Journal: The Journal of Biological Chemistry

Article Title: Redirecting the specificity of tripartite motif containing-21 scaffolds using a novel discovery and design approach

doi: 10.1016/j.jbc.2023.105381

Figure Lengend Snippet: Design and characterization of TRIM21–Tn3 fusion proteins. A , linear representation of the natural TRIM21 protein ( top ) and an engineered TRIM21–Tn3 fusion protein ( bottom ). To construct TRIM21–Tn3 fusion proteins, Tn3 domains were fused to the C terminus of TRIM21ΔPRYSPRY (residues 1–286) via a flexible 15-amino acid (Gly 4 Ser) 3 linker. B , schematic depicting targeted protein degradation (TPD) of a protein of interest (POI) using an engineered dimeric TRIM21–Tn3. C , equilibrium biolayer interferometry (BLI)–based titrations and corresponding equilibrium K D values of TRIM21–HR4, HR4–Fc, and TRIM21 binding to the immobilized extracellular domain of HER4 (residues 26–649). D , flow cytometry–based binding titrations of TRIM21–HR4, HR4–Fc, and TRIM21 to HER4-expressing AD-293 cells and corresponding equilibrium K D values. Error bars represent mean ± SD (n = 3). HER4, human epidermal growth factor receptor 4; TRIM21, tripartite motif containing-21.

Article Snippet: To construct pOE-TRIM21, a gene fragment (Twist Bioscience) encoding full-length human TRIM21 (residues 1–475) was cloned into the pOE vector with an N-terminal 6× His and FLAG tag and no signal peptide. pOE plasmids encoding TRIM21–Tn3 variants were created by gene assembly of TRIM21ΔPRYSPRY (residues 1–286) and the appropriate Tn3 domain using overlap extension PCR with primers that introduced a flexible 15-amino acid (Gly 4 Ser) 3 linker between TRIM21ΔPRYSPRY and the Tn3 domain.

Techniques: Construct, Binding Assay, Flow Cytometry, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Redirecting the specificity of tripartite motif containing-21 scaffolds using a novel discovery and design approach

doi: 10.1016/j.jbc.2023.105381

Figure Lengend Snippet: TRIM21–Tn3 fusion proteins bind to target POIs. A and B , equilibrium biolayer interferometry–based titrations and corresponding equilibrium K D values of MALT1 ( A ) or EED ( B ) binding to immobilized TRIM21–Tn3 variants. C and D , immunoprecipitation studies conducted on HBL-1 ( C ) or Karpas 422 ( D ) cell lysates. Immunoprecipitation was performed by subjecting lysate to anti-FLAG beads that had been preincubated with the indicated recombinant FLAG-tagged TRIM21–Tn3 variant. An equivalent amount of total protein was added for each condition, and immunoblots are representative of two replicate experiments. Multiple bands are observed for EED ( D ) because of the presence of different EED isoforms . EED, embryonic ectoderm development protein; MALT1, mucosa-associated lymphoid tissue lymphoma translocation protein 1; POI, protein of interest; TRIM21, tripartite motif containing-21; Tn3, third fibronectin type III domain of human tenascin-C.

Article Snippet: To construct pOE-TRIM21, a gene fragment (Twist Bioscience) encoding full-length human TRIM21 (residues 1–475) was cloned into the pOE vector with an N-terminal 6× His and FLAG tag and no signal peptide. pOE plasmids encoding TRIM21–Tn3 variants were created by gene assembly of TRIM21ΔPRYSPRY (residues 1–286) and the appropriate Tn3 domain using overlap extension PCR with primers that introduced a flexible 15-amino acid (Gly 4 Ser) 3 linker between TRIM21ΔPRYSPRY and the Tn3 domain.

Techniques: Binding Assay, Immunoprecipitation, Recombinant, Variant Assay, Western Blot, Translocation Assay

Journal: The Journal of Biological Chemistry

Article Title: Redirecting the specificity of tripartite motif containing-21 scaffolds using a novel discovery and design approach

doi: 10.1016/j.jbc.2023.105381

Figure Lengend Snippet: TRIM21–Tn3 fusion proteins degrade tGFP-tagged POIs. A , schematic showing engagement and degradation of a tGFP-tagged POI by a TRIM21–Tn3 fusion protein. Plasmids encoding the tGFP-tagged POI and the TRIM21–Tn3 variant were cotransfected into HEK 293F cells, and degradation was evaluated after 72 h by flow cytometry. B and C , flow cytometry analysis of HEK 293F cells cotransfected with a plasmid encoding MALT1-tGFP ( B ) or EED-tGFP ( C ) as well as a plasmid encoding the indicated TRIM21–Tn3 variant. The background-subtracted tGFP MFI for each sample was normalized to the average background-subtracted tGFP MFI of the control sample. Data represent mean ± SD of biological triplicates, and statistical significance was determined by one-way ANOVA (Dunnett’s multiple-comparison test compared with the control sample). Statistical data are shown in Table S4 . ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, and ∗∗∗∗ p ≤ 0.0001. HEK, human embryonic kidney cell line; MFI, mean fluorescence intensity; POI, protein of interest; tGFP, TurboGFP; Tn3, third fibronectin type III domain of human tenascin-C; TRIM21, tripartite motif containing-21.

Article Snippet: To construct pOE-TRIM21, a gene fragment (Twist Bioscience) encoding full-length human TRIM21 (residues 1–475) was cloned into the pOE vector with an N-terminal 6× His and FLAG tag and no signal peptide. pOE plasmids encoding TRIM21–Tn3 variants were created by gene assembly of TRIM21ΔPRYSPRY (residues 1–286) and the appropriate Tn3 domain using overlap extension PCR with primers that introduced a flexible 15-amino acid (Gly 4 Ser) 3 linker between TRIM21ΔPRYSPRY and the Tn3 domain.

Techniques: Variant Assay, Flow Cytometry, Plasmid Preparation, Control, Comparison, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Redirecting the specificity of tripartite motif containing-21 scaffolds using a novel discovery and design approach

doi: 10.1016/j.jbc.2023.105381

Figure Lengend Snippet: Immunoblot analysis of MALT1 and EED degradation. A and B , immunoblot analysis of cell lysate from HEK 293F cells cotransfected with a plasmid encoding MALT1-tGFP ( A ) or EED-tGFP ( B ) as well as a plasmid encoding the indicated TRIM21–Tn3 variant. Cells were harvested and lysed 72 h after transfection. Blots were probed with an anti-MALT1 antibody ( A ) or an anti-EED antibody ( B ) as well as an anti-FLAG antibody to detect TRIM21–Tn3. An equivalent amount of total protein was added to each lane, and blots were also probed with an anti-β-actin antibody to confirm equivalent loading. Immunoblots are representative of two replicate experiments. Immunoblot images for biological replicates are shown in Fig. S5 , A and B . Bands for MALT1-tGFP and MALT1 are present in A because HEK 293F cells natively express MALT1 ( , ). C and D , relative quantitation of total MALT1 ( C ) or EED ( D ) levels across two biological replicates ( Fig. S5 A ) by densitometry using ImageJ software. Band intensity was normalized to the band intensity of β-actin and then divided by the normalized band intensity of the control sample for each replicate. Data represent the same sample sets analyzed by flow cytometry in Figure 4 , B and C . EED, embryonic ectoderm development protein; HEK, human embryonic kidney cell line; MALT1, mucosa-associated lymphoid tissue lymphoma translocation protein 1; tGFP, TurboGFP; Tn3, third fibronectin type III domain of human tenascin-C; TRIM21, tripartite motif containing-21.

Article Snippet: To construct pOE-TRIM21, a gene fragment (Twist Bioscience) encoding full-length human TRIM21 (residues 1–475) was cloned into the pOE vector with an N-terminal 6× His and FLAG tag and no signal peptide. pOE plasmids encoding TRIM21–Tn3 variants were created by gene assembly of TRIM21ΔPRYSPRY (residues 1–286) and the appropriate Tn3 domain using overlap extension PCR with primers that introduced a flexible 15-amino acid (Gly 4 Ser) 3 linker between TRIM21ΔPRYSPRY and the Tn3 domain.

Techniques: Western Blot, Plasmid Preparation, Variant Assay, Transfection, Quantitation Assay, Software, Control, Flow Cytometry, Translocation Assay

Journal: The Journal of Biological Chemistry

Article Title: Redirecting the specificity of tripartite motif containing-21 scaffolds using a novel discovery and design approach

doi: 10.1016/j.jbc.2023.105381

Figure Lengend Snippet: Fluorescence microscopy and plate reader analysis of MALT1 and EED degradation. HEK 293F cells were cotransfected with a plasmid encoding MALT1-tGFP ( A and B ) or EED-tGFP ( C and D ) as well as a plasmid encoding an mCherry tag fused to the indicated TRIM21–Tn3 variant, separated by a T2A peptide in order to decouple degradation from expression. A and C , after 72 h, 20× images were acquired with a Zeiss Axio Observer with AxioVision 5 software, and composite images were generated with ImageJ. Images are representative of biological triplicates. Scale bar represents 100 μm. B and D , mean tGFP (488 ± 9 nm excitation; 510 ± 9 nm emission) and mCherry fluorescence (580 ± 20 nm excitation; 630 ± 20 nm emission) were also measured after 72 h using a BioTek SynergyMX plate reader (BioTek Instruments, Inc) that analyzed a 3 × 3 grid distributed across the well surface area. Background-subtracted tGFP fluorescence of each well was normalized by the mCherry fluorescence and divided by the mean mCherry-normalized tGFP fluorescence of the appropriate control sample. Data represent mean ± SD of biological triplicates. Statistical significance was determined by a two-tailed unpaired Student’s t test, and statistical data are shown in Table S4 . ∗∗∗ p ≤ 0.001, and ∗∗∗∗ p ≤ 0.0001. EED, embryonic ectoderm development protein; HEK, human embryonic kidney cell line; MALT1, mucosa-associated lymphoid tissue lymphoma translocation protein 1; tGFP, TurboGFP; Tn3, third fibronectin type III domain of human tenascin-C; TRIM21, tripartite motif containing-21.

Article Snippet: To construct pOE-TRIM21, a gene fragment (Twist Bioscience) encoding full-length human TRIM21 (residues 1–475) was cloned into the pOE vector with an N-terminal 6× His and FLAG tag and no signal peptide. pOE plasmids encoding TRIM21–Tn3 variants were created by gene assembly of TRIM21ΔPRYSPRY (residues 1–286) and the appropriate Tn3 domain using overlap extension PCR with primers that introduced a flexible 15-amino acid (Gly 4 Ser) 3 linker between TRIM21ΔPRYSPRY and the Tn3 domain.

Techniques: Fluorescence, Microscopy, Plasmid Preparation, Variant Assay, Expressing, Software, Generated, Control, Two Tailed Test, Translocation Assay

Journal: The Journal of Biological Chemistry

Article Title: Redirecting the specificity of tripartite motif containing-21 scaffolds using a novel discovery and design approach

doi: 10.1016/j.jbc.2023.105381

Figure Lengend Snippet: TRIM21–Tn3 fusion proteins induce targeted degradation through the UPS. A and B , flow cytometry analysis 72 h after cotransfection of HEK 293F cells with a plasmid encoding MALT1-tGFP ( A ) or EED-tGFP ( B ) and a plasmid encoding an mCherry tag fused to the indicated TRIM21–Tn3 variant, separated by a T2A peptide. Inactive TRIM21–Tn3 variants contain mutations in the TRIM21ΔPRYSPRY region that prevent E2 recruitment and subsequent ubiquitination. For each condition, the background-subtracted tGFP MFI was normalized by the background-subtracted mCherry MFI and then divided by the average mCherry-normalized tGFP MFI of the appropriate control sample. Data represent mean ± SD of biological triplicates, and statistical significance was determined by one-way ANOVA with a Tukey post hoc test. Statistical data are shown in Table S4 . ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, and ∗∗∗∗ p ≤ 0.0001. EED, embryonic ectoderm development protein; HEK, human embryonic kidney cell line; MALT1, mucosa-associated lymphoid tissue lymphoma translocation protein 1; MFI, mean fluorescence intensity; tGFP, TurboGFP; Tn3, third fibronectin type III domain of human tenascin-C; TRIM21, tripartite motif containing-21; UPS, ubiquitin proteasome system.

Article Snippet: To construct pOE-TRIM21, a gene fragment (Twist Bioscience) encoding full-length human TRIM21 (residues 1–475) was cloned into the pOE vector with an N-terminal 6× His and FLAG tag and no signal peptide. pOE plasmids encoding TRIM21–Tn3 variants were created by gene assembly of TRIM21ΔPRYSPRY (residues 1–286) and the appropriate Tn3 domain using overlap extension PCR with primers that introduced a flexible 15-amino acid (Gly 4 Ser) 3 linker between TRIM21ΔPRYSPRY and the Tn3 domain.

Techniques: Flow Cytometry, Cotransfection, Plasmid Preparation, Variant Assay, Ubiquitin Proteomics, Control, Translocation Assay, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Redirecting the specificity of tripartite motif containing-21 scaffolds using a novel discovery and design approach

doi: 10.1016/j.jbc.2023.105381

Figure Lengend Snippet: TRIM21–Tn3 fusion proteins degrade untagged and endogenous POI. A and B , immunoblot analysis of cell lysate from HEK 293F cells cotransfected with a plasmid encoding MALT1 ( A ) or EED ( B ) and a plasmid encoding the indicated TRIM21–Tn3 variant. C , immunoblot analysis of cell lysate from HEK 293F cells transfected with a plasmid encoding the indicated TRIM21–Tn3 variant or an empty plasmid (“empty”). Cells were harvested and lysed 72 h after transfection. Blots were probed with an anti-MALT1 antibody ( A and C ) or an anti-EED antibody ( B ), and an anti-FLAG antibody was used to detect TRIM21–Tn3. An equivalent amount of total protein was added to each lane, and blots were probed with an anti-β-actin antibody to confirm equivalent loading. Immunoblots are representative of two replicate experiments. Immunoblot images for biological replicates are shown in Fig. S9 . D–F , relative quantitation of total MALT1 ( D and F ) or EED ( D ) levels across biological replicates by densitometry using ImageJ software. MALT1 or EED band intensity was normalized to the band intensity of β-actin and then divided by the normalized band intensity of the control sample for each replicate. Statistical significance was determined by two-tailed paired Student’s t test. Statistical data are shown in Table S4 . ∗ p ≤ 0.05 and ∗∗ p ≤ 0.01. EED, embryonic ectoderm development protein; HEK, human embryonic kidney cell line; MALT1, mucosa-associated lymphoid tissue lymphoma translocation protein 1; POI, protein of interest; Tn3, third fibronectin type III domain of human tenascin-C; TRIM21, tripartite motif containing-21.

Article Snippet: To construct pOE-TRIM21, a gene fragment (Twist Bioscience) encoding full-length human TRIM21 (residues 1–475) was cloned into the pOE vector with an N-terminal 6× His and FLAG tag and no signal peptide. pOE plasmids encoding TRIM21–Tn3 variants were created by gene assembly of TRIM21ΔPRYSPRY (residues 1–286) and the appropriate Tn3 domain using overlap extension PCR with primers that introduced a flexible 15-amino acid (Gly 4 Ser) 3 linker between TRIM21ΔPRYSPRY and the Tn3 domain.

Techniques: Western Blot, Plasmid Preparation, Variant Assay, Transfection, Quantitation Assay, Software, Control, Two Tailed Test, Translocation Assay