Jon Marles-Wright’s research while affiliated with Newcastle University and other places

The aggregation of α-synuclein is a central neuropathological hallmark in neurodegenerative disorders known as Lewy body diseases, including Parkinson's disease and dementia with Lewy bodies. In the aggregation process, α-synuclein transitions from its native disordered/α-helical form to a β-sheet-rich structure, forming oligomers and protofibrils that accumulate into Lewy bodies, in a process that is thought to underlie neurodegeneration. Lipids are thought to play a critical role in this process by facilitating α-synuclein aggregation and contributing to cell toxicity, possibly through ceramide production. This study aimed to investigate biochemical changes associated with α-synuclein aggregation, focusing on lipid changes, using Raman spectroscopy coupled with machine learning. HEK293, Neuro2a and SH-SY5Y expressing increased levels of α-synuclein were treated with sonicated α-synuclein pre-formed fibrils, to model seeded aggregation. Raman spectroscopy, complemented by an in-house lipid spectral library, was used to monitor the aggregation process and its effects on cellular viability over 14 days. We detected α-synuclein aggregation by assessing β-sheet peaks at 1045 cm⁻1, in cells treated with α-synuclein pre-formed fibrils, using machine learning (principal component analysis and uniform manifold approximation and projection) analysis based on Raman spectral features. Changes in lipid profiles, and especially sphingolipids, including a decrease in sphingomyelin and increase in ceramides, were observed, consistent with oxidative stress and apoptosis. Altogether, our study informs on biochemical alterations that can be considered for the design of therapeutic strategies for Parkinson's disease and related synucleinopathies.

The aggregation of α-synuclein is crucial to the development of Lewy body diseases, including Parkinson’s disease and dementia with Lewy bodies. The aggregation pathway of α-synuclein typically involves a defined sequence of nucleation, elongation, and secondary nucleation, exhibiting prion-like spreading. This study employed Raman spectroscopy and machine learning analysis, alongside complementary techniques, to characterize the biomolecular changes during the fibrillation of purified recombinant wild-type α-synuclein protein. Monomeric α-synuclein was produced, purified, and subjected to a 7-day fibrillation assay to generate preformed fibrils. Stages of α-synuclein fibrillation were analyzed using Raman spectroscopy, with aggregation confirmed through negative staining transmission electron microscopy, mass spectrometry, and light scattering analyses. A machine learning pipeline incorporating principal component analysis and uniform manifold approximation and projection was used to analyze the Raman spectral data and identify significant peaks, resulting in differentiation between sample groups. Notable spectral shifts in α-synuclein were found in various stages of aggregation. Early changes (D1) included increases in α-helical structures (1303, 1330 cm–1) and β-sheet formation (1045 cm–1), with reductions in COO– and CH2 bond regions (1406, 1445 cm–1). By D4, these structural shifts persist with additional β-sheet features. At D7, a decrease in β-sheet H-bonding (1625 cm–1) and tyrosine ring breathing (830 cm–1) indicates further structural stabilization, suggesting a shift from initial helical structures to stabilized β-sheets and aggregated fibrils. Additionally, alterations in peaks related to tyrosine, alanine, proline, and glutamic acid were identified, emphasizing the role of these amino acids in intramolecular interactions during the transition from α-helical to β-sheet conformational states in α-synuclein fibrillation. This approach offers insight into α-synuclein aggregation, enhancing the understanding of its role in Lewy body disease pathophysiology and potential diagnostic relevance.

N‐carbamoyl‐β‐alanine amidohydrolase (CβAA) constitutes one of the most important groups of industrially relevant enzymes used in the production of optically pure amino acids and derivatives. In this study, a CβAA‐encoding gene from Rhizobium radiobacter strain MDC 8606 was cloned and overexpressed in Escherichia coli. The purified recombinant enzyme (RrCβAA) showed a specific activity of 14 U·mg⁻¹ using N‐carbamoyl‐β‐alanine as a substrate with an optimum activity at 55 °C and pH 8.0. In this work, we report also the first prokaryotic CβAA structure at a resolution of 2.0 Å. A discontinuous catalytic domain and a dimerisation domain attached through a flexible hinge region at the domain interface have been revealed. We identify key ligand binding residues, including a conserved glutamic acid (Glu131), histidine (H385) and arginine (Arg291). Our results allowed us to explain the preference of the enzyme for linear carbamoyl substrates, as large and branched carbamoyl substrates cannot fit in the active site of the enzyme. This work envisages the use of RrCβAA from R. radiobacter MDC 8606 for the industrial production of L‐α‐, L‐β‐ and L‐γ‐amino acids. The structural analysis provides new insights on enzyme–substrate interaction, which shed light on engineering of CβAAs for high catalytic activity and broad substrate specificity.

The importance of genomics in the COVID-19 age cannot be overstated and genomics has a key place in the advanced undergraduate microbiology curriculum. The COVID-19 pandemic, and its attendant lockdowns, have necessitated a change in the delivery of our microbial genomics module. The key challenges for delivering a remote computer-based genomics module, are ensuring student engagement and learning; and supporting the technical aspects of remote computer work. In the absence of in-person classes, we have adapted our material covering the principles and theory of microbial genomics, to asynchronous videos and synchronous online workshops. Practical training for all undergraduate microbiologists in the UK has been severely reduced, which has forced us to focus skills training towards computational aspects of genome assembly, analysis, and interpretation. The set up and implementation of a robust and scalable Linux-based genome analysis pipeline for students presents many challenges: from organising remote access to computer clusters; software support; and managing hardware. Assessment for the module is based on the demonstrating learning and skills development through the analysis of SARS-CoV2 genomes to identify spike protein variation and mapping this to published structures. Terminal assessment is through the analysis of newly sequenced bacterial genomes and the preparation of a genome report suitable for publication. We will outline the implementation and management of our Microbial Genomics module as a model for computer-based skills training for undergraduate microbiologists. Furthermore, we will discuss the impact of the COVID-19 pandemic on the module and the opportunities it has presented.

Ubiquitous in eukaryotes, inositol lipids have finely tuned roles in cellular signaling and membrane homeostasis. In Bacteria, however, inositol lipid production is rare. Recently, the prominent human gut bacterium Bacteroides thetaiotaomicron (BT) was reported to produce inositol lipids, including inositol sphingolipids, but the pathways remain ambiguous and their prevalence unclear. Here, we investigated the gene cluster responsible for inositol lipid synthesis in BT using a novel strain with inducible control of sphingolipid synthesis. We characterized the biosynthetic pathway from myo- inositol-phosphate (MIP) synthesis to phosphoinositol-dihydroceramide, including structural and kinetic studies of the enzyme MIP synthase (MIPS). We determined the crystal structure of recombinant BT MIPS with bound NAD cofactor at 2.0 Å resolution, and identified the first reported phosphatase for the conversion of bacterially-derived phosphatidylinositol phosphate (PIP) to phosphatidylinositol (PI). Transcriptomic analysis indicated inositol production is nonessential but its loss alters BT capsule expression. Bioinformatic and lipidomic comparisons of Bacteroidetes species revealed a novel second putative pathway for bacterial PI synthesis without a PIP intermediate. Our results indicate that inositol sphingolipid production, via one of the two pathways, is widespread in host-associated Bacteroidetes, and may be implicated in host interactions both indirectly via the capsule and directly through inositol lipid provisioning.