Memo1 binds reduced copper ions, interacts with copper chaperone Atox1, and protects against copper-mediated redox activity in vitro.

The protein mediator of ERBB2-driven cell motility 1 (Memo1) is connected to many signaling pathways that play key roles in cancer. Memo1 was recently postulated to bind copper (Cu) ions and thereby promote the generation of reactive oxygen species (ROS) in cancer cells. Since the concentration of Cu as well as ROS are increased in cancer cells, both can be toxic if not well regulated. Here, we investigated the Cu-binding capacity of Memo1 using an array of biophysical methods at reducing as well as oxidizing conditions in vitro. We find that Memo1 coordinates two reduced Cu (Cu(I)) ions per protein, and, by doing so, the metal ions are shielded from ROS generation. In support of biological relevance, we show that the cytoplasmic Cu chaperone Atox1, which delivers Cu(I) in the secretory pathway, can interact with and exchange Cu(I) with Memo1 in vitro and that the two proteins exhibit spatial proximity in breast cancer cells. Thus, Memo1 appears to act as a Cu(I) chelator (perhaps shuttling the metal ion to Atox1 and the secretory path) that protects cells from Cu-mediated toxicity, such as uncontrolled formation of ROS. This Memo1 functionality may be a safety mechanism to cope with the increased demand of Cu ions in cancer cells.

Interaction between Copper Chaperone Atox1 and Parkinson's Disease Protein α-Synuclein Includes Metal-Binding Sites and Occurs in Living Cells.

Alterations in copper ion homeostasis appear coupled to neurodegenerative disorders, but mechanisms are unknown. The cytoplasmic copper chaperone Atox1 was recently found to inhibit amyloid formation in vitro of α-synuclein, the amyloidogenic protein in Parkinson's disease. As α-synuclein may have copper-dependent functions, and free copper ions promote α-synuclein amyloid formation, it is important to characterize the Atox1 interaction with α-synuclein on a molecular level. Here we applied solution-state nuclear magnetic resonance spectroscopy, with isotopically labeled α-synuclein and Atox1, to define interaction regions in both proteins. The α-synuclein interaction interface includes the whole N-terminal part up to Gln24; in Atox1, residues around the copper-binding cysteines (positions 11-16) are mostly perturbed, but additional effects are also found for residues elsewhere in both proteins. Because α-synuclein is N-terminally acetylated in vivo, we established that Atox1 also inhibits amyloid formation of this variant in vitro, and proximity ligation in human cell lines demonstrated α-synuclein-Atox1 interactions in situ. Thus, this interaction may provide the direct link between copper homeostasis and amyloid formation in vivo.

Sorption and speciation of selenium in boreal forest soil.

Sorption and speciation of selenium in the initial chemical forms of selenite and selenate were investigated in batch experiments on humus and mineral soil samples taken from a 4-m deep boreal forest soil excavator pit on Olkiluoto Island, on the Baltic Sea coast in southwestern Finland. The HPLC-ICP-MS technique was used to monitor any possible transformations in the selenium liquid phase speciation and to determine the concentrations of selenite and selenate in the samples for calculation of the mass distribution coefficient, Kd, for both species. Both SeO32- and SeO42- proved to be resistant forms in the prevailing soil conditions and no changes in selenium liquid phase speciation were seen in the sorption experiments in spite of variations in the initial selenium species, incubation time or conditions, pH, temperature or microbial activity. Selenite sorption on the mineral soil increased with time in aerobic conditions whilst the opposite trend was seen for the anaerobic soil samples. Selenite retention correlated with the contents of organic matter and weakly crystalline oxides of aluminum and iron, solution pH and the specific surface area. Selenate exhibited poorer sorption on soil than selenite and on average the Kd values were 27-times lower. Mineral soil was more efficient in retaining selenite and selenate than humus, implicating the possible importance of weakly crystalline aluminum and iron oxides for the retention of oxyanions in Olkiluoto soil. Sterilization of the soil samples decreased the retention of selenite, thus implying some involvement of soil microbes in the sorption processes or a change in sample composition, but it produced no effect for selenate. There was no sorption of selenite by quartz, potassium feldspar, hornblende or muscovite. Biotite showed the best retentive properties for selenite in the model soil solution at about pH 8, followed by hematite, plagioclase and chlorite. The Kd values for these minerals were 18, 14, 8 and 7 L/kg, respectively. It is proposed that selenite sorption is affected by the structural Fe(II) in biotite, which is capable of inducing the reduction of SeO32- to Se(0). Selenite probably forms a surface complex with Fe(III) atoms on the surface of hematite, thus explaining its retention on this mineral. None of the minerals retained selenate to any extent.

Shelf-life of ɛ-lysyl-3-(trimethylstannyl)benzamide immunoconjugates, precursors for 211At labeling of antibodies.

Astatine-211 is possibly the most promising radionuclide for targeted α-particle therapy when it comes to the treatment of occult disseminated cancer. Preclinical research has proven effective, and patient studies have been initiated based on these results. However, a lack of production capacity and the complex radiochemistry of (211)At are major obstacles for research and prospective clinical applications. In the present study, astatination of immunoconjugates, already prepared well in advance before radiolabeling, was performed to investigate the possibility of formulating a kit-like reagent for the production of (211)At radiopharmaceuticals. The shelf-life of ɛ-lysyl-3-(trimethylstannyl)benzamide immunoconjugates was evaluated, that is, the effect of different storage times on the quality of the immunoconjugates. The quality being referred to is the capacity to maintain a good radiochemical yield and good cell-binding property after labeling with (211)At. The stability of the conjugates was found to be pH dependent with high stability at pH≥7 and less stability at pH≤5.5. The immunoconjugates (based on trastuzumab) could be kept for more than 3 months in a phosphate buffered saline solution (pH 7.4) at 4°C before labeling, without compromising the quality of the labeled product. The conjugates are also unaffected by storage at -20°C. Conjugates with a good shelf-life compatible with distant shipping as well as improved radiochemistry are important steps to facilitate further clinical progress with (211)At.