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- Ahlgren, Eva Christina, et al.
(författare)
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Iron-induced oligomerization of human FXN81-210 and bacterial CyaY frataxin and the effect of iron chelators
- 2017
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 12:12
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Tidskriftsartikel (refereegranskat)abstract
- Patients suffering from the progressive neurodegenerative disease Friedreich’s ataxia have reduced expression levels of the protein frataxin. Three major isoforms of human frataxin have been identified, FXN42-210, FXN56-210 and FXN81-210, of which FXN81-210 is considered to be the mature form. Both long forms, FXN42-210 and FXN56-210, have been shown to spontaneously form oligomeric particles stabilized by the extended N-terminal sequence. The short variant FXN81-210, on other hand, has only been observed in the monomeric state. However, a highly homologous E. coli frataxin CyaY, which also lacks an N-terminal extension, has been shown to oligomerize in the presence of iron. To explore the mechanisms of stabilization of short variant frataxin oligomers we compare here the effect of iron on the oligomerization of CyaY and FXN81-210. Using dynamic light scattering, small-angle X-ray scattering, electron microscopy (EM) and cross linking mass spectrometry (MS), we show that at aerobic conditions in the presence of iron both FXN81-210 and CyaY form oligomers. However, while CyaY oligomers are stable over time, FXN81-210 oligomers are unstable and dissociate into monomers after about 24 h. EM and MS studies suggest that within the oligomers FXN81-210 and CyaY monomers are packed in a head-to-tail fashion in ring-shaped structures with potential iron-binding sites located at the interface between monomers. The higher stability of CyaY oligomers can be explained by a higher number of acidic residues at the interface between monomers, which may result in a more stable iron binding. We also show that CyaY oligomers may be dissociated by ferric iron chelators deferiprone and DFO, as well as by the ferrous iron chelator BIPY. Surprisingly, deferiprone and DFO stimulate FXN81-210 oligomerization, while BIPY does not show any effect on oligomerization in this case. The results suggest that FXN81-210 oligomerization is primarily driven by ferric iron, while both ferric and ferrous iron participate in CyaY oligomer stabilization. Analysis of the amino acid sequences of bacterial and eukaryotic frataxins suggests that variations in the position of the acidic residues in helix 1, β-strand 1 and the loop between them may control the mode of frataxin oligomerization.
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| 2. |
- Fekry, Mostafa, et al.
(författare)
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SAXS and stability studies of iron-induced oligomers of bacterial frataxin CyaY
- 2017
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 12:9, s. 0184961-0184961
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Tidskriftsartikel (refereegranskat)abstract
- Frataxin is a highly conserved protein found in both prokaryotes and eukaryotes. It is involved in several central functions in cells, which include iron delivery to biochemical processes, such as heme synthesis, assembly of iron-sulfur clusters (ISC), storage of surplus iron in conditions of iron overload, and repair of ISC in aconitase. Frataxin from different organisms has been shown to undergo iron-dependent oligomerization. At least two different classes of oligomers, with different modes of oligomer packing and stabilization, have been identified. Here, we continue our efforts to explore the factors that control the oligomerization of frataxin from different organisms, and focus on E. coli frataxin CyaY. Using small-angle X-ray scattering (SAXS), we show that higher iron-to-protein ratios lead to larger oligomeric species, and that oligomerization proceeds in a linear fashion as a results of iron oxidation. Native mass spectrometry and online size-exclusion chromatography combined with SAXS show that a dimer is the most common form of CyaY in the presence of iron at atmospheric conditions. Modeling of the dimer using the SAXS data confirms the earlier proposed head-to-tail packing arrangement of monomers. This packing mode brings several conserved acidic residues into close proximity to each other, creating an environment for metal ion binding and possibly even mineralization. Together with negative-stain electron microscopy, the experiments also show that trimers, tetramers, pentamers, and presumably higher-order oligomers may exist in solution. Nano-differential scanning fluorimetry shows that the oligomers have limited stability and may easily dissociate at elevated temperatures. The factors affecting the possible oligomerization mode are discussed.
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