full length human trim21  (Addgene inc)

Journal: Cell reports

Article Title: TRIM21-dependent target protein ubiquitination mediates cell-free Trim-Away.

doi: 10.1016/j.celrep.2023.112125

Figure Lengend Snippet: Figure 1. Cell-free Trim-Away in Xenopus egg extracts (A) Domain architectures of full-length (FL) TRIM21 and a minimal TRIM21 construct (TRIM21R-R-PS). R, RING domain; B, B-box domain; CC, coiled-coil domain; PS, PRYSPRY domain; Fc, antibody fragment. (B) Simplified schematic of the current Trim-Away model. The target protein of interest (red) is bound by an antibody (yellow), which in turn is recognized by TRIM21 (shades of blue). Subsequent TRIM21 activation results in sequential mono- and polyubiquitination of its N terminus with K63-linked polyubiquitin, which requires the E2 ubiquitin-conjugating enzymes UBE2W and UBE2N/UBE2V2, respectively. The polyubiquitin chain on TRIM21 has been proposed to promote the proteasomal degradation of all three components. (C) Trim-Away of endogenous TFIIS in Xenopus egg extract (high-speed supernatant [HSS]) comparing recombinant human TRIM21FL and TRIM21R-R-PS (final concentrations of 1 mM). (D) Trim-Away of TFIIS in HSS using TRIM21R-R-PS in the presence of inhibitors targeting E1 enzyme (MLN7243), the proteasome (MG-262), p97 (NMS-873), or the NEDD8-activating enzyme to inhibit Cullin-RING E3 ligases (MLN4924). (E) Endogenous TFIIS was targeted by TRIM21R-R-PS in three types of Xenopus egg extract: low-speed supernatant (LSS), HSS, and nucleoplasmic extract (NPE). Note that TFIIS is more abundant in NPE than in LSS and HSS. (F) Egg extracts were supplemented with 250 nM recombinant HpaII, a bacterial DNA methyltransferase, to compare Trim-Away efficiencies using TRIM21R-R-PS

Article Snippet: The expression plasmid for full-length human TRIM21 (HLTV-hTRIM21, referred to as TRIM21FL) was ordered from Addgene (#104973).

Techniques: Construct, Activation Assay, Ubiquitin Proteomics, Recombinant

Journal: Cell reports

Article Title: TRIM21-dependent target protein ubiquitination mediates cell-free Trim-Away.

doi: 10.1016/j.celrep.2023.112125

Figure Lengend Snippet: Figure 4. Direct polyubiquitination of target proteins is essential for their destruction (A) Schematic of experimental setup. To prevent direct target ubiquitination, proteins were chemically methylated in vitro prior to Trim-Away in egg extract. Methylation (black circle) modifies both lysine residues and the N terminus. (B and C) Trim-Away of recombinant LacI variants (final concentration of 250 nM) by TRIM21R-R-PS (B) or TRIM21FL (C) in HSS. IgG degradation fragments are highlighted. (D) Schematic of experimental setup. As an alternative means of inhibiting ubiquitination, all lysines (black squares) in target proteins were mutated to arginine (DK). Note that lysine-less mutants contain intact N termini. (E and F) Trim-Away of recombinant PEX5 variants (final concentration of 500 nM) by TRIM21R-R-PS (E) or TRIM21FL (F) in HSS. To deplete endogenous PEX5, TRIM21 and antibody were added to egg extract for 15 min (E) or 30 min (F) prior to the addition of recombinant X. laevis PEX5 to start cell-free Trim-Away at 0 min. Note that Trim-Away with TRIM21FL required an extended time course for efficient PEX5 degradation. See also Figure S3.

Article Snippet: The expression plasmid for full-length human TRIM21 (HLTV-hTRIM21, referred to as TRIM21FL) was ordered from Addgene (#104973).

Techniques: Ubiquitin Proteomics, Methylation, In Vitro, Recombinant, Concentration Assay

Journal: Cell reports

Article Title: TRIM21-dependent target protein ubiquitination mediates cell-free Trim-Away.

doi: 10.1016/j.celrep.2023.112125

Figure Lengend Snippet: Figure 5. Direct target polyubiquitination is sufficient for Trim-Away (A) Schematic of experimental setup. To prevent direct ubiquitination of TRIM21 or antibody, reductive methylation (black circle) was performed in vitro prior to Trim-Away in egg extract. Note that this procedure inhibits ubiquitination of both the N terminus and lysine residues. (B and C) Cell-free Trim-Away of recombinant unmethylated HpaII (final concentration of 250 nM) by HpaII antibody and TRIM21R-R-PS (B) or TRIM21FL (C). The methylation state of HpaII antibody and TRIM21 variant is indicated. Trim-Away with TRIM21FL required an extended time course for efficient HpaII degradation. Note that methylation of TRIM21FL resulted in an even stronger non-specific band (asterisks), which is already present at the beginning of the Trim-Away reaction. See also Figure S4.

Article Snippet: The expression plasmid for full-length human TRIM21 (HLTV-hTRIM21, referred to as TRIM21FL) was ordered from Addgene (#104973).

Techniques: Ubiquitin Proteomics, Methylation, In Vitro, Recombinant, Concentration Assay, Variant Assay

Journal: Cell reports

Article Title: TRIM21-dependent target protein ubiquitination mediates cell-free Trim-Away.

doi: 10.1016/j.celrep.2023.112125

Figure Lengend Snippet: Figure 7. TRIM21 is ubiquitinated on lysine residues during cell-free Trim-Away (A) Schematic comparing potential TRIM21 ubiquitination sites (pink): N terminus (left panel) and/or lysine residues (right panel). (B) Cell-free Trim-Away assay targeting endogenous TFIIS in HSS. Comparison of the following TRIM21R-R-PS constructs: (1) wild-type (WT), which can be modified on both the N terminus and on lysines; (2) methylated TRIM21 (meWT), which cannot be ubiquitinated on any primary amine; (3) lysine-less TRIM21 (DK) containing a ubiquitinatable N terminus; and (4) a ubiquitin (K63R)-TRIM21 fusion protein (UbK63R-WT), which can be ubiquitinated on lysines only. See Figure S6 for additional schematics. (C) Reconstitution of antibody-dependent target ubiquitination by TRIM21. Recombinant TFIIS was incubated with ATP, ubiquitin, E1, indicated E2 enzymes, and TRIM21R-R-PS in the absence or presence of TFIIS antibody. (D) Proposed Trim-Away model. Upon TRIM21 recruitment to antibody-bound targets, E3 ligase activation leads to direct polyubiquitination of all components on lysines. Substrate modification with heterotypic K48/K63-mixed or -branched polyubiquitin chains mediates its proteasomal degradation. See also Figures S6 and S7.

Article Snippet: The expression plasmid for full-length human TRIM21 (HLTV-hTRIM21, referred to as TRIM21FL) was ordered from Addgene (#104973).

Techniques: Ubiquitin Proteomics, Comparison, Construct, Methylation, Recombinant, Incubation, Activation Assay

Journal: Cells

Article Title: Endoglin Protein Interactome Profiling Identifies TRIM21 and Galectin-3 as New Binding Partners

doi: 10.3390/cells8091082

Figure Lengend Snippet: Human protein-array analysis of endoglin interactors 1 .

Article Snippet: Human full-length TRIM21 protein (RO52; 6His-tag, N-terminus, LSBio) was diluted to a final concentration of 250 μg/mL in 0.2 M Tris/pH 7.4 + 25% glycerol buffer.

Techniques: Protein Array, Variant Assay, Clinical Proteomics, Membrane, Sequencing, Ubiquitin Proteomics

Journal: Cells

Article Title: Endoglin Protein Interactome Profiling Identifies TRIM21 and Galectin-3 as New Binding Partners

doi: 10.3390/cells8091082

Figure Lengend Snippet: Protein–protein association between TRIM21 and endoglin. ( A – E ) Co-immunoprecipitation of TRIM21 and endoglin. A,B. HUVEC monolayers were lysed and total cell lysates (TCL) were subjected to SDS-PAGE under reducing (for TRIM21 detection) or nonreducing (for endoglin detection) conditions, followed by Western blot (WB) analysis using antibodies to endoglin, TRIM21 or β-actin ( A ). HUVECs lysates were subjected to immunoprecipitation (IP) with anti-TRIM21 or negative control antibodies, followed by WB analysis with anti-endoglin ( B ). C,D. CHO-K1 cells were transiently transfected with pDisplay–HA–Mock (Ø), pDisplay–HA–EngFL ( E ) or pcDNA3.1–HA–hTRIM21 (T) expression vectors, as indicated. Total cell lysates (TCL) were subjected to SDS-PAGE under nonreducing conditions and WB analysis using specific antibodies to endoglin, TRIM21, and β-actin ( C ). Cell lysates were subjected to immunoprecipitation (IP) with anti-TRIM21 or anti-endoglin antibodies, followed by SDS-PAGE under reducing (upper panel) or nonreducing (lower panel) conditions and WB analysis with anti-TRIM21 or anti-endoglin antibodies. Negative controls of appropriate IgG were included ( D ). E. CHO-K1 cells were transiently transfected with pcDNA3.1–HA–hTRIM21 and pDisplay–HA–Mock (Ø), pDisplay–HA–EngFL (FL; full-length), pDisplay–HA–EngEC (EC; cytoplasmic-less) or pDisplay–HA–EngTMEC (TMEC; cytoplasmic-less) expression vectors, as indicated. Cell lysates were subjected to immunoprecipitation with anti-TRIM21, followed by SDS-PAGE under reducing conditions and WB analysis with anti-endoglin antibodies, as indicated. The asterisk indicates the presence of a nonspecific band. Mr, molecular reference; Eng, endoglin; TRIM, TRIM21. ( F ) Protein–protein interactions between TRIM21 and endoglin using Bio-layer interferometry (BLItz). The Ni–NTA biosensors tips were loaded with 5.4 µM recombinant human TRIM21/6xHis at the N-terminus (R052), and protein binding was measured against 0.1% BSA in PBS (negative control) or 4.1 µM soluble endoglin (sEng). Kinetic sensorgrams were obtained using a single channel ForteBioBLItzTM instrument.

Article Snippet: Human full-length TRIM21 protein (RO52; 6His-tag, N-terminus, LSBio) was diluted to a final concentration of 250 μg/mL in 0.2 M Tris/pH 7.4 + 25% glycerol buffer.

Techniques: Immunoprecipitation, SDS Page, Western Blot, Negative Control, Transfection, Expressing, Protein-Protein interactions, Recombinant, Protein Binding

Journal: Cells

Article Title: Endoglin Protein Interactome Profiling Identifies TRIM21 and Galectin-3 as New Binding Partners

doi: 10.3390/cells8091082

Figure Lengend Snippet: TRIM21 and endoglin co-localize in human endothelial cells. HUVEC monolayers were fixed with paraformaldehyde, permeabilized with Triton X-100, incubated with the mouse mAb P4A4 anti-endoglin, washed and incubated with a rabbit monoclonal anti-TRIM21 antibody (#92043). TRIM21 and endoglin were detected by immunofluorescence upon incubation with Alexa 647 goat anti-rabbit IgG (red staining) and Alexa 488 goat anti-mouse IgG (green staining) secondary antibodies, respectively. ( A ) Single staining of TRIM21 (red) and endoglin (green) at the indicated magnifications. ( B ) Merge images plus DAPI (nuclear staining in blue) show co-localization of TRIM21 and endoglin (yellow color). Representative images of four different experiments are shown.

Article Snippet: Human full-length TRIM21 protein (RO52; 6His-tag, N-terminus, LSBio) was diluted to a final concentration of 250 μg/mL in 0.2 M Tris/pH 7.4 + 25% glycerol buffer.

Techniques: Incubation, Immunofluorescence, Staining