Journal: bioRxiv

Article Title: Substrate-induced clustering activates Trim-Away of pathogens and proteins

doi: 10.1101/2020.07.28.225359

Figure Lengend Snippet: (A and C) HEK293T cells stably expressing the indicated TRIM21 mutants (EV = empty vector) were transfected with WT His-ubiquitin and infected with AdV5 ± 9C12 or 9C12(H433A). Ubiquitinated proteins were isolated by denaturing His-pulldown using Ni-NTA beads in 6M Guanidine buffer before immunoblot analysis with anti-TRIM21 antibody. (B) A co-crystal structure of TRIM21 RING:Ube2N∼ubiquitin complex (PDB: 6S53) showing the tri-anionic anchor motif (E12, E13 and D21) and second site residues (R67 and N71). (D) Immunoblot of TRIM21 following denaturing His-ubiquitin pulldown from HEK293T cells overexpressing the indicated ubiquitin mutant at 30 min post infection with AdV5 ± 9C12 or 9C12(H433A). See also

Article Snippet: To generate optogenetic constructs for S2 cell expression, vhhGFP4 , mRFP , mouse TRIM21 (Addgene #105516) and CRY2Clust ( ) coding sequences were inserted into heat-shock inducible Drosophila Gateway expression vectors (Life Technologies) to generate pHR-CRY2Clust-TRIM21, pHR-CRY2Clust and pH-vhhGFP4-RFP-CRY2Clust-TRIM21.

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Transfection, Ubiquitin Proteomics, Infection, Isolation, Western Blot, Mutagenesis

Journal: bioRxiv

Article Title: Substrate-induced clustering activates Trim-Away of pathogens and proteins

doi: 10.1101/2020.07.28.225359

Figure Lengend Snippet: Denaturing TRIM21-His pulldown from HEK293T cells stably expressing TRIM21-His and infected with AdV5 ± 9C12 or 9C12(H433A) which does not bind TRIM21. Cells were lysed in buffer containing 4M urea and where indicated, the Nickel beads were treated with the deubiquitinase USP2 before boiling and immunoblotting with anti-TRIM21 antibody.

Article Snippet: To generate optogenetic constructs for S2 cell expression, vhhGFP4 , mRFP , mouse TRIM21 (Addgene #105516) and CRY2Clust ( ) coding sequences were inserted into heat-shock inducible Drosophila Gateway expression vectors (Life Technologies) to generate pHR-CRY2Clust-TRIM21, pHR-CRY2Clust and pH-vhhGFP4-RFP-CRY2Clust-TRIM21.

Techniques: Stable Transfection, Expressing, Infection, Western Blot

Journal: bioRxiv

Article Title: Substrate-induced clustering activates Trim-Away of pathogens and proteins

doi: 10.1101/2020.07.28.225359

Figure Lengend Snippet: (A) Sequence of TRIM21 with RING domain in gray, B Box in red, coiled-coil in cyan, L2 linker helices in orange & green and PRYSPRY in magenta. (B) SEC-MALS chromatograms of TRIM21 CC (129-235) loaded at concentrations of 15 (black), 5 (green), 0.55 (blue) and 0.25 mg/ml (red) are shown, in which the refractive index is indicated by the solid lines while the molar mass evaluated from the light scattering analysis is indicated with the corresponding coloured dotted lines. (C) Using a MicroCal iTC200 calorimeter, 140 µM TRIM21 coiled-coil was added in 2 µl injections into buffer at 25 °C. Integrated heats were then fit to a dimer dissociation model reveal an enthalpy of 65 kcal/mol and Kd of 7 µM. (D-F) SAXS data on TRIM21 constructs. (D) SAXS data were collected at the beam line P12 of the EMBL at the Petra-III storage ring (DESY, Hamburg). Protein expression, purification, data analysis and collection are in Supplementary Information. Data statistics are shown in Table 1. (D) Concentration-normalised scattering plots and DAM fits for CC235 (cyan, χ = 1.00), MBP-CC235 (green, χ = 1.31) and RBCC (purple, χ = 1.05). (E) The linear Guinier regions from (D). (F) Derived P( r ) curves. The CC235 P( r ) curve is consistent with an elongated rod, while the two peaks observed for MBP-CC235 and RBCC are consistent with ‘dumbbell’-shaped molecules. (G-I). SAXS data on TRIM21:Fc complex. (G) Scattering plot with DAM fit (red line, 1.00), TRIM21:Fc atomic model fit (blue, χ = 1.04), and apo-TRIM21 atomic model fit (brown dashed, χ = 2.00). (H) The linear Guinier regions from (G). (I) Derived P( r ) curves.

Article Snippet: To generate optogenetic constructs for S2 cell expression, vhhGFP4 , mRFP , mouse TRIM21 (Addgene #105516) and CRY2Clust ( ) coding sequences were inserted into heat-shock inducible Drosophila Gateway expression vectors (Life Technologies) to generate pHR-CRY2Clust-TRIM21, pHR-CRY2Clust and pH-vhhGFP4-RFP-CRY2Clust-TRIM21.

Techniques: Sequencing, Refractive Index, Construct, Expressing, Purification, Concentration Assay, Derivative Assay

Journal: bioRxiv

Article Title: Substrate-induced clustering activates Trim-Away of pathogens and proteins

doi: 10.1101/2020.07.28.225359

Figure Lengend Snippet: (A) Dummy atom model (DAM) structures of TRIM21 calculated from SAXS data shown in . Overlay of reconstructions of constructs comprising TRIM21 residues 1-235 (RBCC, purple spheres) and TRIM21 residues 129-235 (CC235, blue spheres). A structural model of the TRIM21 RBCC region based on atomic models of the TRIM21 RING (yellow) and B Box (orange) domains and TRIM25 coiled-coil domain (cyan) using PDBs 5OLM and 4CFG respectively. (B) SAXS-derived model of TRIM21:Fc complex. Averaged and filtered DAM’s (white and green spheres, respectively) overlaid with an atomic model of the TRIM21:Fc complex. The RING, B Box and coiled-coil regions are as above. The PRYSPRY (magenta) and IgG Fc (grey) domains are taken directly from PDB 2IWG. (C) Crystal structure of TRIM21 RING dimer showing the important residues M10 and M72 at the dimer interface. (D) Immunoblot of TRIM21 following denaturing His-ubiquitin pulldown from TRIM21 lentivector reconstituted HEK293T cells overexpressing WT His-ubiquitin at 30 min post infection with AdV5 ± 9C12 or 9C12(H433A). (E) Trim-away of IKKα in TRIM21 lentivector reconstituted HEK293T cell lines by electroporation of anti-IKKα IgG (anti-IKKα). Cell lysates were immunoblotted for the indicated proteins. (F) in vitro ubiquitination assay using the indicated TRIM21 constructs and Ube2N. Samples were taken at the indicated timepoints and analysed by immunoblotting with anti-ubiquitin antibody. (G) Immunoblot analysis of ubiquitin discharge from Ube2N by the indicated TRIM21 constructs over a course of 20 minutes. (H) Quantification of the Ube2N∼Ub band in G relative to time point 0 in each reaction. See also

Article Snippet: To generate optogenetic constructs for S2 cell expression, vhhGFP4 , mRFP , mouse TRIM21 (Addgene #105516) and CRY2Clust ( ) coding sequences were inserted into heat-shock inducible Drosophila Gateway expression vectors (Life Technologies) to generate pHR-CRY2Clust-TRIM21, pHR-CRY2Clust and pH-vhhGFP4-RFP-CRY2Clust-TRIM21.

Techniques: Construct, Derivative Assay, Western Blot, Ubiquitin Proteomics, Infection, Electroporation, In Vitro

Journal: bioRxiv

Article Title: Substrate-induced clustering activates Trim-Away of pathogens and proteins

doi: 10.1101/2020.07.28.225359

Figure Lengend Snippet: (A) Schematic illustration of multimeric TRIM21 assembly on the surface of a virus-antibody complex. (B) Remaining infectivity of AdV5-GFP particles on HEK293T cells following incubation with saturating concentrations of 9C12. For 1/e neutralization, approximately 12% of antibodies must bear an intact TRIM21 binding site which is present on 9C12(WT) but lacking in 9C12(H433A). This equates to 24 out of the maximum ∼200 antibodies bound to AdV5 at saturation. (C, E, J and L) Neutralisation of AdV5 by 9C12 in lentivector reconstituted HEK293T cells expressing the indicated TRIM21 mutants. Data normalised to the virus only condition and presented as the mean ± SEM. (D and F) AdV5-9C12 immune complex-induced NF-kB activation in HEK293T cells stably expressing the indicated TRIM21 mutants. Data normalised to the virus only condition and presented as the mean ± SEM. (G) In vitro ubiquitination assay using the indicated TRIM21 RING-linker-RING (RLR) constructs and Ube2N. Samples were taken at the indicated timepoints and analysed by immunoblotting with anti-ubiquitin antibody. (H) Trim-away of IKKα in TRIM21 lentivector reconstituted HEK293T cell lines by electroporation of anti-IKKα IgG (anti-IKKα). Cell lysates were immunoblotted for the indicated proteins. (I) Crystal structure of TRIM21-RING-B-box (PDB: 5OLM) showing residue S80 at situated at the RING:B Box interface. (K) Structure of the hydrophobic core at the TRIM21 RING:Ube2N interface (PDB: 6S53). See also Figures S3 and S4

Article Snippet: To generate optogenetic constructs for S2 cell expression, vhhGFP4 , mRFP , mouse TRIM21 (Addgene #105516) and CRY2Clust ( ) coding sequences were inserted into heat-shock inducible Drosophila Gateway expression vectors (Life Technologies) to generate pHR-CRY2Clust-TRIM21, pHR-CRY2Clust and pH-vhhGFP4-RFP-CRY2Clust-TRIM21.

Techniques: Virus, Infection, Incubation, Neutralization, Binding Assay, Expressing, Activation Assay, Stable Transfection, In Vitro, Ubiquitin Proteomics, Construct, Western Blot, Electroporation, Residue

Journal: bioRxiv

Article Title: Substrate-induced clustering activates Trim-Away of pathogens and proteins

doi: 10.1101/2020.07.28.225359

Figure Lengend Snippet: (A) Upper panel: TRIM5α (rhesus) residues involved in mediating the three layers of box-box interactions (PDB:5EIA). Lower panel: model of TRIM21 B box domain structure (PDB:5OLM) onto the trimeric box structure of TRIM5α (PDB:5EIA). Residues that could mediate B box: B box interactions are highlighted. (B and C) Neutralisation of AdV5 by 9C12 in lentivector-reconstituted HEK293T cells expressing the indicated TRIM21 mutants. Data normalised to the virus only condition and presented as the mean ± SEM. (D) AdV5-9C12 immune complex-induced NF-kB activation in HEK293T cells stably expressing the indicated TRIM21 mutants. Data normalised to the virus only condition and presented as the mean ± SD. (E) Neutralisation of AdV5 by 9C12 in HEK293T cells stably expressing TRIM21-ΔBox construct with or without MG132 (20 µM). Data normalised to the virus only condition and presented as the mean ± SD.

Article Snippet: To generate optogenetic constructs for S2 cell expression, vhhGFP4 , mRFP , mouse TRIM21 (Addgene #105516) and CRY2Clust ( ) coding sequences were inserted into heat-shock inducible Drosophila Gateway expression vectors (Life Technologies) to generate pHR-CRY2Clust-TRIM21, pHR-CRY2Clust and pH-vhhGFP4-RFP-CRY2Clust-TRIM21.

Techniques: Expressing, Virus, Activation Assay, Stable Transfection, Construct

Journal: bioRxiv

Article Title: Substrate-induced clustering activates Trim-Away of pathogens and proteins

doi: 10.1101/2020.07.28.225359

Figure Lengend Snippet: (A) Schematic of myc-mEGFP constructs. (B-D) HEK293T-mCherry-TRIM21 cells were electroporated with mRNA encoding the indicated myc-mEGFP constructs together with either control IgG (9C12), ant-Myc (9E10) or anti-GFP (polyclonal) antibodies. 8 hours post-electroporation cellular GFP fluorescence was imaged (B) and quantified (C) using the IncuCyte system, or total GFP protein levels analysed by immunoblotting (D) cell extracts with the indicated antibodies. Scale bar 100 µm. (E) HEK293T-mCherry-TRIM21 cells were electroporated with mRNA encoding the indicated myc-mEGFP constructs together with control IgG or increasing concentrations of anti-Myc antibody and GFP fluorescence quantified 8 hours later. (F) HEK293T-mCherry-TRIM21 cells were electroporated with mRNA encoding 2myc-mEGFP together with the indicated antibodies and GFP fluorescence quantified 8 hours later. (G-I) The indicated antibodies (50 nM) either alone, or mixed with GFP protein (100 nM), were analysed by mass photometry. (J) RPE-1 cells expressing mEGFP were electroporated with the indicated antibodies and cell extracts blotted 3 hours later for the indicated proteins. See also

Article Snippet: To generate optogenetic constructs for S2 cell expression, vhhGFP4 , mRFP , mouse TRIM21 (Addgene #105516) and CRY2Clust ( ) coding sequences were inserted into heat-shock inducible Drosophila Gateway expression vectors (Life Technologies) to generate pHR-CRY2Clust-TRIM21, pHR-CRY2Clust and pH-vhhGFP4-RFP-CRY2Clust-TRIM21.

Techniques: Construct, Control, Electroporation, Fluorescence, Western Blot, Expressing

Journal: bioRxiv

Article Title: Substrate-induced clustering activates Trim-Away of pathogens and proteins

doi: 10.1101/2020.07.28.225359

Figure Lengend Snippet: RPE-1 TRIM21 KO cells expressing membrane-localised GFP (mem-mEGFP) were electroporated with PBS, control IgG or the indicated anti-GFP antibodies. Cells were fixed 3 hours post-electroporation and stained with alexa 647-conjugated anti-IgG secondary antibodies and imaged by confocal microscopy. Scale bar 10 µm.

Article Snippet: To generate optogenetic constructs for S2 cell expression, vhhGFP4 , mRFP , mouse TRIM21 (Addgene #105516) and CRY2Clust ( ) coding sequences were inserted into heat-shock inducible Drosophila Gateway expression vectors (Life Technologies) to generate pHR-CRY2Clust-TRIM21, pHR-CRY2Clust and pH-vhhGFP4-RFP-CRY2Clust-TRIM21.

Techniques: Expressing, Membrane, Control, Electroporation, Staining, Confocal Microscopy

Journal: bioRxiv

Article Title: Substrate-induced clustering activates Trim-Away of pathogens and proteins

doi: 10.1101/2020.07.28.225359

Figure Lengend Snippet: (A) Schematic of light-induced clustering of TRIM21. (B-C) Drosophila S2 cells expressing the indicated constructs were incubated with or without MG132 and RFP fluorescence quantified by live imaging. Time shows minutes (min) from onset of blue light exposure. Scale bar 5 µm. Pseudo-coloured kymographs show fluorescence intensity in regions defined by red dotted lines. Graph shows mean fluorescence intensity (± SD) of RFP-CRY2-TRIM21 (n = 23) and RFP-CRY2-TRIM21+MG132 (n = 20) normalised for the respective controls (RFP-CRY2 (n=16) and RFP-CRY2 + MG132 (n=15)). (D-G) RPE-1 cells expressing the indicated constructs together with mem-mEGFP were incubated with or without MG132 and blue light for 3 hours prior to immunoblotting for the indicated proteins. See also Movie S2

Article Snippet: To generate optogenetic constructs for S2 cell expression, vhhGFP4 , mRFP , mouse TRIM21 (Addgene #105516) and CRY2Clust ( ) coding sequences were inserted into heat-shock inducible Drosophila Gateway expression vectors (Life Technologies) to generate pHR-CRY2Clust-TRIM21, pHR-CRY2Clust and pH-vhhGFP4-RFP-CRY2Clust-TRIM21.

Techniques: Expressing, Construct, Incubation, Fluorescence, Imaging, Western Blot

Journal: bioRxiv

Article Title: Substrate-induced clustering activates Trim-Away of pathogens and proteins

doi: 10.1101/2020.07.28.225359

Figure Lengend Snippet: (A) Catalysis of unanchored ubiquitin chains by TRIM21 RING (R), RING-Box (RB), RING-Box-Coiled-Coil (RBCC) and full length MBP-tagged TRIM21 (FL-hT21). (B-C) Catalysis of ubiquitin discharge from ubiquitin-conjugated Ube2N by RING (R), RING Box (RB), RING Box coiled-coiled (RBCC) and full length MBP-tagged TRIM21 (FL-hT21).

Article Snippet: To generate optogenetic constructs for S2 cell expression, vhhGFP4 , mRFP , mouse TRIM21 (Addgene #105516) and CRY2Clust ( ) coding sequences were inserted into heat-shock inducible Drosophila Gateway expression vectors (Life Technologies) to generate pHR-CRY2Clust-TRIM21, pHR-CRY2Clust and pH-vhhGFP4-RFP-CRY2Clust-TRIM21.

Techniques: Ubiquitin Proteomics

Journal: bioRxiv

Article Title: Substrate-induced clustering activates Trim-Away of pathogens and proteins

doi: 10.1101/2020.07.28.225359

Figure Lengend Snippet: (A) Schematic of TRIM21, nanobody-Fc and TRIM21-nanobody chimeric constructs. (B) NIH3T3-Caveolin-1-GFP cells were electroporated with mRNA encoding the indicated constructs. 16 hours later cell extracts were immunoblotted for the indicated proteins. (C-D) NIH3T3-Caveolin-1-GFP and (F-G) RPE-1H2B-mEGFP-FKBP cells were electroporated with water (control) or mRNAs encoding the indicated TRIM21-nanobody chimeric constructs and GFP fluorescence was imaged (C and F) and quantified (D and G) using the IncuCyte system. Time shows hours:minutes (h:min) post-electroporation. Scale bar 50 µm (C) and 30 µm (F). (E) NIH3T3-Caveolin-1-GFP cells and (H) RPE-1-H2B-mEGFP-FKBP cells were electroporated with mRNA encoding mCherry-tagged versions of the indicated constructs and GFP and mCherry fluorescence quantified using the IncuCyte system. TRIM21-nanobody chimera expression levels (mCherry fluorescence) are plotted against GFP fluorescence. See also and Movie S2.

Article Snippet: To generate optogenetic constructs for S2 cell expression, vhhGFP4 , mRFP , mouse TRIM21 (Addgene #105516) and CRY2Clust ( ) coding sequences were inserted into heat-shock inducible Drosophila Gateway expression vectors (Life Technologies) to generate pHR-CRY2Clust-TRIM21, pHR-CRY2Clust and pH-vhhGFP4-RFP-CRY2Clust-TRIM21.

Techniques: Construct, Control, Fluorescence, Electroporation, Expressing

Journal: bioRxiv

Article Title: Substrate-induced clustering activates Trim-Away of pathogens and proteins

doi: 10.1101/2020.07.28.225359

Figure Lengend Snippet: (A-D) NIH3T3-Caveolin-1-GFP (A and B) and RPE-1-H2B-mEGFP-FKBP (C and D) cells were electroporated with mRNA encoding mCherry-T21R-vhhGFP4, incubated with either DMSO (control) or MG132 and imaged (A and C) and GFP fluorescence quantified (B and D) with the IncuCyte system. Scale bar 20 µm. (E and F) NIH3T3 cells (E) and RPE-1 cells (F) were electroporated with mRNA encoding mCherry-tagged versions of the indicated TRIM21-nanobody constructs and mCherry fluorescence quantified using the IncuCyte system. (G and H) NIH3T3-Caveolin-1-GFP cells were electroporated with the indicated concentrations of T21R-vhhGFP4 (G) or T21RB-vhhGFP4 (H) proteins and GFP fluorescence quantified with the IncuCyte system. (I) Data from G and H plotted as protein concentration against GFP fluorescence at 4 hours post-electroporation.

Article Snippet: To generate optogenetic constructs for S2 cell expression, vhhGFP4 , mRFP , mouse TRIM21 (Addgene #105516) and CRY2Clust ( ) coding sequences were inserted into heat-shock inducible Drosophila Gateway expression vectors (Life Technologies) to generate pHR-CRY2Clust-TRIM21, pHR-CRY2Clust and pH-vhhGFP4-RFP-CRY2Clust-TRIM21.

Techniques: Incubation, Control, Fluorescence, Construct, Protein Concentration, Electroporation

Journal: bioRxiv

Article Title: Substrate-induced clustering activates Trim-Away of pathogens and proteins

doi: 10.1101/2020.07.28.225359

Figure Lengend Snippet: (A) Bacterially-expressed 6His-T21R-vhhGFP4 was purified using a two-step protocol of NiNTA-followed by size exclusion-chromatography and analysed by Coomassie blue staining of SDS-PAGE. (B) NIH3T3-Caveolin-1-GFP cells were electroporated with PBS (control) or the indicated concentrations of T21R-vhhGFP4 protein and GFP fluorescence was quantified using the IncuCyte system. (C) H2B-GFP primary MEFs were electroporated with PBS (control) or T21R-vhhGFP4 in the form or mRNA or protein and GFP fluorescence quantified using the IncuCyte system. Time shows hours (h) post-electroporation. (D-G) Drosophila S2 cells expressing GFP-aPKC and (D, E) vhhGFP4-RFP-CRY2-TRIM21 or (F, G) a LARIAT module that includes RFP-CRY2 fused with vhhGFP4 and which enables clustering in the absence of TRIM21. (E, G) Graphs show mean ± SD fluorescence intensity quantified by live imaging (n = 30 in E and n=24 in G). Time shows minutes (min) from onset of blue light exposure. Scale bar 10 µm.

Article Snippet: To generate optogenetic constructs for S2 cell expression, vhhGFP4 , mRFP , mouse TRIM21 (Addgene #105516) and CRY2Clust ( ) coding sequences were inserted into heat-shock inducible Drosophila Gateway expression vectors (Life Technologies) to generate pHR-CRY2Clust-TRIM21, pHR-CRY2Clust and pH-vhhGFP4-RFP-CRY2Clust-TRIM21.

Techniques: Purification, Size-exclusion Chromatography, Staining, SDS Page, Control, Fluorescence, Electroporation, Expressing, Imaging