VCP's Nuclear Journey: Initiated by Interacting with KPNB1 to Repair DNA Damage

Chemistry Medicinal Chemistry Cell Biology Oncology Genetics Biochemistry Life Sciences Medicine Immunology
DNA damage repair VCP KPNB1 nuclear translocation Withaferin A

Academic Background

DNA damage repair (DDR) is a core mechanism for maintaining genome stability, and its dysfunction is closely associated with cancer development. Valosin-containing protein (VCP/p97), a member of the AAA+ ATPase family, plays a critical role in DDR by recognizing ubiquitinated proteins and recruiting repair factors (e.g., 53BP1, BRCA1). However, the mechanism by which VCP, synthesized in the cytoplasm, is transported into the nucleus remains unclear. Meanwhile, the nuclear transport receptor Karyopherin β1 (KPNB1) is highly expressed in various cancers, but its specific regulatory role in DDR is also undefined. This study aims to elucidate the molecular mechanism of VCP nuclear transport and develop small-molecule inhibitors targeting this pathway.

Source of the Paper

This study was collaboratively conducted by Xing Zhichao from the Thyroid Surgery Department of West China Hospital, Sichuan University, and teams from the State Key Laboratory of Biotherapy, including Ye Haoyu and Wu Wenshuang. It was published on May 8, 2025, in PNAS (Proceedings of the National Academy of Sciences), titled “VCP’s nuclear journey: initiated by interacting with KPNB1 to repair DNA damage.”

Research Process and Findings

1. Small-Molecule Compound Screening and Target Identification

Study Subjects:
- A library of 175 natural compounds
- Four anaplastic thyroid carcinoma (ATC) cell lines (C643, OCUT-2C, etc.)

Methods:
- Drug Sensitivity Screening: Identified Withaferin A (WA) as a potent inhibitor of ATC cells (IC50 ≈ 0.5 μM)
- Drug Affinity Responsive Target Stability (DARTS): Protease protection assays and mass spectrometry confirmed KPNB1 as the binding target
- Microscale Thermophoresis (MST): Validated direct binding between WA and KPNB1 (KD = 13.9 nM)

Key Results:
- WA covalently binds to Cys158 of KPNB1 via its α,β-unsaturated ketone group (mutagenesis confirmed loss of activity with C158A mutation)
- Molecular docking revealed WA’s C-3 forms a covalent bond with Cys158, while C-4/C-27 hydroxyl groups hydrogen-bond with Glu203/Lys68

2. Mechanism of KPNB1-Mediated VCP Nuclear Transport

Study Subjects:
- Interaction domains between VCP and KPNB1

Methods:
- Co-Immunoprecipitation (Co-IP): Confirmed VCP as a direct cargo protein of KPNB1
- Bio-Layer Interferometry (BLI): Measured binding affinity (KD = 7.50 nM)
- Truncation Analysis: Identified VCP’s 691-717aa segment inserts into KPNB1’s HEAT repeats 2-5 (33-211aa), forming a “card-slot” binding mode

Key Results:
- Mutations in VCP (E710A/R713A/Q714A/T715A/P717A) significantly reduced KPNB1 binding (KD increased to 348 nM)
- WA competitively inhibits VCP nuclear localization by steric hindrance (BLI validation)

3. Biological Effects of WA on DDR Pathway Disruption

Study Subjects:
- DNA damage markers (γ-H2AX), repair efficiency (HR/NHEJ reporter systems)

Methods:
- Alkaline Comet Assay: Quantified WA-induced DNA breaks
- Immunofluorescence Colocalization: Analyzed 53BP1/RAD51 recruitment to γ-H2AX foci
- Chromatin Fractionation: Examined nuclear distribution of repair proteins (KU70/80, BRCA1, etc.)

Key Results:
- WA treatment led to:
- Nuclear accumulation of K48/K63 ubiquitinated proteins (↑2.5-fold)
- 60% reduction in HR/NHEJ repair efficiency
- Failure of repair factors (53BP1, RAD51) to localize to damage sites
- Nuclear-localized VCP (NLS-VCP) overexpression reversed WA-induced DNA damage

4. In Vivo Antitumor Efficacy Validation

Study Subjects:
- ATC xenograft mouse models (C643, OCUT-2C)
- Patient-derived organoids (PDOs)

Methods:
- Pharmacodynamic Evaluation: Intraperitoneal WA (15 mg/kg) every 2 days
- Immunohistochemistry: Assessed Ki67, TUNEL, etc.

Key Results:
- WA significantly suppressed tumor growth (72–79% volume reduction)
- Safety: No significant weight loss or organ toxicity observed

Research Conclusions and Impact

  1. Scientific Significance:

    • First demonstration of KPNB1-mediated VCP nuclear transport via HEAT repeats 2-5
    • Identified VCP’s 691-717aa as the critical nuclear localization domain

  2. Translational Value:

    • Revealed WA’s novel mechanism of blocking VCP transport by targeting KPNB1-Cys158
    • Provided a theoretical basis for developing nuclear transport-targeted anticancer drugs

  3. Clinical Relevance:

    • KPNB1/VCP overexpression correlates with poor prognosis in thyroid and other cancers
    • WA showed efficacy in both BRAF-mutant (OCUT-2C) and HRAS-mutant (C643) models

Research Highlights

  1. Methodological Innovation:

    • Integrated DARTS, BLI, and molecular docking for target validation
    • Developed VCP nuclear localization reporters (NLS-VCP vs. truncations)

  2. Theoretical Breakthrough:

    • Proposed the “KPNB1-VCP-DNA repair” axis conceptual framework
    • Clarified WA’s unique steric hindrance mechanism without target degradation

  3. Application Potential:

    • WA as a radiosensitizer/chemosensitizer
    • KPNB1-Cys158 as a structural basis for designing specific inhibitors