E3 Ligase TRIM63 Promotes Chondrogenic Differentiation of Mesenchymal Stem Cells by Catalyzing K27-Linked Cysteine Ubiquitination of MYH11

Bioengineering Basic Medical Sciences Cell Biology Biochemistry Life Sciences Medicine Orthopedics
TRIM63 mesenchymal stem cells chondrogenic differentiation ubiquitination MYH11 cartilage repair osteoarthritis

Research Progress on the Role of E3 Ubiquitin Ligase TRIM63 in Promoting Chondrogenic Differentiation of Mesenchymal Stem Cells — An Interpretation of the Paper “E3 ligase TRIM63 promotes the chondrogenic differentiation of mesenchymal stem cells by catalyzing K27-linked cysteine ubiquitination of MYH11”

1. Academic Background and Research Motivation

Osteoarthritis (OA) is a chronic degenerative disease affecting more than 300 million people globally, and its incidence is becoming increasingly severe with the aging population. Articular cartilage defects (ACD) are the direct cause of OA-related pain and functional impairment, making them one of the most challenging problems in clinical orthopedics and regenerative medicine. Articular cartilage itself is poorly vascularized and has limited self-repair capabilities, so conventional treatments such as drugs, physical therapy, and surgery are often limited in efficacy. In recent years, stem cell transplantation has brought new hope for cartilage repair, especially bone marrow-derived mesenchymal stem cells (BMSCs), which have attracted wide attention and are widely used in cartilage tissue engineering due to their multidirectional differentiation and self-renewal abilities.

However, the molecular regulatory mechanisms underlying the efficient differentiation of BMSCs into chondrocytes—especially protein modification-related regulatory pathways—still remain largely unclear. Ubiquitination, as a post-translational modification, plays a central regulatory role in the self-renewal, proliferation, and differentiation of stem cells. Most current studies focus on ubiquitin conjugation to lysine, but much less is known about ubiquitination of non-lysine residues (such as cysteine) and their physiological significance. In addition, the functions of different ubiquitin linkage types (such as K6, K11, K27, K29, K33, K48, K63) in cell fate regulation have not been fully elucidated.

Against this background, the research team aimed to reveal the molecular mechanism of ubiquitin regulation during the chondrogenic differentiation of BMSCs, with a focus on the emerging E3 ubiquitin ligase TRIM63 and its effect and mechanism in chondrogenic differentiation, striving to provide new theoretical basis and molecular targets for cartilage regenerative engineering and osteoarthritis treatment.

2. Source of the Paper and Author Introduction

This paper, entitled “E3 ligase TRIM63 promotes the chondrogenic differentiation of mesenchymal stem cells by catalyzing K27-linked cysteine ubiquitination of MYH11,” was completed by Shanyu Ye, Yanqing Wang, Ziwei Luo, Aijun Liu, Xican Li, Jiasong Guo, Wei Zhao, Dongfeng Chen, Lin Yang, Helu Liu and others. The main authors are from several well-known research and medical institutions in Guangdong, China, including Shenzhen Hospital of Integrated Traditional Chinese and Western Medicine, Department of Anatomy of the School of Basic Medical Sciences at Guangzhou University of Chinese Medicine, School of Chinese Herbal Medicine at Guangzhou University of Chinese Medicine, Department of Histology and Embryology at Southern Medical University, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, etc. The paper was published in 2025 in a high-level academic journal under Oxford University Press.

3. Research Procedure and Methods in Detail

1. Overall Research Design

This study adopted in vitro cell experiments, in vivo animal models, and multiple molecular biology and proteomic techniques, and systematically explored the chondrogenic differentiation of BMSCs and its ubiquitination regulatory mechanisms from the stages of “identification of key ubiquitinating enzymes—functional validation—mechanistic elucidation—verification of therapeutic potential at the animal level”.

1.1 Establishment and Validation of In Vitro BMSC Differentiation Model

  • Sample Source and Processing: BMSCs were extracted from SD rat bone marrow and cultured in standard MSC medium. Fourth-generation cells were used for subsequent experiments.
  • Induced Differentiation Method: DMEM medium containing TGF-β3, dexamethasone, ascorbic acid, insulin-transferrin-selenium, sodium pyruvate, proline and other chondrogenic inducers was applied. After 7 days of continuous induction, intact chondrogenic pellets were collected.
  • Differentiation Validation: Elevated expression of cartilage-related markers such as SOX9 and COL2A1 was detected by qPCR and Western blot. Morphologically, obvious spherical aggregates were formed.

1.2 Screening and Expression Analysis of Key Ubiquitinating Enzymes

  • Whole Transcriptome Sequencing and Proteomic Analysis: High-throughput sequencing and mass spectrometry analysis were performed before and after BMSC chondrogenic differentiation to screen for differentially expressed E3 ligases related to ubiquitination.
  • Key Molecule Identification: E3 ligase TRIM63 was found to be significantly upregulated at both the transcript and protein levels, as confirmed by qPCR and immunoblotting.

1.3 Functional Intervention of TRIM63 and its Impact on Differentiation

  • RNA Interference: Multiple siRNAs targeting TRIM63 were designed, and the most efficient one was used in subsequent experiments.
  • Functional Assays: Under TGF-β3-induced differentiation, TRIM63 was knocked down. Western blot was used to analyze total ubiquitination levels and cartilage-specific gene expression; Alcian blue staining and morphology were used to evaluate the integrity of chondrogenic pellets.
  • Experimental Conclusion: Knockdown of TRIM63 led to reduced overall ubiquitination, inability to form complete cartilage spheres, and decreased expression of cartilage genes—indicating its key role in differentiation.

1.4 Screening and Verification of TRIM63 Substrate

  • Co-IP Combined with Mass Spectrometry: Co-immunoprecipitation using TRIM63 antibody was performed to screen for specific substrate proteins; mass spectrometry and bioinformatics analysis identified the cytoskeletal protein MYH11 (myosin heavy chain 11) as the specific interactor.
  • Functional Validation: Overexpression of MYH11 was shown to inhibit differentiation. Furthermore, interference or overexpression of TRIM63 altered MYH11 expression, revealing regulatory relationships.

1.5 Mechanistic Analysis of Ubiquitination

  • Identification of Ubiquitination Types: HEK293T cells were co-transfected with TRIM63, FLAG-MYH11, and different lysine-mutant HA-Ubiquitin constructs. Immunoprecipitation-immunoblotting determined which ubiquitin chain type was critical. Only the K27R mutant significantly weakened TRIM63-mediated ubiquitination, confirming the involvement of the K27-linked chain.
  • Identification of Ubiquitin Acceptor Sites: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify the specific MYH11 ubiquitination sites; ubiquitination was found at K1520, C382 (cysteine), and T490 residues. The C382 mutant was constructed and functional studies showed that only the C382R mutation affected TRIM63-mediated K27 ubiquitination and blocked pellet formation.

1.6 In Vivo Animal Model—Verification of Cartilage Defect Repair Capability

  • Animal Grouping and Implantation: SD rats were randomly divided into three groups (scaffold-only, AAV-NC control, AAV-TRIM63). A 3mm full-thickness cartilage defect was created in the groove of the knee joint, and each group received injection of BMSCs in hydrogel.
  • Follow-up and Outcome Evaluation: Six weeks after surgery, multiple histological stainings (HE, Safranin O-Fast Green, Toluidine Blue) were used to evaluate cartilage regeneration quality, and the ICRS scoring system was applied.
  • Regeneration Effect: In the AAV-TRIM63 group, defects were filled with newly formed cartilage-like tissue morphologically close to normal cartilage; staining and immunohistochemistry showed enhanced COL2A1 with low COL10A expression, and ICRS scores were significantly higher than other groups.

2. Data Analysis and Methodological Innovations

  • Combination of Large-scale Proteomics and Transcriptomics for screening functionally important E3 ligases.
  • Detailed Functional Screening with Multiple Ubiquitin Chain-Specific Mutants to precisely pinpoint the ubiquitination type.
  • LC-MS/MS for Exact Localization of Substrate Ubiquitination Residues, combined with mutant functional studies to decipher site-specific effects.
  • Integrated In Vitro and In Vivo Validation confirms the multi-level regulatory role of TRIM63 in differentiation and regeneration.

4. Analysis of Main Research Results

1. TRIM63 Is Significantly Upregulated During BMSC Chondrogenic Differentiation

After chondrogenic induction, TRIM63 expression is strongly elevated at both mRNA and protein levels in BMSCs, accompanied by an overall increase in cellular ubiquitination, suggesting this enzyme may be a novel key regulator in the differentiation network.

2. Interference of TRIM63 Blocks BMSC Chondrogenic Differentiation Process

Following siRNA-mediated knockdown of TRIM63, BMSCs failed to form complete chondrogenic spheres under induction, with significant downregulation of marker genes SOX9, COL2A1, etc. Alcian blue staining confirmed incomplete differentiation, reinforcing the essential role of TRIM63.

3. MYH11 is a Specific Substrate for TRIM63 and Must Be Degraded During Differentiation

Mass spectrometry with co-immunoprecipitation showed tighter binding between MYH11 and TRIM63 in the chondrogenic group. Functionally, MYH11 overexpression inhibits chondrogenic differentiation of BMSCs. TRIM63 gene modulation reverses MYH11 expression, forming an upstream-downstream regulatory axis.

4. TRIM63 Specifically Catalyzes K27-Linked Cysteine 382 Ubiquitination of MYH11

Molecular biology experiments precisely identified the ubiquitination pattern as K27-linked ubiquitin chain on MYH11 C382 cysteine residue, rather than conventional lysine ubiquitination. The C382R mutant completely abolished this effect and blocked differentiation, pinpointing the functional site.

5. TRIM63 Overexpression Enhances BMSC Cartilage Repair Capacity In Vivo

AAV-mediated upregulation of TRIM63 enabled BMSC implants in animal ACD models to efficiently generate structurally and functionally normal new cartilage, providing a promising new cellular and molecular strategy for regenerative therapy.

5. Conclusion and Scientific Value

This study clarifies, for the first time, that E3 ligase TRIM63 regulates K27-linked cysteine 382 ubiquitination-driven degradation of MYH11, thus unlocking the regulatory mechanism of BMSC chondrogenic differentiation. This work not only deepens our understanding of the role of ubiquitination in stem cell fate determination, but also offers a novel and specific molecular therapeutic target for cartilage regeneration and osteoarthritis.

  • Theoretical Significance: Expands the boundaries of non-lysine ubiquitination biology and reveals new perspectives for diversified ubiquitin chain regulation of stem cell fate.
  • Application Prospects: TRIM63 is expected to become a drug or gene therapy target for articular cartilage repair, representing a molecular breakthrough for prevalent and burdensome conditions such as osteoarthritis.

6. Summary of Research Highlights

  1. Innovative Molecular Mechanism: First proposal of the regulatory significance of K27-linked cysteine ubiquitination in BMSC differentiation.
  2. Substrate-Enzyme-Residue Central Axis Analysis: Target specificity is made clear, filling a critical gap with respect to MYH11’s role in stem cell differentiation.
  3. Rigorous In Vivo and In Vitro Verification: Theoretical biology and preclinical animal experiments form a closed loop with strong translational potential.
  4. Multi-omics Synergy: Integrated transcriptomics, proteomics, and functional genetics provide both convincing and comprehensive evidence.

7. Supplementary Information and Future Outlook

The author team is experienced and technologically equipped. All original data from the paper can be requested from the corresponding author. The research was supported by multiple science and technology grants from Guangdong and Shenzhen, and all animal experiments were strictly approved by ethics committees.

In the future, further studies are needed to elucidate the upstream and downstream networks regulating TRIM63, and the development of small molecule agonists or inhibitors is worth exploring. In addition, the expansion of ubiquitin chain type diversity and substrate spectra will further enrich the theory and clinical application landscape of regenerative and translational medicine.