A highly prevalent equine glycogen storage disease is explained by constitutive activation of a mutant glycogen synthase

Background: Equine type 1 polysaccharide storage myopathy (PSSM1) is associated with a missense mutation (R309H) in the glycogen synthase (GYS1) gene, enhanced glycogen synthase (GS) activity and excessive glycogen and amylopectate inclusions in muscle. Methods: Equine muscle biochemical and recombinant enzyme kinetic assays in vitro and homology modelling in silico, were used to investigate the hypothesis that higher GS activity in affected horse muscle is caused by higher GS expression, dysregulation, or constitutive activation via a conformational change. Results: PSSM1-affected horse muscle had significantly higher glycogen content than control horse muscle despite no difference in GS expression. GS activity was significantly higher in muscle from homozygous mutants than from heterozygote and control horses, in the absence and presence of the allosteric regulator, glucose 6 phosphate (G6P). Muscle from homozygous mutant horses also had significantly increased GS phosphorylation at sites 2 + 2a and significantly higher AMPKα1 (an upstream kinase) expression than controls, likely reflecting a physiological attempt to reduce GS enzyme activity. Recombinant mutant GS was highly active with a considerably lower Km for UDP-glucose, in the presence and absence of G6P, when compared to wild type GS, and despite its phosphorylation. Conclusions: Elevated activity of the mutant enzyme is associated with ineffective regulation via phosphorylation rendering it constitutively active. Modelling suggested that the mutation disrupts a salt bridge that normally stabilises the basal state, shifting the equilibrium to the enzyme's active state. General significance: This study explains the gain of function pathogenesis in this highly prevalent polyglucosan myopathy

Species-specific consequences of an E40K missense mutation in superoxide dismutase 1 (SOD1)

A glutamic acid to lysine (E40K) residue substitution in superoxide dismutase 1 (SOD1) is associated with canine degenerative myelopathy: the only naturally occurring large animal model of amyotrophic lateral sclerosis (ALS). The E40 residue is highly conserved across mammals, except the horse, which naturally carries the (dog mutant) K40 residue. Here we hypothesized that in vitro expression of mutant dog SOD1 would recapitulate features of human ALS (ie, SOD1 protein aggregation, reduced cell viability, perturbations in mitochondrial morphology and membrane potential, reduced ATP production, and increased superoxide ion levels); further, we hypothesized that an equivalent equine SOD1 variant would share similar perturbations in vitro, thereby explain horses’ susceptibility to certain neurodegenerative diseases. As in human ALS, expression of mutant dog SOD1 was associated with statistically significant increased aggregate formation, raised superoxide levels (ROS), and altered mitochondrial morphology (increased branching (form factor)), when compared to wild‐type dog SOD1‐expressing cells. Similar deficits were not detected in cells expressing the equivalent horse SOD1 variant. Our data helps explain the ALS‐associated cellular phenotype of dogs expressing the mutant SOD1 protein and reveals that species‐specific sequence conservation does not necessarily predict pathogenicity. The work improves understanding of the etiopathogenesis of canine degenerative myelopathy

Chimeric Vitronectin : Insulin-like Growth Factor Proteins Enhance Cell Growth and Migration through Co-Activation of Receptors

Complexes comprised of IGF-I, IGF-binding proteins and the ECM protein vitronectin (VN) stimulate cell migration and growth and can replace the requirement for serum for the ex vivo expansion of cells, as well as promote wound healing in vivo. Moreover, the activity of the complexes is dependent on co-activation of the IGF-I receptor and VN-binding integrins. In view of this we sought to develop chimeric proteins able to recapitulate the action of the multiprotein complex within a single molecular species. We report here the production of two recombinant chimeric proteins, incorporating domains of VN linked to IGF-I, which mimic the functions of the complex. Further, the activity of the chimeric proteins is dependent on co-activation of the IGF-I- and VN-binding cell surface receptors. Clearly the use of chimeras that mimic the activity of growth factor:ECM complexes, such as these, offer manufacturing advantages that ultimately will facilitate translation to cost-effective therapies

Integrated analysis of acetyl-CoA and histone modification via mass spectrometry to investigate metabolically driven acetylation

Acetylation is a highly abundant and dynamic post-translational modification (PTM) on histone proteins which, when present on chromatin-bound histones, facilitates the accessibility of DNA for gene transcription. The central metabolite, acetyl-CoA, is a substrate for acetyltransferases, which catalyze protein acetylation. Acetyl-CoA is an essential intermediate in diverse metabolic pathways, and cellular acetyl-CoA levels fluctuate according to extracellular nutrient availability and the metabolic state of the cell. The Michaelis constant (Km) of most histone acetyltransferases (HATs), which specifically target histone proteins, falls within the range of cellular acetyl-CoA concentrations. As a consequence, global levels of histone acetylation are often restricted by availability of acetyl-CoA. Such metabolic regulation of histone acetylation is important for cell proliferation, differentiation, and a variety of cellular functions. In cancer, numerous oncogenic signaling events hijack cellular metabolism, ultimately inducing an extensive rearrangement of the epigenetic state of the cell. Understanding metabolic control of the epigenome through histone acetylation is essential to illuminate the molecular mechanisms by which cells sense, adapt, and occasionally disengage nutrient fluctuations and environmental cues from gene expression. In particular, targeting metabolic regulators or even dietary interventions to impact acetyl-CoA availability and histone acetylation is a promising target in cancer therapy. Liquid chromatography coupled to mass spectrometry (LC-MS) is the most accurate methodology to quantify protein PTMs and metabolites. In this chapter, we present state-of-the-art protocols to analyze histone acetylation and acetyl-CoA. Histones are extracted and digested into short peptides (4–20 aa) prior to LC-MS. Acetyl-CoA is extracted from cells and analyzed using an analogous mass spectrometry-based procedure. Model systems can be fed with isotopically labeled substrates to investigate the metabolic preference for acetyl-CoA production and the metabolic dependence and turnover of histone acetylation. We also present an example of data integration to correlate changes in acetyl-CoA production with histone acetylation

Flagellin Treatment Prevents Increased Susceptibility to Systemic Bacterial Infection after Injury by Inhibiting Anti-Inflammatory IL-10+ IL-12- Neutrophil Polarization

Severe trauma renders patients susceptible to infection. In sepsis, defective bacterial clearance has been linked to specific deviations in the innate immune response. We hypothesized that innate immune modulations observed during sepsis also contribute to increased bacterial susceptibility after severe trauma. A well-established murine model of burn injury, used to replicate infection following trauma, showed that wound inoculation with P. aeruginosa quickly spreads systemically. The systemic IL-10/IL-12 axis was skewed after burn injury with infection as indicated by a significant elevation in serum IL-10 and polarization of neutrophils into an anti-inflammatory (“N2”; IL-10(+) IL-12(−)) phenotype. Infection with an attenuated P. aeruginosa strain (ΔCyaB) was cleared better than the wildtype strain and was associated with an increased pro-inflammatory neutrophil (“N1”; IL-10(−)IL-12(+)) response in burn mice. This suggests that neutrophil polarization influences bacterial clearance after burn injury. Administration of a TLR5 agonist, flagellin, after burn injury restored the neutrophil response towards a N1 phenotype resulting in an increased clearance of wildtype P. aeruginosa after wound inoculation. This study details specific alterations in innate cell populations after burn injury that contribute to increased susceptibility to bacterial infection. In addition, for the first time, it identifies neutrophil polarization as a therapeutic target for the reversal of bacterial susceptibility after injury

Genome-wide survey of protein kinases required for cell cycle progression

Cycles of protein phosphorylation are fundamental in regulating the progression of the eukaryotic cell through its division cycle. Here we test the complement of Drosophila protein kinases (kinome) for cell cycle functions after gene silencing by RNA-mediated interference. We observed cell cycle dysfunction upon downregulation of 80 out of 228 protein kinases, including most kinases that are known to regulate the division cycle. We find new enzymes with cell cycle functions; some of these have family members already known to phosphorylate microtubules, actin or their associated proteins. Additionally, depletion of several signalling kinases leads to specific mitotic aberrations, suggesting novel roles for familiar enzymes. The survey reveals the inter-digitation of systems that monitor cellular physiology, cell size, cellular stress and signalling processes with the basic cell cycle regulatory machinery