Biology subjects

Currie, M. J.

Publications and source records attributed to Currie, M. J..

4 recordsLinked to original sources

bioRxiv2025

Development of a 3D in vitro wound healing model to assess the effect of ADSC-EVs on vascularisation

Angiogenesis is critical for effective wound healing and relies on the successful coordination of various cell types including endothelial cells, macrophages, and fibroblasts. Adipose-derived stem cell extracellular vesicles (ADSC-EVs) have demonstrated pro-angiogenic properties and have been posited as a novel therapeutic to aid wound healing; however, their functional impact within human-derived multicellular models remains largely uncharacterised. This study explores the development and application of a 3D multicellular in vitro model to assess the effects of ADSC-EVs on vascularisation in the context of wound healing. 3D multicellular in vitro models were developed by co-culturing human umbilical vein endothelial cells (HUVECs), monocyte-derived macrophages (MDMs), and fibroblasts within Matrigel to recapitulate the in vivo wound healing microenvironment. A five-colour confocal microscopy panel was developed to visualise each cell type and EVs within the models. The optimised models were then treated ADSC-EVs or control to determine their impact on angiogenesis and cell co-localisation. We determined that vessel formation was significantly enhanced when HUVECs were co-cultured in multicellular models compared to monocultures, with the greatest effect observed in the full three-cell-type model. This effect was even more pronounced with the addition of ADSC-EVs. ADSC-EV treatment also enhanced macrophage co-localisation within endothelial structures. This study developed a multicellular model that can be used for future work assessing wound healing in vitro and will be additive to currently used single-cell and in vivo models. We have applied these models to demonstrate that ADSC-EVs significantly enhance tube formation in HUVECs, and the development of tissue-like structures in multicell systems, highlighting their potential as a promising therapeutic approach for improving wound healing.

cell biology↗

bioRxiv2024

Structural and biophysical characterisation of ubiquitin variants that specifically inhibit the ubiquitin conjugating enzyme Ube2d2

The ubiquitin conjugating E2 enzymes play a central role in ubiquitin transfer. Disruptions to the ubiquitin system are implicated in multiple diseases and as a result, molecules that modulate the activity of the ubiquitin system are of interest. E2 enzyme function is reliant on interactions with partner proteins and disruption of these is an effective way of modulating activity. Here, we report the discovery of ubiquitin variants (UbVs) that inhibit the E2 enzyme, Ube2d2 (UbcH5b). The six UbVs identified inhibit ubiquitin chain building, and structural and biophysical characterisation of two of these demonstrate they bind to Ube2d2 with low micromolar affinity and high specificity within the Ube2d family of E2 enzymes. Both characterised UbVs bind at a site that overlaps with E1 binding, while the more inhibitory UbV blocks a critical non-covalent ubiquitin binding site on the E2 enzyme. The discovery of novel protein-based ubiquitin derivatives that inhibit protein-protein interactions is an important step towards discovery of small molecules that inhibit the activity of E2 enzymes. Furthermore, the specificity of the UbVs within the Ube2d family suggests that it may be possible to develop tools to selectively inhibit highly related E2 enzymes.

biochemistry↗

bioRxiv2023

Tuneable hydrogel stiffness in a 3D in vitro model induces epithelial to mesenchymal transition in MCF7 but not MDA-MB-231 breast cancer cells

The study of in vitro models of breast cancer is crucial for understanding and treating the malignancy in patients, with 3D in vitro models providing researchers with more biomimetic systems to overcome limitations of current to 2D cultures and in vivo animal models. Ex vivo patient tissues have shown that malignant breast tissues are stiffer than healthy or benign tissues, and that the stiffness corresponds with increasing tumour grade. Stiffening of the breast tumour environment alters tumour cell phenotype and facilitates tumour progression, invasion and metastasis. Better understanding of the relationship between extracellular matrix stiffness and breast cancer cell phenotype, and how that is important in the initiation of metastasis, should lead to designing 3D models that mimic the breast tumour microenvironment at different stages of breast cancer progression. This study investigated phenotypic response of two breast cancer cell lines that are representative of clinical breast cancer subtypes (MCF7, Luminal A; MDA-MB-231, Triple Negative Breast Cancer) in gelatin-methacryloyl (GelMA) hydrogels of varying stiffness. A visible light photoinitiation system was adopted to provide a tuneable photocrosslinking platform to systematically control hydrogel stiffness and tumour microenvironment. This allowed rapid fabrication of biocompatible hydrogels supporting high cell viability over long-term culture. The impact of a clinically relevant range of microenvironmental stiffness on breast cancer cell behaviour and phenotype was examined over a 21-day culture period using GelMA hydrogels. Results showed that MCF7 cells cultured for 21 days in high stiffness hydrogels (10 wt%; 28 kPa) responded by downregulating the epithelial marker E-cadherin and upregulating mesenchymal markers N-cadherin and Vimentin, whereas MDA-MB-231 cells showed no changes in EMT-markers when cultured in hydrogels of corresponding stiffness (10 wt%; 33 kPa). Culturing both cell lines in soft hydrogels (5 wt%; 11 kPa) maintained their phenotype over 21 days, highlighting the importance of controlling hydrogel mechanical properties when studying breast cancer cell phenotype.

cancer biology↗

bioRxiv2022

Structure and mechanism of the tripartite ATP-independent periplasmic (TRAP) transporter

In bacteria and archaea, tripartite ATP-independent periplasmic (TRAP) transporters uptake essential carboxylate- and sulfonate-containing nutrients into the cytoplasm. Unlike other secondary active transporters, TRAP transporters cannot receive their substrates directly, but do so indirectly via a secreted soluble substrate-binding protein. How a sodium-driven secondary active transporter is strictly coupled to a passenger-carrying substrate-binding domain is poorly understood. Here, we report the cryo-EM structure of the sialic acid TRAP transporter SiaQM from Photobacterium profundum at 2.97 [A] resolution. SiaM has 12-TMs that come together to form a "transport" domain and a "scaffold" domain, with the transport domain consisting of helical hairpins as seen in the sodium-coupled elevator transporter VcINDY. Interestingly, the SiaQ protein forms intimate contacts with SiaM to extend the size of the scaffold domain, indicating TRAP transporters may operate as monomers, rather than the typically observed oligomers. We have identified the Na+ and sialic acid binding sites in SiaM and confirmed a strict dependence on the substrate-binding protein SiaP for uptake. We have determined the SiaP crystal structure that, together with co-evolution driven docking studies, provides a molecular basis for how sialic acid is delivered to the SiaQM transporter complex. We conclude that TRAP proteins are conceptually a marriage between an ABC importer and a secondary active transporter, which we describe herein as an elevator-with-an-operator mechanism.

molecular biology↗