S100A6 amyloid fibril formation is calcium-modulated and enhances superoxide dismutase-1 (SOD1) aggregation

Abstract

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.

FIGURE 1.

Amyloidogenic propensity analysis of S100A6. The sequence-dependent amyloidogenic propensity at neutral pH was computed by the Zyggregator and Waltz algorithms. Zyggregator was used to retrieve intrinsic fibril formation propensity (Z_agg) and protofibril/oligomer formation propensity scores (Z_tox). Significant scores for amyloidogenesis are above 1. Waltz reports on fibrillization propensity. Horizontal gray bars represent significant Waltz scores (∼98%). The top scheme represents S100A6 topology.

FIGURE 2.

S100A6 forms amyloid fibrils. A, formation of S100A6 amyloid at pH 2.5 and 57 °C (3 mg/ml) was monitored by the ThT fluorescence assay. Lysozyme (10 mg/ml) is a positive control, owing to the well characterized amyloidogenesis at the same conditions. The plot represents the normalized variation of the ThT emission in respect to the end point. The solid lines guide the eye as a clear plateau is not defined. B, after 28 days of incubation at 37 °C, TEM analysis revealed a polymorphic fibril structure encompassing 4–5 nm wide fibrils (black arrows), 15 nm wide fibrils (black arrowhead), short thick fibrils composed by the association of several 4–5 nm wide fibrils (white arrows) and several other thicker fibrils. C, magnification of panel B (white rectangle), highlighting the association of 15 nm of wide structures (black arrows) into a larger fibril.

FIGURE 3.

Calcium modulates structural changes at early phases of fibril formation The kinetics of amyloid formation by S100A6 (3 mg/ml) at pH 2. 5 and 57 °C was determined by DLS (A) and the ThT fluorescence assay (B). In both cases, amyloidogenesis occurred without a lag phase. Light scattering aggregates were formed irrespective of the presence of Ca²⁺ (A). In the ThT assay (B), aggregation kinetics was equivalent for S100A6 in the apo form (□) or in the presence of Zn²⁺ (2.9 mm). Ca²⁺ (2.9 mm, ○) completely inhibited amyloidogenesis in this time scale. However, Ca²⁺ chelation by excess EDTA (●, EDTA addition indicated by arrow) restored apo-like amyloidogenesis. The plot represents the normalized variation of the ThT emission in respect to end points. The solid lines are used to guide the eye. C, amide I FT-IR absorption spectra of apo (- - -) and Ca²⁺-S100A6 (——) after 2 h incubation at pH 2.5 and 57 °C. D, difference spectrum derived from C (Ca²⁺-apo). Incubation in the presence of Ca²⁺ leads to the accumulation of anti-parallel β-sheets (contributions at 1625 cm⁻¹ and 1690 cm⁻¹) and the loss of α-helices (1650 cm⁻¹), indicating structurally distinct conformations in the two preparations.

FIGURE 4.

Amyloid fibril formation by S100A6 under physiological conditions. A, amyloid formation kinetics for apo (●) and Ca²⁺-S100A6 (■) at pH 7, 37 °C, 3 mg/ml and 1000 rpm, as derived from the ThT fluorescence assay. Under these conditions, Ca²⁺ partially inhibits amyloid formation. TEM analysis of apo S100A6 at 28 (B) and 65 days incubation (C). TEM analysis of Ca²⁺-S100A6 at 28 (D) and 65 days incubation (E). At lower incubation times large amorphous aggregates are the prevalent structures, which partially assemble into long fibrils in apo but not Ca²⁺-S100A6. Scale bars: 200 nm.

FIGURE 5.

Morphology of S100A6 amyloid fibrils. Upon prolonged incubation of S100A6 (3 mg/ml) at pH 7, 37 °C and 1000 rpm, the amyloid population became homogenous. A, after incubating S100A6 for 84 days 7–8 nm wide fibrils were observed, which formed bundles when further incubated until 116 days (B). Scale bars: 100 nm.

FIGURE 6.

Cytotoxicity of native and amyloid forms of S100A6. SHSY-5Y neuroblastoma cells were incubated with apo or Ca²⁺-S100A6 (5 μm) in distinct conformations raised at pH 2.5: native, early oligomer (0.5 h at 57 °C), late oligomer (1 h at 57 °C) or mature aggregate/amyloid (1 week at 57 °C) in the apo or Ca²⁺-bound states (Fig. 3). Cell viability was assessed after 48 h by the WST-1 reduction assay. Unperturbed cell controls were used to calibrate the measurements. Error bars represent the standard deviation (n = 3).*: p < 0.05. Ca²⁺ completely reverts the toxicity of intermediate S100A6 amyloid species.

FIGURE 7.

Native S100A6 nucleates SOD1 fibrillization. The ThT fluorescence assay was used to monitor SOD1 aggregation (50 μm SOD1 at pH 7, 37 °C and under 600 rpm orbital agitation) in the absence (○) and presence of 2% native apo S100A6 (□). Both seeded and un-seeded SOD1 aggregated according to a sigmoidal kinetics with similar slope but the lag phase was shortened in the presence of S100A6 (from 45 h to 31 h). In this assay, S100A6 has a negligible contribution to the ThT fluorescence signal, as judged from the baseline fluorescence signal when SOD1 was omitted (♢).