SRSF2 is a prototypical SR protein which plays important roles in the alternative splicing of pre-mRNA. It has been shown to be involved in regulatory pathways for maintaining genomic stability and play important roles in regulating key receptors in the heart. We report here the solution structure of the RNA recognition motifs (RRM) domain of free human SRSF2 (residues 9–101). Compared with other members of the SR protein family, SRSF2 structure has a longer L3 loop region. The conserved aromatic residue in the RNP2 motif is absent in SRSF2. Calorimetric titration shows that the RNA sequence 5′AGCAGAGUA3′ binds SRSF2 with a K_d of 61 ± 1 nM and a 1:1 stoichiometry. NMR and mutagenesis experiments reveal that for SFSF2, the canonical β1 and β3 interactions are themselves not sufficient for effective RNA binding; the additional loop L3 is crucial for RNA complex formation. A comparison is made between the structures of SRSF2–RNA complex with other known RNA complexes of SR proteins. We conclude that interactions involving the L3 loop, N- and C-termini of the RRM domain are collectively important for determining selectivity between the protein and RNA.

Neutrophils play a key role in the pathophysiology of rheumatoid arthritis (RA) where release of ROS and proteases directly causes damage to joints and tissues. Neutrophil function can be modulated by Janus Kinase (JAK) inhibitor drugs, including tofacitinib and baricitinib, which are clinically effective treatments for RA. However, clinical trials have reported increased infection rates and transient neutropenia during therapy. The subtle differences in the mode of action, efficacy and safety of JAK inhibitors have been the primary research topic of many clinical trials and systematic reviews, to provide a more precise and targeted treatment to patients. The aim of this study was to determine both the differences in the metabolome of neutrophils from healthy controls and people with RA, and the effect of different JAK inhibitors on the metabolome of healthy and RA neutrophils. Isolated neutrophils from healthy controls (HC) (n = 6) and people with RA (n = 7) were incubated with baricitinib, tofacitinib or a pan-JAK inhibitor (all 200 ng/mL) for 2 h. Metabolites were extracted, and ¹H nuclear magnetic resonance (NMR) was applied to study the metabolic changes. Multivariate analyses and machine learning models showed a divergent metabolic pattern in RA neutrophils compared to HC at 0 h (F1 score = 86.7%) driven by energy metabolites (ATP, ADP, GTP and glucose). No difference was observed in the neutrophil metabolome when treated with JAK inhibitors. However, JAK inhibitors significantly inhibited ROS production and baricitinib decreased NET production (p < 0.05). Bacterial killing was not impaired by JAK inhibitors, indicating that the effect of JAK inhibitors on neutrophils can inhibit joint damage in RA without impairing host defence. This study highlights altered energy metabolism in RA neutrophils which may explain the cause of their dysregulation in inflammatory disease.

MUPs (major urinary proteins) play an important role in chemical signalling in rodents and possibly other animals. In the house mouse (Mus musculus domesticus) MUPs in urine and other bodily fluids trigger a range of behavioural responses that are only partially understood. There are at least 21 Mup genes in the C57BL/6 mouse genome, all located on chromosome 4, encoding sequences of high similarity. Further analysis separates the MUPs into two groups, the ‘central’ near-identical MUPs with over 97% sequence identity and the ‘peripheral’ MUPs with a greater degree of heterogeneity and approximately 20–30% non-conserved amino acids. This review focuses on differences between the two MUP sub-groups and categorizes these changes in terms of molecular structure and pheromone binding. As small differences in amino acid sequence can result in marked changes in behavioural response to the signal, we explore the potential of single amino acid changes to affect chemical signalling and protein stabilization. Using analysis of existing molecular structures available in the PDB we compare the chemical and physical properties of the ligand cavities between the MUPs. Furthermore, we identify differences on the solvent exposed surfaces of the proteins, which are characteristic of protein–protein interaction sites. Correlations can be seen between molecular heterogeneity and the specialized roles attributed to some MUPs.

The antiepileptic drug ethosuximide has recently been shown to be neuroprotective in various Caenorhabditis elegans and rodent neurodegeneration models. It is therefore a promising repurposing candidate for the treatment of multiple neurodegenerative diseases. However, high concentrations of the drug are required for its protective effects in animal models, which may impact on its translational potential and impede the identification of its molecular mechanism of action. Therefore, we set out to develop more potent neuroprotective lead compounds based on ethosuximide as a starting scaffold. Chemoinformatic approaches were used to identify compounds with structural similarity to ethosuximide and to prioritise these based on good predicated blood-brain barrier permeability and C. elegans bioaccumulation properties. Selected compounds were initially screened for anti-convulsant activity in a C. elegans pentylenetetrazol-induced seizure assay, as a rapid primary readout of bioactivity; and then assessed for neuroprotective properties in a C. elegans TDP-43 proteinopathy model based on pan-neuronal expression of human A315T mutant TDP-43. The most potent compound screened, α-methyl-α-phenylsuccinimide (MPS), ameliorated the locomotion defects and extended the shortened lifespan of TDP-43 mutant worms. MPS also directly protected against neurodegeneration by reducing the number of neuronal breaks and cell body losses in GFP-labelled GABAergic motor neurons. Importantly, optimal neuroprotection was exhibited by external application of 50 μM MPS, compared to 8 mM for ethosuximide. This greater potency of MPS was not due to bioaccumulation to higher internal levels within the worm, based on ¹H-nuclear magnetic resonance analysis. Like ethosuximide, the activity of MPS was abolished by mutation of the evolutionarily conserved FOXO transcription factor, daf-16, suggesting that both compounds act via the same neuroprotective pathway(s). In conclusion, we have revealed a novel neuroprotective activity of MPS that is >100-fold more potent than ethosuximide. This increased potency will facilitate future biochemical studies to identify the direct molecular target(s) of both compounds, as we have shown here that they share a common downstream DAF-16-dependent mechanism of action. Furthermore, MPS is the active metabolite of another approved antiepileptic drug, methsuximide. Therefore, methsuximide may have repurposing potential for treatment of TDP-43 proteinopathies and possibly other human neurodegenerative diseases.

The metabolic perturbations caused by competitive rugby are not well characterized. Our aim is to utilize untargeted metabolomics to develop appropriate interventions, based on the metabolic fluctuations that occur in response to this collision-based team sport. Seven members of an English Premiership rugby squad consented to provide blood, urine, and saliva samples daily, over a competitive week including gameday (GD), with physical demands and dietary intake also recorded. Sample collection, processing and statistical analysis were performed in accordance with best practice set out by the metabolomics standards initiative employing 700 MHz NMR spectroscopy. Univariate and multivariate statistical analysis were employed to reveal the acute energy needs of this high intensity sport are met via glycolysis, the TCA cycle and gluconeogenesis. The recovery period after cessation of match play and prior to training recommencing sees a re-entry to gluconeogenesis, coupled with markers of oxidative stress, structural protein degradation, and reduced fatty acid metabolism. This novel insight leads us to propose that effective recovery from muscle damaging collisions is dependent upon the availability of glucose. An adjustment in the periodisation of carbohydrate to increase GD+1 provision may prevent the oxidation of amino acids which may also be crucial to allay markers of structural tissue degradation. Should we expand the ‘Fuel for the work required’ paradigm in collision-based team sports to include ‘Fuel for the damage induced’?

Aortic medial amyloid is the most prevalent amyloid found to date, but remarkably little is known about it. It is characterised by aberrant deposition of a 5.4 kDa protein called medin within the medial layer of large arteries. Here we employ a combined approach of ab initio protein modelling and ¹³C-direct detection NMR to generate a model for soluble monomeric medin comprising a stable core of three β-strands and shorter more labile strands at the termini. Molecular dynamics simulations suggested that detachment of the short, C-terminal β-strand from the soluble fold exposes key amyloidogenic regions as a potential site of nucleation enabling dimerisation and subsequent fibril formation. This mechanism resembles models proposed for several other amyloidogenic proteins suggesting that despite variations in sequence and protomer structure these proteins may share a common pathway for amyloid nucleation and subsequent protofibril and fibril formation.