Biology subjects

Crowe, C.

Publications and source records attributed to Crowe, C..

6 recordsLinked to original sources

bioRxiv2025

Native Mass Spectrometry Analysis of a Cullin RING Ubiquitin E3 Ligase Complex in the Context of Targeted Protein Degradation

The binding of PROTACs to their partner ubiquitin E3 ligase (E3) and a protein of interest (POI) is critical for PROTAC development and validation. Characterisation of PROTAC complexes by cryo-electron microscopy and X-ray crystallography is not always feasible, especially where species may be transient and protein structures may not resolve due to flexible domains or intrinsically disordered regions. More routine biophysical methods with broader applicability to varied samples is desirable to support the rapidly expanding targeted protein degradation field. The majority of PROTACs in development and in the clinic act through a Cullin RING E3 Ligase (CRL) of which the pentameric von Hippel-Lindau (VHL) Cullin 2 RING E3 complex (CRL2VHL) is the foundational example. Native mass spectrometry (nMS) can be used to characterise protein complexes but has not previously been used to characterise a full E3 or any E3-E2 interactions. Here, we show that CRL2VHL is amenable to characterisation by nMS and its interactions with the other protein components integral to the targeted protein degradation mechanism can be observed. Specifically, we characterise binary, ternary and higher order complexes that comprise CRL2VHL, including the multiprotein systems of POI-PROTAC-CRL2VHL, CRL2VHL-E2-Ub, and POI-PROTAC-CRL2VHL-E2-Ub, all of which are essential in facilitating productive POI ubiquitination and degradation. We benchmarked the nMS with two POI examples (BRD4BD2 and KRAS) with their respective PROTACs (MZ1 and ACBI3) and were able to observe all relevant complexes across both systems. We anticipate that our findings will open avenues for nMS to integrate as an alternative experimental method enabling scalable characterisation of the intricate high mass multiprotein interactions central to the PROTAC mechanism of action.

biophysics↗

bioRxiv2025

Linker-rigidified VHL homodimerizers convert degraders into stabilizers of non-ubiquitinable ternary complexes

Protein degraders recruit E3 ligases to targets for ubiquitination and subsequent proteasomal degradation. Efficient degradation typically correlates with long-lived ternary complexes that position target lysines for productive ubiquitination. Here, we determine a cryo-EM structure of the Cullin 2 RING VHL (CRL2VHL) ligase dimerized by the VHL homo-PROTAC CM11, wherein one CRLVHL acts as the ligase and a second VHL as the neo-substrate. Guided by this structure, we design side-by-side, linker-rigidified VHL homodimerizers that bias the relative VHL orientation away from the E2[~]Ub active site, contrasting the flexible, head-to-head PEG linkage of CM11. Biophysical binding and in vitro ubiquitination assays show that these compounds form stable, long-lived and compact ternary complexes that are incompatible with VHL cross-ubiquitination. In cells, the compounds stabilize VHL and concomitantly inhibit it to elevate HIF-1 levels. Thus, a stable ternary complex can be non-productive for ubiquitination, and linker architecture can reprogram degraders into stabilizers by controlling target ubiquitinability.

biochemistry↗

bioRxiv2025

Induced ubiquitination of the partially disordered Estrogen Receptor alpha protein via a 14-3-3-directed molecular glue-based PROTAC design

Proteins lacking defined ligandable pockets remain challenging drug targets. Here, we develop a molecular glue-based PROTAC (MGPROTACs) approach that chemically conjugates a molecular glue stabilizer to a VHL-recruiting ligand to capture and ubiquitinate the 14-3-3/Estrogen Receptor (ER) complex. Our designed MGPROTACs engage a composite interface between 14-3-3 and the disordered F-domain of ER, promoting cooperative complex formation and target ubiquitination. Biophysical characterization revealed distinct linker-dependent cooperativities across the MGPROTAC series, which influenced both cellular permeability and ubiquitination efficiency. Cryo-EM of the most cooperative MGPROTAC uncovers de novo VHL-14-3-3{zeta} contacts, while molecular dynamics simulations rationalize the stabilizing interactions underlying cooperativity. Strikingly, fine-tuning linker design enables selective ubiquitination of distinct complex subunits. These findings establish a structural and mechanistic framework for integrating molecular glue and PROTAC principles, expanding the scope of drug discovery to previously intractable protein complexes.

biochemistry↗

bioRxiv2025

LRRC58 defines an E3 ubiquitin ligase complex sensitive to cysteine abundance

Adaptation to fluctuating nutrient supply is essential for organismal survival, but how cells sense and respond to changes in the abundance of many critical nutrients remains undefined. One such example is the amino acid cysteine, whose reactive thiol group is exploited for diverse cellular functions. Here, by characterizing the machinery required for the conditional degradation of cysteine dioxygenase type I (CDO1), the critical enzyme responsible for cysteine catabolism, we identify a Cul2 E3 ligase complex containing the uncharacterized substrate adaptor LRRC58 that is sensitive to cysteine abundance. When cysteine is replete, LRRC58 activity is restrained through auto-ubiquitination and proteasomal degradation; upon cysteine deprivation, LRRC58 is stabilized to permit CDO1 degradation. Through saturation mutagenesis stability profiling, we systematically validate a structural model of the CDO1-LRRC58 interaction and identify residues at the LRRC58 C-terminus required for cysteine-dependent instability. The LRRC58-mediated degradation of CDO1 is essential to prevent ferroptotic cell death under conditions of cysteine scarcity, and mutations in CDO1 which cause neurodevelopmental defects in humans encode dominant-active proteins refractory to LRRC58 recognition. Altogether, these data reveal the CDO1-LRRC58 axis as a critical regulator of cysteine homeostasis that safeguards neural development.

molecular biology↗

bioRxiv2024

Mechanism of degrader-targeted protein ubiquitinability

Small molecule degraders of disease-driving proteins offer a clinically proven modality with enhanced therapeutic efficacy and the potential to tackle previously undrugged targets. Thermodynamically stable and kinetically long-lived degrader-mediated ternary complexes can drive faster, more profound and durable target degradation, however the mechanistic features by which they impact on target ubiquitination remain elusive. Here, we solve cryo-EM structures of the VHL Cullin 2 RING E3 ligase complexed with degrader MZ1, target protein Brd4BD2 and primed for catalysis with its cognate E2-ubiquitin bound. We find that Brd4BD2 adopts a favourable orientation towards the E2 active site. In vitro ubiquitination coupled with mass spectrometry illuminates a patch of ubiquitinable lysines on one face of Brd4BD2, with Lys456 showing optimal distance and geometry for nucleophilic attack. Our results demonstrate the proficiency of MZ1 in directing the substrate towards catalysis, explains the favourability of Brd4BD2 for ubiquitination, and reveals the flexibility of the enzyme in capturing sub-optimal lysines. We propose a model for ubiquitinability of degrader-recruited targets that provides a mechanistic blueprint for further rational drug design and optimization. One-Sentence SummaryStructural assembly a PROTAC-mediated complex of whole Cullin RING E3 ligase with bound target and E2-ubiquitin reveals structural and mechanistic insights of specificity for target protein ubiquitination.

biochemistry↗

bioRxiv2018

Narcolepsy risk loci are enriched in immune cells and suggest autoimmune modulation of the T cell receptor repertoire

Type 1 narcolepsy (T1N) is a neurological condition, in which the death of hypocretin-producing neurons in the lateral hypothalamus leads to excessive daytime sleepiness and symptoms of abnormal Rapid Eye Movement (REM) sleep. Known triggers for narcolepsy are influenza-A infection and associated immunization during the 2009 H1N1 influenza pandemic. Here, we genotyped all remaining consented narcolepsy cases worldwide and assembled this with the existing genotyped individuals. We used this multi-ethnic sample in genome wide association study (GWAS) to dissect disease mechanisms and interactions with environmental triggers (5,339 cases and 20,518 controls). Overall, we found significant associations with HLA (2 GWA significant subloci) and 11 other loci. Six of these other loci have been previously reported (TRA, TRB, CTSH, IFNAR1, ZNF365 and P2RY11) and five are new (PRF1, CD207, SIRPG, IL27 and ZFAND2A). Strikingly, in vaccination-related cases GWA significant effects were found in HLA, TRA, and in a novel variant near SIRPB1. Furthermore, IFNAR1 associated polymorphisms regulated dendritic cell response to influenza-A infection in vitro (p-value =1.92*10-25). A partitioned heritability analysis indicated specific enrichment of functional elements active in cytotoxic and helper T cells. Furthermore, functional analysis showed the genetic variants in TRA and TRB loci act as remarkable strong chain usage QTLs for TRAJ*24 (p-value = 0.0017), TRAJ*28 (p-value = 1.36*10-10) and TRBV*4-2 (p-value = 3.71*10-117). This was further validated in TCR sequencing of 60 narcolepsy cases and 60 DQB1*06:02 positive controls, where chain usage effects were further accentuated. Together these findings show that the autoimmune component in narcolepsy is defined by antigen presentation, mediated through specific T cell receptor chains, and modulated by influenza-A as a critical trigger.

genetics↗