Protein complexes from mouse and chick brain that interact with phospho-KXGS motif tau/microtubule associated protein antibody.

Mouse monoclonal 12E8 antibody, which recognises conserved serine phosphorylated KXGS motifs in the microtubule binding domains of tau/tau-like microtubule associated proteins (MAPs), shows elevated binding in brain during normal embryonic development (mammals and birds) and at the early stages of human Alzheimer's disease (AD). It also labels ADF/cofilin-actin rods that form in neurites during exposure to stressors. We aimed to identify direct and indirect 12E8 binding proteins in postnatal mouse brain and embryonic chick brain by immunoprecipitation (IP), mass spectrometry and immunofluorescence. Tau and/or MAP2 were major direct 12E8-binding proteins detected in all IPs, and actin and/or tubulin were co-immunoprecipitated in most samples. Additional proteins were different in mouse versus chick brain IP. In mouse brain IPs, FSD1l and intermediate filament proteins - vimentin, α-internexin, neurofilament polypeptides - were prominent. Immunofluorescence and immunoblot using recombinant intermediate filament subunits, suggests an indirect interaction of these proteins with the 12E8 antibody. In chick brain IPs, subunits of eukaryotic translation initiation factor 3 (EIF3) were found, but no direct interaction between 12E8 and recombinant Eif3e protein was detected. Fluorescence microscopy in primary cultured chick neurons showed evidence of co-localisation of Eif3e and tubulin labelling, consistent with previous data demonstrating cytoskeletal organisation of the translation apparatus. Neither total tau or MAP2 immunolabelling accumulated at ADF/cofilin-actin rods generated in primary cultured chick neurons, and we were unable to narrow down the major antigen recognised by 12E8 antibody on ADF/cofilin-actin rods.

Arabidopsis Iron Superoxide Dismutase FSD1 Protects Against Methyl Viologen-Induced Oxidative Stress in a Copper-Dependent Manner.

Iron superoxide dismutase 1 (FSD1) was recently characterized as a plastidial, cytoplasmic, and nuclear enzyme with osmoprotective and antioxidant functions. However, the current knowledge on its role in oxidative stress tolerance is ambiguous. Here, we characterized the role of FSD1 in response to methyl viologen (MV)-induced oxidative stress in Arabidopsis thaliana. In accordance with the known regulation of FSD1 expression, abundance, and activity, the findings demonstrated that the antioxidant function of FSD1 depends on the availability of Cu2+ in growth media. Arabidopsis fsd1 mutants showed lower capacity to decompose superoxide at low Cu2+ concentrations in the medium. Prolonged exposure to MV led to reduced ascorbate levels and higher protein carbonylation in fsd1 mutants and transgenic plants lacking a plastid FSD1 pool as compared to the wild type. MV induced a rapid increase in FSD1 activity, followed by a decrease after 4 h long exposure. Genetic disruption of FSD1 negatively affected the hydrogen peroxide-decomposing ascorbate peroxidase in fsd1 mutants. Chloroplastic localization of FSD1 is crucial to maintain redox homeostasis. Proteomic analysis showed that the sensitivity of fsd1 mutants to MV coincided with decreased abundances of ferredoxin and photosystem II light-harvesting complex proteins. These mutants have higher levels of chloroplastic proteases indicating an altered protein turnover in chloroplasts. Moreover, FSD1 disruption affects the abundance of proteins involved in the defense response. Collectively, the study provides evidence for the conditional antioxidative function of FSD1 and its possible role in signaling.

In vivo light-sheet microscopy resolves localisation patterns of FSD1, a superoxide dismutase with function in root development and osmoprotection.

Superoxide dismutases (SODs) are enzymes detoxifying superoxide to hydrogen peroxide while temporal developmental expression and subcellular localisation are linked to their functions. Therefore, we aimed here to reveal in vivo developmental expression, subcellular, tissue- and organ-specific localisation of iron superoxide dismutase 1 (FSD1) in Arabidopsis using light-sheet and Airyscan confocal microscopy. FSD1-GFP temporarily accumulated at the site of endosperm rupture during seed germination. In emerged roots, it showed the highest abundance in cells of the lateral root cap, columella, and endodermis/cortex initials. The largest subcellular pool of FSD1-GFP was localised in the plastid stroma, while it was also located in the nuclei and cytosol. The majority of the nuclear FSD1-GFP is immobile as revealed by fluorescence recovery after photobleaching. We found that fsd1 knockout mutants exhibit reduced lateral root number and this phenotype was reverted by genetic complementation. Mutant analysis also revealed a requirement for FSD1 in seed germination during salt stress. Salt stress tolerance was coupled with the accumulation of FSD1-GFP in Hechtian strands and superoxide removal. It is likely that the plastidic pool is required for acquiring oxidative stress tolerance in Arabidopsis. This study suggests new developmental and osmoprotective functions of SODs in plants.

Stability and folding behavior analysis of zinc-finger using simple models.

Zinc-fingers play crucial roles in regulating gene expression and mediating protein-protein interactions. In this article, two different proteins (Sp1f2 and FSD-1) are investigated using the Gaussian network model and anisotropy elastic network model. By using these simple coarse-grained methods, we analyze the structural stabilization and establish the unfolding pathway of the two different proteins, in good agreement with related experimental and molecular dynamics simulation data. From the analysis, it is also found that the folding process of the zinc-finger motif is predominated by several factors. Both the zinc ion and C-terminal loop affect the folding pathway of the zinc-finger motif. Knowledge about the stability and folding behavior of zinc-fingers may help in understanding the folding mechanisms of the zinc-finger motif and in designing new zinc-fingers. Meanwhile, these simple coarse-grained analyses can be used as a general and quick method for mechanistic studies of metalloproteins.