Tim Clausen

RNF213 is a giant AAA+ ubiquitin ligase that serves as a central hub of innate immunity, responding to diverse stress signals with broad reactivity ^1-6 . This versatility demands tight regulation, and its failure causes disease, such as Moyamoya angiopathy ^7-10 and cancer progression ^11-13 . How the mega-enzyme is restrained and how mutations override this control is unknown. Here, we show that RNF213 activity is regulated by a dimeric switch — a constitutive dimer that alternates between auto-inhibited and activation-competent states. In the closed dimer, intermolecular contacts bury the E3 machinery, whereas ATP binding remodels the AAA+ core to open the dimer and prime ubiquitination. Dimer conversion is tuned by ubiquitination factors, redox conditions and post-translational modifications, allowing multiple stress signals to act through the same activation mechanism. Moyamoya mutations subvert the same open-closed transition, destabilizing the autoinhibited dimer in a graded manner that tracks with clinical severity. Structural, biochemical and cellular data reveal how disease mutations allosterically disrupt the dimer interface and bypass ATP-dependent control to drive NF-κB activation and caspase-dependent apoptosis. Together, our findings reveal a tuneable dimeric switch that converts diverse stressors into graded inflammatory signalling and provide a mechanistic link between genetic predisposition, environmental triggers and Moyamoya disease.

A major strength of forward genetic screens is their unbiased nature: the plant reveals which genes underlie a physiological process. Yet this strength often presents a new challenge: determining the biochemical basis of the mutant phenotype. BIG/DARK OVEREXPRESSION OF CAB1/TRANSPORT INHIBITOR RESPONSE3 encodes an exceptionally large (∼0.5 MDa) protein identified in diverse genetic screens. big mutants have smaller stature, reduced elongation growth and apical dominance, and defects in lateral root formation, phenotypes consistent with disrupted auxin gradients. However, they also display altered stomatal behaviour, circadian regulation, pathogen responses, and changes in other hormone pathways. This broad pleiotropy points to wide-ranging roles in signalling and development, yet the biochemical function of BIG remained unresolved for more than three decades. Recently, we showed that BIG participates in the Arg/N-degron pathways, a specialized form of proteostasis in which the half-lives of proteins are influenced by their N-terminal residues. However, this does not explain the pleiotropic phenotypes of big mutants. New structural and functional clues from the mammalian homologue ubiquitin amino-end recognizing protein 4 (UBR4) point to wider proteostatic roles for BIG, providing a potential explanation for the impacts of BIG on plant physiology and a rich source of hypotheses for future studies.

Targeting the protein quality control system in Mycobacterium tuberculosis represents a promising and underexplored opportunity for antibiotic development. The ClpC1:ClpP1P2 protease is an essential component of the system that mediates both regulatory and stress-related protein degradation. Several non-ribosomal peptide natural products, including ecumicin, ilamycins (rufomycins) and cyclomarins, have been discovered that bind to the ClpC1 chaperone of the complex and exhibit potent antimycobacterial activity, leading to significant interest in the ClpC1:ClpP1P2 system as a bona fide target for the new tuberculosis drugs. In this study, we combine quantitative proteomics, bioinformatics, transcriptomics, CRISPRi knockdown, and targeted biochemical and biophysical assays to dissect the mechanisms of ecumicin, ilamycin and cyclomarin in clinically relevant Mycobacterium tuberculosis . Strikingly, despite exhibiting similar binding modes to ClpC1, each compound induces distinct effects on protein degradation. Notably, ilamycin and ecumicin do not trigger the ClpC2 rescue mechanism that mitigates cyclomarin-induced mycobacterial toxicity. In addition, we identify a novel interaction between ecumicin and stress-response chaperone Hsp20. The differential disruption of ClpC1 substrates, stress-response chaperones, and distinct reshaping of the Mycobacterium tuberculosis proteome by the three natural products, unveils new opportunities for the development of protein quality control-targeted antimycobacterials.

BORC and BLOC-1 are multisubunit complexes that regulate endolysosomal trafficking. Although they are presumed to be distinct, their paralogous origins and shared subunits suggest the potential for higher-order assembly. Here, we reveal the conserved octameric architecture of BORC formed by two intertwined tetramers and present the structure of C. elegans BORC. Through cross-linking mass spectrometry of endogenous complexes, we validate this model for human BORC and demonstrate that the integrity of the complex, which is essential for lysosomal transport, relies on specific interfacial residues. We also clarify the disruptive nature of disease-causing mutations and propose that the formation and function of BORC are likely regulated by specific cues. These cues might include the phosphorylation of Snapin and a pH-sensitive histidine residue in BORCS5. Additionally, we present direct biochemical and structural evidence of BORC–BLOC-1 hybrid complexes. Finally, we link a specific hybrid complex to the regulation of transferrin receptor recycling via interaction with the EARP complex. Our work challenges the paradigm of BORC and BLOC-1 as separate entities, establishing a model of dynamic complex formation wherein modular assembly creates functional specialization to meet diverse cellular demands.

APOBEC family members play crucial roles in antiviral restriction. However, certain APOBEC3 (A3) proteins drive harmful hypermutation in humans, contributing to cancer. The cancer-associated A3 proteins are capable of transiting from the cytosol to the nucleus, where they can cause genome mutations. Here, we uncover a specific set of cellular pathways that protect genomic DNA from the major cancer-associated A3 proteins. Through genetic and proteomic screening, we identify UBR4, UBR5, and HUWE1 as key ubiquitin E3 ligases marking cancer-associated A3B and A3H-I for degradation, thereby limiting A3-driven hypermutation. Mechanistically, UBR5 and HUWE1 recognize A3s in the absence of their RNA binding partner, thus promoting proteasomal degradation of APOBEC3 protein that is not engaged in its antiviral cellular function. Depletion or mutation of the E3 ligases in cells and human cancer samples increases A3-driven genome mutagenesis. Our findings reveal that UBR4, UBR5, and HUWE1 are crucial factors in a ubiquitination cascade that maintains human genome stability.

Eukaryotic cells have evolved sophisticated quality control mechanisms to eliminate aggregation-prone proteins that compromise cellular health. Central to this defense is the ubiquitin-proteasome system, where UBR4 acts as an essential E4 ubiquitin ligase, amplifying degradation marks on defective proteins. Cryo–electron microscopy analysis of UBR4 in complex with its cofactors KCMF1 and CALM1 reveals a massive 1.3-megadalton ring structure, featuring a central substrate-binding arena and flexibly attached catalytic units. Our structure shows how UBR4 binds substrate and extends lysine-48–specific ubiquitin chains. Efficient substrate targeting depends on both preubiquitination and specific N-degrons, with KCMF1 acting as a key substrate filter. The architecture of the E4 megacomplex is conserved across eukaryotes, but species-specific adaptations allow UBR4 to perform its precisely tuned quality control function in diverse cellular environments.

The giant E3 ubiquitin ligase RNF213 is a conserved component of mammalian cell-autonomous immunity, limiting the replication of bacteria, viruses and parasites. To understand how RNF213 reacts to these unrelated pathogens, we employ chemical and structural biology to find that ATP binding to its ATPases Associated with diverse cellular Activities (AAA) core activates its E3 function. We develop methodology for proteome-wide E3 activity profiling inside living cells, revealing that RNF213 undergoes a reversible switch in E3 activity in response to cellular ATP abundance. Interferon stimulation of macrophages raises intracellular ATP levels and primes RNF213 E3 activity, while glycolysis inhibition depletes ATP and downregulates E3 activity. These data imply that ATP bears hallmarks of a danger/pathogen associated molecular pattern, coordinating cell-autonomous defence. Furthermore, quantitative labelling of RNF213 with E3-activity probes enabled us to identify the catalytic cysteine required for substrate ubiquitination and obtain a cryo-EM structure of the RNF213-E2-ubiquitin conjugation enzyme transfer intermediate, illuminating an unannotated E2 docking site. Together, our data demonstrate that RNF213 represents a new class of ATP-dependent E3 enzyme, employing distinct catalytic and regulatory mechanisms adapted to its specialised role in the broad defence against intracellular pathogens.