Biology subjects

Hughes, D. J.

Publications and source records attributed to Hughes, D. J..

9 recordsLinked to original sources

bioRxiv2026

Dissecting the interactions of the ISG15-USP18-STAT2 inhibitory complex

The suppression of type I interferon (IFN) signalling by the ISG15-USP18-STAT2 inhibitory complex (ISG15 IC) is an established regulatory mechanism of the antiviral response. However, a molecular understanding of how the ISG15 IC forms to suppress IFN signalling is still emerging. Here, we use AlphaFold modelling in conjunction with biochemical and biophysical approaches to elucidate the interactions of this multiprotein assembly. Our analysis identified a unique STAT2 binding loop (SBL) in USP18, which is critical for the USP18-STAT2 association. Further biochemical characterisation through site-directed mutagenesis confirmed the importance of residues within and surrounding the SBL, enabling the design of mutants with both increased and decreased binding affinities. Moreover, several USP18 and STAT2 patient mutations severely disrupted this interaction. Lastly, using influenza B virus (IBV) and Zika virus (ZIKV) proteins, we investigated the influence of these viral effector proteins on these interactions. Taken together, these results provide much-needed insights into a key aspect of IFN signalling control.

biochemistry↗

bioRxiv2024

Controlled 'out-of-season' spawning of reef-forming corals in aquaria using offset environmental profiles

The global climate crisis has heightened the urgency for developing interventions to enhance resilience and recovery of coral reef ecosystems. However, research programs are often bottlenecked by availability of coral early life stage material due to the seasonal (annual) nature of coral mass spawning events. Here, we present a proof-of-concept of "out-of-season" coral spawning, utilising advanced aquarium control technology to induce spawning in multiple key Great Barrier Reef (GBR) coral species held in long-term indoor aquaria. By developing a 6-month offset environmental profile encompassing seasonal temperature, photoperiod, and lunar profiles, we successfully induced synchronised mass coral spawning during austral autumn/winter (May - June) over consecutive years in 2022 and 2023. We also "phase-shifted" the hour of sunset by five hours on spawning nights, creating a more favourable time window (i.e., minimising late nights) for gamete fertilisation. The timing of gamete release in captive corals closely matched previous GBR field observations for both nights after full moon (NAFM) and time after sunset (TAS). Gamete fertilisation was successful for six GBR species: Acropora millepora, Acropora loripes, Acropora hyacinthus, Acropora elseyi, Acropora austera, and Montipora aequituberculata producing a total of 1.75 million larvae. We outline key physiological insights gained into environmental regulation of coral spawning synchronicity and discuss the potential for out-of-season spawning to accelerate various aspects of coral reproduction research and enhance the growing toolbox of active restoration strategies aimed at reversing global coral reef decline.

ecology↗

bioRxiv2024

Highly-sensitive reporter cell line for detection of interferon types I-III and their neutralization by antibodies

Interferons (IFNs) are a critical component of innate immune defenses and limit viral disease severity. To advance studies on IFNs and their neutralization by pathogenic autoantibodies, we generated a Renilla luciferase-based reporter cell line capable of detecting the activities of IFN-Is, IFN-II, and IFN-IIIs. The reporter cell line exhibits a 125-to 2000-fold higher sensitivity to IFNs than a commonly-used alternative biological reporter system, and allows for a rapid and simple live-cell workflow for detecting low titer amounts of neutralizing anti-IFN antibodies.

immunology↗

bioRxiv2024

Microscale sampling of the coral gastric cavity reveals a gut-like microbial community

Animal guts contain numerous microbes, which are critical for nutrient assimilation and pathogen defence. While corals and other Cnidaria lack a true differentiated gut, they possess gastrovascular cavities (GVCs), semi-enclosed compartments where vital processes such as digestion, reproduction and symbiotic exchanges take place. The microbiome harboured in GVCs is therefore likely key to holobiont fitness, but remains severely understudied due to challenges of working in these small compartments. Here, we developed minimally invasive methodologies to sample the GVC of coral polyps and characterise the microbial communities harboured within. We used glass capillaries, low dead volume microneedles, or nylon microswabs to sample the gastric microbiome of individual polyps from six species of corals, then applied low-input DNA extraction to characterise the microbial communities from these microliter volume samples. Microsensor measurements of GVCs revealed anoxic or hypoxic micro-niches, which persist even under prolonged illumination with saturating irradiance. These niches harboured microbial communities enriched in putatively microaerophilic or facultatively anaerobic taxa, such as Epsilonproteobacteria. Some core taxa found in the GVC of Lobophyllia hemprichii from the Great Barrier Reef were also detected in conspecific colonies held in aquaria, indicating that these associations are unlikely to be transient. Our findings suggest that the coral GVC is chemically and microbiologically similar to the gut of higher Metazoa. Given the importance of gut microbiomes in mediating animal health, harnessing the coral "gut microbiome" may foster novel active interventions aimed at increasing the resilience of coral reefs to the climate crisis.

microbiology↗

bioRxiv2024

Spatial proteomics identifies a novel CRTC-dependent viral sensing pathway that stimulates production of Interleukin-11

Appropriate cellular recognition of viruses is essential for the generation of effective innate and adaptive antiviral immunity. Viral sensors and their signalling components thus provide a crucial first line of host defence. Many exhibit subcellular relocalisation upon activation, triggering expression of interferon and antiviral genes. To identify novel signalling factors we analysed protein relocalisation on a global scale during viral infection. CREB Regulated Transcription Coactivators-2 and 3 (CRTC2/3) exhibited early cytoplasmic-to-nuclear translocation upon a diversity of viral stimuli, in diverse cell types. This movement was depended on Mitochondrial Antiviral Signalling Protein (MAVS), cyclo-oxygenase proteins and protein kinase A. We identify a key effect of transcription stimulated by CRTC2/3 translocation as production of the pro-fibrogenic cytokine interleukin-11. This may be important clinically in viral infections associated with fibrosis, including SARS-CoV-2.

immunology↗

bioRxiv2022

Modelling the within-host spread of SARS-CoV-2 infection, and the subsequent immune response, using a hybrid, multiscale, individual-based model. Part I: Macrophages.

Individual responses to SARS-CoV-2 infection vary significantly, ranging from mild courses of infection that do not require hospitalisation to the development of disease which not only requires hospitalisation but can be fatal. Whilst many immunological studies have revealed fundamental insights into SARS-CoV-2 infection and COVID-19, mathematical and computational modelling can offer an additional perspective and enhance understanding. The majority of mathematical models for the within-host spread of SARS-CoV-2 infection are ordinary differential equations, which neglect spatial variation. In this article, we present a hybrid, multiscale, individual-based model to study the within-host spread of SARS-CoV-2 infection. The model incorporates epithelial cells (each containing a dynamical model for viral entry and replication), macrophages and a subset of cytokines. We investigate the role of increasing initial viral deposition, increasing delay in type I interferon secretion from epithelial cells (as well as the magnitude of secretion), increasing macrophage virus internalisation rate and macrophage activation, on the spread of infection.

systems biology↗

bioRxiv2021

Kv1.3 induced hyperpolarisation and Cav3.2-mediated calcium entry are required for efficientKaposi's sarcoma-associated herpesvirus lytic replication

Understanding the host factors critical for Kaposis sarcoma-associated herpesvirus (KSHV) lytic replication can identify new targets for therapeutic intervention. Using pharmacological and genetic silencing approaches, we reveal for the first time that KSHV requires a B cell expressed voltage-gated K+ channel, Kv1.3, to enhance lytic replication. We show that the KSHV replication and transcription activator (RTA) protein upregulates Kv1.3 expression, leading to enhanced K+ channel activity and hyperpolarisation of the B cell membrane. Enhanced Kv1.3 activity then promotes intracellular Ca2+ influx through Cav3.2, a T-type Ca2+ channel, leading to the Ca2+ driven nuclear localisation of NFAT and the subsequent NFAT1-responsive gene expression. Importantly, KSHV lytic replication and infectious virion production could be inhibited by both Kv1.3 and Cav3.2 blockers or through Kv1.3 silencing. These findings provide new mechanistic insight into the essential role of host ion channels during KSHV infection and highlight Kv1.3 and Cav3.2 as new druggable host factors that are key to the successful completion of KSHV lytic replication.

microbiology↗

bioRxiv2021

A co-opted ISG15-USP18 binding mechanism normally reserved for deISGylation controls type I IFN signalling

Type I interferon (IFN) signalling induces the expression of several hundred IFN-stimulated genes (ISGs) that provide an unfavourable environment for viral replication. To prevent an overexuberant response and autoinflammatory disease, IFN signalling requires tight control. One critical regulator is the ubiquitin-like protein ISG15, evidenced by autoinflammatory disease in patients with inherited ISG15 deficiencies. Current models suggest that ISG15 stabilises USP18, a well-established negative regulator of IFN signalling. USP18 also functions as an ISG15-specific peptidase that cleaves ISG15 from ISGylated proteins; however, USP18s catalytic activity is dispensable for controlling IFN signalling. Here, we show that the ISG15-dependent stabilisation of USP18 involves transient hydrophobic interactions. Nonetheless, while USP18 stabilisation is necessary, it is not sufficient for regulation of IFN signalling. USP18 requires non-covalent interactions with the ISG15 C-terminal diGlycine motif to promote its regulatory function. This trait may have been acquired in humans through co-option of a binding mechanism normally reserved for deISGylation, identifying an unexpected new function for human ISG15.

immunology↗

bioRxiv2019

Direct antiviral activity of interferon stimulated genes is responsible for resistance to paramyxoviruses in ISG15-deficient cells

Interferons (IFNs), produced during viral infections, induce the expression of hundreds of IFN- stimulated genes (ISGs). Some ISGs have specific antiviral activity while others regulate the cellular response. Besides functioning as an antiviral effector, IFN-stimulated gene 15 (ISG15) is a negative regulator of IFN signalling and inherited ISG15-deficiency leads to autoinflammatory interferonopathies where individuals exhibit elevated ISG expression in the absence of pathogenic infection. We have recapitulated these effects in cultured human A549-ISG15-/- cells and (using A549-UBA7-/- cells) confirmed that posttranslational modification by ISG15 (ISGylation) is not required for regulation of the type-I IFN response. ISG15-deficient cells pre-treated with IFN- were resistant to paramyxovirus infection. We also showed that IFN- treatment of ISG15-deficient cells led to significant inhibition of global protein synthesis leading us to ask whether resistance was due to the direct antiviral activity of ISGs or whether cells were non-permissive due to translation defects. We took advantage of the knowledge that IFN-induced protein with tetratricopeptide repeats 1 (IFIT1) is the principal antiviral ISG for parainfluenza virus 5 (PIV5). Knockdown of IFIT1 restored PIV5 infection in IFN--pre-treated ISG15-deficient cells, confirming that resistance was due to the direct antiviral activity of the IFN response. However, resistance could be induced if cells were pre-treated with IFN- for longer times, presumably due to inhibition of protein synthesis. These data show that the cause of virus resistance is two-fold; ISG15-deficiency leads to the early over-expression of specific antiviral ISGs, but the later response is dominated by an unanticipated, ISG15- dependent, loss of translational control. Key pointsCell culture model of ISG15-deficiency replicate findings in ISG15-/- patient cells Cause of resistance in ISG15-/- cells differs depending on duration of IFN treatment ISG15-/- patients without serious viral disease dont prove ISGylation is unimportant

immunology↗