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