231175).

Techniques: Binding Assay, Cryo-EM Sample Prep

Journal: Science Advances

Article Title: Type II kinase inhibitors that target Parkinson’s disease–associated LRRK2

doi: 10.1126/sciadv.adt2050

Figure Lengend Snippet: ( A ) Kinome phylogenetic tree, with 96 kinases screened in the DSF assay against Rebastinib highlighted in blue or light blue. The 18.5 K ∆ T m shift of LRRK2 KW is highlighted in red. For all screened kinases, the bubble size and color correlates with the degree of ∆ T m shift, as indicated in the legend. ( B ) Kinome phylogenetic tree, with 103 kinases screened in the DSF assay against RN341 highlighted in blue. The 20-K ∆ T m shift of LRRK2 KW is highlighted in red. The bubble size or color for each kinase correlates with the ∆ T m shifts, as indicated in the legend (as in A). Kinases with ∆ T m > 6 K are labeled. ( C ) Waterfall plots of the ReactionBiology 33 PanQinase screen of RN341 at 1 and 10 μM against 350 wild-type kinases. Kinases with decreased activity in the presence of RN341 to <22% of the control value are labeled. ( D ) Off-target validation from both screens via in cellulo nanoBRET assay in two biological replicates, error bars ± SD, EC 50 (JNK2) = 2.7 μM, EC 50 (STK10) = 1.5 μM, EC 50 (MAPK14) = 1.7 μM, EC 50 (TTK) = 3.2 μM, EC 50 (CDKL1) = 17 μM, EC 50 (CLK1) = 6.0 μM, EC 50 (JNK3) = 15 μM, EC 50 (DYRK2) ≥ 20 μM, EC 50 (SLK) > 20 μM, EC 50 (DDR2) > 20 μM, and EC 50 (STK17B) ≥ 20 μM. ( E ) Representative immunoblot from 293T cells transiently co-transfected with LRRK1 and its substrate GFP-Rab7 before treatment with a dilution series of RN277 and RN341. Lysed cells were immunoblotted for LRRK1, GFP-Rab7, phospho-Rab7 (pS72), and GAPDH. ( F ) Quantification of the GFP-pRab7/GFP-Rab7/LRRK1 ratio from three independent Western blots. Statistical analysis performed using one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons of means. P < 0.0001 for all inhibitor concentrations versus DMSO; error bars ± SEM.

Article Snippet: For analysis of inhibitor activity against wild-type and G2019S mutant LRRK2, 293T cells were transfected with 500 ng of GFP-Rab8a and either 1000 ng of GFP-11 tagged wild-type LRRK2 (pcDNA5-FRT-TO-GFP11-LRRK2, RRID: Addgene_231174) or GFP-11 tagged G2019S LRRK2 (pcDNA5-FRT-TO-GFP11-LRRK2-G2019S, RRID: Addgene_231175).

Techniques: Labeling, Activity Assay, Control, Biomarker Discovery, Western Blot, Transfection

Journal: Science Advances

Article Title: Type II kinase inhibitors that target Parkinson’s disease–associated LRRK2

doi: 10.1126/sciadv.adt2050

Figure Lengend Snippet: ( A and B ) Dose-response curve of RN277 (A) and RN341 (B) inhibiting LRRK2 RCKW -mediated phosphorylation of Rab8a. Activity was calculated as the percentage (%) of phosphorylated Rab8a versus non-phosphorylated Rab8a detected in the presence of different concentrations of RN277/RN341. ( C ) Representative immunoblot from 293T cells transiently co-transfected with LRRK2 and GFP-Rab8a, treated with the indicated inhibitors. Lysed cells were immunoblotted for LRRK2, GFP-Rab8a, phospho-Rab8a (pT72), and GAPDH. ( D ) Sample from (C) run separately under identical conditions and immunoblotted for phospho-S935 LRRK2 and GAPDH. ( E ) Quantification of the GFP-pRab8a/GFP-Rab8a/LRRK2 ratio from three independent immunoblots (C). Statistical analysis performed using one-way ANOVA with Tukey’s multiple comparisons of means. ** P = 0.0049, DMSO versus MLi-2; *** P = 0.0004, DMSO versus Ponatinib; *** P = 0.0006, DMSO versus 5 μM RN277; *** P = 0.0003, DMSO versus 10 μM RN277; * P = 0.0406, DMSO versus 5 μM RN341; ** P = 0.0065, DMSO versus 10 μM RN341; error bars ± SEM. ( F ) Quantification of the pS935 LRRK2/LRRK2 ratio (run under identical conditions on separate blots) from three independent immunoblots (D). Statistical analysis performed using one-way ANOVA with Tukey’s multiple comparisons of means. **** P < 0.0001 for all conditions versus MLi-2; error bars ± SEM. ( G ) Representative immunoblot from 293T cells transiently co-transfected with GFP-Rab8a and either GFP-11 tagged wild-type (WT) or GFP-11 tagged G2019S LRRK2, treated with the indicated inhibitors. Lysed cells were immunoblotted for LRRK2, GFP-Rab8a, phospho-Rab8a (pT72), and GAPDH. ( H ) Quantification of the GFP-pRab8a/GFP-Rab8a/LRRK2 ratio from four independent immunoblots (G). Statistical analysis performed using one-way ANOVA with Tukey’s multiple comparisons of means. ** P = 0.0077, WT LRRK2 DMSO versus MLi-2; * P = 0.0324, WT LRRK2 DMSO versus 5 μM RN277; * P = 0.0461, WT LRRK2 DMSO versus 5 μM RN341; **** P < 0.0001 for all inhibitor treatments versus G2019S LRRK2 DMSO; error bars ± SEM.

Article Snippet: For analysis of inhibitor activity against wild-type and G2019S mutant LRRK2, 293T cells were transfected with 500 ng of GFP-Rab8a and either 1000 ng of GFP-11 tagged wild-type LRRK2 (pcDNA5-FRT-TO-GFP11-LRRK2, RRID: Addgene_231174) or GFP-11 tagged G2019S LRRK2 (pcDNA5-FRT-TO-GFP11-LRRK2-G2019S, RRID: Addgene_231175).

Techniques: Phospho-proteomics, Activity Assay, Western Blot, Transfection

Journal: Science Advances

Article Title: Type II kinase inhibitors that target Parkinson’s disease–associated LRRK2

doi: 10.1126/sciadv.adt2050

Figure Lengend Snippet: ( A ) Schematic of the single-molecule in vitro motility assay. ( B ) Example kymographs from single-molecule motility assays showing kinesin motility with DMSO or the type I inhibitor MLi-2 (5 μM) in the presence or absence of LRRK2 RCKW . Scale bars, 5 μm ( x ) and 30 s ( y ). ( C ) Quantification of the percentage (means ± SEM) of motile events per microtubule as a function of LRRK2 RCKW concentration in the absence (DMSO) or presence of MLi-2 (5 μM). Three technical replicates were collected per condition, with data points represented as circles, triangles, and squares corresponding to single data points (microtubules) within each replicate. Statistical analysis was performed using the mean of each technical replicate; *** P = 0.0007, DMSO condition; *** P = 0.0003, MLi-2 condition, one-way ANOVA with Šidák’s multiple comparisons test within drug only. ( D ) Example kymographs from single-molecule motility assays showing kinesin motility with DMSO or the type II inhibitors Ponatinib, RN277, and RN341 (10 μM) in the presence or absence of LRRK2 RCKW . Scale bars, 5 μm ( x ) and 30 s ( y ). ( E ) Quantification of the percentage (means ± SEM) of motile events per microtubule as a function of LRRK2 RCKW concentration in the absence (DMSO) or presence of type II inhibitors Ponatinib, RN277, and RN341 (10 μM). Three technical replicates were collected per condition, with data points represented as circles, triangles, and squares corresponding to single data points (microtubules) within each replicate. Statistical analysis was performed using the mean of each technical replicate; *** P = 0.0003, one-way ANOVA with Šidák’s multiple comparisons test within drug only.

Article Snippet: For analysis of inhibitor activity against wild-type and G2019S mutant LRRK2, 293T cells were transfected with 500 ng of GFP-Rab8a and either 1000 ng of GFP-11 tagged wild-type LRRK2 (pcDNA5-FRT-TO-GFP11-LRRK2, RRID: Addgene_231174) or GFP-11 tagged G2019S LRRK2 (pcDNA5-FRT-TO-GFP11-LRRK2-G2019S, RRID: Addgene_231175).

Techniques: In Vitro, Motility Assay, Concentration Assay

Journal: eLife

Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7

doi: 10.7554/eLife.102277

Figure Lengend Snippet: ( A ) Knockdown of MARCHF7 or UBR5 resulted in nsp16 restoration. HEK293T cells were transfected with small interfering RNA (siRNA) of E3 ligase candidates for 24 hr, followed by co-incubation with the nsp16-Flag-expressing plasmids for 48 hr, treated with MG132 (10 µM) for 16 hr before harvesting, lysed, and subjected to immunoblotting (IB) assay using anti-Flag antibody. RT-qPCR was conducted to determine the mRNA expression levels of E3 ligase candidates. The siRNA targeting regions for the candidate E3 ubiquitin ligase proteins and the targeted regions for RT-qPCR are shown in . Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by a two-tailed t-test: ***p<0.001. ( B ) RNA levels of UBR5 or MARCHF7 from HEK293T cells infected with lentivirus containing control or shRNA targeting UBR5 or MARCHF7 for 48 hr and screened with antibiotics for 48 hr. Knockdown cell lines were transfected with plasmids expressing nsp16-Flag, collected at the indicated times, and the protein levels of nsp16, MARCHF7, and UBR5 were detected by IB. ( C ) MARCHF7 and UBR5 acted separately and did not depend on each other. HEK293T cells stably expressing UBR5 shRNA or MARCHF7 shRNA were transfected with siRNA of MARCHF7 or UBR5 for 24 hr, respectively, followed by co-incubation with the nsp16-Flag-expressing plasmids for 48 hr. The protein levels and the RNA levels of nsp16, UBR5, and MARCHF7 were measured by IB and RT-qPCR, respectively. Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by a two-tailed t-test: ***p<0.001. ( D, E ) In HEK293T cells stably expressing UBR5 shRNA or MARCHF7 shRNA, nsp16 was degraded by overexpressed UBR5 or MARCHF7, respectively, whereas the mutant failed to degrade nsp16. The cell lysates were analyzed by anti-Flag antibody. Figure 2—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 2—source data 2. Original files for western blot analysis displayed in . Figure 2—source data 3. Numerical data obtained during experiments represented in .

Article Snippet: UBR5 (Gene ID: 51366) and its mutants (UBR5-ΔHECT, UBR5-ΔPABC, UBR5-ΔUBR) expression plasmids were constructed using purchased plasmids from Addgene (Watertown, MA, USA) as templates with no tag or a MYC tag at the N-terminus.

Techniques: Knockdown, Transfection, Small Interfering RNA, Incubation, Expressing, Western Blot, Quantitative RT-PCR, Ubiquitin Proteomics, Two Tailed Test, Infection, Control, shRNA, Stable Transfection, Mutagenesis

Journal: eLife

Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7

doi: 10.7554/eLife.102277

Figure Lengend Snippet: ( A, B ) UBR5 small interfering RNA (siRNA) was transfected into shMARCHF7 cells to knock down UBR5 . After 24 hr, MARCHF7 and nsp16 expression vectors were co-transfected, and the cells were harvested 72 hr later. The levels of nsp16 were characterized by immunoblotting (IB) with anti-Flag antibody. Whether MARCHF7 was dependent on UBR5 to degrade nsp16 was determined by further transfection of MARCHF7 siRNA into shUBR5 cells, followed by co-transfection of UBR5 and nsp16 expression vectors 24 hr later. The other operations are the same as above. Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by a two-tailed t-test. p>0.05; **p<0.01; ***p<0.001. ( C ) The siRNA targeting regions and RT-qPCR targeting regions for the E3 ubiquitin ligases—HECTD1, UBR5, MYCBP2, TRIM21, TRIM32, and MARCHF7—are shown. Figure 2—figure supplement 1—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 2—figure supplement 1—source data 2. Original files for western blot analysis displayed in . Figure 2—figure supplement 1—source data 3. Numerical data obtained during experiments represented in .

Article Snippet: UBR5 (Gene ID: 51366) and its mutants (UBR5-ΔHECT, UBR5-ΔPABC, UBR5-ΔUBR) expression plasmids were constructed using purchased plasmids from Addgene (Watertown, MA, USA) as templates with no tag or a MYC tag at the N-terminus.

Techniques: Small Interfering RNA, Transfection, Knockdown, Expressing, Western Blot, Cotransfection, Two Tailed Test, Quantitative RT-PCR, Ubiquitin Proteomics

Journal: eLife

Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7

doi: 10.7554/eLife.102277

Figure Lengend Snippet: ( A ) Nsp16 can be ubiquitinated. HEK293T cells co-transfected with ubiquitin-Myc and nsp16-Flag or transfected with nsp16-Flag alone. The cells were treated with MG132 for 12 hr before collection. The whole-cell lysates were incubated with anti-Flag beads and used for immunoblotting (IB) with anti-Myc or anti-Flag antibodies to detect the polyubiquitination chain of nsp16. ( B ) Assess the endogenous ubiquitination level of nsp16 protein. Cells were transfected with nsp16-Flag or an empty vector and collected 48 hr later. Prior to harvesting, cells were treated with MG132 for 16 hr. Co-immunoprecipitation (Co-IP) experiments were then performed to analyze the endogenous ubiquitination level of nsp16. ( C ) The level of ubiquitination of nsp16 decreased with decreasing the protein levels of MARCHF7 or UBR5. E3 was knocked down by transfection with small interfering RNA (siRNA) targeting UBR5 or MARCHF7 , and 24 hr later, ubiquitin-Myc and nsp16-HA were co-transfected or nsp16-HA alone. Cells were treated with MG132 for 16 hr before collection. Whole-cell lysates were incubated with anti-HA beads, and polyubiquitinated chains of nsp16 were detected by IB with anti-Myc or anti-HA antibodies. ( D ) Nsp16 can be modified by a variety of ubiquitin chains. HEK293T cells were transfected with either nsp16-HA alone or together with plasmids encoding various mutants of ubiquitin (K6 only, K11 only, K27 only, K29 only, K33 only, K48 only, K63 only). Thirty-six hours later, cells were treated with MG132 for 12 hr. Cell lysates were then subjected to immunoprecipitation, followed by IB to analysis. ( E, F ) MARCHF7 or UBR5 causes nsp16 to be modified by the K27-type or K48-type ubiquitin chain. 293T cell lines with or without MARCHF7 or UBR5 knockdown were co-transfected with plasmids encoding ubiquitin-WT or various mutants of ubiquitin (K6 only, K11 only, K27 only, K29 only, K33 only, K48 only, K63 only). The other experimental methods were the same as C. Figure 3—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 3—source data 2. Original files for western blot analysis displayed in .

Article Snippet: UBR5 (Gene ID: 51366) and its mutants (UBR5-ΔHECT, UBR5-ΔPABC, UBR5-ΔUBR) expression plasmids were constructed using purchased plasmids from Addgene (Watertown, MA, USA) as templates with no tag or a MYC tag at the N-terminus.

Techniques: Transfection, Ubiquitin Proteomics, Incubation, Western Blot, Plasmid Preparation, Immunoprecipitation, Co-Immunoprecipitation Assay, Small Interfering RNA, Modification, Knockdown

Journal: eLife

Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7

doi: 10.7554/eLife.102277

Figure Lengend Snippet: ( A ) HEK293T cells were transfected with either nsp16-Flag alone or together with MARCHF7-Myc. Thirty-six hours after transfection, the cells were treated with MG132 (10 µM) for 12 hr. Cell lysates were subjected to immunoprecipitation with anti-Flag antibody. Using immunoblotting (IB) to analyze the precipitates and input. ( B ) HEK293T cells were transfected with nsp16-Flag. Cell lysates were subjected to immunoprecipitation with anti-UBR5 or IgG antibody. ( C, D ) HeLa cells were co-transfected with YFP-nsp16 and CFP-UBR5 or CFP-MARCHF7. A representative image of YFP-nsp16 (yellow) and ECFP-MARCHF7 (cyan) or ECFP-UBR5 (cyan) expressing cells before and after photobleaching the acceptor fluorophore, YFP. The region chosen for photobleaching is marked (white open box). Scale bars, 10 µm. The quantization of fluorescence brightness was analyzed by ImageJ. Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by a two-tailed t-test. p>0.05; **p<0.01; ***p<0.001. ( E ) HeLa cells transfected with nsp16-Flag were analyzed by confocal microscopy. The Flag-tagged nsp16 labeled with anti-Flag antibody (red). MARCHF7 or UBR5 were labeled with endogenous antibodies (green). Cell nuclei were stained using DAPI (4′,6-diamidino-2-phenylindole) (blue). Representative images were shown. Scale bars, 20 µm. The ratio of colocalization was quantified by measuring the fluorescence intensities using ImageJ. ( F ) nsp16 was stably transfected into HEK293T cells. The cells were analyzed by confocal microscopy. The other operations are the same as above. Figure 4—figure supplement 1—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 4—figure supplement 1—source data 2. Original files for western blot analysis displayed in . Figure 4—figure supplement 1—source data 3. Numerical data obtained during experiments represented in .

Article Snippet: UBR5 (Gene ID: 51366) and its mutants (UBR5-ΔHECT, UBR5-ΔPABC, UBR5-ΔUBR) expression plasmids were constructed using purchased plasmids from Addgene (Watertown, MA, USA) as templates with no tag or a MYC tag at the N-terminus.

Techniques: Transfection, Immunoprecipitation, Western Blot, Expressing, Fluorescence, Two Tailed Test, Confocal Microscopy, Labeling, Staining, Stable Transfection

Journal: eLife

Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7

doi: 10.7554/eLife.102277

Figure Lengend Snippet: ( A, B ) The binding of MARCHF7 or UBR5 to nsp16 was not mutually dependent. The binding of nsp16 to UBR5 or MARCHF7 was identified by co-immunoprecipitation in HEK293T cells transfected into si MARCHF7 or si UBR5 , respectively. The immunoprecipitates and input were analyzed by immunoblotting (IB). The knockdown efficiency was detected by RT-qPCR and IB. Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by a two-tailed t-test: ***p<0.001. ( C, D ) MARCHF7 or UBR5 colocalized with nsp16 in the endoplasmic reticulum. Hela cells were co-transfected with YFP-nsp16 (yellow) and CFP-UBR5 (cyan) or CFP-MARCHF7 (cyan). The organelles were labeled with antibodies against marker proteins of endoplasmic reticulum, Golgi apparatus, and mitochondria respectively (red). The cells were analyzed by confocal microscopy ( C ). Scale bars, 20 µm. The ratio of colocalization was quantified by measuring the fluorescence intensities using ImageJ ( D ). Figure 4—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 4—source data 2. Original files for western blot analysis displayed in . Figure 4—source data 3. Numerical data obtained during experiments represented in .

Article Snippet: UBR5 (Gene ID: 51366) and its mutants (UBR5-ΔHECT, UBR5-ΔPABC, UBR5-ΔUBR) expression plasmids were constructed using purchased plasmids from Addgene (Watertown, MA, USA) as templates with no tag or a MYC tag at the N-terminus.

Techniques: Binding Assay, Immunoprecipitation, Transfection, Western Blot, Knockdown, Quantitative RT-PCR, Two Tailed Test, Labeling, Marker, Confocal Microscopy, Fluorescence

Journal: eLife

Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7

doi: 10.7554/eLife.102277

Figure Lengend Snippet: ( A ) The schematic represents UBR5 wild-type (WT) or mutants used in the study. ( B ) The homologous to the E6AP carboxyl terminus (HECT) domain of UBR5 is required for nsp16 degradation. After co-transfection with UBR5 WT or mutants and nsp16-HA, cells were harvested 48 hr later, and cell lysates were examined by immunoblotting (IB). ( C ) The HECT domain of UBR5 affects K48-type ubiquitin chain of nsp16. HEK293T cells were transfected with the assigned plasmids. After 36 hr, cells were treated with 10 µM MG132 for 12 hr, harvested, and cell lysates were incubated with protein G agarose beads conjugated with anti-HA antibodies. Cell lysates and precipitated samples were analyzed by IB. ( D ) The schematic represents WT and truncated forms of MARCHF7 used in the study. (E) Only MARCHF7 WT degraded nsp16. ( F ) The N-terminal region of MARCHF7 interacted with nsp16, and only the WT could catalyze the K27-type ubiquitin chain of nsp16. Figure 4—figure supplement 2—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 4—figure supplement 2—source data 2. Original files for western blot analysis displayed in .

Article Snippet: UBR5 (Gene ID: 51366) and its mutants (UBR5-ΔHECT, UBR5-ΔPABC, UBR5-ΔUBR) expression plasmids were constructed using purchased plasmids from Addgene (Watertown, MA, USA) as templates with no tag or a MYC tag at the N-terminus.

Techniques: Cotransfection, Western Blot, Ubiquitin Proteomics, Transfection, Incubation

Journal: eLife

Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7

doi: 10.7554/eLife.102277

Figure Lengend Snippet: ( A, B ) Knocking down MARCHF7 or UBR5 enhances SARS-CoV-2 trVLP infectivity. MARCHF7 or UBR5 was knocked down by small interfering RNA (siRNA) in Caco2 cells with stable expression of SARS-CoV-2 N protein. Twenty-four hours later, cells were infected with SARS-CoV-2 virus-like particles (MOI: 0.1), the medium was changed 2 hr after infection, and the eGFP-positive cells were detected by flow cytometry 48 hr later ( A ). Protein content was determined by RT-qPCR ( B ). Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by one-way ANOVA, followed by a Tukey’s multiple comparisons posttest: *p<0.05; **p<0.01; ***p<0.001. Figure 5—figure supplement 1—source data 1. Numerical data obtained during experiments represented in .

Article Snippet: UBR5 (Gene ID: 51366) and its mutants (UBR5-ΔHECT, UBR5-ΔPABC, UBR5-ΔUBR) expression plasmids were constructed using purchased plasmids from Addgene (Watertown, MA, USA) as templates with no tag or a MYC tag at the N-terminus.

Techniques: Infection, Small Interfering RNA, Expressing, Virus, Flow Cytometry, Quantitative RT-PCR

Journal: eLife

Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7

doi: 10.7554/eLife.102277

Figure Lengend Snippet: ( A ) The virus-encoded nsp16 protein interacts with endogenous MARCHF7 and UBR5 and undergoes ubiquitination modification. In 293T-ACE2 cells, with or without IME-BJ01 strain infection (MOI: 0.01), the medium was changed 2 hr post-infection, and cells were harvested 48 hr later, with MG132 treatment added 16 hr before harvesting. nsp16 protein was enriched using protein G beads coupled with the nsp16 antibody, and interactions and ubiquitination were analyzed by immunoblotting (IB) with endogenous antibodies against MARCHF7, UBR5, and ubiquitination. ( B–I ) MARCHF7 and UBR5 were knocked down by small interfering RNA (siRNA) in Caco2 cells. 24 hr after transfection, the cells were infected with IME-BJ01 strain (MOI: 0.01) ( C–E ) or Omicron BA.1 strain (MOI: 0.001) ( F–H ), respectively. 2 hr post-infection, the supernatant was discarded, and the cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 3% fetal bovine serum for 48 hr. The mRNA levels of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) M and E genes in the cells ( C, F ) and E genes in supernatant ( D, G ) were detected by RT-qPCR, and the viral titers in supernatant ( E, H ) were measured. The N protein levels of IME-BJ01 or Omicron viruses were detected by IB ( I ). Knockdown efficiencies of MARCHF7 and UBR5 were detected by RT-qPCR or IB ( B, I ). Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by one-way ANOVA, followed by a Tukey’s multiple comparisons posttest: *p<0.05; **p<0.01; ***p<0.001. Figure 5—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 5—source data 2. Original files for western blot analysis displayed in . Figure 5—source data 3. Numerical data obtained during experiments represented in .

Article Snippet: UBR5 (Gene ID: 51366) and its mutants (UBR5-ΔHECT, UBR5-ΔPABC, UBR5-ΔUBR) expression plasmids were constructed using purchased plasmids from Addgene (Watertown, MA, USA) as templates with no tag or a MYC tag at the N-terminus.

Techniques: Virus, Ubiquitin Proteomics, Modification, Infection, Western Blot, Small Interfering RNA, Transfection, Cell Culture, Quantitative RT-PCR, Knockdown

Journal: eLife

Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7

doi: 10.7554/eLife.102277

Figure Lengend Snippet: ( A–H ) UBR5 or MARCHF7 was transfected in 293T cells stably overexpressed with ACE2, and the increased doses of nsp16-Flag were transfected simultaneously. After 24 hr, the cells were infected with IME-BJ01 strains. The mRNA levels of M and E genes of the IME-BJ01 strain in the cells ( A, D ) and E gene in supernatant ( B, E ) were detected by RT-qPCR, as well as the detection of viral titers in supernatant ( C, F ). The N protein of the virus and the overexpression efficiency were detected by IB ( G, H ). Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by one-way ANOVA, followed by a Tukey’s multiple comparisons posttest. p>0.05; **p<0.01; ***p<0.001. shows data related to infection with Omicron BA.1. Figure 6—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 6—source data 2. Original files for western blot analysis displayed in . Figure 6—source data 3. Numerical data obtained during experiments represented in .

Article Snippet: UBR5 (Gene ID: 51366) and its mutants (UBR5-ΔHECT, UBR5-ΔPABC, UBR5-ΔUBR) expression plasmids were constructed using purchased plasmids from Addgene (Watertown, MA, USA) as templates with no tag or a MYC tag at the N-terminus.

Techniques: Transfection, Stable Transfection, Infection, Quantitative RT-PCR, Virus, Over Expression, Western Blot

Journal: eLife

Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7

doi: 10.7554/eLife.102277

Figure Lengend Snippet: ( A–H ) In 293T-ACE2 cells, the Really Interesting New Gene (RING) domain deletion mutant of MARCHF7 (MARCHF7-aa 1–542) or the homologous to the E6AP carboxyl terminus (HECT) domain inactivated mutant of UBR5 (UBR5-ΔHECT) were transfected, along with a gradient of nsp16-Flag overexpression. The cells were infected with the IME-BJ01 strain (MOI: 0.01), medium was changed 2 hr post-infection, and cells and supernatants were collected 48 hr after infection. Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by one-way ANOVA, followed by a Tukey’s multiple comparisons posttest. p>0.05; **p<0.01; ***p<0.001. Figure 6—figure supplement 2—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 6—figure supplement 2—source data 2. Original files for western blot analysis displayed in . Figure 6—figure supplement 2—source data 3. Numerical data obtained during experiments represented in .

Article Snippet: UBR5 (Gene ID: 51366) and its mutants (UBR5-ΔHECT, UBR5-ΔPABC, UBR5-ΔUBR) expression plasmids were constructed using purchased plasmids from Addgene (Watertown, MA, USA) as templates with no tag or a MYC tag at the N-terminus.

Techniques: Mutagenesis, Transfection, Over Expression, Infection, Western Blot

Journal: eLife

Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7

doi: 10.7554/eLife.102277

Figure Lengend Snippet: ( A ) This diagram shows the mutation of nsp16 in different virus subtypes. The amino acid sequences of different SARS-CoV-2 strains were obtained from the National Center for Biotechnology Information (NCBI), and the amino acid sequences of nsp16 of different strains were compared by DNAMAN software. ( B ) Nsp16 mutants can still be regulated by MG132. The mutated nsp16 plasmids were transfected into HEK293T cells. After 36 hr of culture, cells were treated with 10 µm MG132 or DMSO, harvested 12 hr later, and cell lysates were examined by immunoblotting (IB). ( C ) MARCHF7 or UBR5 can degrade nsp16 mutants. After transfecting MARCHF7 or UBR5 small interfering RNA (siRNA) and the mutated nsp16 plasmids, the cells were harvested 48 hr later. The cell lysates were detected by IB. Figure 6—figure supplement 3—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 6—figure supplement 3—source data 2. Original files for western blot analysis displayed in .

Article Snippet: UBR5 (Gene ID: 51366) and its mutants (UBR5-ΔHECT, UBR5-ΔPABC, UBR5-ΔUBR) expression plasmids were constructed using purchased plasmids from Addgene (Watertown, MA, USA) as templates with no tag or a MYC tag at the N-terminus.

Techniques: Mutagenesis, Virus, Software, Transfection, Western Blot, Small Interfering RNA

Journal: eLife

Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7

doi: 10.7554/eLife.102277

Figure Lengend Snippet: ( A–C ) The protein and mRNA levels of MARCHF7 or UBR5 upon infection with different titers. Endogenous MARCHF7 and UBR5 RNA levels were detected by RT-qPCR 48 hr after infection with different titers of IME-BJ01 strain (MOI: 0, 0.0001, 0.001, 0.01) or Omicron BA.1 strain (MOI: 0, 0.0001, 0.001). Protein levels were examined by immunoblotting (IB). ( D ) The expression level of UBR5 was negatively correlated with the severity of the disease, but MARCHF7 expression levels were not. Peripheral blood mononuclear cells (PBMCs) were extracted from common, severe, and critical coronavirus disease 2019 (COVID-19) patients. RT-qPCR was used to detect the mRNA level of UBR5 or MARCHF7 in patients. Significance was determined by one-way ANOVA, followed by a Tukey’s multiple comparisons posttest. ns, p>0.05; **p<0.01; ***p<0.001. Figure 6—figure supplement 4—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 6—figure supplement 4—source data 2. Original files for western blot analysis displayed in . Figure 6—figure supplement 4—source data 3. Numerical data obtained during experiments represented in .

Article Snippet: UBR5 (Gene ID: 51366) and its mutants (UBR5-ΔHECT, UBR5-ΔPABC, UBR5-ΔUBR) expression plasmids were constructed using purchased plasmids from Addgene (Watertown, MA, USA) as templates with no tag or a MYC tag at the N-terminus.

Techniques: Infection, Quantitative RT-PCR, Western Blot, Expressing

Journal: eLife

Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7

doi: 10.7554/eLife.102277

Figure Lengend Snippet: ( A–G ) BLAB/C mice were injected with the corresponding plasmids at 40 µg/500 µl via the high-pressure tail vein, followed by nasal inoculation with 50 µl severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus at a dosage of 10 5.5 TCID50/ml (created with BioRender.com and the agreement no. is OO281XWHNA). Immunoblotting (IB) was used to detect the expression of MARCHF7 or UBR5 in the lung tissues ( B ). Viral RNA loads in mouse lung tissues were detected by measuring the mRNA levels of the E genes by RT-qPCR ( C ). Lung tissue was collected, homogenized, and the residue was removed by centrifugation to collect the supernatant. The viral titer was then measured using the TCID50 method ( D ). Mouse body weight was monitored during the experimental period ( E ). Representative images of hematoxylin and eosin (H&E) staining of lungs of mice with different treatments. Magnification, ×40. Scale bars, 20 µm ( F ). The staining of viral N proteins. Magnification, ×63. Scale bars, 20 µm. n=3 in each group ( G ). RT-qPCR was used to measure the expression of cytokines and chemokines in the spleens of mice in each group ( H ). Statistical significance was analyzed using a one-way analysis of variance with Tukey’s multiple comparisons test (NS, no significance, *p<0.05, **p<0.01, ***p<0.001). Figure 7—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 7—source data 2. Original files for western blot analysis displayed in . Figure 7—source data 3. Numerical data obtained during experiments represented in .

Article Snippet: UBR5 (Gene ID: 51366) and its mutants (UBR5-ΔHECT, UBR5-ΔPABC, UBR5-ΔUBR) expression plasmids were constructed using purchased plasmids from Addgene (Watertown, MA, USA) as templates with no tag or a MYC tag at the N-terminus.

Techniques: Injection, Virus, Western Blot, Expressing, Quantitative RT-PCR, Residue, Centrifugation, Staining

Journal: eLife

Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7

doi: 10.7554/eLife.102277

Figure Lengend Snippet: Schematic diagram of MARCHF7 and UBR5 ubiquitinate the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nonstructural protein nsp16, leading to its degradation via the proteasomal pathway, thereby affecting viral replication (created with BioRender.com and the agreement no. is EV281XWATL).

Article Snippet: UBR5 (Gene ID: 51366) and its mutants (UBR5-ΔHECT, UBR5-ΔPABC, UBR5-ΔUBR) expression plasmids were constructed using purchased plasmids from Addgene (Watertown, MA, USA) as templates with no tag or a MYC tag at the N-terminus.

Techniques:

Journal: eLife

Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7

doi: 10.7554/eLife.102277

Figure Lengend Snippet: ( A ) Single-lysine mutation of nsp16 protein can be restored by MG132. Single-lysine mutants of nsp16 protein were obtained through mutagenesis and overexpressed in 293T cells. After 36 hr, cells were treated with either MG132 or DMSO for 16 hr and then harvested. Protein levels were detected by immunoblotting (IB) using the HA antibody. ( B ) nsp16 protein truncates can be restored by MG132. nsp16 protein truncates were obtained through structural analysis and mutations. IB was performed to determine whether the truncates could be restored by MG132. ( C ) The mass spectrometry analysis identified the ubiquitination modification site at lysine 76. nsp16-Flag was overexpressed in 293T cells, followed by MG132 treatment and cell harvest. nsp16 protein was enriched using Flag antibody-conjugated protein G beads. Flag-peptide competition was used to obtain the nsp16-containing solution. The protein and ubiquitination status were visualized by SDS-PAGE and Coomassie staining. Mass spectrometry was used for further analysis. ( D ) Degradation of nsp16-K76R is still regulated by MARCHF7 or UBR5. A plasmid with a mutation at lysine 76 of nsp16 to arginine (nsp16-K76R) was constructed. MARCHF7 or UBR5 was knocked down by small interfering RNA (siRNA) in 293T cells. nsp16-WT or nsp16-K76R was transfected the next day, and cells were harvested 48 hr later. Protein levels were detected by IB. ( E ) Ubiquitination levels of nsp16-K76R are reduced but still regulated by MARCHF7 or UBR5. MARCHF7 or UBR5 was knocked down using siRNA in 293T cells. The cells were co-transfected with Ub-K27 or K48, and nsp16-WT or nsp16-K76R mutant. Cells were harvested 48 hr later, with MG132 treatment 16 hr before harvesting. Co-immunoprecipitation (Co-IP) experiments were performed to analyze the ubiquitination status of nsp16-WT or its mutant. Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by one-way ANOVA, followed by a Tukey’s multiple comparisons posttest. p>0.05; **p<0.01; ***p<0.001. Figure 8—figure supplement 1—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 8—figure supplement 1—source data 2. Original files for western blot analysis displayed in . Figure 8—figure supplement 1—source data 3. Numerical data obtained during experiments represented in .

Article Snippet: UBR5 (Gene ID: 51366) and its mutants (UBR5-ΔHECT, UBR5-ΔPABC, UBR5-ΔUBR) expression plasmids were constructed using purchased plasmids from Addgene (Watertown, MA, USA) as templates with no tag or a MYC tag at the N-terminus.

Techniques: Mutagenesis, Western Blot, Mass Spectrometry, Ubiquitin Proteomics, Modification, SDS Page, Staining, Plasmid Preparation, Construct, Small Interfering RNA, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: PLoS ONE

Article Title: p300 Alters Keratinocyte Cell Growth and Differentiation through Regulation of p21 Waf1/CIP1

doi: 10.1371/journal.pone.0008369

Figure Lengend Snippet: HFKs were induced to differentiate in CaCl 2 medium and harvested at the indicated time points (t = 0, 6, 16, 48 hrs). ( A ) Western blot analysis of levels of p300 in control and p300 depleted HFKs using scrambled shRNA (scr) two shRNA molecules (shp300A and shp300B) directed against p300. ( B ) Cells stably expressing p300 targeting shRNAs were induced to differentiate and harvested at the indicated time points. Western blot analysis indicates that knocking down p300 dramatically reduces the p300 mediated acetylation and expression of p53. There is also a decrease in p21 Waf1/CIP1 and K1 levels in p300 depleted HFKs. The results shown are representative of three independent experiments.

Article Snippet: All these constructs were sub-cloned into the XbaI site of pGL-3 basic (Promega), except the 0-luc that was inserted into a blunt-ended Xho1 site of pGL3-basic. p21 Waf1/Cip1 -mut1 luciferase report vector construct was kindly provided by Professor Wei-Guo Zhu . pMT5-p21 Waf1/CIP1 -Flag over-expression vector was purchased from Addgene.

Techniques: Western Blot, shRNA, Stable Transfection, Expressing

Journal: PLoS ONE

Article Title: p300 Alters Keratinocyte Cell Growth and Differentiation through Regulation of p21 Waf1/CIP1

doi: 10.1371/journal.pone.0008369

Figure Lengend Snippet: Stably expressing p300 targeted shRNAs cells were transfected with p21 Waf1/CIP1 promoter luciferase construct. Transfected cells were either kept in low calcium (0.5 mM) or high calcium (1.5 mM) for the indicated times prior to the termination of the experiment (72 hours after transfection). Doxorubicin (0.1 µg/ml) was used as a positive control in this experiment. (A) Depletion of p300 down-regulates p21 Waf1/CIP1 promoter activity after 16 hours of calcium treatment (Mean +/− SE, three independent biological replicates; asterisk (*) p<0.01 relative to relevant control, Student's t test). (B) Real time quantification confirms that depletion of p300 inhibits the transcription of p21 Waf1/CIP1 during differentiation (Mean +/− SE, two independent biological replicated; asterisk (*) p<0.05 relative to relevant control, Student's t test). (C) ChIP assay for p53 and p300 binding to two p53 response elements located in the p21 Waf1/CIP1 promoter in HFKs induced to differentiate by addition of calcium. PCR analysis of DNA precipitated by a p300 or p53 antibodies indicates that p300 only interacts with both p53 response elements within the p21 Waf1/CIP1 promoter after 6 hours calcium treatment. Left panel: p53 binding to the p53 respond elements (RE1 and RE2) increases over a 16 hour periods. Right panel: Depletion of p53 results absence of p53 and p300 at both p53 response sites. Mouse (IgGM) and Rabbit IgG (IgGR) are included as negative control. (D) Western blot of the proteins from the same extracts as in (C).

Article Snippet: All these constructs were sub-cloned into the XbaI site of pGL-3 basic (Promega), except the 0-luc that was inserted into a blunt-ended Xho1 site of pGL3-basic. p21 Waf1/Cip1 -mut1 luciferase report vector construct was kindly provided by Professor Wei-Guo Zhu . pMT5-p21 Waf1/CIP1 -Flag over-expression vector was purchased from Addgene.

Techniques: Stable Transfection, Expressing, Transfection, Luciferase, Construct, Positive Control, Activity Assay, Binding Assay, Negative Control, Western Blot

Journal: PLoS ONE

Article Title: p300 Alters Keratinocyte Cell Growth and Differentiation through Regulation of p21 Waf1/CIP1

doi: 10.1371/journal.pone.0008369

Figure Lengend Snippet: HFKs were transiently tranfected with p21 Waf1/Cip1 targeting siRNA molecules. ( A ) Immunoblotting of the whole cell lysates with p21 Waf'1/CIP1 antibody shows that both siRNA molecules, sip21 Waf'1/CIP1 A and sip21 Waf'1/CIP1 B, were able to knockdown p21 Waf'1/CIP1 protein levels efficiently (∼90%) ( B ) Western blot shows p21 Waf1/CIP1 depletion with sip21 Waf1/CIP1 A and a decreased in K1 levels after 48 hours of differentiation. ( C ) Transfected cells were seeded onto coverslips, incubated with 1.5 mM calcium for 48 hours, and were pulsed with BrdU prior to fixation. BrdU uptake indicates a 90% increased proliferation of p21 Waf1/CIP1 knockdown cells prior to the addition differentiation (t = 0). After 48 hours calcium treatment, around 40% of the p21 Waf1/CIP1 knock-down cells remained BrdU positive. ( D ) H&E staining shows an increase in thickness of epithelia in p21 Waf1/CIP1 depleted cells (upper panel). Immunohistochemisty staining of organotypic raft cultures indicates that the differentiation marker K1 is reduced in p21 Waf1/CIP1 knockdown rafts with low levels of K1 (middle panel). Rafts were pulsed with BrdU for 16 hours prior to harvest and BrdU positive cells counted (lower panel). BrdU positive cells were observed in most parabasal cells. ( E ) Graph represents BrdU uptake expressed as percentage of scrambled control (Mean +/− SE, two independent experiments). (F) Stably expressing p300 targeted shRNA cells were transiently transfected with either 1 µg pMT5-p21 Waf1/CIP1 -Flag vector or pCMV empty vector (negative control) for 24 hours. Transfected cells were then incubated with calcium for 48 hours. Western blot analysis indicates that exogenous expression of p21 Waf1/CIP1 rescued the expression of K1 in p300 depleted cells.

Article Snippet: All these constructs were sub-cloned into the XbaI site of pGL-3 basic (Promega), except the 0-luc that was inserted into a blunt-ended Xho1 site of pGL3-basic. p21 Waf1/Cip1 -mut1 luciferase report vector construct was kindly provided by Professor Wei-Guo Zhu . pMT5-p21 Waf1/CIP1 -Flag over-expression vector was purchased from Addgene.

Techniques: Western Blot, Transfection, Incubation, Staining, Marker, Stable Transfection, Expressing, shRNA, Plasmid Preparation, Negative Control