Selenocysteine is a rare amino acid in protein that is encoded by UGA with the requirement of a downstream mRNA stem-loop structure, the selenocysteine insertion sequence element. To detect selenoproteins in Drosophila, the entire genome was analyzed with a novel program that searches for selenocysteine insertion sequence elements, followed by selenoprotein gene signature analyses. This computational screen and subsequent metabolic labeling with ⁷⁵Se and characterization of selenoprotein mRNA expression resulted in identification of three selenoproteins: selenophosphate synthetase 2 and novel G-rich and BthD selenoproteins that had no homology to known proteins. To assess a biological role for these proteins, a simple chemically defined medium that supports growth of adult Drosophila and requires selenium supplementation for optimal survival was devised. Flies survived on this medium supplemented with 10⁻⁸ to 10⁻⁶ mselenium or on the commonly used yeast-based complete medium at about twice the rate as those on a medium without selenium or with >10⁻⁶ m selenium. This effect correlated with changes in selenoprotein mRNA expression. The number of eggs laid by Drosophila was reduced approximately in half in the chemically defined medium compared with the same medium supplemented with selenium. The data provide evidence that dietary selenium deficiency shortens, while supplementation of the diet with selenium normalizes the Drosophila life span by a process that may involve the newly identified selenoproteins.

Store-operated calcium entry (SOCE) is an important Ca²⁺ entry pathway that regulates many cell functions. Upon store depletion, STIM1, a transmembrane protein located in the endoplasmic reticulum (ER), aggregates and relocates close to the plasma membrane (PM) where it activates store-operated calcium channels (SOCs). Although STIM1 was early defined as a phosphoprotein, the contribution of the phosphorylation has been elusive. In the present work, STIM1 was found to be a target of extracellular-signal-regulated kinases 1 and 2 (ERK1/2) in vitro, and we have defined the ERK1/2-phosphorylated sites on the STIM1 sequence. Using HEK293 cells stably transfected for the expression of tagged STIM1, we found that alanine substitution mutants of ERK1/2 target sites reduced SOCE significantly, suggesting that phosphorylation of these residues are required to fully accomplish SOCE. Indeed, the ERK1/2 inhibitors PD184352 and PD0325901 decreased SOCE in transfected cells. Conversely, 12-O-tetradecanoylphorbol-13-acetate, which activates ERK1/2, enhanced SOCE in cells expressing wild-type tagged STIM1, but did not potentiate Ca²⁺ influx in cells expressing serine to alanine mutations in ERK1/2 target sites of STIM1. Alanine substitution mutations decreased Ca²⁺ influx without disturbing the aggregation of STIM1 upon store depletion and without affecting the relocalization in ER–PM punctae. However, our results suggest that STIM1 phosphorylation at ERK1/2 target sites can modulate SOCE by altering STIM1 binding to SOCs, because a significant decrease in FRET efficiency was observed between alanine substitution mutants of STIM1–GFP and ORAI1–CFP.

Hydrogen sulfide (H₂S) concentration can be maintained in cell cultures within the range reported for rat brain by repetitive pulses of sodium hydrogen sulfide. Less than 2 h exposure to H₂S concentrations within 50 and 120 μM (i.e., within the upper segment of the reported physiological range of H₂S in rat brain), produces a large shift of the intracellular calcium homeostasis in cerebellar granule neurons (CGN) in culture, leading to a large and sustained increase of cytosolic calcium concentration. Only 1 h exposure to H₂S concentrations within 100 and 300 μM raises intracellular calcium to the neurotoxic range, with nearly 50% cell death after 2 h. L-type Ca²⁺ channels antagonists nimodipine and nifedipine block both the H₂S-induced rise of cytosolic calcium and cell death. The N-methyl-D-aspartate receptor antagonists (+)-MK-801 and DL-2-amino-5-phosphonovaleric acid afforded a nearly complete protection against H₂S-induced CGN death and largely attenuated the rise of cytosolic calcium. Thus, H₂S-induced rise of cytosolic calcium eventually reaches the neurotoxic cytosolic calcium range, leading to glutamate-induced excitotoxic CGN death. The authors conclude that H₂S is a major modulator of calcium homeostasis in neurons as it induces activation of Ca²⁺ entry through L-type Ca²⁺ channels, and thereby of neuronal activity.