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Characterization of the oligomerization and aggregation of human Serum Amyloid A.

Patke S, Srinivasan S, Maheshwari R, Srivastava SK, Aguilera JJ, Colón W, Kane RS - PLoS ONE (2013)

Bottom Line: We found that hSAA1.1 formed alpha helix-rich, marginally stable oligomers in vitro on refolding and cross-beta-rich aggregates following incubation at 37°C.Strikingly, while hSAA1.1 was not highly amyloidogenic in vitro, the addition of a single N-terminal methionine residue significantly enhanced the fibrillation propensity of hSAA1.1 and modulated its fibrillation pathway.A deeper understanding of the oligomerization and fibrillation pathway of hSAA1.1 may help elucidate its pathological role.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York, USA.

ABSTRACT
The fibrillation of Serum Amyloid A (SAA) - a major acute phase protein - is believed to play a role in the disease Amyloid A (AA) Amyloidosis. To better understand the amyloid formation pathway of SAA, we characterized the oligomerization, misfolding, and aggregation of a disease-associated isoform of human SAA - human SAA1.1 (hSAA1.1) - using techniques ranging from circular dichroism spectroscopy to atomic force microscopy, fluorescence spectroscopy, immunoblot studies, solubility measurements, and seeding experiments. We found that hSAA1.1 formed alpha helix-rich, marginally stable oligomers in vitro on refolding and cross-beta-rich aggregates following incubation at 37°C. Strikingly, while hSAA1.1 was not highly amyloidogenic in vitro, the addition of a single N-terminal methionine residue significantly enhanced the fibrillation propensity of hSAA1.1 and modulated its fibrillation pathway. A deeper understanding of the oligomerization and fibrillation pathway of hSAA1.1 may help elucidate its pathological role.

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Characterization of aggregation of MetSAA1.1 and hSAA1.1 by the ThT Fluorescence assay, Congo red binding assay, far UV CD, and solubility assay.(A) ThT fluorescence intensity profile for MetSAA1.1 (black bars) and hSAA1.1 (red bars); (B) Congo red absorbance spectra for Congo red only (blue solid line); Congo red plus MetSAA1.1 sample (red solid line); MetSAA1.1 difference spectra (red dash line); Congo red plus hSAA1.1 sample (black solid line); hSAA1.1 difference spectra (black dash line); (C) Far UV CD spectra of MetSAA1.1 samples incubated at 37°C for 6 h (red solid line), 24 h (blue solid line), and 72 h (green solid line); (D) Far UV CD spectra of hSAA1.1 samples incubated at 37°C for 6 h (red solid line), 24 h (blue solid line), and 72 h (green solid line); (E) solubility profile for MetSAA1.1 (black solid line) and hSAA1.1 (red solid line). The starting concentration of protein was 20 µM. All assays were performed after the proteins were allowed to aggregate at 37°C.
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pone-0064974-g002: Characterization of aggregation of MetSAA1.1 and hSAA1.1 by the ThT Fluorescence assay, Congo red binding assay, far UV CD, and solubility assay.(A) ThT fluorescence intensity profile for MetSAA1.1 (black bars) and hSAA1.1 (red bars); (B) Congo red absorbance spectra for Congo red only (blue solid line); Congo red plus MetSAA1.1 sample (red solid line); MetSAA1.1 difference spectra (red dash line); Congo red plus hSAA1.1 sample (black solid line); hSAA1.1 difference spectra (black dash line); (C) Far UV CD spectra of MetSAA1.1 samples incubated at 37°C for 6 h (red solid line), 24 h (blue solid line), and 72 h (green solid line); (D) Far UV CD spectra of hSAA1.1 samples incubated at 37°C for 6 h (red solid line), 24 h (blue solid line), and 72 h (green solid line); (E) solubility profile for MetSAA1.1 (black solid line) and hSAA1.1 (red solid line). The starting concentration of protein was 20 µM. All assays were performed after the proteins were allowed to aggregate at 37°C.

Mentions: We induced aggregation of the “native-like” protein oligomers by incubating the proteins (starting concentration 20 µM) at 37°C and monitored the intensity of ThT fluorescence as a function of time. As seen in Figure 2A, MetSAA1.1 aggregation was characterized by a time-dependent increase in the ThT fluorescence intensity suggesting a transformation of the “native-like” oligomers into aggregates rich in cross-beta structure. A measurable “lag time” of ca. 3 h was observed, and the ThT fluorescence intensity reached its maximum value by ca. 24 h, after which there was no significant change in the fluorescence intensity. Next, we performed similar ThT fluorescence experiments using hSAA1.1. hSAA1.1 showed similar ThT binding kinetics, with a time-dependent increase in ThT fluorescence intensity followed by saturation of ThT signal by ca. 24 h (Fig. 2A). Similar ThT fluorescence intensity profiles for both proteins suggested the formation of cross-beta-rich aggregates with similar aggregation kinetics.


Characterization of the oligomerization and aggregation of human Serum Amyloid A.

Patke S, Srinivasan S, Maheshwari R, Srivastava SK, Aguilera JJ, Colón W, Kane RS - PLoS ONE (2013)

Characterization of aggregation of MetSAA1.1 and hSAA1.1 by the ThT Fluorescence assay, Congo red binding assay, far UV CD, and solubility assay.(A) ThT fluorescence intensity profile for MetSAA1.1 (black bars) and hSAA1.1 (red bars); (B) Congo red absorbance spectra for Congo red only (blue solid line); Congo red plus MetSAA1.1 sample (red solid line); MetSAA1.1 difference spectra (red dash line); Congo red plus hSAA1.1 sample (black solid line); hSAA1.1 difference spectra (black dash line); (C) Far UV CD spectra of MetSAA1.1 samples incubated at 37°C for 6 h (red solid line), 24 h (blue solid line), and 72 h (green solid line); (D) Far UV CD spectra of hSAA1.1 samples incubated at 37°C for 6 h (red solid line), 24 h (blue solid line), and 72 h (green solid line); (E) solubility profile for MetSAA1.1 (black solid line) and hSAA1.1 (red solid line). The starting concentration of protein was 20 µM. All assays were performed after the proteins were allowed to aggregate at 37°C.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3672174&req=5

pone-0064974-g002: Characterization of aggregation of MetSAA1.1 and hSAA1.1 by the ThT Fluorescence assay, Congo red binding assay, far UV CD, and solubility assay.(A) ThT fluorescence intensity profile for MetSAA1.1 (black bars) and hSAA1.1 (red bars); (B) Congo red absorbance spectra for Congo red only (blue solid line); Congo red plus MetSAA1.1 sample (red solid line); MetSAA1.1 difference spectra (red dash line); Congo red plus hSAA1.1 sample (black solid line); hSAA1.1 difference spectra (black dash line); (C) Far UV CD spectra of MetSAA1.1 samples incubated at 37°C for 6 h (red solid line), 24 h (blue solid line), and 72 h (green solid line); (D) Far UV CD spectra of hSAA1.1 samples incubated at 37°C for 6 h (red solid line), 24 h (blue solid line), and 72 h (green solid line); (E) solubility profile for MetSAA1.1 (black solid line) and hSAA1.1 (red solid line). The starting concentration of protein was 20 µM. All assays were performed after the proteins were allowed to aggregate at 37°C.
Mentions: We induced aggregation of the “native-like” protein oligomers by incubating the proteins (starting concentration 20 µM) at 37°C and monitored the intensity of ThT fluorescence as a function of time. As seen in Figure 2A, MetSAA1.1 aggregation was characterized by a time-dependent increase in the ThT fluorescence intensity suggesting a transformation of the “native-like” oligomers into aggregates rich in cross-beta structure. A measurable “lag time” of ca. 3 h was observed, and the ThT fluorescence intensity reached its maximum value by ca. 24 h, after which there was no significant change in the fluorescence intensity. Next, we performed similar ThT fluorescence experiments using hSAA1.1. hSAA1.1 showed similar ThT binding kinetics, with a time-dependent increase in ThT fluorescence intensity followed by saturation of ThT signal by ca. 24 h (Fig. 2A). Similar ThT fluorescence intensity profiles for both proteins suggested the formation of cross-beta-rich aggregates with similar aggregation kinetics.

Bottom Line: We found that hSAA1.1 formed alpha helix-rich, marginally stable oligomers in vitro on refolding and cross-beta-rich aggregates following incubation at 37°C.Strikingly, while hSAA1.1 was not highly amyloidogenic in vitro, the addition of a single N-terminal methionine residue significantly enhanced the fibrillation propensity of hSAA1.1 and modulated its fibrillation pathway.A deeper understanding of the oligomerization and fibrillation pathway of hSAA1.1 may help elucidate its pathological role.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York, USA.

ABSTRACT
The fibrillation of Serum Amyloid A (SAA) - a major acute phase protein - is believed to play a role in the disease Amyloid A (AA) Amyloidosis. To better understand the amyloid formation pathway of SAA, we characterized the oligomerization, misfolding, and aggregation of a disease-associated isoform of human SAA - human SAA1.1 (hSAA1.1) - using techniques ranging from circular dichroism spectroscopy to atomic force microscopy, fluorescence spectroscopy, immunoblot studies, solubility measurements, and seeding experiments. We found that hSAA1.1 formed alpha helix-rich, marginally stable oligomers in vitro on refolding and cross-beta-rich aggregates following incubation at 37°C. Strikingly, while hSAA1.1 was not highly amyloidogenic in vitro, the addition of a single N-terminal methionine residue significantly enhanced the fibrillation propensity of hSAA1.1 and modulated its fibrillation pathway. A deeper understanding of the oligomerization and fibrillation pathway of hSAA1.1 may help elucidate its pathological role.

Show MeSH
Related in: MedlinePlus