Acta Biochimica et Biophysica Sinica•2026•DOI: 10.3724/abbs.2026006
Protein homeostasis serves as the foundation for every cellular decision—division, differentiation, stress adaptation, or death—by precisely balancing the proteome across abundance, quality, spatial distribution, and temporal dynamics; its dysregulation drives numerous human pathologies, including cancers and neurological disorders. In the traditional ubiquitin-dependent degradation cascade, target proteins are marked by covalent attachment of polyubiquitin chains, a process requiring E1 activating enzymes, E2 conjugating enzymes, and E3 ligases that confer substrate specificity. This ubiquitin signal is then recognized by the 19S regulatory particle of the proteasome, which unfolds and translocates the tagged protein into the 20S core for proteolytic destruction. The ubiquitin-independent proteasomal degradation pathway mediated by midnolin (MIDN) has recently emerged as a distinct and biologically important mechanism for regulating nuclear protein turnover. While earlier genetic, biochemical, and cryo-electron microscopy studies established MIDN as a proteasome-associated adaptor for immediate-early gene (IEG) products, the molecular logic underlying its broad yet selective substrate recognition remains unresolved. In the current study, Zhong et al. provide a comprehensive structural and biochemical analysis of the MIDN Catch domain bound to IRF4-derived peptides, substantially advancing our understanding of MIDN substrate recognition. By solving multiple crystal structures of the Catch-IRF4 complex, including wild-type and systematically engineered mutants, the authors demonstrate that MIDN recognizes substrates through a conserved β-strand insertion mechanism at the interface of the Catch1 and Catch2 subdomains. This investigation generalizes and expands prior structural observations of MIDN-IRF4 to diverse substrates, demonstrating that β-strand complementation constitutes a universal recognition mechanism utilized by MIDN. A major conceptual advance of this study is the identification of a minimal and generalizable recognition principle centered on two highly conserved positions within the substrate motif. The authors show that a reciprocal phenylalanine-glycine interaction between the substrate and Catch2—forming an “F-G zipper”—constitutes the dominant energetic determinant for binding. Disruption of this zipper severely compromises protein stability and binding, explaining prior functional observations that single-point mutations in IRF4 or EGR1 abolish MIDN-mediated degradation. In contrast, flanking residues within the binding motif display remarkable tolerance to substitution. Through combined mutagenesis, thermostability analysis, AlphaFold3 modeling, and structural determination, the study demonstrates that these positions occupy large and plastic hydrophobic pockets in the Catch domain. This architectural flexibility provides a direct molecular explanation for how MIDN can accommodate a wide spectrum of substrates while preserving selectivity. From these data, the authors derive a concise consensus recognition motif (G/S-x-F/Y) embedded within an unstructured or loop region, offering a predictive framework for identifying additional MIDN targets. Importantly, this work bridges a critical gap between previous cryo-EM studies describing MIDN-proteasome engagement and earlier substrate-specific structural analyses. Whereas prior studies clarified how MIDN delivers captured substrates to the proteasome, the present study elucidates how MIDN initially selects and binds those substrates. Together, these findings unify MIDN biology across structural, biochemical and functional dimensions. Overall, this study represents a significant advance in the field of proteostasis by revealing how a ubiquitin-independent adaptor achieves broad substrate specificity through a simple yet robust structural principle. Beyond MIDN, the work provides a paradigm for short-linear-motif-based proteasomal targeting and has important implications for immune regulation, neurodegeneration and cancer biology.
Acta Biochimica et Biophysica Sinica•2025•DOI: 10.3724/abbs.2025048
As a classic tumor suppressor gene, p53 has been extensively studied since its discovery in the mid-1980s. Research findings have revealed that p53 protein expression is suppressed in various cancers [1]. For example, in cervical cancer, p53 predominantly exists in a wild-type form to maintain its biological function [2]. Nevertheless, its tumor-suppressive activity is significantly impaired because of rapid protein degradation, short half-life, and low levels. Post-translational modifications (PTMs) of p53, such as ubiquitination, acetylation, phosphorylation and methylation, are critical regulators of its stability, activity, conformation, localization, and interactions with cofactors [3]. Among these, ubiquitination and acetylation play central roles in controlling p53 protein stability and activity [4]. Therefore, targeting p53 PTMs to modulate its ubiquitination and acetylation levels represents an effective strategy to increase its stability and tumor-suppressive function, offering a promising avenue for cervical cancer drug development. In 99% of cervical cancers (high-risk human papillomavirus-positive), E3 ubiquitin ligase E6-associated protein (E6AP) mediates the ubiquitination degradation of p53 [5], whereas histone deacetylase 6 (HDAC6) deacetylates p53. In this study, we explored the possibility of combining the natural product withferin A (WA) with the HDAC6 inhibitor ricolinostat (RIC) to treat cervical cancer cells, with a focus on the ubiquitination and acetylation of p53 and the consequences for its stability. These results suggested that the combination of WA and RIC is more effective than either treatment alone in inhibiting the degradation and increasing the stability of p53, thereby synergistically slowing the onset and progression of cervical cancer.
Acta Biochimica et Biophysica Sinica•2025•DOI: 10.3724/abbs.2024179
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.
Acta Biochimica et Biophysica Sinica•2025•DOI: 10.3724/abbs.2024104
Carfilzomib (CFZ) is the second-generation proteasome inhibitor that is approved by Food and Drug Administration (FDA) of USA for the treatment of relapsed and refractory multiple myeloma. Although the preclinical and clinical efficacy of CFZ is obvious, the mechanism by which CFZ leads to cell death has not been fully elucidated. Since CFZ primarily functions as a proteasome inhibitor, profiling CFZ-induced changes in protein turnover at the systematic level is sufficient and necessary. In this study, we characterize the effects of CFZ on the stability of 15,000 human proteins using Protein Turnover Assay (ProTA). CFZ affects fundamental cellular glycolysis, nitric oxide production and proteasome subunit homeostasis in multiple myeloma cells. In addition, LY294002 or KU-0063794 has synergistic effects with CFZ in multiple myeloma treatment. A profound understanding of how cells respond to chemotherapeutic agents provides insights into the basic mechanism of drug function and the rationale for CFZ combination therapy.
Acta Biochimica et Biophysica Sinica•2026•DOI: 10.3724/abbs.2026006
Protein homeostasis is fundamental to cellular decisions, and its dysregulation drives numerous pathologies. The ubiquitin-independent proteasomal degradation pathway mediated by midnolin (MIDN) has emerged as a distinct mechanism for regulating nuclear protein turnover. In the current study, Zhong et al. provide a comprehensive structural and biochemical analysis of the MIDN Catch domain bound to IRF4-derived peptides, substantially advancing our understanding of MIDN substrate recognition. By solving multiple crystal structures of the Catch-IRF4 complex, the authors demonstrate that MIDN recognizes substrates through a conserved β-strand insertion mechanism at the interface of the Catch1 and Catch2 subdomains. A major conceptual advance is the identification of a minimal and generalizable recognition principle centered on two highly conserved positions within the substrate motif, forming an 'F-G zipper' that constitutes the dominant energetic determinant for binding. In contrast, flanking residues display remarkable tolerance to substitution, occupying large and plastic hydrophobic pockets in the Catch domain. This architectural flexibility provides a direct molecular explanation for how MIDN can accommodate a wide spectrum of substrates while preserving selectivity. The authors derive a concise consensus recognition motif (G/S-x-F/Y) embedded within an unstructured or loop region, offering a predictive framework for identifying additional MIDN targets. Importantly, this work bridges a critical gap between previous cryo-EM studies describing MIDN-proteasome engagement and earlier substrate-specific structural analyses. Overall, this study represents a significant advance in the field of proteostasis by revealing how a ubiquitin-independent adaptor achieves broad substrate specificity through a simple yet robust structural principle, with important implications for immune regulation, neurodegeneration, and cancer biology.
Acta Biochimica et Biophysica Sinica•2026•DOI: 10.3724/abbs.2025232
Midnolin (MIDN) is a newly recognized master regulator that drives ubiquitin-independent proteasomal degradation, yet the mechanisms governing its own turnover remain enigmatic. Here, we demonstrate that MIDN is ubiquitinated and identify RNF126 as the cognate E3 ligase. RNF126 physically associates with MIDN and catalyzes its ubiquitination, and mass spectrometry mapping reveals that this process occurs primarily at non-canonical cysteine, serine, and threonine residues (C230, C236, S237, T239, and S241) rather than at lysine residues. This non-classical ubiquitination targets MIDN for 26S-proteasomal degradation. In vivo dissection of the RNF126-MIDN axis shows that it governs EGR1 abundance and, consequently, the tumor-suppressor proteins PTEN and p53, thereby restraining the progression of testicular germ-cell tumors (TGCTs). Our findings reveal an unappreciated layer of MIDN regulation and identify the RNF126-MIDN ubiquitination cascade as a potential therapeutic vulnerability in TGCTs and related malignancies.
Acta Biochimica et Biophysica Sinica•2025•DOI: 10.3724/abbs.2025048
Cervical cancer remains a leading cause of cancer-related mortality among women, with high-risk HPV-positive cases constituting 99% of instances. Despite wild-type p53 expression, its tumor-suppressive function is crippled by E6AP-mediated ubiquitination and HDAC6-driven deacetylation, resulting in rapid degradation and low steady-state levels. This study evaluates a combinatorial strategy employing the natural product withaferin A (WA) and the HDAC6 inhibitor ricolinostat (RIC) to simultaneously target p53 ubiquitination and acetylation in HeLa, SiHa, and Caski cervical cancer cells. Dose-response CCK-8 assays established that both agents inhibit proliferation in a dose-dependent manner. Combination treatment significantly reduced cell viability compared to monotherapies, with CompuSyn analysis yielding a combination index (CI) below 1, confirming synergy. Colony formation assays further demonstrated a marked decrease in clonogenic survival. Mechanistically, WA disrupted the p53-E6AP interaction, reducing p53 ubiquitination, while RIC inhibited HDAC6-mediated deacetylation, increasing p53 acetylation. The dual treatment stabilized p53, as evidenced by extended half-life in cycloheximide chase assays. These findings suggest that concurrent modulation of p53 post-translational modifications via WA and RIC offers a potent therapeutic avenue for cervical cancer, meriting further preclinical development.
Acta Biochimica et Biophysica Sinica•2025•DOI: 10.3724/abbs.2025062
p62/SQSTM1 is the archetypal selective autophagy receptor, bridging ubiquitinated cargo to LC3 on phagophores. Its abundance is a critical determinant of autophagic flux, yet the E3 ligase governing its turnover remained incompletely defined. Using Flag-p62 Co-IP coupled to mass spectrometry in HEK293T cells, we identified the E3 ligases TRIM25 and ITCH as highest-confidence interactors. Endogenous and ectopic p62 formed complexes with both ligases; GST pull-down confirmed direct binding, and mCherry-TRIM25 co-localized with GFP-p62 in HeLa cytoplasm. In vitro ubiquitination demonstrated that TRIM25, but not ITCH, efficiently ubiquitinates p62. A reconstituted E. coli system mapped fifteen lysine residues, with K7 and K189 validated as the dominant TRIM25-mediated ubiquitination sites. Functionally, TRIM25 destabilized wild-type p62 but not the K7/189R mutant; TRIM25 knockdown stabilized p62 in HeLa and Caski cells. Degradation proceeded primarily via the lysosomal pathway, as bafilomycin (20 nM) but not bortezomib (1 μM) blocked p62 loss. TRIM25 knockdown enhanced GFP-LC3 puncta formation (P < 0.01) and elevated autophagic markers, whereas TRIM25 overexpression suppressed p62-mediated GFP-LC3 puncta (P < 0.05) and autophagy. These data establish TRIM25 as the principal E3 ligase targeting p62 for lysosomal degradation, defining a negative feedback node in selective autophagy with therapeutic implications for cancers and neurodegenerative disorders characterized by p62 accumulation.