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A covalent homodimer probing early oligomers along amyloid aggregation.

Halabelian L, Relini A, Barbiroli A, Penco A, Bolognesi M, Ricagno S - Sci Rep (2015)

Bottom Line: In this study, by mutating Ser33 to Cys, and assembling the disulphide-stabilized β2m homodimer (DimC33), such DD strand interface was locked.Three distinct crystal structures of DimC33 suggest that dimerization through the DD interface is instrumental for enhancing DimC33 aggregation propensity.Furthermore, the crystal structure of DimC33 in complex with the amyloid-specific dye Thioflavin-T pinpoints a second interface, which likely participates in the first steps of β2m aggregation.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Bioscienze, Università degli Studi di Milano, Via Celoria 26, 20133 Milan, Italy.

ABSTRACT
Early oligomers are crucial in amyloid aggregation; however, due to their transient nature they are among the least structurally characterized species. We focused on the amyloidogenic protein beta2-microglobulin (β2m) whose early oligomers are still a matter of debate. An intermolecular interaction between D strands of facing β2m molecules was repeatedly observed, suggesting that such interface may be relevant for β2m dimerization. In this study, by mutating Ser33 to Cys, and assembling the disulphide-stabilized β2m homodimer (DimC33), such DD strand interface was locked. Although the isolated DimC33 display a stability similar to wt β2m under native conditions, it shows enhanced amyloid aggregation propensity. Three distinct crystal structures of DimC33 suggest that dimerization through the DD interface is instrumental for enhancing DimC33 aggregation propensity. Furthermore, the crystal structure of DimC33 in complex with the amyloid-specific dye Thioflavin-T pinpoints a second interface, which likely participates in the first steps of β2m aggregation. The present data provide new insight into β2m early steps of amyloid aggregation.

No MeSH data available.


Related in: MedlinePlus

DimC33: fibrillogenesis at pH7.4.(A) Kinetics of DimC33 fibril formation in 100 mM NaCl, 50 mM Na phosphate buffer (pH 7.4) at 37 °C incubation for two weeks, monitored by measuring ThT fluorescence at 0, 4, 48, 96, 144, 216, 312 hours. Four samples were tested: DimC33_20% TFE and wtβ2m_20% TFE (1 mg mL−1 protein in 20% TFE); DimC33_10% TFE and wtβ2m_10% TFE (5 mg mL−1 protein in 10% TFE). Values represent the average of three independent experiments and error bars represent standard deviation (SD). (B) Tapping mode AFM images (height data) of DimC33 fibrillar aggregates obtained in 20% TFE (left) and 10% TFE (right). Scan size 500 nm, Z range: left, 37 nm; right, 15 nm. (C) DimC33 aggregation analysis monitored by ThT fluorescence after one week of incubation at 37 °C, using the same aggregation conditions as tested under DimC33_20% TFE in three conditions; 0 μM doxycycline, in the presence of 100 μM and 400 μM doxycycline. Values represent the average of three independent experiments and error bars represent SD.
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f3: DimC33: fibrillogenesis at pH7.4.(A) Kinetics of DimC33 fibril formation in 100 mM NaCl, 50 mM Na phosphate buffer (pH 7.4) at 37 °C incubation for two weeks, monitored by measuring ThT fluorescence at 0, 4, 48, 96, 144, 216, 312 hours. Four samples were tested: DimC33_20% TFE and wtβ2m_20% TFE (1 mg mL−1 protein in 20% TFE); DimC33_10% TFE and wtβ2m_10% TFE (5 mg mL−1 protein in 10% TFE). Values represent the average of three independent experiments and error bars represent standard deviation (SD). (B) Tapping mode AFM images (height data) of DimC33 fibrillar aggregates obtained in 20% TFE (left) and 10% TFE (right). Scan size 500 nm, Z range: left, 37 nm; right, 15 nm. (C) DimC33 aggregation analysis monitored by ThT fluorescence after one week of incubation at 37 °C, using the same aggregation conditions as tested under DimC33_20% TFE in three conditions; 0 μM doxycycline, in the presence of 100 μM and 400 μM doxycycline. Values represent the average of three independent experiments and error bars represent SD.

Mentions: We studied DimC33 aggregation propensity in vitro at physiological pH conditions (pH 7.4) in the absence of any pre-fibrillar seeds. In 20% (v/v) TFE, DimC33 at a final concentration of 1 mg mL−1 aggregates promptly without a lag phase, reaching equilibrium within the first 4 hours, and yielding a high ThT binding signal (Fig. 3a); under the same conditions wt β2m remains soluble and does not aggregate. Interestingly, in 10% (v/v) TFE—where DimC33 and wt β2m display CD spectra indicative of native-like conformations—a 5 mg mL−1 solution of DimC33 also aggregated. The AFM analysis of the samples after 1 week aggregation showed that DimC33 formed fibrils both in 20% TFE (Fig. 3b, left) and in 10% TFE (Fig. 3b, right). The fibril heights measured in the two conditions were similar and were in the range between 2.0 and 5.5 nm.


A covalent homodimer probing early oligomers along amyloid aggregation.

Halabelian L, Relini A, Barbiroli A, Penco A, Bolognesi M, Ricagno S - Sci Rep (2015)

DimC33: fibrillogenesis at pH7.4.(A) Kinetics of DimC33 fibril formation in 100 mM NaCl, 50 mM Na phosphate buffer (pH 7.4) at 37 °C incubation for two weeks, monitored by measuring ThT fluorescence at 0, 4, 48, 96, 144, 216, 312 hours. Four samples were tested: DimC33_20% TFE and wtβ2m_20% TFE (1 mg mL−1 protein in 20% TFE); DimC33_10% TFE and wtβ2m_10% TFE (5 mg mL−1 protein in 10% TFE). Values represent the average of three independent experiments and error bars represent standard deviation (SD). (B) Tapping mode AFM images (height data) of DimC33 fibrillar aggregates obtained in 20% TFE (left) and 10% TFE (right). Scan size 500 nm, Z range: left, 37 nm; right, 15 nm. (C) DimC33 aggregation analysis monitored by ThT fluorescence after one week of incubation at 37 °C, using the same aggregation conditions as tested under DimC33_20% TFE in three conditions; 0 μM doxycycline, in the presence of 100 μM and 400 μM doxycycline. Values represent the average of three independent experiments and error bars represent SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4588566&req=5

f3: DimC33: fibrillogenesis at pH7.4.(A) Kinetics of DimC33 fibril formation in 100 mM NaCl, 50 mM Na phosphate buffer (pH 7.4) at 37 °C incubation for two weeks, monitored by measuring ThT fluorescence at 0, 4, 48, 96, 144, 216, 312 hours. Four samples were tested: DimC33_20% TFE and wtβ2m_20% TFE (1 mg mL−1 protein in 20% TFE); DimC33_10% TFE and wtβ2m_10% TFE (5 mg mL−1 protein in 10% TFE). Values represent the average of three independent experiments and error bars represent standard deviation (SD). (B) Tapping mode AFM images (height data) of DimC33 fibrillar aggregates obtained in 20% TFE (left) and 10% TFE (right). Scan size 500 nm, Z range: left, 37 nm; right, 15 nm. (C) DimC33 aggregation analysis monitored by ThT fluorescence after one week of incubation at 37 °C, using the same aggregation conditions as tested under DimC33_20% TFE in three conditions; 0 μM doxycycline, in the presence of 100 μM and 400 μM doxycycline. Values represent the average of three independent experiments and error bars represent SD.
Mentions: We studied DimC33 aggregation propensity in vitro at physiological pH conditions (pH 7.4) in the absence of any pre-fibrillar seeds. In 20% (v/v) TFE, DimC33 at a final concentration of 1 mg mL−1 aggregates promptly without a lag phase, reaching equilibrium within the first 4 hours, and yielding a high ThT binding signal (Fig. 3a); under the same conditions wt β2m remains soluble and does not aggregate. Interestingly, in 10% (v/v) TFE—where DimC33 and wt β2m display CD spectra indicative of native-like conformations—a 5 mg mL−1 solution of DimC33 also aggregated. The AFM analysis of the samples after 1 week aggregation showed that DimC33 formed fibrils both in 20% TFE (Fig. 3b, left) and in 10% TFE (Fig. 3b, right). The fibril heights measured in the two conditions were similar and were in the range between 2.0 and 5.5 nm.

Bottom Line: In this study, by mutating Ser33 to Cys, and assembling the disulphide-stabilized β2m homodimer (DimC33), such DD strand interface was locked.Three distinct crystal structures of DimC33 suggest that dimerization through the DD interface is instrumental for enhancing DimC33 aggregation propensity.Furthermore, the crystal structure of DimC33 in complex with the amyloid-specific dye Thioflavin-T pinpoints a second interface, which likely participates in the first steps of β2m aggregation.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Bioscienze, Università degli Studi di Milano, Via Celoria 26, 20133 Milan, Italy.

ABSTRACT
Early oligomers are crucial in amyloid aggregation; however, due to their transient nature they are among the least structurally characterized species. We focused on the amyloidogenic protein beta2-microglobulin (β2m) whose early oligomers are still a matter of debate. An intermolecular interaction between D strands of facing β2m molecules was repeatedly observed, suggesting that such interface may be relevant for β2m dimerization. In this study, by mutating Ser33 to Cys, and assembling the disulphide-stabilized β2m homodimer (DimC33), such DD strand interface was locked. Although the isolated DimC33 display a stability similar to wt β2m under native conditions, it shows enhanced amyloid aggregation propensity. Three distinct crystal structures of DimC33 suggest that dimerization through the DD interface is instrumental for enhancing DimC33 aggregation propensity. Furthermore, the crystal structure of DimC33 in complex with the amyloid-specific dye Thioflavin-T pinpoints a second interface, which likely participates in the first steps of β2m aggregation. The present data provide new insight into β2m early steps of amyloid aggregation.

No MeSH data available.


Related in: MedlinePlus