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Biology subjects

Vere, G.

Publications and source records attributed to Vere, G..

5 recordsLinked to original sources

Mgl2+ cDC2 triggering of fungal allergic inflammation depends on a spore induced glycolytic shift fuelled by local availability of glucose

Fungal spores are a major cause of severe asthmatic disease. However, the precise events that cause individuals to become sensitised to spores are poorly understood. Mgl2+ type 2 conventional dendritic cells (Mgl2+ cDC2s) are critical in coordinating allergic airway inflammation in mice following repeated exposure to inhaled spores. Yet, whether these DCs are directly acquiring spores from the airway, and the downstream mechanism(s) upon fungal uptake causing DCs to trigger allergic inflammation are unknown. Here we find that spores are acquired by lung DCs after inhalation although these events are rare ([~] 0.5% of the cDC2 population). Transcriptomics on isolated spore+ Mgl2+ cDC2s, compared to spore- Mgl2+ cDC2s from the same environment, revealed that a major consequence of fungal uptake was a boost in metabolic activity. Single-cell metabolic profiling revealed this increase in Mgl2+ cDC2 metabolism upon spore acquisition was fuelled by a glycolytic shift. To pinpoint if nutrient availability and acquisition is an important determinant of this response, mass spectrometry-based metabolomics revealed that, during fungal allergic inflammation, the local airway nutrient environment is altered. To ascertain which of these could be fuelling DC responses, we identified which substrates that feed into glycolysis were crucial. Of these, we found that glucose availability acts as a key rheostat in shaping cDC2 responses to spores. These data highlight a crucial role for glycolytic metabolism in driving cDC2 responses to spores, which is governed by glucose availability, defining novel targets for future therapeutic development of fungal allergic inflammation.

immunology↗

Aspergillus-mediated allergic airway inflammation is triggered by dendritic cell recognition of a defined spore morphotype, a process that can be targeted via antifungal therapeutics.

BackgroundExposure to fungi, especially Aspergillus fumigatus (A.f.), can elicit potent allergic inflammation that triggers and worsens asthmatic disease. Dendritic cells (DCs), initiate allergic inflammatory responses to allergic stimuli. However, it is unclear if A.f. spores during isotropic growth (early spore swelling) can activate DCs to initiate allergic responses or if germination is required. This lack of basic understanding of how A.f. causes disease is a barrier to the development of new treatments. ObjectiveTo show that a precise A.f. morphotype stage during spore swelling can trigger DCs to mediate allergic inflammatory responses and ascertain if antifungal therapeutics can be effective at suppressing this process. MethodsWe employed an A.f. strain deficient in pyrimidine biosynthesis ({Delta}pyrG) to generate populations of A.f. spores arrested at different stages of isotropic growth (swelling) via temporal removal of uracil and uridine from growth media. These arrested spore stages were cultured with bone marrow derived DCs (BMDCs), and their activation measured via flow cytometry and ELISA to interrogate which growth stage was able to activate BMDCs. These BMDCs were then adoptively transferred into the airways, to assess if they were able to mediate allergic inflammation in naive recipient mice. Allergic airway inflammation in vivo was determined via flow cytometry, ELISA and qPCR. This system was also used to determine if antifungal drug (itraconazole) treatment could alter early stages of spore swelling and therefore BMDC activation and in vivo allergic inflammation upon adoptive transfer. ResultsWe found that A.f. isotropic growth is essential to trigger BMDC activation and mediate allergic airway inflammation. Furthermore, using time arrested A.f. stages, we found that least 3h in growth media enabled spores to swell sufficiently to activate BMDCs to elicit allergic airway inflammation in vivo. Incubation of germinating A.f. with itraconazole reduced spore swelling and partially reduced their ability to activate BMDCs to elicit in vivo allergic airway inflammation. ConclusionIn summary, our results have pinpointed the precise stage of A.f. development when germinating spores are able to activate DCs to mediate downstream allergic airway inflammation. Furthermore, we have identified that antifungal therapeutics can be effective in reducing the potential of A.f. spores to stimulate allergic responses, highlighting a potential mechanism by which antifungal treatment might help to prevent the development of fungal allergy.

immunology↗

Interplay Between KRAS and LZTR1 Protein Turnover, Controlled by CUL3/LZTR1 E3 Ubiquitin Ligase, is Disrupted by KRAS Mutations

KRAS is a proto-oncogene encoding a small GTPase. Mutations contribute up to 30% of human solid tumours including lung adenocarcinoma, pancreatic and colorectal carcinomas. Most KRAS activating mutations interfere with GTP hydrolysis, essential for its role as a molecular switch, leading to alterations in their molecular environment and oncogenic signalling. Here, APEX-2 proximity labelling was used to profile the molecular environment of wild type and G12D, G13D and Q61H activating mutants of KRAS under both, starvation and stimulation conditions. We demonstrate by quantitative proteomics the presence of known interactors of KRAS including a-RAF and LZTR1, which varied in abundance with wildtype and KRAS mutants. Notably, the KRAS mutations G12D, G13D and Q61H abrogate association with LZTR1. Wildtype KRAS and LZTR1, as part of the CUL3 ubiquitin E3 ligase complex, affect each others protein stability, revealing a direct feedback loop mechanism. KRAS mutations disconnect this regulatory circuit, thereby contributing to oncogenesis.

molecular biology↗

USP28 deletion and small molecule inhibition destabilises c-Myc and elicitsregression of squamous cell lung carcinoma

Lung squamous cell carcinoma (LSCC) is a considerable global health burden, with an incidence of over 600,000 cases per year. Treatment options are limited, and patient 5-year survival rate is less than 5%. The ubiquitin specific protease 28 (USP28) has been implicated in tumorigenesis through its stabilization of the oncoprotein c-MYC. Here, we show that genetic inactivation of Usp28 induced regression of established murine LSCC lung tumors. We developed a small molecule that inhibits USP28 activity in the low nanomole range. While displaying cross-reactivity against the closest homologue USP25, this inhibitor showed a high degree of selectivity over other deubiquitinases. USP28 inhibitor treatment resulted in a dramatic decrease in c-Myc proteins levels and consequently induced substantial regression of autochthonous murine LSCC tumors and human LSCC xenografts, thereby phenocopying the effect observed by genetic deletion. Thus, USP28 may represent a promising therapeutic target for the treatment of squamous cell lung carcinoma.

cancer biology↗

Deep analysis of the USP18-dependent ISGylome and proteome unveils important roles for USP18 in tumour cell antigenicity and radiosensitivity

The deubiquitylating enzyme USP18 is a major negative regulator of the interferon (IFN) signalling cascade. IFN pathways contribute to resistance to conventional chemotherapy, radiotherapy, and immunotherapy and are often deregulated in cancer. USP18 is the predominant human protease that cleaves interferon-stimulated gene ISG15, a ubiquitin-like protein tightly regulated in the context of innate immunity, from its modified substrate proteins in vivo. In this study, using advanced proteomic techniques, we have expanded the USP18-dependent ISGylome and proteome in a chronic myeloid leukaemia (CML)-derived cell line (HAP1) treated with type I IFN. Novel ISGylation targets were characterised that modulate the sensing of innate ligands, antigen presentation and secretion of cytokines. Consequently, CML USP18-deficient cells are more antigenic, driving increased activation of cytotoxic T lymphocytes (CTLs) and are more susceptible to irradiation. Our results suggest USP18 as a pharmacological target in cancer immunotherapy and radiotherapy.

cancer biology↗