Biology subjects

McDonnell, E.

Publications and source records attributed to McDonnell, E..

3 recordsLinked to original sources

bioRxiv2023

A bespoke analytical workflow for the confident identification of sulfopeptides and their discrimination from phosphopeptides

Protein tyrosine sulfation (sY) is a post-translational modification (PTM) catalysed by Golgi-resident Tyrosyl Protein SulfoTransferases (TPSTs). Information on protein tyrosine sulfation is currently limited to [~]50 human proteins with only a handful having verified sites of sulfation. The contribution of this chemical moiety for the regulation of biological processes, both inside and outside the cell, remains poorly defined, in large part due to analytical limitations. Mass spectrometry-based proteomics is the method of choice for PTM analysis, but has yet to be applied for the systematic investigation and large-scale analysis of biomolecular sulfation (constituting the sulfome), primarily due to issues associated with discrimination of sY-from phosphotyrosine (pY)-containing peptides. In this study, we developed a mass spectrometry (MS)-based workflow centred on the characterization of sY-peptides, incorporating optimised Zr4+-IMAC and TiO2 enrichment strategies. Extensive characterization of a panel of sY- and pY-peptides using an array of MS fragmentation regimes (CID, HCD, EThcC, ETciD, UVPD) highlights differences in the ability to generate site-determining product ions, which can be exploited to differentiate sulfated peptides from nominally isobaric phosphopeptides based on precursor ion neutral loss at low collision energy. Application of our analytical workflow to a HEK-293 cell extracellular secretome facilitated identification of 21 new sulfotyrosine-containing proteins, several of which we validate enzymatically using in vitro sulfation assays. This study demonstrates the applicability of our strategy for confident, high-throughput, sulfomics studies, and reveals new sY interplay between enzymes relevant to both protein and glycan sulfation.

biochemistry↗

bioRxiv2021

CircHIPK3 dysregulation of the miR-30c/DLL4 axis is essential for KSHV lytic replication

Non-coding RNA (ncRNA) regulatory networks are emerging as critical regulators of gene expression. These intricate networks of ncRNA:ncRNA interactions modulate multiple cellular pathways and impact the development and progression of multiple diseases. Herpesviruses, including Kaposis sarcoma-associated herpesvirus, are adept at utilising ncRNAs, encoding their own as well as dysregulating host ncRNAs to modulate virus gene expression and the host response to infection. Research has mainly focused on unidirectional ncRNA-mediated regulation of target protein-coding transcripts; however, we have identified a novel host ncRNA regulatory network essential for KSHV lytic replication in B cells. KSHV-mediated upregulation of the host cell circRNA, circHIPK3, is a key component of this network, functioning as a competing endogenous RNA of miR-30c, leading to increased levels of the miR-30c target, DLL4. Dysregulation of this network highlights a novel mechanism of cell cycle control during KSHV lytic replication in B cells. Importantly, disruption at any point within this novel ncRNA regulatory network has a detrimental effect on KSHV lytic replication, highlighting the essential nature of this network and potential for therapeutic intervention.

microbiology↗

bioRxiv2020

Functional characterization of clinical isolates of the opportunistic fungal pathogen Aspergillus nidulans

Aspergillus nidulans is an opportunistic fungal pathogen in patients with immunodeficiency and virulence of A. nidulans isolates has mainly been studied in the context of the chronic granulomatous disease (CGD), with characterization of clinical isolates obtained from non-CGD patients remaining elusive. This study therefore carried out a detailed biological characterization of two A. nidulans clinical isolates (CIs), obtained from a patient with breast carcinoma and pneumonia and from a patient with cystic fibrosis that underwent lung transplantation, and compared them to the reference, non-clinical A4 strain. Both CIs presented increased growth in comparison to the reference strain in the presence of physiologically-relevant carbon sources. Metabolomic analyses showed that the three strains are metabolically very different from each other in these carbon sources. Furthermore, the CIs were highly susceptible to cell wall perturbing agents but not to other physiologically-relevant stresses. Genome analyses identified several frame-shift variants in genes encoding cell wall integrity (CWI) signalling components. Significant differences in CWI signalling were confirmed by western blotting among the three strains. In vivo virulence studies using several different models revealed that strain MO80069 had significantly higher virulence in hosts with impaired neutrophil function when compared to the other strains. In summary, this study presents detailed biological characterization of two A. nidulans sensu stricto clinical isolates. Just like in A. fumigatus, strain heterogeneity exists in A. nidulans clinical strains that can define virulence traits. Further studies are required to fully characterize A. nidulans strain-specific virulence traits and pathogenicity. ImportanceImmunocompromised patients are susceptible to infections with opportunistic filamentous fungi from the genus Aspergillus. Although A. fumigatus is the main etiological agent of Aspergillus spp.-related infections, other species, such as A. nidulans are prevalent in a condition-specific manner. A. nidulans is a predominant infective agent in patients suffering from chronic granulomatous disease (CGD). A. nidulans isolates have mainly been studied in the context of CGD, although infection with A. nidulans also occurs in non-CGD patients. This study carried out a detailed biological characterization of two non-CGD A. nidulans clinical isolates and compared it to a reference strain. Phenotypic, metabolomic and genomic analyses highlight fundamental differences in carbon source utilization, stress responses and maintenance of cell wall integrity among the strains. One clinical strain had increased virulence in models with impaired neutrophil function. Just as in A. fumigatus, strain heterogeneity exists in A. nidulans clinical strains that can define virulence traits.

microbiology↗