Nanogel-facilitated Protein Intracellular Specific Degradation through Trim-Away.
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ABSTRACT:
SUBMITTER: Sui B
PROVIDER: S-EPMC8376022 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Tripartite motif-containing protein 21 (TRIM21) is a cytosolic antibody receptor and E3 ubiquitin ligase that promotes destruction of a broad range of pathogens. TRIM21 also underlies the antibody-dependent protein targeting method Trim-Away. Current evidence suggests that TRIM21 binding to antibodies leads to formation of a self-anchored K63 ubiquitin chain on the N terminus of TRIM21 that triggers the destruction of TRIM21, antibody, and target protein. Here, we report that addition of antibody and TRIM21 to Xenopus egg extracts promotes efficient degradation of endogenous target proteins, establishing cell-free Trim-Away as a powerful tool to interrogate protein function. Chemical methylation of TRIM21 had no effect on target proteolysis, whereas deletion of all lysine residues in targets abolished their ubiquitination and proteasomal degradation. These results demonstrate that target protein, but not TRIM21, polyubiquitination is required for Trim-Away, and they suggest that current models of TRIM21 function should be fundamentally revised.
| S-EPMC10435667 | biostudies-literature
Project description:Most recently, a technology termed TRIM-Away has allowed acute and rapid destruction of endogenous target proteins in cultured cells using specific antibodies and endogenous/exogenous tripartite motif 21 (TRIM21). However, the relatively large size of the full-size mAbs (150 kDa) results in correspondingly low tissue penetration and inaccessibility of some sterically hindered epitopes, which limits the target protein degradation. In addition, exogenous introduction of TRIM21 may cause side effects for treated cells. To tackle these limitations, we sought to replace full-size mAbs with the smaller format of antibodies, a nanobody (VHH, 15 kDa), and construct a new type of fusion protein named TRIMbody by fusing the nanobody and RBCC motif of TRIM21. Next, we introduced enhanced green fluorescent protein (EGFP) as a model substrate and generated αEGFP TRIMbody using a bispecific anti-EGFP (αEGFP) nanobody. Remarkably, inducible expression of αEGFP TRIMbody could specifically degrade intracellular EGFP in HEK293T cells in a time-dependent manner. By treating cells with inhibitors, we found that intracellular EGFP degradation by αEGFP TRIMbody relies on both ubiquitin-proteasome and autophagy-lysosome pathways. Taken together, these results suggested that TRIMbody-Away technology could be utilized to specifically degrade intracellular protein and could expand the potential applications of degrader technologies.
| S-EPMC8533776 | biostudies-literature
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.
2019-07-10 | GSE124844 | GEO
Project description:Trim-Away is a versatile intracellular protein degradation pathway that has been extensively explored in vitro . However, the in vivo application of Trim-Away is limited at oocyte and zygote stages due to the lack of an in vivo practical approach for intracellular antibody delivery. To broaden the application of Trim-Away, especially for clinical use, we developed a nanogel-based Nano-ERASER system. Here, we demonstrated that the intracellular delivery of anti-programmed cell death ligand 1 (PD-L1) antibody through Nano-ERASER could effectively deplete PD-L1 in triple negative breast cancer (TNBC) cells and induce cancer cell death. Furthermore, with the help of a tumor tissue-targeted nanogel, anti-PD-L1 antibody-loaded Nano-ERASER effectively inhibited tumor progression in a TNBC mouse model. These results confirmed that Nano-ERASER realized Trim-Away in adult animals for the first time, which could be an effective tool for disease treatment and studying gene/protein function both in vitro and in vivo.
| S-EPMC9882598 | biostudies-literature
Project description:Trim-Away is a recent technique to rapidly deplete a protein from any cell type. Guided by antibodies, TRIM21 selects proteins for destruction. However, the applicability of this method in model organisms has not been investigated. Here, we show that Trim-Away can degrade proteins in zebrafish embryos. Trim-Away depletes proteins faster than morpholinos, which enables analysis of protein function during early embryogenesis. Furthermore, Trim-Away can be applied to evaluate the role of maternally contributed proteins in zebrafish embryos. Our findings indicate that Trim-Away is a powerful tool to perform functional analysis of proteins during zebrafish development.
| S-EPMC6343325 | biostudies-literature
Project description:Trim-Away is a recently developed technology that exploits off-the-shelf antibodies and the RING E3 ligase and cytosolic antibody receptor TRIM21 to carry out rapid protein depletion. How TRIM21 is catalytically activated upon target engagement, either during its normal immune function or when repurposed for targeted protein degradation, is unknown. Here we show that a mechanism of target-induced clustering triggers intermolecular dimerization of the RING domain to switch on the ubiquitination activity of TRIM21 and induce virus neutralization or drive Trim-Away. We harness this mechanism for selective degradation of disease-causing huntingtin protein containing long polyglutamine tracts and expand the Trim-Away toolbox with highly active TRIM21-nanobody chimeras that can also be controlled optogenetically. This work provides a mechanism for cellular activation of TRIM RING ligases and has implications for targeted protein degradation technologies.
| S-EPMC7611929 | biostudies-literature
Project description:Cataracts are a leading cause of blindness worldwide. Surgical removal of cataracts is a safe and effective procedure to restore vision. However, a large number of patients later develop vision loss due to regrowth of lens cells and subsequent degradation of the visual axis leading to visual disability. This postsurgical complication, known as posterior capsular opacification (PCO), occurs in up to 30% of cataract patients and has no clinically proven pharmacological means of prevention. Despite the availability of many compounds capable of preventing early steps in PCO development, there is currently no effective means to deliver such therapies into the eye for a suitable duration. To model a solution to this unmet medical need, we fabricated acrylic substrates as intraocular lens (IOL) mimics scaled to place into the capsular bag of the mouse lens following a mock-cataract surgery. Substrates were coated with a hydrophilic crosslinked acrylate nanogel designed to elute Sorbinil, an aldose reductase inhibitor previously shown to suppress PCO. Insertion of the Sorbinil-eluting device into the lens capsule at the time of cataract surgery resulted in substantial prevention of cellular changes associated with PCO development. This model demonstrates that a cataract inhibitor can be delivered into the postsurgical lens capsule at therapeutic levels.
| S-EPMC8391309 | biostudies-literature
Project description:Mutation studies always defined the functions of the zona pellucida (ZP) as extracellular, namely: to encase the oocytes in ovarian follicles, to ensure species-specific sperm binding, and to dampen shear stress on the embryo surface. Therefore, mutations in the three ZP mouse genes ZP1, ZP2 or ZP3 cause primary infertility due to empty follicles, polyspermic fertilization or harmful contact between embryos and oviductal epithelium. However, the concepti of ZP2-null and ZP3-null oocytes were still unviable also when the defects were obviated by monospermic fertilization in vitro and blastocyst transfer to uterus (PMID 11245577). This suggests that the tasks of ZPs don’t end in the extracellular space as previously assumed, but there may be also intracellular functions yet to be discovered. The present study tested if experimentally induced degradation of intracellular ZP3 impacted on the development and transcriptome of mouse embryos. To this end we degraded ZP3 using its antibody in conjunction with the ubiquitin-protein ligase TRIM21. This method is known as 'Trim-away' (PMID 29153837). Briefly, in this method a cell (e.g. oocyte) expressing TRIM21 is supplied e.g. injected with a specific antibody to a protein of interest, in this case ZP3. As a result, the ternary complex (target protein-antibody-TRIM21) is destroyed in the proteasome. TRIM21 is here always to be understood as translation product of microinjected mCherry-Trim21 mRNA. We compared two experimental groups, as follows. Pronuclear-stage oocytes (B6C3F1 x CD1) were microinjected with approx. 100 picoliters of mix comprised of mCherry-Trim21 mRNA 0.2 mg/mL + anti-ZP3 antibody (Proteintech 21279-1-AP) 1 mg/mL + dextran beads 0.02 mg/mL, forming a group named 'Trim-away ZP3' group, in triplicate. As a reference, pronuclear-stage oocytes were microinjected with the same mixture as above, except that the antibody buffer was used in lieu of the antibody itself, in triplicate, forming a group named ‚no Trim'. To identify differently expressed genes we compared group 'Trim-away ZP3' with group ‘no Trim’. Ten hours after microinjection, embryos were collected and lysed for transcriptome analysis. Transcriptome analysis revealed that embryos of group 'Trim-away ZP3' and group ‘no Trim' differed in gene expression and were resolved in principal component analysis. The data support a conclusion that ZP3 found inside the embryo was not merely a remnant from oogenesis, but served an intracellular, post-fertilization role during mouse preimplantation development.
2023-10-12 | GSE232142 | GEO
Project description:Mutation studies always defined the functions of the zona pellucida (ZP) as extracellular, namely: to encase the oocytes in ovarian follicles, to ensure species-specific sperm binding, and to dampen shear stress on the embryo surface. Therefore, mutations in the three ZP mouse genes ZP1, ZP2 or ZP3 cause primary infertility due to empty follicles, polyspermic fertilization or harmful contact between embryos and oviductal epithelium. However, the concepti of ZP2-null and ZP3-null oocytes were still unviable also when the defects were obviated by monospermic fertilization in vitro and blastocyst transfer to uterus (PMID 11245577). This suggests that the tasks of ZPs don’t end in the extracellular space as previously assumed, but there may be also intracellular functions yet to be discovered. The present study tested if experimentally induced degradation of intracellular ZP3 impacted on the development and transcriptome of mouse embryos. To this end we degraded ZP3 using its antibody in conjunction with the ubiquitin-protein ligase TRIM21. This method is known as 'Trim-away' (PMID 29153837). Briefly, in this method a cell (e.g. oocyte) expressing TRIM21 is supplied e.g. injected with a specific antibody to a protein of interest, in this case ZP3. As a result, the ternary complex (target protein-antibody-TRIM21) is destroyed in the proteasome. TRIM21 is here always to be understood as translation product of microinjected mCherry-Trim21 mRNA. We compared two experimental groups, as follows. Pronuclear-stage oocytes (B6C3F1 x CD1) were microinjected with approx. 100 picoliters of mix comprised of mCherry-Trim21 mRNA 0.2 mg/mL + buffer of ZP3 antibody + dextran beads 0.02 mg/mL as tracer, forming a group named 'Trim21 overexpression', in quadruplicate. As a reference, pronuclear-stage oocytes were microinjected with mCherry-Trim21 mRNA 0.2 mg/mL + anti-ZP3 antibody (Proteintech 21279-1-AP) 1 mg/mL + dextran beads, forming a group named 'Trim-away ZP3' group, in triplicate. To identify differently expressed genes we compared group 'Trim-away ZP3' with group 'Trim21 overexpression'. In addition, a single 'non-manipulated' sample was also incuded merely to confirm that Trim21 was detectable in the two experimental groups, but not in the non-microinjected embryos. On the day after microinjection, embryos were collected and lysed for transcriptome analysis. Those of group 'Trim21 overexpression' were at the 2-cell stage so as the non-manipulated embryos, whereas those of group 'Trim-away ZP3' were arrested at the 1-cell stage. Transcriptome analysis revealed that embryos of group 'Trim-away ZP3' and 'group 'Trim21 overexpression' differed in the expression of 197 of 11137 genes (t test, FDR<0.05). The data support a conclusion that ZP3 found inside the embryo was not merely a remnant from oogenesis, but served an intracellular, post-fertilization role during mouse preimplantation development.
2023-10-12 | GSE203626 | GEO
Project description: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.
| S-EPMC10105713 | biostudies-literature