Caught the ‘Catch’ of midnolin: structural basis for broad substrate specificity in ubiquitin-independent proteasomal degradation

Abstract

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.

1. Introduction

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 [1–3]. 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 [3].

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 [4–7]. 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.