S100A6 amyloid fibril formation is calcium-modulated and enhances superoxide dismutase-1 (SOD1) aggregation.
S100A6 is a small EF-hand calcium- and zinc-binding protein involved in the regulation of cell proliferation and cytoskeletal dynamics. It is overexpressed in neurodegenerative disorders and a proposed marker for Amyotrophic Lateral Sclerosis (ALS). Following recent reports of amyloid formation by S100 proteins, we investigated the aggregation properties of S100A6. Computational analysis using aggregation predictors Waltz and Zyggregator revealed increased propensity within S100A6 helices H(I) and H(IV). Subsequent analysis of Thioflavin-T binding kinetics under acidic conditions elicited a very fast process with no lag phase and extensive formation of aggregates and stacked fibrils as observed by electron microscopy. Ca(2+) exerted an inhibitory effect on the aggregation kinetics, which could be reverted upon chelation. An FT-IR investigation of the early conformational changes occurring under these conditions showed that Ca(2+) promotes anti-parallel β-sheet conformations that repress fibrillation. At pH 7, Ca(2+) rendered the fibril formation kinetics slower: time-resolved imaging showed that fibril formation is highly suppressed, with aggregates forming instead. In the absence of metals an extensive network of fibrils is formed. S100A6 oligomers, but not fibrils, were found to be cytotoxic, decreasing cell viability by up to 40%. This effect was not observed when the aggregates were formed in the presence of Ca(2+). Interestingly, native S1006 seeds SOD1 aggregation, shortening its nucleation process. This suggests a cross-talk between these two proteins involved in ALS. Overall, these results put forward novel roles for S100 proteins, whose metal-modulated aggregation propensity may be a key aspect in their physiology and function.
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, 2781-901 Oeiras, Portugal.
Hugo M
HM
Botelho
Sónia S
SS
Leal
Isabel
I
Cardoso
Kiran
K
Yanamandra
Ludmilla A
LA
Morozova-Roche
Günter
G
Fritz
Cláudio M
CM
Gomes
D000682:Amyloid / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D000690:Amyotrophic Lateral Sclerosis / Q000378:metabolism; D052160:Benzothiazoles; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000378:metabolism; D045744:Cell Line, Tumor; D006801:Humans; D007700:Kinetics; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D000075383:S100 Calcium Binding Protein A6; D009418:S100 Proteins / Q000737:chemistry* / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000378:metabolism; D000072105:Superoxide Dismutase-1; D013844:Thiazoles / Q000737:chemistry*
D000682:Amyloid; D052160:Benzothiazoles; D018797:Cell Cycle Proteins; D000075383:S100 Calcium Binding Protein A6; D009418:S100 Proteins; C000606290:SOD1 protein, human; D013844:Thiazoles; C055351:S100A6 protein, human; C009462:thioflavin T; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; D002118:Calcium