Biology subjects

Bronowska, A.

Publications and source records attributed to Bronowska, A..

3 recordsLinked to original sources

bioRxiv2026

AMPK senses cellular levels of nicotinamide adenine dinucleotide

The electron shuttle and coenzyme nicotinamide adenine nucleotide (NAD) is essential for cellular metabolism and homeostasis. NAD levels significantly fluctuate in cells, whilst several age-related diseases are associated with depletion of this metabolite. However, how NAD changes are monitored by nutrient/energy sensing signalling pathways remains poorly understood. We found that at physiological concentrations NAD controls the activity of the AMP-activated protein kinase (AMPK) in vitro and in human cells. Mechanistically, NAD binds gamma subunit of AMPK, and mutagenesis of the putative binding site renders the holoenzyme insensitive to NAD inhibition. Hyperactivation of AMPK in response to NAD depletion suppresses metabolic pathways including mammalian Target of Rapamycin Complex I (mTORC1) and autophagy. These results demonstrate that in addition to monitoring cellular energy levels AMPK functions as a NAD sensor, providing novel insight into how cells and tissues detect and respond to metabolic fluctuations with implications for stress resistance and ageing.

molecular biology↗

bioRxiv2025

Structural basis of quinone-sensing by the MarR-type repressor MhqR in Staphylococcus aureus

The MarR-family regulator MhqR of Staphylococcus aureus (SaMhqR) was previously characterized as quinone-sensing repressor of the mhqRED operon. Here, we resolved the crystal structures of apo-SaMhqR and the 2-methylbenzoquinone (MBQ)-bound SaMhqR complex. AlphaFold3 modelling was used to predict the structure of the SaMhqR in complex with its operator DNA. In the DNA-bound SaMhqR state, S65 and S66 of an allosteric 3-4 loop adapted a helically wound conformation to elongate helix 4 for optimal DNA binding. Key residues for MBQ interaction were identified as F11, F39, E43, and H111, forming the MBQ-binding pocket. MBQ binding prevented the formation of the extended helix 4 in the allosteric loop, leading to steric clashes with the DNA. Molecular dynamics (MD) simulations revealed an increased intrinsic dynamics within the allosteric loop and the {beta}1/{beta}2-wing regions after MBQ binding, to prevent DNA binding. Using mutational analyses, we validated that F11, F39, and H111 are required for quinone sensing in vivo, whereas S65 and S66 of the allosteric loop and D88, K89, V91 and Y92 of the {beta}1/{beta}2-wing are essential for DNA binding in vitro and in vivo. In conclusion, our structure-guided modelling and mutational analyses identified a quinone-binding pocket of SaMhqR and the mechanism of SaMhqR inactivation, which involves local structural rearrangements of an allosteric loop and a high intrinsic dynamics to prevent DNA interactions. Our results provide novel insights into the redox-mechanism of the conserved SaMhqR repressor, that functions as an important determinant of quinone and antimicrobial resistance in S. aureus. IMPORTANCES. aureus is a major human pathogen, which can cause life-threatening infections in humans. However, treatment options are limited due to the prevalence of antimicrobial resistant isolates in the hospital and the community. The MarR-type repressor SaMhqR was described to control resistance towards quinones and quinone-like antimicrobials. However, the redox-regulatory mechanism of SaMhqR by quinones was unknown. In this work, we explored the DNA-binding and quinone-sensing mechanism of SaMhqR and identified a quinone-binding pocket and an allosteric loop, which facilitates DNA binding activity via a helical wound conformation and adapts an unstructured coiled conformation upon quinone binding to inhibit DNA binding. A similar mechanism has been recently discovered for regulation of uric acid resistance by UrtR family repressors (1). Our results contribute to a better understanding of antimicrobial resistance regulation, which can be exploited for future drug-design to eradicate multidrug-resistant S. aureus.

microbiology↗

bioRxiv2022

Determining the toxicity and potential for environmental transport of pyridine using the brown crab Cancer pagurus (L.)

1A series of mass mortalities (wash-ups) of marine life were documented along Englands north east coastline with peaks in September and October 2021, coincident with a programme of intensified maintenance dredging of the River Tees. Decapod crustaceans were the worst affected fauna, with brown crab (Cancer pagurus), European lobster (Homarus gammarus, L.), green shore crab (Carcinus maenas, L.) and velvet swimming crab (Necora puber, L.) populations severely affected. Moribund animals presented with twitching behaviours and paralysis. A potential release of the industrial pollutant pyridine was forwarded as one explanation; however, toxicology data for pyridine in decapods is lacking. In this study, we address this knowledge gap by executing a programme of immersion exposure experiments (pyridine at 2 - 100 mg L-1) using C. pagurus, measuring toxicity effects at the individual (survival) and cellular levels (cellular, mitochondrial, and lipid peroxidation reactive oxygen species (ROS) formation in the gills, hepatopancreas and claw muscle). Highest mortality rates were seen after 72 hours of exposure, returning an LC50 value of 2.75 mg L-1. Exposed crabs presented with patterns of convulsions, limb twitching, paralysis, and death. Crabs exposed to the lowest pyridine dose (2 mg L-1) were noticeably more docile than controls. Concentration was a significant factor influencing mitochondrial ROS formation at low concentrations, with tissue type, time, and their interaction all significant at 100 mg L-1. Computer simulations were used to model the transport of any pyridine released from the dredging work, demonstrating the potential for a pyridine plume to extend from Seaham to the north of the Tees to Whitby and Robin Hoods Bay to the south. This range corresponds well with the reported wash-ups and subsequent declines in catch rates.

pharmacology and toxicology↗