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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 “seeding” properties of MetSAA1.1 and hSAA1.1 by ThT fluorescence assay.(A) ThT fluorescence intensity profile for freshly refolded MetSAA1.1 only (black bars) and MetSAA1.1+ MetSAA1.1 “seed” (gray bars); (B) ThT fluorescence intensity profile for freshly refolded hSAA1.1 only (black bars) and hSAA1.1+ hSAA1.1 “seed” (gray bars). The concentration of protein was 20 µM. ThT fluorescence intensities were recorded by incubating the samples at 37°C.
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pone-0064974-g004: Characterization of “seeding” properties of MetSAA1.1 and hSAA1.1 by ThT fluorescence assay.(A) ThT fluorescence intensity profile for freshly refolded MetSAA1.1 only (black bars) and MetSAA1.1+ MetSAA1.1 “seed” (gray bars); (B) ThT fluorescence intensity profile for freshly refolded hSAA1.1 only (black bars) and hSAA1.1+ hSAA1.1 “seed” (gray bars). The concentration of protein was 20 µM. ThT fluorescence intensities were recorded by incubating the samples at 37°C.

Mentions: After analyzing the fibrillation pathways of the two proteins, we then tested the ability of the aggregates formed by both the proteins to seed aggregation. Specifically, we attempted to “seed” the “native-like” oligomers of both MetSAA1.1 and hSAA1.1 with amyloid aggregates formed by the same protein following 72 h of incubation at 37°C. The solutions for these seeding experiments contained 18 µM freshly refolded MetSAA1.1 or hSAA1.1 and 2 µM MetSAA1.1 or hSAA1.1 aggregates obtained after 72 h incubation at 37°C. The final concentration of proteins in each of these mixtures was thus 20 µM (the same as that used for all previous studies) and the percentage of “seed” was effectively 10% of the total protein concentration. We monitored the aggregation kinetics of the “seeded” protein solutions using the ThT fluorescence assay and compared them with those for protein solutions containing 20 µM MetSAA1.1 or hSAA1.1 alone. As shown in Figure 4A (black bars) and also as observed previously (Fig. 2A), MetSAA1.1 aggregation was a gradual process with ThT fluorescence intensity saturating after ca. 24 h. “Seeding” freshly refolded MetSAA1.1 with MetSAA1.1 amyloid fibrils however significantly enhanced the rate of formation of cross-beta-rich aggregates with ThT fluorescence intensities saturating by ca. 1 h (Fig. 4A). On the other hand, we observed that hSAA1.1 “seed” did not have a significant impact on the aggregation kinetics of hSAA1.1 and ThT fluorescence intensities for both, hSAA1.1 alone (black bars) and hSAA1.1 plus hSAA1.1 “seed” (gray bars), saturated by ca. 24 h (Fig. 4B). Interestingly, “cross-seeding” freshly refolded hSAA1.1 with MetSAA1.1 amyloid fibrils marginally enhanced the rate of formation of cross-beta-rich aggregates with ThT fluorescence intensities saturating by ca. 6 h (Fig. S7 in File S1). Taken together these results show that while the late stage amyloid aggregates formed by MetSAA1.1 promote the conversion of MetSAA1.1 to cross-beta-rich aggregates, the late-stage aggregates formed by hSAA1.1 do not have a significant effect on hSAA1.1 aggregation.


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 “seeding” properties of MetSAA1.1 and hSAA1.1 by ThT fluorescence assay.(A) ThT fluorescence intensity profile for freshly refolded MetSAA1.1 only (black bars) and MetSAA1.1+ MetSAA1.1 “seed” (gray bars); (B) ThT fluorescence intensity profile for freshly refolded hSAA1.1 only (black bars) and hSAA1.1+ hSAA1.1 “seed” (gray bars). The concentration of protein was 20 µM. ThT fluorescence intensities were recorded by incubating the samples at 37°C.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3672174&req=5

pone-0064974-g004: Characterization of “seeding” properties of MetSAA1.1 and hSAA1.1 by ThT fluorescence assay.(A) ThT fluorescence intensity profile for freshly refolded MetSAA1.1 only (black bars) and MetSAA1.1+ MetSAA1.1 “seed” (gray bars); (B) ThT fluorescence intensity profile for freshly refolded hSAA1.1 only (black bars) and hSAA1.1+ hSAA1.1 “seed” (gray bars). The concentration of protein was 20 µM. ThT fluorescence intensities were recorded by incubating the samples at 37°C.
Mentions: After analyzing the fibrillation pathways of the two proteins, we then tested the ability of the aggregates formed by both the proteins to seed aggregation. Specifically, we attempted to “seed” the “native-like” oligomers of both MetSAA1.1 and hSAA1.1 with amyloid aggregates formed by the same protein following 72 h of incubation at 37°C. The solutions for these seeding experiments contained 18 µM freshly refolded MetSAA1.1 or hSAA1.1 and 2 µM MetSAA1.1 or hSAA1.1 aggregates obtained after 72 h incubation at 37°C. The final concentration of proteins in each of these mixtures was thus 20 µM (the same as that used for all previous studies) and the percentage of “seed” was effectively 10% of the total protein concentration. We monitored the aggregation kinetics of the “seeded” protein solutions using the ThT fluorescence assay and compared them with those for protein solutions containing 20 µM MetSAA1.1 or hSAA1.1 alone. As shown in Figure 4A (black bars) and also as observed previously (Fig. 2A), MetSAA1.1 aggregation was a gradual process with ThT fluorescence intensity saturating after ca. 24 h. “Seeding” freshly refolded MetSAA1.1 with MetSAA1.1 amyloid fibrils however significantly enhanced the rate of formation of cross-beta-rich aggregates with ThT fluorescence intensities saturating by ca. 1 h (Fig. 4A). On the other hand, we observed that hSAA1.1 “seed” did not have a significant impact on the aggregation kinetics of hSAA1.1 and ThT fluorescence intensities for both, hSAA1.1 alone (black bars) and hSAA1.1 plus hSAA1.1 “seed” (gray bars), saturated by ca. 24 h (Fig. 4B). Interestingly, “cross-seeding” freshly refolded hSAA1.1 with MetSAA1.1 amyloid fibrils marginally enhanced the rate of formation of cross-beta-rich aggregates with ThT fluorescence intensities saturating by ca. 6 h (Fig. S7 in File S1). Taken together these results show that while the late stage amyloid aggregates formed by MetSAA1.1 promote the conversion of MetSAA1.1 to cross-beta-rich aggregates, the late-stage aggregates formed by hSAA1.1 do not have a significant effect on hSAA1.1 aggregation.

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