Phosphoproteomics reveals conserved exercise-stimulated signaling and AMPK regulation of store-operated calcium entry.

Exercise stimulates cellular and physiological adaptations that are associated with widespread health benefits. To uncover conserved protein phosphorylation events underlying this adaptive response, we performed mass spectrometry-based phosphoproteomic analyses of skeletal muscle from two widely used rodent models: treadmill running in mice and in situ muscle contraction in rats. We overlaid these phosphoproteomic signatures with cycling in humans to identify common cross-species phosphosite responses, as well as unique model-specific regulation. We identified > 22,000 phosphosites, revealing orthologous protein phosphorylation and overlapping signaling pathways regulated by exercise. This included two conserved phosphosites on stromal interaction molecule 1 (STIM1), which we validate as AMPK substrates. Furthermore, we demonstrate that AMPK-mediated phosphorylation of STIM1 negatively regulates store-operated calcium entry, and this is beneficial for exercise in Drosophila. This integrated cross-species resource of exercise-regulated signaling in human, mouse, and rat skeletal muscle has uncovered conserved networks and unraveled crosstalk between AMPK and intracellular calcium flux.

An exo-ß-(1→3)-D-galactanase from Streptomyces sp. provides insights into type II arabinogalactan structure.

An exo-ß-(1→3)-D-galactanase (SGalase1) that specifically cleaves the ß-(1→3)-D-galactan backbone of arabinogalactan-proteins (AGPs) was isolated from culture filtrates of a soil Streptomyces sp. Internal peptide sequence information was used to clone and recombinantly express the gene in E. coli. The molecular mass of the isolated enzyme was ~45 kDa, similar to the 48.2 kDa mass predicted from the amino acid sequence. The pI, pH and temperature optima for the enzyme were ~7.45, 3.8 and 48 °C, respectively. The native and recombinant enzymes specifically hydrolysed ß-(1→3)-D-galacto-oligo- or poly-saccharides from the upstream (non-reducing) end, typical of an exo-acting enzyme. A second homologous Streptomyces gene (SGalase2) was also cloned and expressed. SGalase2 was similar in size (47.9 kDa) and enzyme activity to SGalase1 but differed in its pH optimum (pH 5). Both SGalase1 and SGalase2 are predicted to belong to the CAZy glycosyl hydrolase family GH 43 based on activity, sequence homology and phylogenetic analysis. The K(m) and V(max) of the native exo-ß-(1→3)-D-galactanase for de-arabinosylated gum arabic (dGA) were 19 mg/ml and 9.7 µmol D-Gal/min/mg protein, respectively. The activity of these enzymes is well suited for the study of type II galactan structures and provides an important tool for the investigation of the biological role of AGPs in plants. De-arabinosylated gum arabic (dGA) was used as a model to investigate the use of these enzymes in defining type II galactan structure. Exhaustive hydrolysis of dGA resulted in a limited number of oligosaccharide products with a trisaccharide of Gal(2)GlcA(1) predominating.

Preparation of a new chromogenic substrate to assay for beta-galactanases that hydrolyse type II arabino-3,6-galactans.

A chromogenic assay using RB5-dGA, Reactive Black 5 (RB5) dye covalently coupled to de-arabinosylated gum arabic (dGA), was developed for rapid screening of beta-galactanases. dGA was prepared by partial acid hydrolysis (0.25M trifluoroacetic acid for 2h at 90-95 degrees C) of gum Arabic (GA) from Acacia senegal. The dGA exhibited a median molecular mass of approximately 10kDa, corresponding to a degree of polymerisation (DP) approximately 60. It was devoid of Ara residues, and contained mostly Galp (68mol%) together with GlcpA (30mol%). The Galp residues were (1,6)- (34mol%), (1,3)- (3mol%) and (1,3,6)- (26mol%) linked, and the GlcAp residues were primarily terminal (28mol%) together with a small amount of (1,4)-linked (2mol%), as expected for a type II (3,6)-galactan. The new chromogenic assay is simple, cost effective, relatively sensitive, and is specific for either beta-(1-->3)- and/or beta-(1-->6)-d-galactanases. It will enable routine large-scale screening of beta-galactanases from crude enzyme preparations and microorganism cultures, and is suitable for profiling activity during purification processes.