There are four isoforms of the ␣ subunit (␣1-4) and three isoforms of the ␤ subunit (␤1-3) of Na,K-ATPase, with distinct tissue-specific distribution and physiological functions. ␣2 is thought to play a key role in cardiac and smooth muscle contraction and be an important target of cardiac glycosides. An ␣2-selective cardiac glycoside could provide important insights into physiological and pharmacological properties of ␣2. The isoform selectivity of a large number of cardiac glycosides has been assessed utilizing ␣1␤1, ␣2␤1, and ␣3␤1 isoforms of human Na,K-ATPase expressed in Pichia pastoris and the purified detergent-soluble isoform proteins. Binding affinities of the digitalis glycosides, digoxin, ␤-methyl digoxin, and digitoxin show moderate but highly significant selectivity (up to 4-fold) for ␣2/␣3 over ␣1 (K D ␣1 > ␣2 ‫؍‬ ␣3). By contrast, ouabain shows moderate selectivity (≈2.5-fold) for ␣1 over ␣2 (K D ␣1 < ␣3 < ␣2). Binding affinities for the three isoforms of digoxigenin, digitoxigenin, and all other aglycones tested are indistinguishable (K D ␣1 ‫؍‬ ␣3 ‫؍‬ ␣2), showing that the sugar determines isoform selectivity. Selectivity patterns for inhibition of Na,K-ATPase activity of the purified isoform proteins are consistent with binding selectivities, modified somewhat by different affinities of K ؉ ions for antagonizing cardiac glycoside binding on the three isoforms. The mechanistic insight on the role of the sugars is strongly supported by a recent structure of Na,K-ATPase with bound ouabain, which implies that aglycones of cardiac glycosides cannot discriminate between isoforms. In conclusion, several digitalis glycosides, but not ouabain, are moderately ␣2-selective. This supports a major role of ␣2 in cardiac contraction and cardiotonic effects of digitalis glycosides.

Deg1 is a Ser protease peripherally attached to the lumenal side of the thylakoid membrane. Its physiological function is unknown, but its localization makes it a suitable candidate for participation in photoinhibition repair by degradation of the photosystem II reaction center protein D1. We transformed Arabidopsis thaliana with an RNA interference construct and obtained plants with reduced levels of Deg1. These plants were smaller than wild-type plants, flowered earlier, were more sensitive to photoinhibition, and accumulated more of the D1 protein, probably in an inactive form. Two C-terminal degradation products of the D1 protein, of 16 and 5.2 kD, accumulated at lower levels compared with the wild type. Moreover, addition of recombinant Deg1 to inside-out thylakoid membranes isolated from the mutant could induce the formation of the 5.2-kD D1 C-terminal fragment, whereas the unrelated proteases trypsin and thermolysin could not. Immunoblot analysis revealed that mutants containing less Deg1 also contain less FtsH protease, and FtsH mutants contain less Deg1. These results suggest that Deg1 cooperates with the stroma-exposed proteases FtsH and Deg2 in degrading D1 protein during repair from photoinhibition by cleaving lumen-exposed regions of the protein. In addition, they suggest that accumulation of Deg1 and FtsH proteases may be coordinated.

A hallmark of the ERK1/2 functioning is their nuclear translocation, which is mainly required for the induction of proliferation. Activated ERK1/2 molecules that remain in the cytoplasm initiate other activities, including immediate feedback loops. Prevention of the nuclear translocation should therefore inhibit proliferation, without affecting cytoplasm-induced cellular processes. Here we present an NTS-derived myristoylated phosphomimetic peptide, which blocks the interaction of importin7 and ERK1/2, and consequently the nuclear translocation of the latter. In culture, the peptide induces apoptosis of melanoma cells inhibits the viability of other cancer cells, but has no effect on non-transformed, immortalized cells. It even inhibits the viability of PLX4032-and U0126-resistant melanoma cells. In xenograft models, the peptide inhibits several cancers, and acts much better than PLX4032 in preventing melanoma recurrence. This study provides a proof of concept for using the nuclear translocation of ERK1/2 as a drug target for the combat of various ERK1/2-related cancers.

Background: Na,K-ATPase is stabilized by phosphatidylserine/cholesterol and is stimulated by neutral phospholipids. Results: Three specific lipid-Na,K-ATPase interactions are detectable that either (a) stabilize the protein or (b) stimulate or (c) inhibit Na,K-ATPase activity, with distinct kinetic mechanisms. Conclusion: There are separate binding sites for phosphatidylserine/cholesterol (stabilizing), polyunsaturated phosphatidylethanolamine (stimulatory), and sphingomyelin/cholesterol (inhibitory). Significance: In physiological conditions, specifically bound lipids may regulate Na,K-ATPase activity.

The Arabidopsis genome contains 14 genes encoding the serine protease DegP. Products of four of these genes are located in the chloroplast: three in the thylakoid lumen and one on the stromal side of the membrane. We expressed the gene encoding DegP1 as a His-tagged fusion protein in Escherichia coli, purified the protein by affinity chromatography, and characterized it biochemically. Size-exclusion chromatography suggested that DegP1 eluted from the column as a mixture of monomers and hexamers. Proteolytic activity was characterized using ␤-casein as a model substrate. DegP1 demonstrated concentration-dependent activity, a pH optimum of 6.0 and increasing activity at elevated temperatures. DegP1 was capable of degrading two lumenal proteins, plastocyanin and OE33, suggesting a role as a general-purpose protease in the thylakoid lumen. The results of this work are discussed in the context of the recent elucidation of the structure of the E. coli homolog and the possible physiological role of the protease in the chloroplast lumen.