Journal: The Journal of Biological Chemistry

Article Title: Molecular basis of cullin-3 (Cul3) ubiquitin ligase subversion by vaccinia virus protein A55

doi: 10.1074/jbc.RA118.006561

Figure Lengend Snippet: A55 directly binds to cullin-3 via its N-terminal BB domain. A–C, representative immunoblots following immunoprecipitation ( IP ) of cleared lysates from HEK293T–REx cell lines ( A ) expressing empty vector ( EV ), B14–TAP ( B14 ), or TAP–A55 ( A55 ), the TAP tag comprising STREP and FLAG epitopes; ( B ) expressing B14–TAP or TAP–A55 and transfected with plasmids encoding myc-Cul3 or myc-Cul5; ( C ) expressing EV, B14–TAP, TAP–A55, TAP–A55BB, or TAP–A55 Kelch. Cells were lysed in Nonidet P-40 ( A and C ) or RIPA buffer ( B ). Immunoprecipitates were subjected to SDS-PAGE and immunoblotting. A and C, FLAG IP and immunoblotting for co-IP of endogenous Cul3. B, Myc IP and immunoblotting for co-IP of TAP-tagged B14 or A55. Input, cleared lysate. Data shown are representative of at least three independent experiments. Signals arising from the light chain (*) or heavy chain (**) of the antibody used for IP are marked. D, SEC-MALS analyses showing the SEC elution profiles ( thin lines ) and molecular mass distribution ( thick lines ) across the elution peaks for A55BB ( peak 1, green , theoretical molecular mass 30 kDa and observed molecular mass 60 kDa), Cul3NΔ22 ( peak 2, blue, theoretical molecular mass 46 kDa and observed molecular mass 45 kDa), and A55BB and Cul3NΔ22 together ( peak 3, red, theoretical molecular mass 76 kDa and observed molecular mass 141 kDa) when eluting from a Superdex 200 10/300 GL column. Peak 4 is assumed to be excess Cul3NΔ22. E, Coomassie-stained SDS-PAGE analysis of peaks 1–3 from D .

Article Snippet: The mammalian expression vectors pcDNA-myc- CUL3 (19893) and pcDNA-myc- CUL5 (19895) were purchased from Addgene.

Techniques: Western Blot, Immunoprecipitation, Expressing, Plasmid Preparation, Transfection, SDS Page, Co-Immunoprecipitation Assay, Staining

Journal: The Journal of Biological Chemistry

Article Title: Molecular basis of cullin-3 (Cul3) ubiquitin ligase subversion by vaccinia virus protein A55

doi: 10.1074/jbc.RA118.006561

Figure Lengend Snippet: ITC studies show that A55 binds to Cul3 with nanomolar to sub-nanomolar affinity. A–D, representative ITC titration curves showing interactions between A55BB and Cul3NΔ22 ( A ) or Cul3N ( B ) and between KLHL3, a human BTB related to A55, and Cul3NΔ22 ( C ) or Cul3N ( D ). The top figure in each panel shows the baseline-corrected differential power ( DP ) versus time. The bottom figure of each panel is the normalized binding curve showing integrated changes in enthalpy (Δ H ) against molar ratio. The corresponding dissociation constant ( K D ), number of binding sites ( N ), enthalpy change (Δ H ), and change in Gibbs free energy (Δ G ) for each representative experiment are shown. All experiments were performed at least twice independently.

Article Snippet: The mammalian expression vectors pcDNA-myc- CUL3 (19893) and pcDNA-myc- CUL5 (19895) were purchased from Addgene.

Techniques: Titration, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Molecular basis of cullin-3 (Cul3) ubiquitin ligase subversion by vaccinia virus protein A55

doi: 10.1074/jbc.RA118.006561

Figure Lengend Snippet: Comparison of the dissociation constants ( K D ), enthalpic change (Δ H ), and entropic change ( T Δ S ) for BB–Cul3 interaction Experiments for this study were performed at least twice, and mean ± S.E. is shown.

Article Snippet: The mammalian expression vectors pcDNA-myc- CUL3 (19893) and pcDNA-myc- CUL5 (19895) were purchased from Addgene.

Techniques: Comparison

Journal: The Journal of Biological Chemistry

Article Title: Molecular basis of cullin-3 (Cul3) ubiquitin ligase subversion by vaccinia virus protein A55

doi: 10.1074/jbc.RA118.006561

Figure Lengend Snippet: A55 and cellular BB domains share conserved modes of dimerization and Cul3 binding. A, structure of the A55/Cul3NΔ22 heterodimer in the asymmetric unit as ribbon diagram . Cul3 is in cyan and the three domains of A55 (BTB, three-box and BACK) are in green, orange, and gray , respectively. Helices α1–α12 from A55 are labeled in black with the exception of α10, which is hidden behind α9 in the picture. Helices α1–α16 from Cul3 are labeled in red. B, A55/Cul3 dimer formed by applying crystallographic 2-fold symmetry. C, overlay of three BB/Cul3 complex structures (KLHL3/Cul3, PDB code 4HXI ; KLHL11/Cul3, PDB code 4APF ; and A55/Cul3). The structures are aligned to the Cul3 part of the A55/Cul3 complex only. A55, KLHL3, KLHL11, and Cul3 are in green, purple, magenta, and cyan , respectively, and the three sub-domains are marked. Additional helices at the C terminus of the KLHL11 BACK domain are shown as semi-transparent helices. D, comparison of the dimers formed by A55, KLHL3, and KLHL11 BB domains, colored as in C .

Article Snippet: The mammalian expression vectors pcDNA-myc- CUL3 (19893) and pcDNA-myc- CUL5 (19895) were purchased from Addgene.

Techniques: Binding Assay, Labeling, Comparison

Journal: The Journal of Biological Chemistry

Article Title: Molecular basis of cullin-3 (Cul3) ubiquitin ligase subversion by vaccinia virus protein A55

doi: 10.1074/jbc.RA118.006561

Figure Lengend Snippet: Structure-based sequence alignment of the A55, KLHL3, and KLHL11 BB domains. Columns are shaded based on amino acid similarity. Secondary structural elements for A55 are shown above the aligned sequences and colored as in A . Residues at the A55/Cul3 interface are underlined in blue . Residues selected for subsequent mutagenesis studies of A55 are marked by stars at bottom: the two conserved sites (Phe-54 and Asp-56) are marked by purple stars , and the nonconserved site (Ile-48) is marked by a red star . Residues from KLHL11 that are involved in Cul3–NTE binding are marked by magenta triangles .

Article Snippet: The mammalian expression vectors pcDNA-myc- CUL3 (19893) and pcDNA-myc- CUL5 (19895) were purchased from Addgene.

Techniques: Sequencing, Mutagenesis, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Molecular basis of cullin-3 (Cul3) ubiquitin ligase subversion by vaccinia virus protein A55

doi: 10.1074/jbc.RA118.006561

Figure Lengend Snippet: Conserved and nonconserved interactions at the interface between A55 and Cul3. A, A55BB/Cul3NΔ22 complex structure with two key Cul3-binding sites in the BTB domain boxed in black (enlarged in B–F ) and red (enlarged in G ). B, structural overlay of the φ X (D/E) motifs from A55, KLHL3, KLHL11, and SPOP. C–F, surface of Cul3 colored by residue hydrophobicity from yellow (hydrophobic) to white (polar) . Hydrophobic binding pockets are shown for Phe-54 of A55, Met-83 of KLHL3, Phe-130 of KLHL11, and Met-233 of SPOP, which are equivalent to the φ residue of the φ X (D/E) motif, and for Ile-48 of A55 and its equivalent residues Ala-77, Pro-124, and Ala-227 in KLHL3, KLHL11, and SPOP, respectively. G, overlay of the hydrogen bond formed between Tyr-125 of Cul3 and Asp-99 of A55 with equivalent residues Ser-128, Asp-181, and Asp-278 in KLHL3, KLHL11, and SPOP, respectively.

Article Snippet: The mammalian expression vectors pcDNA-myc- CUL3 (19893) and pcDNA-myc- CUL5 (19895) were purchased from Addgene.

Techniques: Binding Assay, Residue

Journal: The Journal of Biological Chemistry

Article Title: Molecular basis of cullin-3 (Cul3) ubiquitin ligase subversion by vaccinia virus protein A55

doi: 10.1074/jbc.RA118.006561

Figure Lengend Snippet: I48E mutation significantly impairs A55 binding to Cul3. A, representative thermal melt curves of WT A55BB and mutants F54E, D99A, I48A, and I48E from DSF studies. Curves are offset along the vertical axis for clarity. All experiments were performed in triplicate. B, comparison of the melting temperatures for WT A55BB ( green ), F54E ( red ), D99A ( blue ), I48A ( orange ), and I48E ( black ) mutants. Upper and lower panels display T m values for the first and second melting events, respectively. Error bars show the standard errors of the mean from experiments performed in triplicate. C–J, representative ITC titration curves showing binding of A55BB mutants F54E ( C and D ), D99A ( E and F ), I48A ( G and H ), and I48E ( I and J ) to Cul3NΔ22 and Cul3N, respectively. Integrated changes in enthalpy (Δ H ) are plotted against molar ratio of titrant. The corresponding dissociation constant ( K D ), number of binding sites ( N ), enthalpy change (Δ H ), and change in Gibbs free energy (Δ G ) for each representative experiment are shown. All experiments were performed at least twice independently. Raw data for C–J are shown in Fig. S7 .

Article Snippet: The mammalian expression vectors pcDNA-myc- CUL3 (19893) and pcDNA-myc- CUL5 (19895) were purchased from Addgene.

Techniques: Mutagenesis, Binding Assay, Comparison, Titration

Journal: The Journal of Biological Chemistry

Article Title: Molecular basis of cullin-3 (Cul3) ubiquitin ligase subversion by vaccinia virus protein A55

doi: 10.1074/jbc.RA118.006561

Figure Lengend Snippet: Comparison of the dissociation constants ( K D ) for WT and mutant A55BB for Cul3 Experiments were performed n times and the mean ± S.E. is shown.

Article Snippet: The mammalian expression vectors pcDNA-myc- CUL3 (19893) and pcDNA-myc- CUL5 (19895) were purchased from Addgene.

Techniques: Comparison, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Molecular basis of cullin-3 (Cul3) ubiquitin ligase subversion by vaccinia virus protein A55

doi: 10.1074/jbc.RA118.006561

Figure Lengend Snippet: Cul3-binding residues of A55 are conserved across orthopoxvirus orthologues but not in VACV paralogues C2 and F3. Multiple sequence alignment of the A55 BB domains against its orthologues from selected poxviruses and two other VACV BTB–Kelch proteins, C2 and F3. Columns are shaded based on amino acid similarity. Secondary structural elements for A55 are shown above the aligned sequences and colored as in A . Residues at the A55/Cul3 interface are underlined in blue . Residues aligned with A55-Ile-48 and the φ X (D/E) motif are boxed in red .

Article Snippet: The mammalian expression vectors pcDNA-myc- CUL3 (19893) and pcDNA-myc- CUL5 (19895) were purchased from Addgene.

Techniques: Binding Assay, Sequencing

Journal: Cells

Article Title: Potassium Effects on NCC Are Attenuated during Inhibition of Cullin E3–Ubiquitin Ligases

doi: 10.3390/cells11010095

Figure Lengend Snippet: High dietary K + supplementation effects on NCC phosphorylation is attenuated in Cul3-Δ9 mice. ( A ) Representative immunoblots of pNCC, NCC, and actin in kidney homogenates of control and Cul3-Δ9 mice that were on either normal diet (control, n = 5, vs. Cul3-Δ9, n = 6) or high K + diet (control, n = 5, vs. Cul3-Δ9, n = 5). Molecular weight markers (kDa) are shown on the right. ( B ) Summarized relative protein abundance data. Each dot represents value from an individual mouse and bars represents mean ± SEM. * indicates p < 0.05 relative to mice that were on control diet in their respective group.

Article Snippet: Primary antibodies used in immunoblotting include phosphorylated NCC (pT58) [ ], NCC (SPC-402D, StressMarq), pSPAK/OSR1 (07-2273, Millipore), SPAK/OSR1 [ ], Cul1 (sc-17775, Santa Cruz), Cul2 (sc-166506, Santa Cruz), Cul3 (sc-166110, Santa Cruz), Cul4 (sc-377188, Santa Cruz), Cul5 (sc-373822, Santa Cruz), CSN5/Jab1 (sc-13157, Santa Cruz), NAE (sc-390002, Santa Cruz), and Proteasome 20S (ab3325, Abcam).

Techniques: Phospho-proteomics, Western Blot, Control, Molecular Weight, Quantitative Proteomics

Journal: Cells

Article Title: Potassium Effects on NCC Are Attenuated during Inhibition of Cullin E3–Ubiquitin Ligases

doi: 10.3390/cells11010095

Figure Lengend Snippet: Pharmacological inhibition of cullin neddylation increases NCC phosphorylation in isolated renal tubules ex vivo. ( A ) Representative immunoblots of Cul1, Cul3, Cul4, Cul5, pSPAK, SPAK, pNCC, NCC, and P20s in isolated renal tubules that were incubated with either vehicle or the cullin inhibitor (MLN4924; 0.5 uM) for various time points (1, 2, or 4 h) ex vivo. Molecular weight markers (kDa) are shown on the right. ( B ) Summarized relative protein abundance data. Each dot represents value from an individual well of independent tubules and each bar represents mean ± SEM obtained from two independent experiments ( n = 6). * indicates p < 0.05 relative to 3.5 mM condition.

Article Snippet: Primary antibodies used in immunoblotting include phosphorylated NCC (pT58) [ ], NCC (SPC-402D, StressMarq), pSPAK/OSR1 (07-2273, Millipore), SPAK/OSR1 [ ], Cul1 (sc-17775, Santa Cruz), Cul2 (sc-166506, Santa Cruz), Cul3 (sc-166110, Santa Cruz), Cul4 (sc-377188, Santa Cruz), Cul5 (sc-373822, Santa Cruz), CSN5/Jab1 (sc-13157, Santa Cruz), NAE (sc-390002, Santa Cruz), and Proteasome 20S (ab3325, Abcam).

Techniques: Inhibition, Phospho-proteomics, Isolation, Ex Vivo, Western Blot, Incubation, Molecular Weight, Quantitative Proteomics

Journal: Journal of Virology

Article Title: Rotavirus NSP1 Associates with Components of the Cullin RING Ligase Family of E3 Ubiquitin Ligases

doi: 10.1128/JVI.00704-16

Figure Lengend Snippet: Full-length HaloTag-NSP1 proteins associate with endogenous (A and B) and exogenously expressed (C and D) Cul1 and Cul3. (A and B) 293T cells were transfected with a plasmid encoding one of the HaloTag-NSP1 proteins or with the HaloTag vector alone as indicated and harvested at 24 h p.t. (A) or medium was removed from the cells and replaced with fresh medium containing 20 μM the proteasome inhibitor MG132 at 8 h p.t., and the cells were incubated for an additional 16 h and then harvested (B). (C and D) 293T cells were cotransfected with a plasmid encoding one of the HaloTag-NSP1 proteins and HA-tagged Cul3 (C) or HA-tagged Cul1 (D). For all the panels, the HaloTag-NSP1 proteins, along with their interacting partners, were captured using HaloLink resin. Proteins in the WCL and HaloTag pulldown (PD) were resolved by SDS-PAGE and analyzed by immunoblotting for Halo, Cul3, Cul1, IRF3, and β-TrCP. Actin or GAPDH was used as a loading control. The upper band of the Cul1 triplet and the Cul3 doublet represents the Nedd8-conjugated form of Cul1 and Cul3, respectively. The blots are representative of the results of three independent experiments.

Article Snippet: To assess the binding between NSP1(SA11-4F) and Cul3, amylose resin (NEB) was incubated at RT overnight with 1% BSA to block nonspecific binding.

Techniques: Transfection, Plasmid Preparation, Incubation, SDS Page, Western Blot

Journal: Journal of Virology

Article Title: Rotavirus NSP1 Associates with Components of the Cullin RING Ligase Family of E3 Ubiquitin Ligases

doi: 10.1128/JVI.00704-16

Figure Lengend Snippet: HaloTag-NSP1 proteins associate with dominant-negative (DN) Cul3 and Cul1 mutants. 293T cells were cotransfected with a plasmid encoding one of the HaloTag-NSP1 proteins or the HaloTag vector alone and Flag-tagged DNCul3 (A) or DNCul1 (B). Cells were harvested at 48 h p.t., and the HaloTag fusion proteins, along with their interacting partners, were captured using HaloLink resin. Proteins in the WCL and HaloTag PD were resolved by SDS-PAGE and analyzed by immunoblotting for Halo, Flag, and GAPDH (loading control). The blots are representative of the results of three independent experiments.

Article Snippet: To assess the binding between NSP1(SA11-4F) and Cul3, amylose resin (NEB) was incubated at RT overnight with 1% BSA to block nonspecific binding.

Techniques: Dominant Negative Mutation, Plasmid Preparation, SDS Page, Western Blot

Journal: Journal of Virology

Article Title: Rotavirus NSP1 Associates with Components of the Cullin RING Ligase Family of E3 Ubiquitin Ligases

doi: 10.1128/JVI.00704-16

Figure Lengend Snippet: Recombinant NSP1 directly interacts with Cul3. MBP-NSP1(SA11-4F) (750 ng) was immobilized on amylose resin by incubating for 1 h at 4°C. Recombinant His-Cul3-Rbx1 protein was incubated with (+) or without (−) MBP-NSP1(SA11-4F) for 1 h at 4°C. In the reciprocal experiment, 20 μg of recombinant His-Cul3-Rbx1 was immobilized on cobalt resin by incubating for 1 h at 4°C. Recombinant MBP-NSP1 (SA11-4F) protein was incubated with (+) or without (−) His-Cul3-Rbx1for 1 h at 4°C. After incubation, bound proteins were eluted from the resin using SDS loading buffer and analyzed by immunoblotting for Cul3 and the viral protein NSP1 (SA11-4F). The same protein concentrations used in binding assays were resolved by SDS-PAGE and immunoblotted for NSP1 and Cul3 as shown (Input). The blots are representative of the results of three independent experiments.

Article Snippet: To assess the binding between NSP1(SA11-4F) and Cul3, amylose resin (NEB) was incubated at RT overnight with 1% BSA to block nonspecific binding.

Techniques: Recombinant, Incubation, Western Blot, Binding Assay, SDS Page

Journal: Journal of Virology

Article Title: Rotavirus NSP1 Associates with Components of the Cullin RING Ligase Family of E3 Ubiquitin Ligases

doi: 10.1128/JVI.00704-16

Figure Lengend Snippet: Rotavirus infection does not alter the cellular level of Cul3 or Cul1. MA104 cells were mock infected or infected with rotavirus strain SA11-4F, SA11-5S, OSU, WI61, UK, or DS-1 (MOI = 5). Cells were harvested at 10 h p.i., and total cellular protein was resolved by SDS-PAGE, followed by immunoblotting for endogenous Cul3, Cul1, IRF3, β-TrCP, and GAPDH proteins. An antibody to detect the viral protein VP6 was used to verify that cells were infected. The blots are representative of the results of three independent experiments.

Article Snippet: To assess the binding between NSP1(SA11-4F) and Cul3, amylose resin (NEB) was incubated at RT overnight with 1% BSA to block nonspecific binding.

Techniques: Infection, SDS Page, Western Blot

Journal: Journal of Virology

Article Title: Rotavirus NSP1 Associates with Components of the Cullin RING Ligase Family of E3 Ubiquitin Ligases

doi: 10.1128/JVI.00704-16

Figure Lengend Snippet: Rotavirus NSP1 associates with Cul3 during viral infection. 293T cells were either mock infected or infected with the indicated strains of rotavirus (MOI = 5) and harvested at 10 h p.i. The cell lysates were incubated with Cul3 antibodies cross-linked to protein A agarose for 1 h at 4°C. The protein complexes that immunoprecipitated with Cul3 were eluted from the protein A agarose. Proteins in the WCL and immunoprecipitation elution were resolved by SDS-PAGE, followed by immunoblot analysis using antibodies specific for Cul3 and individual NSP1 proteins. GAPDH was used as a loading control for WCL fractions. The blots are representative of the results of two independent experiments.

Article Snippet: To assess the binding between NSP1(SA11-4F) and Cul3, amylose resin (NEB) was incubated at RT overnight with 1% BSA to block nonspecific binding.

Techniques: Infection, Incubation, Immunoprecipitation, SDS Page, Western Blot

Journal: Journal of Virology

Article Title: Rotavirus NSP1 Associates with Components of the Cullin RING Ligase Family of E3 Ubiquitin Ligases

doi: 10.1128/JVI.00704-16

Figure Lengend Snippet: Knockdown of Cul1 or Cul3 by siRNA does not prevent NSP1-induced degradation of IRF3 and β-TrCP. (A) MA104 cells were transfected twice (24 and 48 h postseeding) with no siRNA (None), control nontarget siRNA (siNT), or siRNA targeting Cul3 (siCul3). At 62 h p.t., the cells were mock infected or infected with rotavirus strain SA11-4F, SA11-5S, OSU, WI61, UK, or DS-1 at an MOI of 5. (B) MA104 cells were transfected twice (24 and 48 h postseeding) with no siRNA (None), control nontarget siRNA (siNT), or siRNA targeting Cul1 (siCul1). At 62 h p.t., the cells were mock infected or infected with rotavirus strain SA11-4F, SA11-5S, OSU, or UK at an MOI of 5. For both panels, cells were harvested at 10 h p.i., and total cellular proteins were resolved by SDS-PAGE, followed by quantitative immunoblot analysis for endogenous Cul1, Cul3, IRF3, β-TrCP, and GAPDH (loading control). The band intensities were normalized to the loading control (GAPDH) and expressed as a percentage of those of the proteins in the siNT-transfected cells and mock-infected cells, which were set at 100%. An antibody to detect the viral protein VP6 was used to verify that cells were infected. The blots are representative of the results of three independent experiments.

Article Snippet: To assess the binding between NSP1(SA11-4F) and Cul3, amylose resin (NEB) was incubated at RT overnight with 1% BSA to block nonspecific binding.

Techniques: Transfection, Infection, SDS Page, Western Blot

Journal: Journal of Virology

Article Title: Rotavirus NSP1 Associates with Components of the Cullin RING Ligase Family of E3 Ubiquitin Ligases

doi: 10.1128/JVI.00704-16

Figure Lengend Snippet: A DN Cul3 mutant does not affect the ability of NSP1 to induce degradation of target proteins. Plasmids encoding one of the HaloTag-NSP1 proteins or the HaloTag vector alone were cotransfected into 293T cells with Flag-tagged Cul3 (wild type) or DNCul3. Cells were harvested at 24 h p.t., and total cellular protein was resolved by SDS-PAGE and analyzed by immunoblotting for HaloTag and Flag-tagged proteins, as well as endogenous IRF3, β-TrCP, and GAPDH (loading control). IRF3 and β-TrCP protein levels were quantified using the Odyssey infrared imaging system. The band intensities were normalized to the loading control (GAPDH) and expressed as a percentage of the protein in HaloTag empty-vector (Vector)-transfected cells, which was set at 100% (denoted by the dashed lines). The mean values from three experiments are shown, and the error bars represent the standard errors of the mean (SEM).

Article Snippet: To assess the binding between NSP1(SA11-4F) and Cul3, amylose resin (NEB) was incubated at RT overnight with 1% BSA to block nonspecific binding.

Techniques: Mutagenesis, Plasmid Preparation, SDS Page, Western Blot, Imaging, Transfection