Dataset Information

Trim-Away ubiquitinates and degrades lysine-less and N-terminally acetylated substrates.

ABSTRACT: TRIM proteins are the largest family of E3 ligases in mammals. They include the intracellular antibody receptor TRIM21, which is responsible for mediating targeted protein degradation during Trim-Away. Despite their importance, the ubiquitination mechanism of TRIM ligases has remained elusive. Here we show that while Trim-Away activation results in ubiquitination of both ligase and substrate, ligase ubiquitination is not required for substrate degradation. N-terminal TRIM21 RING ubiquitination by the E2 Ube2W can be inhibited by N-terminal acetylation, but this doesn't prevent substrate ubiquitination nor degradation. Instead, uncoupling ligase and substrate degradation prevents ligase recycling and extends functional persistence in cells. Further, Trim-Away degrades substrates irrespective of whether they contain lysines or are N-terminally acetylated, which may explain the ability of TRIM21 to counteract fast-evolving pathogens and degrade diverse substrates.

SUBMITTER: Kiss L

PROVIDER: S-EPMC10105713 | biostudies-literature | 2023 Apr

REPOSITORIES: biostudies-literature

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Publications

Trim-Away ubiquitinates and degrades lysine-less and N-terminally acetylated substrates.

Kiss Leo L Rhinesmith Tyler T Luptak Jakub J Dickson Claire F CF Weidenhausen Jonas J Smyly Shannon S Yang Ji-Chun JC Maslen Sarah L SL Sinning Irmgard I Neuhaus David D Clift Dean D James Leo C LC

Nature communications 20230415 1

TRIM proteins are the largest family of E3 ligases in mammals. They include the intracellular antibody receptor TRIM21, which is responsible for mediating targeted protein degradation during Trim-Away. Despite their importance, the ubiquitination mechanism of TRIM ligases has remained elusive. Here we show that while Trim-Away activation results in ubiquitination of both ligase and substrate, ligase ubiquitination is not required for substrate degradation. N-terminal TRIM21 RING ubiquitination b ...[more]

PMID: 37061529

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Project description:In order to study early developmental events in the mammalian embryo it is often desirable to be able to impair expression of specific genes. While DNA and RNA methods are routine, protein methods are still at the very beginning. When a specific antibody is supplied to mouse oocytes expressing the ubiquitin-protein ligase TRIM21, a ternary complex forms with the target protein, leading to its rapid and acute degradation - hence the name 'Trim-away' (PMID 29153837). However, there are many unknowns in this new endeavour. First and foremost, the extent to which endogenous proteins can be depleted depends on their amount, in relation to the amount of exogenous antibody, which is limited by the microinjection procedure. Secondly, the depletion of the protein must be sustained over days. Using the iBAQ algorithm we show that proteins found in preimplantation mouse embryos range from 3,5E-10 to 2,6E-02 picomoles. These amounts are tractable with our microinjection method, which supplies up to 100 picoliters and up to 6,7E-4 picomoles antibody before incurring in toxic effects on mouse development. Building on these data, we demonstrate the feasibility of protein knock-down for a gene which is essential in the preimplantation mouse embryo, namely TEAD4 (TEA domain family member 4). Protein knock-down persists for sufficient time to result in a phenotype which is entirely consistent with that of the null mutation (Tead4 -/-) and of the RNA interference, namely: significantly reduced mRNA expression of TEAD4 target genes Cdx2 and Gata3, and embryo’s inability to implant. We conclude that for a time window of 3-4 days of preimplantation development the protein depletion method can be a valid alternative to DNA and RNA methods. After in vivo fertilization and short culture in KSOM(aa) medium, pronuclear-stage oocytes (B6C3F1 x CD1) were microinjected with Trim21 mRNA and dextran beads as tracer (named 'group 4'), or Trim21 mRNA, dextran beads and anti-GFP antibody (named 'group 5'), or Trim21 mRNA, dextran beads and anti-TEAD4 antibody (named 'group 6'), two replicates each ('a' and 'b'). On day 4 after microinjection, the most advanced embryos were examined for phenotype or lysed for transcriptome analysis. TEAD4-depleted embryos formed only 30% blastocysts and these were not able to implant in the outgrowth assay, in contrast to the almost full rates of the embryos injected with Trim21 mRNA or Trim21 mRNA + anti-GFP antibody. Transcriptome analysis revealed that the TEAD4 target genes Cdx2 and Gata3 are significantly reduced in the embryos that received the anti-TEAD4 antibody compared to the embryos that received Trim21 mRNA only, while the embryos that received anti-GFP antibody were much similar to those that received Trim21 mRNA only.

Dataset Information

Trim-Away ubiquitinates and degrades lysine-less and N-terminally acetylated substrates.

Publications

Trim-Away ubiquitinates and degrades lysine-less and N-terminally acetylated substrates.

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