Key Takeaways & Executive Findings
- •• TRIM21 (ΔBB), a B-box domain deletion variant, significantly enhances targeted protein degradation efficiency compared to wild-type TRIM21 and other engineered constructs. • The TRIM21 (ΔBB) system effectively degrades HPV oncoproteins E6 and E7 in both exogenous and endogenous (CaSki) settings, offering a potential therapeutic strategy for HPV-associated cancers. • The study demonstrates that domain engineering of E3 ligases can optimize antibody-based TPD technologies, providing a versatile platform for targeted protein degradation. • The Fc-nanobody approach enables specific targeting of proteins for degradation, with potential applications in research and clinical interventions.
Abstract
The ubiquitin-proteasome pathway is a highly selective protein degradation pathway that is capable of efficiently degrading intracellular proteins and plays an important role in various life processes. Dysfunction of this pathway has been associated with numerous problems, including cancer and neurodegenerative diseases. Targeted protein degradation (TPD) technologies have emerged as promising tools for use in a number of different areas, including biological research and clinical interventions. Recently, a technology named Trim-Away was developed for the rapid degradation of proteins in mammalian cells. Briefly, an antibody is designed against a target protein, and the E3 ligase TRIM21 is used to recognize the Fc region of the antibody and subsequently mediate antibody-dependent protein degradation via the proteasome. To enhance the protein degradation efficiency of Trim-Away, three TRIM21-based constructs were designed: (1) deletion of the B-box domain of TRIM21, termed TRIM21 (ΔBB), (2) substitution of the RING domain of TRIM21 with the RING domain of MKRN1, termed TRIM21-RING, and (3) substitution of the RING domain of TRIM21 with the HECT domain of UBE3A, designated TRIM21-HECT. The antibody was designed as a human IgG Fc region-fused nanobody. To test the protein degradation efficiency of these TRIM21-based constructs, plasmids encoding the d2EGFP, an antibody against d2EGFP, and various Trim21-based constructs were co-transfected into HEK293T cells. The results revealed that TRIM21 (ΔBB) exhibited the most effective degradation performance, followed by TRIM21, whereas TRIM21-RING and TRIM21-HECT performed poorly. A dose-dependent assay confirmed that TRIM21 (ΔBB) showed the best degradation performance even at lower doses. Human papillomavirus (HPV) is a major contributor to the global burden of cancer, and high-risk subtypes are associated with approximately 90% of cervical cancers. Two viral oncoproteins, E6 and E7, play a role in carcinogenesis. Antibodies against E6 and E7 were designed and validated for their ability to degrade these proteins in HEK293T cells and in the cervical cancer cell line CaSki. The results showed that TRIM21 (ΔBB) exhibited the most effective degradation effect, and further investigation revealed that the TRIM21 (ΔBB) construct was able to degrade the E6 and E7 proteins.
1. Introduction
The ubiquitin-proteasome pathway is a highly selective protein degradation pathway that is capable of efficiently degrading intracellular proteins and plays an important role in various life processes [1,2]. Dysfunction of this pathway has been associated with numerous problems, including cancer and neurodegenerative diseases [3,4]. Targeted protein degradation (TPD) technologies have emerged as promising tools for use in a number of different areas, including biological research and clinical interventions [5].
Recently, a technology named Trim-Away was developed for the rapid degradation of proteins in mammalian cells [6]. Briefly, an antibody is designed against a target protein, and the E3 ligase TRIM21 is used to recognize the Fc region of the antibody and subsequently mediate antibody-dependent protein degradation via the proteasome (Figure 1A).
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Weikang Hu, Xiang Qiu, Yun Yang, Yaoyao Wu, Chengcheng Wang, Ronggui Hu, Chuanyin Li (2026). A TRIM21-based method for targeted protein degradation. Acta Biochimica et Biophysica Sinica. https://doi.org/10.3724/abbs.2024179
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Frequently Asked Questions
What is the TRIM21-based method for targeted protein degradation?
The method uses the E3 ligase TRIM21 to recognize antibodies bound to target proteins, leading to their degradation via the proteasome. The study engineered TRIM21 variants to enhance degradation efficiency.
Which TRIM21 variant showed the best degradation performance?
TRIM21 (ΔBB), which lacks the B-box domain, exhibited the most effective degradation of d2EGFP and HPV E6/E7 proteins compared to wild-type TRIM21 and other variants.
How does the TRIM21 (ΔBB) system degrade HPV E6 and E7?
The system uses Fc-nanobodies specific to E6 and E7, which are recognized by TRIM21 (ΔBB), leading to their ubiquitination and proteasomal degradation, thereby reducing oncoprotein levels.
What are the potential applications of this technology?
This technology can be used for targeted protein degradation in research and therapy, particularly for diseases like cancer where specific oncoproteins need to be eliminated.
What is the significance of using a nanobody in this system?
Nanobodies are small, stable antibody fragments that can be easily fused to Fc regions, allowing TRIM21 to recognize and degrade the target protein with high specificity.
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