Structural Insights into the Ubiquitin-Independent Midnolin-Proteasome Pathway

Biophysics Cell Biology Oncology Biochemistry Life Sciences Medicine Immunology
proteasome ubiquitin-independent degradation Midnolin cryo-EM B-cell malignancies

Academic Background

Protein homeostasis (proteostasis) is a core mechanism for maintaining normal cellular function, with the ubiquitin-proteasome system (UPS) responsible for degrading approximately 80% of abnormal proteins. Traditionally, proteins were thought to require ubiquitination for recognition and degradation by the 26S proteasome. However, recent studies have revealed that certain transcription factors (e.g., EGR1, FOSB) can be degraded independently of ubiquitination, a phenomenon closely linked to lymphocyte development and malignancies. Midnolin has been identified as a key mediator of this process, but its structural basis and molecular mechanisms remained unclear.

This study, led by Bruce Beutler’s team at UT Southwestern Medical Center (winner of the 2011 Nobel Prize in Physiology or Medicine), aimed to elucidate the structural basis of ubiquitin-independent degradation by resolving the three-dimensional structure of the Midnolin-proteasome complex using cryo-electron microscopy (cryo-EM). The research was published in PNAS (Proceedings of the National Academy of Sciences) on May 8, 2025, under the title “Structural insights into the ubiquitin-independent midnolin-proteasome pathway.”

Research Process and Findings

1. Complex Reconstitution and Cryo-EM Analysis

The research team first constructed a human Midnolin-proteasome-IRF4 (transcription factor) ternary complex using an in vitro reconstitution system:
- Sample Preparation: Purified 26S proteasomes from HEK293 cells were mixed with recombinant Midnolin (468 amino acids) and IRF4 at a 1:50 molar ratio, supplemented with ATPγS (a non-hydrolyzable ATP analog) and the proteasome inhibitor MG-132.
- Cryo-Sample Preparation: Quantifoil copper grids were used for rapid freezing (−196°C liquid ethane) at 100% humidity.
- Data Collection: 20,794 movie frames were collected using an FEI Titan Krios microscope (300 kV), yielding 939,289 valid particles.

Technical Highlights:
- Developed a “focused local refinement” strategy to optimize interactions between Midnolin and the regulatory particle (RP) of the proteasome.
- Applied DeepEMhancer for density map sharpening, improving the resolution of the RPN11-Midnolin binding interface to 2.8 Å.

2. 3D Classification and Model Building

Using cryoSPARC v4 for 3D classification, seven conformational states were identified, with two key states analyzed in detail:
- EBmidn State (66% of particles): The proteasome core particle (CP) gate is closed, with Midnolin simultaneously bound to RPN1 and RPN11.
- Resolution: 3.0 Å, clearly revealing the C-terminal α-helix (αhelix-c, residues 381–409) of Midnolin binding to the T2 site of RPN1.
- The ubiquitin-like (UBL) domain (residues 31–105) inserts into the catalytic cleft of RPN11, mimicking ubiquitin binding.
- EDmidn State (15% of particles): The CP gate is open, with only weak binding of the UBL domain to RPN11 detected.

Structural Discoveries:
- Midnolin acts as a “molecular bridge” connecting RPN1 (PSMD2) and RPN11 (PSMD14), forming a stable ternary complex.
- Although the catch domain lacks clear density, low-pass filtered maps (6 Å) show it positioned above the AAA-ATPase ring, suggesting its role as a “delivery platform” to guide substrates into the degradation channel.

3. Functional Validation Experiments

Key findings were validated through mutagenesis:
- Binding Assays:
- L395K/L399K mutations in αhelix-c reduced RPN1 binding by 80% (co-immunoprecipitation data).
- UBL domain deletion (ΔUBL) did not affect RPN11 binding, but the G105R mutation significantly impaired proteasome activation.
- Proteasome Activity Assays:
- Wild-type Midnolin enhanced LLVY-AMC substrate hydrolysis activity by 3–4-fold (fluorometric detection).
- Δαhelix-c mutants retained only 30% activity, confirming the necessity of the dual-anchoring mechanism.

Mechanistic Model and Scientific Significance

The study proposes a “ubiquitin-mimicry” mechanism:
1. Initial Binding: Midnolin anchors to the T2 site of RPN1 via αhelix-c.
2. Conformational Activation: The UBL domain binds RPN11, inducing an EB-state conformational change in the proteasome (40° expansion of the AAA-ATPase ring).
3. Substrate Delivery: The catch domain positions ubiquitin-free substrates at the ATPase channel entrance, initiating translocation.

Therapeutic Implications:
- The R381D mutation boosts Midnolin activity by 75%, offering a target for enhanced degradation strategies.
- Inhibitors targeting the Midnolin-RPN1 interface (e.g., L395K-mimicking peptides) may selectively suppress B-cell malignancies.

Key Highlights

  1. Methodological Innovation: First atomic-resolution capture of Midnolin-proteasome dynamics, establishing a “cryo-EM + functional mutagenesis” paradigm.
  2. Theoretical Breakthrough: Unveils the “molecular bridge” mechanism of ubiquitin-independent degradation, expanding UPS understanding.
  3. Translational Potential: Provides novel precision therapy targets for B-cell malignancies like multiple myeloma (MM).

Supplementary Findings

  • Comparative proteomics showed Midnolin expression in lymphocytes is 5–8 times higher than in other tissues (validated via ENU-mutagenized mouse models).
  • Divergence from a concurrent preprint (Gu et al., 2023): This study found excessive Midnolin may suppress MM by degrading IRF4, suggesting dose-dependent regulation.

The study lays the foundation for understanding the “third pathway” of protein degradation. Structural data are deposited in EMDB (IDs: 49507–49510) and PDB (IDs: 9NKF–9NKJ).