Biology subjects

McKie, S. J.

Publications and source records attributed to McKie, S. J..

4 recordsLinked to original sources

bioRxiv2026

FLiPA: A versatile platform for quantitative analysis of protein-glycosphingolipid interactions

Interactions between proteins and glycosphingolipids (GSLs) regulate various cellular processes and altered GSL metabolism contributes to numerous diseases. The diverse glycan headgroups and ceramide backbones of GSLs shape membrane organisation, fluidity, curvature, and tension. As protein recognition frequently depends on both glycan specificity and the organisation of GSLs within the membrane, these interactions remain challenging to characterise in vitro. Here, we introduce FLiPA (Fluorescent Liposome Plate Assay), a versatile method that utilises fluorescent agarose-embedded giant liposomes for the quantitative analysis of protein-GSL interactions. By enabling systematic control of membrane and buffer composition, FLiPA provides an accessible and robust platform for dissecting the molecular determinants of protein-GSL interactions, including the roles of cholesterol, membrane order, protein oligomerisation and ionic strength.

molecular biology↗

bioRxiv2024

Highly sensitive mapping of in vitro type II topoisomerase DNA cleavage sites with SHAN-seq

Type II topoisomerases (topos) are a ubiquitous and essential class of enzymes that form transient enzyme-bound double-stranded breaks on DNA called cleavage complexes. The location and frequency of these cleavage complexes on DNA is important for cellular function, genomic stability, and a number of clinically important anticancer and antibacterial drugs, e.g., quinolones. We developed a simple high-accuracy end-sequencing (SHAN-seq) method to sensitively map type II topo cleavage complexes on DNA in vitro. Using SHAN-seq, we detected Escherichia coli gyrase and topoisomerase IV cleavage complexes at hundreds of sites on supercoiled pBR322 DNA, approximately one site every ten bp, with frequencies that varied by two-to-three orders of magnitude. These sites included previously identified sites and 20-50 fold more new sites. We show that the location and frequency of cleavage complexes at these sites are enzyme-specific and vary substantially in the presence of the quinolone, ciprofloxacin, but not with DNA supercoil chirality, i.e., negative vs. positive supercoiling. SHAN-seqs exquisite sensitivity provides an unprecedented single-nucleotide resolution view of the distribution of gyrase and topoisomerase IV cleavage complexes on DNA. Moreover, the discovery that these enzymes can cleave DNA at orders of magnitude more sites than the relatively few previously known sites resolves the apparent paradox of how these enzymes resolve topological problems throughout the genome.

biochemistry↗

bioRxiv2022

Altered plasma membrane abundance of the sulfatide-binding protein NF155 links glycosphingolipid imbalances to demyelination

Myelin is a multi-layered membrane that tightly wraps neuronal axons enabling efficient, high-speed signal propagation. The axon and myelin sheath form tight contacts, mediated by specific plasma membrane proteins and lipids, and disruption of these contacts causes devastating demyelinating diseases. Using two cell-based models of demyelinating sphingolipidoses, we demonstrate that altered lipid metabolism changes the abundance of specific plasma membrane proteins. These altered membrane proteins have known roles in cell adhesion and signalling, with several implicated in neurological diseases. The cell surface abundance of the adhesion molecule Neurofascin, a protein critical for the maintenance of myelin-axon contacts, changes following disruption to sphingolipid metabolism. This provides a direct molecular link between altered lipid abundance and myelin stability. We show that the Neurofascin isoform NF155, but not NF186, interacts directly and specifically with the sphingolipid sulfatide via multiple binding sites and that this interaction requires the full-length extracellular domain of NF155. We demonstrate that NF155 adopts an S-shaped conformation and preferrentially binds sulfatide-containing membranes in cis, with important implications for protein arrangement in the tight axon-myelin space. Our work links glycosphingolipid imbalances to disturbance of membrane protein abundance and demonstrates how this may be driven by direct protein-lipid interactions, providing a mechanistic framework to understand the pathogenesis of galactosphingolipidoses.

neuroscience↗

bioRxiv2021

Topoisomerase VI is a chirally-selective, preferential DNA decatenase

DNA topoisomerase VI (topo VI) is a type IIB DNA topoisomerase found predominantly in archaea and some bacteria, but also in plants and algae. Since its discovery, topo VI has been proposed to be a DNA decatenase, however robust evidence and a mechanism for its preferential decatenation activity was lacking. Using single-molecule magnetic tweezers measurements and supporting ensemble biochemistry, we demonstrate that Methanosarcina mazei topo VI preferentially unlinks, or decatenates, DNA crossings, in comparison to relaxing supercoils, through a preference for certain DNA crossing geometries. In addition, topo VI demonstrates a dramatic increase in ATPase activity, DNA binding and rate of strand passage, with increasing DNA writhe, providing further evidence that topo VI is a DNA crossing sensor. Our study strongly suggests that topo VI has evolved an intrinsic preference for the unknotting and decatenation of interlinked chromosomes by sensing and preferentially unlinking DNA crossings with geometries close to 90{degrees}.

biochemistry↗