Biology subjects

Squair, D. R.

Publications and source records attributed to Squair, D. R..

4 recordsLinked to original sources

bioRxiv2025

ZNFX1 uses two-component ubiquitin circuitry to quarantine viral RNA

The detection of viral RNA inside cells triggers a diverse range of antiviral responses, including global translation inhibition, interferon secretion and RNA sequestration. Mutations in the gene ZNFX1 cause severe paediatric immunodeficiencies, including chronic viral infection and autoinflammation. Here, we show that ZNFX1 is an RNA helicase with cryptic and unusual bifurcating E3 ubiquitin ligase activity. Nucleotide-dependent RNA binding stimulates ZNFX1 to generate complex ubiquitin chains via a two-component ubiquitin circuit wired in parallel, with ubiquitin flux occurring via either of two competing paths. One route produces K63-linked polyubiquitin that drives ZNFX1 aggregation and RNA entrapment; the other route produces K48-linked polyubiquitin that drives ZNFX1 turnover. RNA entrapment restricts RNA virus replication, and is reversible by deubiquitination. Patient ZNFX1 variants are defective for viral restriction, linking RNA entrapment to antiviral immunity in vivo.

biochemistry↗

bioRxiv2023

An Atypical E3 Ligase Module in UBR4 Mediates Destabilization of N-degron Substrates

UBR4 is an E3 ligase (E3) of the N-degron pathway and is involved in neurodevelopment, age-associated muscular atrophy and cancer progression. The location and mechanistic classification of the E3 module within the 600 kDa protein UBR4 remains unknown. Herein, we identify and characterize, at a biochemical and structural level, a distinct E3 module within human UBR4 consisting of a novel "hemiRING" zinc finger, a helical-rich UBR Zinc-finger Interacting (UZI) subdomain, and a predicted backside interacting N-terminal helix. A structure of an E2 conjugating enzyme (E2)-E3 complex provides atomic level insight into the exquisite specificity of the hemiRING towards the E2s UBE2A/B. The UZI subdomain can be considered a component of the E3 module as it has a modest activating effect on the ubiquitin loaded E2 (E2[~]Ub), which is complemented by the intrinsically high lysine reactivity of UBE2A. These findings reveal the mechanistic underpinnings of a neuronal N-degron E3 ligase, its specific recruitment of UBE2A, and highlight the underappreciated architectural diversity of cross-brace domains associated with ubiquitin E3 activity.

biochemistry↗

bioRxiv2020

Targeting a critical step in fungal hexosamine biosynthesis

Aspergillus fumigatus is a human opportunistic fungal pathogen with a cell wall that protects it from the extracellular environment. Chitin, an essential cell wall component, is synthesised from UDP-GlcNAc that is produced by the hexosamine biosynthetic pathway. Here, we provide genetic and chemical evidence that glucosamine 6-phosphate N-acetyltransferase (Gna1), a key enzyme in this pathway, is an exploitable antifungal drug target. Deletion of GNA1 results in loss of viability and disruption of the cell wall, phenotypes that can be rescued by the product of the enzyme. In a murine model of aspergillosis, the{Delta} gna1 mutant strain attenuates virulence. Using a fragment-based approach, we discovered a small heterocyclic scaffold that binds proximal to the active site and can be optimised to a selective sub-micromolar binder. Taken together, we have provided genetic, structural and chemical evidence for Gna1 as an antifungal target in Aspergillus fumigatus.

microbiology↗

bioRxiv2019

Salt-inducible kinases (SIKs) regulate TGFβ-mediated transcriptional and apoptotic responses

AbstractThe signalling pathways initiated by members of the transforming growth factor-{beta} (TGF{beta}) family of cytokines control many metazoan cellular processes, including proliferation and differentiation, epithelial-mesenchymal transition (EMT), and apoptosis. TGF{beta} signalling is therefore strictly regulated to ensure appropriate context-dependent physiological responses. In an attempt to identify novel regulatory components of the TGF{beta} signalling pathway, we performed a pharmacological screen using a cell line engineered to report the endogenous transcription of the TGF{beta}-responsive target gene PAI-1. The screen revealed that small-molecular inhibitors of salt-inducible kinases (SIKs) attenuate TGF{beta}-mediated transcription of PAI-1 without affecting receptor-mediated SMAD phosphorylation, SMAD complex formation or nuclear translocation. We provide evidence that genetic inactivation of SIK isoforms also attenuates TGF{beta}-dependent transcriptional responses. Pharmacological inhibition of SIKs using multiple small-molecule inhibitors potentiated apoptotic cell death induced by TGF{beta} stimulation. Our data therefore provides evidence for a novel function of SIKs in modulating TGF{beta}-mediated transcriptional and cellular responses.

cancer biology↗