Journal: Journal of Virology

Article Title: Porcine MKRN1 Modulates the Replication and Pathogenesis of Porcine Circovirus Type 2 by Inducing Capsid Protein Ubiquitination and Degradation

doi: 10.1128/JVI.00100-18

Figure Lengend Snippet: Porcine MKRN1 interaction with PCV2 Cap. (A) Schematic comparison of the functional domains of MKRN1-long (Human-long; GenBank accession no. NP_038474), putative porcine full-size MKRN1 protein (Porcine-long; GenBank accession no. XM_003134608.4), the human MKRN1 variants human-MKRN1-short 1 (Human-short1; GenBank accession no. NP_001138597.1), human MKRN1-short 2 (Human-short2; GenBank accession no. XP_011514299.1, XP_011514300.1), and human MKRN1-short 3 (Human-short3; GenBank accession no. NP_001278592.1), and the putative porcine MKRN1 variant pMKRN1-short (Putative porcine-short; UniProtKB accession no. A0A287ALB7). (B) Molecular expression patterns of MKRN1 in PK-15 cells and ST cells. PK-15 cells and ST cells were lysed by RIPA, and the lysates were subjected to Western blotting with MKRN1 antibodies. (C) The putative porcine MKRN1 variant (pMKRN1-short) interacts with PCV2 Cap protein. PK-15 cells were infected with PCV2 (MOI = 1) for 24 h, and lysates were immunoprecipitated (IP) using an anti-Cap antibody; an anti-MKRN1 antibody was used for immunoblotting. (D, E) Cap interacts with pMKRN1 in transfected cells. HEK293T cells were cotransfected with GFP-Cap and Flag-pMKRN1 expression plasmids or cotransfected with the pEGFP control vector and the Flag-pMKRN1 expression plasmid. The cells transfected with the pEGFP control vector, the GFP-Cap expression plasmid, or the Flag-pMKRN1 expression plasmid alone served as controls. Immunoprecipitation was performed to detect the GFP-Cap interaction with Flag-pMKRN1 using anti-Flag antibodies or anti-GFP antibodies. (F) Endogenous MKRN1 interacts with GFP-Cap. PK-15 cells were transfected with GFP-Cap, cells were lysed for immunoprecipitation with GFP monoclonal antibodies, and anti-MKRN1 antibodies were used for immunoblotting. The cells untransfected with GFP-Cap served as a control. (G) The C terminus (aa 201 to 418) of pMKRN1 is responsible for interacting with Cap. HEK293T cells were transfected with plasmids to express the indicated proteins, an immunoprecipitation assay was performed using anti-Flag monoclonal antibodies, and anti-Cap antibodies were used for immunoblotting. These results were confirmed by three independent experiments.

Article Snippet: Antibodies included monoclonal mouse anti-green fluorescent protein (anti-GFP) antibody (catalog number CSB-MA000051M0m; Cusabio), monoclonal rabbit anti-GFP antibody (catalog number {"type":"entrez-nucleotide","attrs":{"text":"G10362","term_id":"942211","term_text":"G10362"}} G10362 ; Invitrogen), mouse monoclonal anti-Flag (M2) antibody (catalog number F1804; Sigma-Aldrich), rabbit monoclonal anti-Flag antibody (catalog number 701629; Invitrogen), rabbit polyclonal anti-MKRN1 antibody (catalog number A300-990A; Bethyl Laboratories, Inc.), mouse monoclonal anti-ubiquitin antibody (Cell Signaling Technology, 3936), and mouse monoclonal anti-HA tag antibody (catalog number AE008; ABclonal).

Techniques: Functional Assay, Variant Assay, Expressing, Western Blot, Infection, Immunoprecipitation, Transfection, Plasmid Preparation

Journal: Journal of Virology

Article Title: Porcine MKRN1 Modulates the Replication and Pathogenesis of Porcine Circovirus Type 2 by Inducing Capsid Protein Ubiquitination and Degradation

doi: 10.1128/JVI.00100-18

Figure Lengend Snippet: Overexpression of pMKRN1 decreases ORF2 protein levels and inhibits the replication of PCV2. (A) A stable cell line, PK-15pMKRN1, expresses the pMKRN1 protein. PK-15 cells were transfected with linearized pCI-Flag-pMKRN1 or the control vector, pCI-neo, to establish PK-15pMKRN1 and PK-15PCI cells selected by G418, respectively. The cells were analyzed by immunoblotting using an anti-Flag antibody. β-Actin was detected as an internal control. (B) The level of Cap was decreased in cells overexpressing pMKRN1. (Left) The pcDNA-Cap and pEGFP vectors were cotransfected into PK-15, PK-15PCI, or PK-15pMKRN1 cells, and Cap, pMKRN1, and GFP levels were detected by immunoblotting. (Right) The ImageJ program was used to quantify the relative intensities of the bands. **, P < 0.01 versus the Cap level of PK-15 cells. (C and D) Increased pMKRN1 expression is correlated with decreased Cap. (Left) Different doses of pCI-Flag-pMKRN1 plasmids (0, 1, 3, 5 μg) were transfected with a fixed amount of Cap expression vectors into PK-15 cells (C) or ST cells (D), and Western blotting was used to examine Cap expression. GFP was used as a control. (Right) Relative intensities of the bands. *, P < 0.05 versus Cap levels in PK-15 cells or ST cells with 0 μg of pCI-Flag-pMKRN1 transfection; **, P < 0.01 versus Cap levels in PK-15 cells or ST cells with 0 μg of pCI-Flag-pMKRN1 transfection. (E to G) PK-15, PK-15PCI, and PK-15pMKRN1 cells were infected with PCV2 at an MOI of 1 for the indicated periods, and PCV2 Rep, Cap, and ORF3 protein levels were determined by Western blotting (E); the mRNA levels of Cap (F) and PCV2 copy numbers (G) were determined by quantitative PCR. The Cap mRNA levels of PK-15 cells at each time point were defined as 1 in panel F. The data shown are representatives from three independent experiments. *, P < 0.05 versus PCV2-infected PK-15 cells at the same time points; **, P < 0.01 versus PCV2-infected PK-15 cells at the same time points.

Article Snippet: Antibodies included monoclonal mouse anti-green fluorescent protein (anti-GFP) antibody (catalog number CSB-MA000051M0m; Cusabio), monoclonal rabbit anti-GFP antibody (catalog number {"type":"entrez-nucleotide","attrs":{"text":"G10362","term_id":"942211","term_text":"G10362"}} G10362 ; Invitrogen), mouse monoclonal anti-Flag (M2) antibody (catalog number F1804; Sigma-Aldrich), rabbit monoclonal anti-Flag antibody (catalog number 701629; Invitrogen), rabbit polyclonal anti-MKRN1 antibody (catalog number A300-990A; Bethyl Laboratories, Inc.), mouse monoclonal anti-ubiquitin antibody (Cell Signaling Technology, 3936), and mouse monoclonal anti-HA tag antibody (catalog number AE008; ABclonal).

Techniques: Over Expression, Stable Transfection, Transfection, Plasmid Preparation, Western Blot, Expressing, Infection, Real-time Polymerase Chain Reaction

Journal: Journal of Virology

Article Title: Porcine MKRN1 Modulates the Replication and Pathogenesis of Porcine Circovirus Type 2 by Inducing Capsid Protein Ubiquitination and Degradation

doi: 10.1128/JVI.00100-18

Figure Lengend Snippet: pMKRN1 induces Cap ubiquitination, resulting in its degradation via the proteasome pathway. (A to D) pMKRN1 promotes the degradation of Cap via the proteasome pathway. PK-15 cells were transfected with HA-Cap; at 24 h posttransfection, the cells were treated with CHX (100 μg/ml) for another 24 h; and the expression of HA-Cap was tested at the indicated times by immunoblotting (A). 64PKpmkrn1−/− cells were transfected with pCI-Flag-pMKRN1, pcDNA-Cap, and pEGFP, as indicated; the cells were treated with 10 μM MG132 (a proteasome inhibitor), 10 μM LLnL (an MG132 analogue) (B), or 10 μM E64 (a lysosome inhibitor) (C); and the expression of Flag-pMKRN1, Cap, or GFP was analyzed by Western blotting (WB). The lysates were immunoprecipitated with anti-Flag antibodies, and Western blotting was used to detect Cap (D). (E) pMKRN1 induces Cap ubiquitination. 64PKpmkrn1−/− cells were transfected with the indicated expression plasmids, followed by treatment of the cells with MG132 (10 μM) for 6 h. Cap proteins were immunoprecipitated using an anti-Flag antibody, and ubiquitinated proteins were immunoblotted using an anti-HA antibody. (F) The ubiquitination of Cap is lost in pMKRN1 knockout cells. Wild-type PK-15, 2211PKpmkrn1+/+, or 64PKpmkrn1−/− cells were infected with PCV2, cell lysates were immunoprecipitated using anti-Cap antibodies, and polyubiquitinated Cap [Cap(Ub)n] was detected by immunoblotting using anti-ubiquitin antibodies. These results were confirmed in three independent experiments.

Article Snippet: Antibodies included monoclonal mouse anti-green fluorescent protein (anti-GFP) antibody (catalog number CSB-MA000051M0m; Cusabio), monoclonal rabbit anti-GFP antibody (catalog number {"type":"entrez-nucleotide","attrs":{"text":"G10362","term_id":"942211","term_text":"G10362"}} G10362 ; Invitrogen), mouse monoclonal anti-Flag (M2) antibody (catalog number F1804; Sigma-Aldrich), rabbit monoclonal anti-Flag antibody (catalog number 701629; Invitrogen), rabbit polyclonal anti-MKRN1 antibody (catalog number A300-990A; Bethyl Laboratories, Inc.), mouse monoclonal anti-ubiquitin antibody (Cell Signaling Technology, 3936), and mouse monoclonal anti-HA tag antibody (catalog number AE008; ABclonal).

Techniques: Transfection, Expressing, Western Blot, Immunoprecipitation, Knock-Out, Infection

Journal: Journal of Virology

Article Title: Porcine MKRN1 Modulates the Replication and Pathogenesis of Porcine Circovirus Type 2 by Inducing Capsid Protein Ubiquitination and Degradation

doi: 10.1128/JVI.00100-18

Figure Lengend Snippet: PCV2 Cap lysine residues 164, 179, and 191 are the sites of ubiquitination by pMKRN1. (A) Schematic diagram of N- and C-terminal truncation mutants of PCV2 Cap. (B) Mapping the regions of PCV2 Cap that interact with pMKRN1. HEK293T cells were transfected with plasmids to express Flag-pMKRN1 and Cap truncation mutants fused with the GFP tag, and lysates were immunoprecipitated with an anti-GFP antibody, followed by immunoblotting using an anti-MKRN1 antibody, anti-GFP antibody, or anti-β-actin antibody. The middle white space divides the samples that were resolved by two SDS-PAGEs due to the limit of the gel lanes. (C) PCV2 Cap containing amino acid residues 162 to 198 is degraded by pMKRN1. HEK293T cells were transfected with plasmids to express Flag-pMKRN1 and the indicated Cap mutants, and Western blotting was used to detect the levels of Cap mutant expression with anti-HA antibodies. (D to F) K164, K179, and K191 were the ubiquitination sites of the PCV2 Cap protein. The Cap lysine residues at residues 164, 179, and 191 were replaced with alanine (D). HEK293T cells were transfected to express Flag-pMKRN1 and the indicated Cap lysine mutants, Western blotting was used to detect the expression levels of Cap or mutants using anti-Cap antibodies (E), and the ubiquitination of Cap mutants was detected (F). These results were confirmed in three independent experiments.

Article Snippet: Antibodies included monoclonal mouse anti-green fluorescent protein (anti-GFP) antibody (catalog number CSB-MA000051M0m; Cusabio), monoclonal rabbit anti-GFP antibody (catalog number {"type":"entrez-nucleotide","attrs":{"text":"G10362","term_id":"942211","term_text":"G10362"}} G10362 ; Invitrogen), mouse monoclonal anti-Flag (M2) antibody (catalog number F1804; Sigma-Aldrich), rabbit monoclonal anti-Flag antibody (catalog number 701629; Invitrogen), rabbit polyclonal anti-MKRN1 antibody (catalog number A300-990A; Bethyl Laboratories, Inc.), mouse monoclonal anti-ubiquitin antibody (Cell Signaling Technology, 3936), and mouse monoclonal anti-HA tag antibody (catalog number AE008; ABclonal).

Techniques: Transfection, Immunoprecipitation, Western Blot, Mutagenesis, Expressing

Journal: Nature Communications

Article Title: Ufd2p synthesizes branched ubiquitin chains to promote the degradation of substrates modified with atypical chains

doi: 10.1038/ncomms14274

Figure Lengend Snippet: ( a ) Schematic representation of the stepwise ubiquitination experiment. ( b ) Ufd2p promotes ubiquitination independent of Ufd4p. As illustrated in ( a ), either Ub-V-GFP or Ub2-GFP synthesized by Ufd4p was subjected to immunoprecipitation (IP) with the anti-GFP antibody. E1, E2, ubiquitin, ATP and Ufd2p were then added to the mixture. The reaction products were detected using the anti-GFP antibody; the Ufd2p and Ufd4p proteins were detected using the anti-Ufd2 and anti-Xpress antibodies, respectively. Input was from step I reaction products. To exclude the influence of Ufd4p in the second step, we used two controls: one contained Ufd4p alone (set a), and the other did not include Ub-V-GFP in step I (set b). ( c ) Ufd2p transfers one ubiquitin to the proximal ubiquitin of Ub2-GFP to form branched ubiquitin chains on the substrate. As illustrated in ( a ), K29R Ub, K29/48R Ub or wild-type Ub was used in step I, and wild-type Ub, K48R Ub or methylated Ub was used in step II. Input was from step I reaction products. ( d ) As in ( c ), except that the ubiquitin mutants in step I were replaced by K29R-Wt di-Ub, K29R-K48R di-Ub, K29/48R-Wt di-Ub, or K29/48R-K48R di-Ub. ( e ) As in ( c ), except that the ubiquitin mutants in step I were replaced with K29R Ub, K29R-Wt di-Ub and K29R-Wt-Wt tri-Ub. ( f ) As in ( e ), K29R-Wt-Wt tri-Ub was used in step I and wild-type Ub, K48R Ub or methylated Ub was used in step II, but the incubation time was 2 h. ( g ) A model for the functional role of Ufd2p and Ufd4p in the stepwise ubiquitination assay. First, Ufd4p assembles K29-linked ubiquitin chains on Ub-V-GFP; Ufd2p then catalyses multi-monoubiquitination on Ub-V-GFP modified with K29-linked ubiquitin chains to form branched ubiquitin chains. See also .

Article Snippet: The FLAG (1:2,000, M20008L), GST (1:2,000, M20007L), Myc (1:1,000, M20002M) and mouse GFP (1:1,000, M20004M) antibodies were purchased from Abmart (Shanghai, China), and the Xpress (1:2,000, R910-25) antibody was purchased from Invitrogen (Carlsbad, CA).

Techniques: Synthesized, Immunoprecipitation, Methylation, Incubation, Functional Assay, Ubiquitin Assay, Modification

Journal: Nature Communications

Article Title: Ufd2p synthesizes branched ubiquitin chains to promote the degradation of substrates modified with atypical chains

doi: 10.1038/ncomms14274

Figure Lengend Snippet: ( a ) Schematic representation of the method used to monitor the synthesis of branched ubiquitin chains on Ub-V-GFP. ( b ) In vitro validation of the method used to monitor branched ubiquitin chains on Ub-V-GFP. As illustrated in ( a ), Ub-V-GFP was ubiquitinated by Ufd4p or Ufd4p-Ufd2p. Reaction products were treated with the TEV enzyme and analysed by immunoblotting with the anti-GFP antibody. ( c ) Ub-V-GFP was modified with branched ubiquitin chains in yeast cells. Ub-V-GFP and its modified forms were expressed in a wild-type strain expressing FLAG-Ub 53TEV and Myc-Ub 64TEV/FLAG and purified under denaturing conditions in the presence or absence of the proteasome inhibitor MG132. The pull-down products were treated with the TEV enzyme and analysed by immunoblotting with anti-FLAG and anti-GFP antibodies. ( d ) The addition of branched ubiquitin chains to Ub-V-GFP is mainly dependent on UFD2 in vivo . Ub-V-GFP and its modified forms were purified from either a wild-type or ufd2Δ strain expressing both FLAG-Ub 53TEV and Myc-Ub 64TEV/FLAG in the presence of MG132 under denaturing conditions. The pull-down products were treated with the TEV enzyme and analysed by immunoblotting with the anti-FLAG and anti-GFP antibodies. ( e ) K29 and K48 on Ub-V-GFP are required for the formation of branched ubiquitin chains in yeast cells. Wild-type, K29R, K48R or K29/48R Ub-V-GFP and their modified forms were purified from a wild-type strain expressing FLAG-Ub 53TEV and Myc-Ub 64TEV/FLAG . The pull-down products were treated with the TEV enzyme and analysed by immunoblotting with the anti-FLAG and anti-GFP antibodies.

Article Snippet: The FLAG (1:2,000, M20008L), GST (1:2,000, M20007L), Myc (1:1,000, M20002M) and mouse GFP (1:1,000, M20004M) antibodies were purchased from Abmart (Shanghai, China), and the Xpress (1:2,000, R910-25) antibody was purchased from Invitrogen (Carlsbad, CA).

Techniques: In Vitro, Western Blot, Modification, Expressing, Purification, In Vivo

Journal: Nature Communications

Article Title: Ufd2p synthesizes branched ubiquitin chains to promote the degradation of substrates modified with atypical chains

doi: 10.1038/ncomms14274

Figure Lengend Snippet: ( a ) Interactions between the substrate and Ufd2p. Biotinylated Ufd2p was immobilized on streptavidin-coated beads, which were then incubated with GFP, Ub-V-GFP or Ubn-GFP that was synthesized by Ufd4p. The precipitated proteins were analysed by immunoblotting with the indicated antibodies. ( b ) Quantification of the relative binding affinity between Ufd2p and Ubn-GFP. Relative amounts of Ub1-4-GFP were quantified using Odyssey software and compared with the input. Error bars denote the s.e.m. of three independent replicates. * indicated P <0.05, ** indicated P <0.01. ( c ) A summary of the Ufd2p variants used to map the Ubn-GFP binding motif, which include Ufd2p (amino acids 1–961), Ufd2p CF1 (amino acids 519–961), Ufd2p CF2 (amino acids 760–961), Ufd2p CF3 (amino acids 879–961), Ufd2p MFD1(amino acids Δ111-879), Ufd2p MFD2(amino acids Δ75-879), Ufd2p MFD3(amino acids Δ55-879), Ufd2p MFD4(amino acids Δ55-75&111-879), Ufd2p NHD1(amino acids Δ35-55) and Ufd2p MFD2(amino acids Δ75-111). ( d ) Amino acids 35–55 and 75–111 of Ufd2p are necessary to bind to Ubn-GFP. GST-Ufd2p and its variants were purified and used to pull down Ubn-GFP, which was synthesized by Ufd4p; GST was used as a control. Asterisks in the bottom panel indicate GST products cleaved from the fused proteins. ( e ) Ufd2p binds to Ubn-GFP via its two N-terminal loops. GST-Ufd2p and GST-Ufd2p NLM (E51A K52A L53A D54A K55A E105A N106A M109A N110A) were purified and used to pull down Ubn-GFP. Asterisks in the bottom panel indicate GST products cleaved from GST-Ufd2p NLM. ( f ) SPR sensorgrams for the binding of GST-Ufd2p to K29-linked Ub2-GFP. A series of two-fold GST-Ufd2p dilutions was applied to the K29-linked Ub2-GFP surface (GFP served as a control). ( g ) SPR sensorgrams for the binding of GST-Ufd2p NLM to K29-linked Ub2-GFP. ( h ) The interaction between Ufd2p and Ubn-GFP is necessary for the E4 activity of Ufd2p. Ufd2p or Ufd2p NLM was incubated with E1, E2, Ufd4p, ATP, ubiquitin and Ub-V-GFP and detected using the anti-GFP antibody. See also .

Article Snippet: The FLAG (1:2,000, M20008L), GST (1:2,000, M20007L), Myc (1:1,000, M20002M) and mouse GFP (1:1,000, M20004M) antibodies were purchased from Abmart (Shanghai, China), and the Xpress (1:2,000, R910-25) antibody was purchased from Invitrogen (Carlsbad, CA).

Techniques: Incubation, Synthesized, Western Blot, Binding Assay, Software, Purification, Activity Assay

Journal: Nature Communications

Article Title: Ufd2p synthesizes branched ubiquitin chains to promote the degradation of substrates modified with atypical chains

doi: 10.1038/ncomms14274

Figure Lengend Snippet: ( a ) Rad23p prefers to bind to branched ubiquitin chains on Ub-V-GFP over Ub-V-GFP with K29-linked ubiquitin chains or unmodified Ub-V-GFP. Ub-V-GFP, Ub-V-GFP with K29-linked ubiquitin chains (synthesized via Ufd4p) or Ub-V-GFP with branched ubiquitin chains (synthesized via Udf4p-Ufd2p) immobilized on protein A-sepharose beads were used to immunoprecipitate GST-Rad23p. The precipitated proteins were analysed by immunoblotting with the indicated antibodies. The asterisks indicate GST products cleaved from GST-Rad23p. ( b ) SPR sensorgrams for the binding of GST-Rad23p to Ub-V-GFP. A series of two-fold GST-Rad23p dilutions was applied to the Ub-V-GFP surface (GFP served as a control). ( c ) SPR sensorgrams for the binding of GST-Rad23p to K29-linked Ub2-GFP. ( d ) SPR sensorgrams for the binding of GST-Rad23p to branched Ub3-GFP. ( e ) Proteasome adaptor proteins such as Rad23p fail to recognize Ufd2p NLM-mediated ubiquitination products. Either wild-type Ufd2p-mediated ubiquitination products or Ufd2p NLM-mediated ubiquitination products immobilized on protein A-sepharose beads were used to immunoprecipitate GST-Rad23p. Ubn-GFP modified by Ufd2p NLM could not immunoprecipitate GST-Rad23p efficiently. Asterisks indicate nonspecific background signals from the IgG chains. ( f ) The ubiquitin chain linkage switch is necessary for substrate degradation. Protein expression in wild-type or ufd2Δ strains expressing Ub-V-GFP under the control of a galactose-induced promoter was stopped upon transfer to 2% glucose. The ufd2Δ strains harboured empty vector, UFD2 or UFD2 NLM under the control of a galactose-induced promoter. The degradation of Ub-V-GFP over time was analysed by immunoblotting. Pgk1p served as a loading control. ( g ) Quantification of relative Ub-V-GFP levels in ( f ) using Odyssey software. Error bars denote the s.e.m. of three independent replicates. See also .

Article Snippet: The FLAG (1:2,000, M20008L), GST (1:2,000, M20007L), Myc (1:1,000, M20002M) and mouse GFP (1:1,000, M20004M) antibodies were purchased from Abmart (Shanghai, China), and the Xpress (1:2,000, R910-25) antibody was purchased from Invitrogen (Carlsbad, CA).

Techniques: Synthesized, Western Blot, Binding Assay, Modification, Expressing, Plasmid Preparation, Software