Mutagenesis studies on the phototropin-related protein YtvA from Bacillus subtilis have revealed the role of selected structural elements in interdomain communication. The LOV (light, oxygen, voltage) domain of YtvA undergoes light-driven reactions similar to that of phot-LOV, with reversible formation of a covalent flavin-cysteine adduct. The mutated proteins Ytva-E105L and YtvA-E56Q have been studied by UV fluorescence and circular dichroism (CD) spectroscopy. E105 (L in phototropin) is located at the solvent-exposed surface of the LOV domain central beta-sheet, demonstrated to participate in interdomain interaction in phototropin. CD data show that YtvA-E105L has a lower alpha-helix content in the dark and undergoes larger light-driven conformational changes than YtvA-WT. The E56Q mutation breaks the E56-K97 salt bridge, a structural element highly conserved within the LOV series. In YtvA-E56Q the CD spectrum is the same as in YtvA-WT, although the conserved W103 becomes more exposed to the solvent and the dark-recovery kinetics is slower. These results indicate that the E56-K97 salt bridge stabilizes locally the protein structure and participates in the regulation of the photocycle but has negligible effects on the overall structure. The E105L mutation, instead, highlights the involvement of the central beta-sheet in the light-driven conformational changes in LOV proteins.

No abstract

Zinc ions are essential, but at elevated concentrations, they also have toxic effects on mammalian cells. Zinc plays a crucial role in cell proliferation and differentiation and it even protects cells against apoptosis caused by various reagents. On the other hand, zinc at high concentrations causes cell death that was characterized as apoptotic by internucleosomal DNA fragmentation, formation of apoptotic bodies, and breakdown of the mitochondrial membrane potential. In the present work, a clone of rat C6 glioma cells that was resistant to toxic effects of ZnCl2 up to 250 microM was employed to study the effect of the ionophore A23187 on zinc-induced apoptosis. Neither 150 microM Zn2+ nor 100 nM A23187 alone caused apoptosis as measured by internucleosomal DNA fragmentation. However, combined exposure of C6 cells to 100 nM A23187 and 150 microM Zn2+ for 48 h was effective in inducing apoptosis. Because the so-called calcium ionophore A23187 is not specific for Ca2+ ions but also transports Zn2+ with high selectivity over Ca2+, we investigated whether this substance promoted the uptake of Zn2+ ions into C6 cells. Employing the zinc-specific fluorescence probe Zinquin, we observed that the very low concentration of 1.9 nM A23187 significantly and rapidly raised the intracellular mobile Zn2+ content. Analysis by atomic absorption spectroscopy revealed that incubation with 1.9 nM A23187 caused a doubling of the total intracellular zinc level within 60 min. We conclude that the apoptosis evoked by the combined action of Zn2+ and A23187 was the result of enhanced Zn2+ influx evoked by the ionophore, resulting in higher intracellular zinc levels.

No abstract

Zinc homeostasis in mammalian cells is precisely regulated by cellular signal transduction mechanisms. The main result of this study is the finding that modulators of phospholipase C (PLC) activity affect cellular zinc export. Two different PLC inhibitors caused an increase of the total cellular zinc level whereas two different PLC activators caused a decrease. Furthermore, both the inhibition of cyclic nucleotide phosphodiesterases as well as the administration of 8-bromo-cAMP evoked a drop in the intracellular zinc level, indicating the involvement of cAMP in the control of cellular zinc export. It is concluded that the activity of PLC controls cellular zinc transport and that the effect of elevated zinc concentrations on PLC activity might be mediated by cAMP. However, modulation of other major signaling enzymes did not affect the cellular zinc homeostasis. These include activation and inhibition of guanylate cyclase, activation of protein kinase G, activation of protein kinase A, and activation or inhibition of protein kinase C. Furthermore there was no evidence for the existence of a zinc-sensing receptor in C6 glioma cells, which would stimulate PLC activity and evoke a mobilization of intracellular free-calcium levels.