Highly sensitive biological assay for determining the photoprotective efficacy of sunscreen.

The protective effect of sunscreens has been extensively evaluated in vivo as a measure of erythema induced in human skin and is expressed as Sun Protection Factor (SPF). In vitro alternatives that use human cells might overcome the limitations of testing on human beings. Here is proposed a broad and accurate in vitro approach for evaluating the efficacy of commercial sunscreens even under environmental conditions. This Cell dosimeter allowed the determination of Sun Protection Factor for DNA (DNA-SPF), using specific DNA repair enzymes and antibodies, and Sun Protection Factor for Lethal Damage (LD-SPF), by measuring cell viability and apoptosis induced after the irradiation of human cells. The use of xeroderma pigmentosum (XP) cells, which are deficient in DNA repair, rendered this assay more sensitive. The results revealed significant protection against the effects elicited by UVB radiation; however, there was no efficient protection from DNA lesions and cell death induced by UVA radiation or natural sunlight. This work demonstrates the environmental application of this biodosimeter for measuring UV-induced biological damage to human cells and supports the need for better evaluation of the UVA protection efficacy conferred by commercial sunscreens, in terms of induction of DNA lesions and cell death.

Both XPA and DNA polymerase eta are necessary for the repair of doxorubicin-induced DNA lesions.

Doxorubicin (DOX) is an important tumor chemotherapeutic agent, acting mainly by genotoxic action. This work focus on cell processes that help cell survival, after DOX-induced DNA damage. In fact, cells deficient for XPA or DNA polymerase eta (pol eta, XPV) proteins (involved in distinct DNA repair pathways) are highly DOX-sensitive. Moreover, LY294002, an inhibitor of PIKK kinases, showed a synergistic killing effect in cells deficient in these proteins, with a strong induction of G2/M cell cycle arrest. Taken together, these results indicate that XPA and pol eta proteins participate in cell resistance to DOX-treatment, and kinase inhibitors can selectively enhance its killing effects, probably reducing the cell ability to recover from breaks induced in DNA.

Gcn1 and actin binding to Yih1: implications for activation of the eIF2 kinase GCN2.

Yeast Yih1 protein and its mammalian ortholog IMPACT, abundant in neurons, are inhibitors of Gcn2, a kinase involved in amino acid homeostasis, stress response, and memory formation. Like Gcn2, Yih1/IMPACT harbors an N-terminal RWD domain that mediates binding to the Gcn2 activator Gcn1. Yih1 competes with Gcn2 for Gcn1 binding, thus inhibiting Gcn2. Yih1 also binds G-actin. Here, we show that Yih1-actin interaction is independent of Gcn1 and that Yih1-Gcn1 binding does not require actin. The Yih1 RWD (residues 1-132) was sufficient for Gcn2 inhibition and Gcn1 binding, but not for actin binding, showing that actin binding is dispensable for inhibiting Gcn2. Actin binding required Yih1 residues 68-258, encompassing part of the RWD and the C-terminal "ancient domain"; however, residues Asp-102 and Glu-106 in helix3 of the RWD were essential for Gcn1 binding and Gcn2 inhibition but dispensable for actin binding. Thus, the Gcn1- and actin-binding sites overlap in the RWD but have distinct binding determinants. Unexpectedly, Yih1 segment 68-258 was defective for inhibiting Gcn2 even though it binds Gcn1 at higher levels than does full-length Yih1. This and other results suggest that Yih1 binds with different requirements to distinct populations of Gcn1 molecules, and its ability to disrupt Gcn1-Gcn2 complexes is dependent on a complete RWD and hindered by actin binding. Modeling of the ancient domain on the bacterial protein YigZ showed peculiarities to the eukaryotic and prokaryotic lineages, suggesting binding sites for conserved cellular components. Our results support a role for Yih1 in a cross-talk between the cytoskeleton and translation.

Novel membrane-bound eIF2alpha kinase in the flagellar pocket of Trypanosoma brucei.

Translational control mediated by phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIF2alpha) is central to stress-induced programs of gene expression. Trypanosomatids, important human pathogens, display differentiation processes elicited by contact with the distinct physiological milieu found in their insect vectors and mammalian hosts, likely representing stress situations. Trypanosoma brucei, the agent of African trypanosomiasis, encodes three potential eIF2alpha kinases (TbeIF2K1 to -K3). We show here that TbeIF2K2 is a transmembrane glycoprotein expressed both in procyclic and in bloodstream forms. The catalytic domain of TbeIF2K2 phosphorylates yeast and mammalian eIF2alpha at Ser51. It also phosphorylates the highly unusual form of eIF2alpha found in trypanosomatids specifically at residue Thr169 that corresponds to Ser51 in other eukaryotes. T. brucei eIF2alpha, however, is not a substrate for GCN2 or PKR in vitro. The putative regulatory domain of TbeIF2K2 does not share any sequence similarity with known eIF2alpha kinases. In both procyclic and bloodstream forms TbeIF2K2 is mainly localized in the membrane of the flagellar pocket, an organelle that is the exclusive site of exo- and endocytosis in these parasites. It can also be detected in endocytic compartments but not in lysosomes, suggesting that it is recycled between endosomes and the flagellar pocket. TbeIF2K2 location suggests a relevance in sensing protein or nutrient transport in T. brucei, an organism that relies heavily on posttranscriptional regulatory mechanisms to control gene expression in different environmental conditions. This is the first membrane-associated eIF2alpha kinase described in unicellular eukaryotes.