Biology subjects

Currie, M.

Publications and source records attributed to Currie, M..

4 recordsLinked to original sources

bioRxiv2024

On the function of TRAP substrate-binding proteins: conformational variation of the sialic acid binding protein SiaP

Tripartite ATP-independent periplasmic (TRAP) transporters are analogous to ABC transporters in that they use a substrate-binding proteins to scavenge metabolites (e.g., N-acetylneuraminate) and deliver them to the membrane components for import. TRAP substrate-binding proteins are thought to bind the substrate using a two-state (open and closed) induced-fit mechanism. We solved the structure of the TRAP N-acetylneuraminate substrate-binding protein from Aggregatibacter actinomycetemcomitans (AaSiaP) in both the open ligand-free and closed liganded conformations. Surprisingly, we also observed an intermediate conformation, where AaSiaP is mostly closed and is bound to a non-cognate ligand, acetate, which hints at how N-acetylneuraminate binding stabilises a fully closed state. AaSiaP preferentially binds N-acetylneuraminate (KD = 0.4 {micro}M) compared to N-glycolylneuraminate (KD = 4.4 {micro}M), which is explained by the closed-N-acetylneuraminate bound structure. Small-angle X-ray scattering data alongside molecular dynamics simulations suggest the AaSiaP adopts a more open state in solution than in crystal. However, the open unliganded conformation can also sample closed conformations. Molecular dynamics simulations also demonstrate the importance of water molecules for stabilising the closed conformation. Although our data is consistent with an induced fit model of binding, it is likely that the open unliganded conformation encompasses multiple states capable of binding substrate. The mechanism by which the ligand is released for import remains to be determined.

biochemistry↗

bioRxiv2023

Structural and biophysical analysis of a Haemophilus influenzae tripartite ATP-independent periplasmic (TRAP) transporter

Tripartite ATP-independent periplasmic (TRAP) transporters are secondary-active transporters that receive their substrates via a soluble binding protein to move bioorganic acids across bacterial or archaeal cell membranes. Recent cryo-EM structures of TRAP transporters provide a broad framework to understand how they work, but the mechanistic details of transport are not yet defined. Here we report the cryo-EM structure of the Haemophilus influenzae N-acetylneuraminate TRAP transporter (HiSiaQM) at 2.99 [A] resolution (extending to 2.2 [A] at the core), revealing new features. The improved resolution (the previous HiSiaQM structure is 4.7 [A] resolution) permits accurate assignment of two Na+ sites and the architecture of the substrate binding site, consistent with mutagenic and functional data. Moreover, rather than a monomer, the HiSiaQM structure is a homodimer. We observe lipids at the dimer interface, as well as a lipid trapped within the fusion that links the SiaQ and SiaM subunits. We show that the affinity (KD) for the complex between the soluble HiSiaP protein and HiSiaQM is in the micromolar range and that a related SiaP can bind HiSiaQM. This work provides key data that enhances our understanding of the elevator-with-an-operator mechanism of TRAP transporters.

biochemistry↗

bioRxiv2023

ADSC-EVs modulate primary human macrophages to an anti-inflammatory phenotype in vitro

BackgroundEVs released by adipose derived stem cells (ADSCs) have shown promise as a therapeutic for tissue repair and regeneration because of their purported immune-regulatory properties. In this capacity, ADSC-EVs could be beneficial in improving graft retention rates for autologous fat grafting (AFG) post-mastectomy as, currently, grafted tissue rates are reported to be variable and low. Enriching grafted tissue with ADSC-EVs may improve retention rates by modulating macrophages resident within both the breast and lipoaspirate. We aimed to identify key macrophage phenotypes that are modulated by ADSC-EVs in vitro. MethodsADSCs were isolated from lipoaspirates of women undergoing AFG and characterised by flow cytometry and differentiation potential. ADSC-EVs were isolated from cell culture media and characterised by tunable resistive pulse sensing (TRPS), transmission electron microscopy (TEM), and Western blot. Primary monocyte-derived macrophages were polarized to an M1-like (GM-CSF, IFN{gamma}) or M2-like phenotype (M-CSF, IL-4) or maintained (M0-like; M-CSF) and, at the time of polarization, ADSC-EVs were co-cultured with macrophages for 48 hrs. Flow cytometry coupled with high-dimensional analysis was used to cluster macrophages post co-culture. A manual gating strategy was generated to recapitulate these clusters and was applied to a repeat experimental run. Both runs were analysed to examine the prevalence of each cluster, representing a unique macrophage phenotype, with and without ADSC-EVs. ResultsFollowing the addition of ADSC-EVs, M0-like macrophages demonstrated a reciprocal shift of cell distribution from a cluster defined as having a high inflammatory profile (CD36+++CD206+++CD86+++; 38.6{+/-}14.8% of M1-like macrophages without ADSC-EVs; 16.5{+/-}7.0% with ADSC-EVs; p<0.0001) to a cluster with a lower inflammatory profile (CD36+CD206+CD86+; 16.6{+/-}11.2% to 35{+/-}21.5%; p<0.05). There was no shift in M2-like clusters following treatment with ADSC-EVs. ConclusionsADSC-EVs are complex regulators of macrophage phenotype that can shift macrophages away from a heightened pro-inflammatory state.

molecular biology↗

bioRxiv2018

An open source platform for analyzing and sharing worm behavior data

Animal behavior is increasingly being recorded in systematic imaging studies that generate large data sets. To maximize the usefulness of these data there is a need for improved resources for analyzing and sharing behavior data that will encourage re-analysis and method development by computational scientists1. However, unlike genomic or protein structural data, there are no widely used standards for behavior data. It is therefore desirable to make the data available in a relatively raw form so that different investigators can use their own representations and derive their own features. For computational ethology to approach the level of maturity of other areas of bioinformatics, we need to address at least three challenges: storing and accessing video files, defining flexible data formats to facilitate data sharing, and making software to read, write, browse, and analyze the data. We have developed an open resource to begin addressing these challenges using worm tracking as a model.

animal behavior and cognition↗