Biology subjects

Shirran, S. L.

Publications and source records attributed to Shirran, S. L..

6 recordsLinked to original sources

bioRxiv2026

Direct targeting of the WAVE-regulatory complex and the actin cytoskeleton by the Anaplasma phagocytophilum effector, AnkA

The obligate intracellular tick-borne pathogen Anaplasma phagocytophilum is unusual in its tropism for neutrophils, within which it survives and replicates. Central to the ability of this bacterium to colonize this hostile niche are secreted effector proteins. One such effector protein, AnkA, interacts with multiple host ligands in both the cytoplasm and the nucleus, yet how these interactions promote infection is largely unknown. We discovered that AnkA directly binds both the WAVE Regulatory Complex (WRC), a signal-integration hub that regulates the formation of branched actin networks underpinning diverse cell functions, and to monomeric and filamentous actin. We determined that AnkA uses two forms of molecular mimicry, first by binding to the Abi1 component of the WRC via a WRC-Recruiting Abi-binding Peptide (WRAP), and second by binding to actin via a WH2-like Actin Binding Motif (ABM). Via these interactions, AnkA can exert multi-factorial influence on the actin cytoskeleton and signaling pathways converging on this host structure. AnkA and the WRC co-localise during bacterial internalization and during the late stages of infection, suggesting a central role in both host-cell colonization and bacterial dissemination. Our work reveals AnkA as the first example of a bacterial effector that directly targets the WRC. The co-occurrence of WRAP- and ABM-like motifs in other bacterial effector proteins suggests that this mechanism of actin cytoskeleton subversion may represent a conserved strategy used by other obligate intracellular bacteria.

microbiology↗

bioRxiv2026

Bacterial Aminopeptidase-Activated Peptide Prodrug Enables Species-Selective Targeting of Pseudomonas aeruginosa

Pseudomonas aeruginosa is an adaptable organism, frequently found in chronic infections, and for which antimicrobial resistance is a growing concern. Therefore, there is an urgent need for alternative therapeutic strategies. Cationic antimicrobial peptides (AMPs) offer potent bactericidal activity but suffer from limited selectivity and potential host toxicity. To enhance species-specific targeting, we designed two prodrug variants of the AMP D-Bac8CLeu2,5 - EEEE{square}-Bac8CLeu2,5 and ELE{square} D-Bac8CLeu2,5 --engineered for activation by the P. aeruginosa extracellular aminopeptidase PaAP. While both prodrug motifs effectively neutralized the positive charge of D-Bac8CLeu2,5 and prevented DNA-peptide complex formation, EEEE{square}-Bac8CLeu2,5 showed negligible antimicrobial activity due to slow and incomplete activation. In contrast, ELEG{square}-Bac8CLeu2,5 underwent rapid PaAP{square}mediated activation, restoring bactericidal activity in planktonic cultures and biofilms. PaAP contributed significantly to complete prodrug activation, particularly within biofilms, where the accumulation of partially activated intermediates correlated with biphasic killing kinetics. The prodrug showed reduced activity against other ESKAPEE pathogens, demonstrating selective activation by P. aeruginosa. Experiments selecting resistant bacteria revealed distinct mutations in lipopolysaccharide biosynthesis pathways for D-Bac8CLeu2,5 and the prodrug, with limited cross{square}resistance. These findings establish aminopeptidase{square}activated AMP prodrugs as a promising approach for species{square}selective antimicrobial therapy and highlight the feasibility of exploiting bacterial enzymes for controlled antimicrobial peptide activation.

microbiology↗

bioRxiv2025

Dimerization and Threonine-Dependent Stabilization Govern Human YRDC Catalysis

Addition of N6-threonylcarbamoyladenosine occurs at position 37 (tA37) of five mitochondrial tRNA species, influencing translation fidelity and efficiency. Mutations in YRDC, the enzyme catalyzing the first step in tA37 synthesis, cause severe neurological and renal diseases in humans, including in Galloway-Mowatt disease (GAMOS). YRDC generates threonylcarbamoyl-AMP (TC-AMP) using ATP, threonine and bicarbonate as substrates, yet their binding order, residues involved in amino-acid selectivity, and precisely how mutations contribute to disease remain unclear. Here, we combine protein biophysics, mass spectrometry, NMR, mutagenesis and kinetics to define how the human enzyme operates. Differential scanning fluorimetry and circular dichroism identify L-threonine as the gatekeeper ligand. It binds with millimolar affinity, stabilizing YRDC and enhancing ATP binding nearly fortyfold, enabling bicarbonate association. Several amino acids can form aminoacylcarbamoyl-AMP adducts in vitro, but threonine yields superior protein stabilization and product formation. Native mass spectrometry and single-molecule mass photometry corroborate YRDCs dimeric state. GAMOS-linked mutations destabilize folding, disrupting dimerization and uncoupling substrate binding from catalysis, explaining functional losses. Supported by kinetic data and NMR, we propose a chemical mechanism for the reaction and identify dimerization and conformational stability as control points for activity. This work provides a foundation for designing selective YRDC inhibitors or enhancers.

biochemistry↗

bioRxiv2025

Investigating the Interactomic Landscape of Survival Motor Neurons (SMN) and the SMN Δ7 truncated protein

This protocol describes a methodology combining TurboID - a recently developed proximity biotinylation technique - with conventional epitope-tag based co-immunoprecipitation (Co-IP) to analyse protein-protein interactions (PPIs) in cell culture systems. This integrated approach allows for the targeted examination of both transient and stable protein interactors, enhancing our understanding of protein dynamics. TurboID captures transient interactions often missed by Co-IP, which captures high affinity, stable interactors. Combination of both techniques enables direct comparison of interaction strengths, providing insights into the dynamic nature of protein interactions within cells. The rapid biotinylation capability of TurboID reduces background noise and false positives while Co-IP enriches stable interactors, together improving data quality and interpretation of the interactomic landscape. Demonstrating the efficacy of this methodology, proteins relevant to the pathology of Spinal Muscular Atrophy were utilised to explore variations at the interactome level. The use of identical starting cell lysates for both TurboID and Co-IP minimised variability and ensured datasets were comparable, allowing for consistency and enhancing the reliability of findings regarding the nature and strength of protein interactions. This novel framework effectively combines both innovative and classical techniques while maintaining consistency in sample handling, advancing our understanding of the intricate networks that govern cellular processes.

cell biology↗

bioRxiv2022

Identification of plasma proteins associated with oesophageal cancer chemotherapeutic treatment outcomes using SWATH-MS

Oesophageal adenocarcinoma (OAC) is an aggressive cancer with a five-year survival of <15%. Current chemotherapeutic strategies only benefit a minority (20-30%) of patients and there are no methods available to differentiate between responders and non-responders. We performed quantitative proteomics using Sequential Window Acquisition of all THeoretical fragment-ion spectra-Mass Spectrometry (SWATH-MS) on albumin/IgG-depleted and non-depleted plasma samples from 23 patients with locally advanced OAC prior to treatment. Individuals were grouped based on tumour regression (TRG) score (TRG1/2/3 vs TRG4/5) after chemotherapy, and differentially abundant proteins were compared. Protein depletion of highly abundant proteins led to the identification of around twice as many proteins. SWATH-MS revealed significant quantitative differences in the abundance of several proteins between the two groups. These included complement c1q subunit proteins, C1QA, C1QB and C1QC, which were of higher abundance in the low TRG group. Of those that were found to be of higher abundance in the high TRG group, GSTP1 was found to exhibit the lowest p-value and highest classification accuracy and Cohens kappa value. Concentrations of these proteins were further examined using ELISA-based assays. This study provides quantitative information relating to differences in the plasma proteome that underpin response to chemotherapeutic treatment in oesophageal cancers.

cancer biology↗

bioRxiv2021

Structure, dynamics, and inhibition of Staphylococcus aureus m1A22-tRNA methyltransferase

The enzyme m1A22-tRNA methyltransferase (TrmK) catalyses the transfer of a methyl group from SAM to the N1 of adenine 22 in tRNAs. TrmK is essential for Staphylococcus aureus survival during infection, but has no homologue in mammals, making it a promising target for antibiotic development. Here we describe the structural and functional characterisation of S. aureus TrmK. Crystal structures are reported for S. aureus TrmK apoenzyme and in complexes with SAM and SAH. Isothermal titration calorimetry showed that SAM binds to the enzyme with favourable but modest enthalpic and entropic contributions, whereas SAH binding leads to an entropic penalty compensated by a large favourable enthalpic contribution. Molecular dynamics simulations point to specific motions of the C-terminal domain being altered by SAM binding, which might have implications for tRNA recruitment. Activity assays for S. aureus TrmK-catalysed methylation of WT and position 22 mutants of tRNALeu demonstrate that the enzyme requires an adenine at position 22 of the tRNA. Intriguingly, a small RNA hairpin of 18 nucleotides is methylated by TrmK depending on the position of the adenine. In-silico screening of compounds suggested plumbagin as a potential inhibitor of TrmK, which was confirmed by activity measurements. Furthermore, LC-MS indicated the protein was covalently modified by one equivalent of the inhibitor, and proteolytic digestion coupled with LC-MS identified Cys92, in the vicinity of the SAM-binding site, as the sole residue modified. These results these results identify a cryptic binding pocket of S. aureus TrmK and lay the foundation for future structure-based drug discovery.

biochemistry↗