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The chaperone-like activity of α-synuclein attenuates aggregation of its alternatively spliced isoform, 112-synuclein in vitro: plausible cross-talk between isoforms in protein aggregation.

Manda KM, Yedlapudi D, Korukonda S, Bojja S, Kalivendi SV - PLoS ONE (2014)

Bottom Line: The effects were dose and time-dependent and a significant aggregation of 112-syn was evident at as low as 45 °C following 10 min of incubation.The heat-induced aggregates were found to be ill-defined structures and weakly positive towards Thioflavin-T (ThT) staining as compared to clearly distinguishable ThT positive extended fibrils resulting upon 24 h of incubation at 37 °C.Further, the chaperone-like activity of WT-syn significantly attenuated heat-induced aggregation of 112-syn in a dose and time-dependent manner.

View Article: PubMed Central - PubMed

Affiliation: Centre for Academy of Scientific & Innovative Research, CSIR-Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad, Andhra Pradesh, India; Centre for Chemical Biology, CSIR-Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad, Andhra Pradesh, India.

ABSTRACT
Abnormal oligomerization and aggregation of α-synuclein (α-syn/WT-syn) has been shown to be a precipitating factor in the pathophysiology of Parkinson's disease (PD). Earlier observations on the induced-alternative splicing of α-syn by Parkinsonism mimetics as well as identification of region specific abnormalities in the transcript levels of 112-synuclein (112-syn) in diseased subjects underscores the role of 112-syn in the pathophysiology of PD. In the present study, we sought to identify the aggregation potential of 112-syn in the presence or absence of WT-syn to predict its plausible role in protein aggregation events. Results demonstrate that unlike WT-syn, lack of 28 aa in the C-terminus results in the loss of chaperone-like activity with a concomitant gain in vulnerability to heat-induced aggregation and time-dependent fibrillation. The effects were dose and time-dependent and a significant aggregation of 112-syn was evident at as low as 45 °C following 10 min of incubation. The heat-induced aggregates were found to be ill-defined structures and weakly positive towards Thioflavin-T (ThT) staining as compared to clearly distinguishable ThT positive extended fibrils resulting upon 24 h of incubation at 37 °C. Further, the chaperone-like activity of WT-syn significantly attenuated heat-induced aggregation of 112-syn in a dose and time-dependent manner. On contrary, WT-syn synergistically enhanced fibrillation of 112-syn. Overall, the present findings highlight a plausible cross-talk between isoforms of α-syn and the relative abundance of these isoforms may dictate the nature and fate of protein aggregates.

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Temperature dependent aggregation of 112-syn.(A) 112-syn at different concentrations (0.2, 0.4, 0.6 mg/mL in PBS) was heated at 45°C, 55°C, and 65°C and the light scattering was monitored at 360 nm for a period of 10 min as described under “Methods” section. (B) Fold change in the net absorbance values from (A) at 360 nm before and after heating the reaction mixtures. (C) ThT fluorescence of reaction mixtures as indicated in (A) before (0 min) and after exposing the samples at indicated temperatures for 10 min.*p<0.01 as compared to 0 min readings for both (B) and (C). (D) Negatively stained TEM images of WT and 112-syn before and after exposure to 65°C for 10 min.
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pone-0098657-g002: Temperature dependent aggregation of 112-syn.(A) 112-syn at different concentrations (0.2, 0.4, 0.6 mg/mL in PBS) was heated at 45°C, 55°C, and 65°C and the light scattering was monitored at 360 nm for a period of 10 min as described under “Methods” section. (B) Fold change in the net absorbance values from (A) at 360 nm before and after heating the reaction mixtures. (C) ThT fluorescence of reaction mixtures as indicated in (A) before (0 min) and after exposing the samples at indicated temperatures for 10 min.*p<0.01 as compared to 0 min readings for both (B) and (C). (D) Negatively stained TEM images of WT and 112-syn before and after exposure to 65°C for 10 min.

Mentions: Next, we analyzed the kinetics of apparent light scattering of 112-syn (0.2, 0.4, 0.6 mg/mL) against heat-induced aggregation at three different temperatures (45, 55 and 65°C) over a period of 10 min. Results indicate a dose-dependent increase in the turbidity of 112-syn at all the temperatures examined [Fig. 2A and B]. While, 0.2 mg/mL of 112-syn displayed nearly 2-fold increase in the absorbance following 10 min of incubation at 45°C, the corresponding values for 0.4 and 0.6 mg/mL of protein were found to be 5 and 9 fold respectively. Similarly at 55°C, the three different concentrations of 112-syn (0.2, 0.4, 0.6 mg/mL) exhibited 4, 11 and 16 fold increase in the absorbance and at 65°C the fold increase in absorbance was found to be 11, 16 and 18 fold respectively [Fig. 2B]. The obtained data clearly demonstrate the enhanced vulnerability of 112-syn to variations in temperature and a significant increase in turbidity was evident at as low as 45°C. The binding of ThT to the crossed β-sheet structures (characteristic feature of amyloid fibrils of proteins) enhances its fluorescence and is being widely used to study protein fibrillation [43]. ThT staining of 112-syn before and after exposure to different temperatures though found significantly increased [Fig. 2C], but, the fold increase was not proportional to that of the observed turbidity under same experimental conditions (Fig. 2A and B). TEM images of 112-syn exposed to heat (65°C for 10 min) revealed the presence of ill-defined aggregates, however, under similar conditions no aggregates were found with WT-syn [Fig. 2D].


The chaperone-like activity of α-synuclein attenuates aggregation of its alternatively spliced isoform, 112-synuclein in vitro: plausible cross-talk between isoforms in protein aggregation.

Manda KM, Yedlapudi D, Korukonda S, Bojja S, Kalivendi SV - PLoS ONE (2014)

Temperature dependent aggregation of 112-syn.(A) 112-syn at different concentrations (0.2, 0.4, 0.6 mg/mL in PBS) was heated at 45°C, 55°C, and 65°C and the light scattering was monitored at 360 nm for a period of 10 min as described under “Methods” section. (B) Fold change in the net absorbance values from (A) at 360 nm before and after heating the reaction mixtures. (C) ThT fluorescence of reaction mixtures as indicated in (A) before (0 min) and after exposing the samples at indicated temperatures for 10 min.*p<0.01 as compared to 0 min readings for both (B) and (C). (D) Negatively stained TEM images of WT and 112-syn before and after exposure to 65°C for 10 min.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4043908&req=5

pone-0098657-g002: Temperature dependent aggregation of 112-syn.(A) 112-syn at different concentrations (0.2, 0.4, 0.6 mg/mL in PBS) was heated at 45°C, 55°C, and 65°C and the light scattering was monitored at 360 nm for a period of 10 min as described under “Methods” section. (B) Fold change in the net absorbance values from (A) at 360 nm before and after heating the reaction mixtures. (C) ThT fluorescence of reaction mixtures as indicated in (A) before (0 min) and after exposing the samples at indicated temperatures for 10 min.*p<0.01 as compared to 0 min readings for both (B) and (C). (D) Negatively stained TEM images of WT and 112-syn before and after exposure to 65°C for 10 min.
Mentions: Next, we analyzed the kinetics of apparent light scattering of 112-syn (0.2, 0.4, 0.6 mg/mL) against heat-induced aggregation at three different temperatures (45, 55 and 65°C) over a period of 10 min. Results indicate a dose-dependent increase in the turbidity of 112-syn at all the temperatures examined [Fig. 2A and B]. While, 0.2 mg/mL of 112-syn displayed nearly 2-fold increase in the absorbance following 10 min of incubation at 45°C, the corresponding values for 0.4 and 0.6 mg/mL of protein were found to be 5 and 9 fold respectively. Similarly at 55°C, the three different concentrations of 112-syn (0.2, 0.4, 0.6 mg/mL) exhibited 4, 11 and 16 fold increase in the absorbance and at 65°C the fold increase in absorbance was found to be 11, 16 and 18 fold respectively [Fig. 2B]. The obtained data clearly demonstrate the enhanced vulnerability of 112-syn to variations in temperature and a significant increase in turbidity was evident at as low as 45°C. The binding of ThT to the crossed β-sheet structures (characteristic feature of amyloid fibrils of proteins) enhances its fluorescence and is being widely used to study protein fibrillation [43]. ThT staining of 112-syn before and after exposure to different temperatures though found significantly increased [Fig. 2C], but, the fold increase was not proportional to that of the observed turbidity under same experimental conditions (Fig. 2A and B). TEM images of 112-syn exposed to heat (65°C for 10 min) revealed the presence of ill-defined aggregates, however, under similar conditions no aggregates were found with WT-syn [Fig. 2D].

Bottom Line: The effects were dose and time-dependent and a significant aggregation of 112-syn was evident at as low as 45 °C following 10 min of incubation.The heat-induced aggregates were found to be ill-defined structures and weakly positive towards Thioflavin-T (ThT) staining as compared to clearly distinguishable ThT positive extended fibrils resulting upon 24 h of incubation at 37 °C.Further, the chaperone-like activity of WT-syn significantly attenuated heat-induced aggregation of 112-syn in a dose and time-dependent manner.

View Article: PubMed Central - PubMed

Affiliation: Centre for Academy of Scientific & Innovative Research, CSIR-Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad, Andhra Pradesh, India; Centre for Chemical Biology, CSIR-Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad, Andhra Pradesh, India.

ABSTRACT
Abnormal oligomerization and aggregation of α-synuclein (α-syn/WT-syn) has been shown to be a precipitating factor in the pathophysiology of Parkinson's disease (PD). Earlier observations on the induced-alternative splicing of α-syn by Parkinsonism mimetics as well as identification of region specific abnormalities in the transcript levels of 112-synuclein (112-syn) in diseased subjects underscores the role of 112-syn in the pathophysiology of PD. In the present study, we sought to identify the aggregation potential of 112-syn in the presence or absence of WT-syn to predict its plausible role in protein aggregation events. Results demonstrate that unlike WT-syn, lack of 28 aa in the C-terminus results in the loss of chaperone-like activity with a concomitant gain in vulnerability to heat-induced aggregation and time-dependent fibrillation. The effects were dose and time-dependent and a significant aggregation of 112-syn was evident at as low as 45 °C following 10 min of incubation. The heat-induced aggregates were found to be ill-defined structures and weakly positive towards Thioflavin-T (ThT) staining as compared to clearly distinguishable ThT positive extended fibrils resulting upon 24 h of incubation at 37 °C. Further, the chaperone-like activity of WT-syn significantly attenuated heat-induced aggregation of 112-syn in a dose and time-dependent manner. On contrary, WT-syn synergistically enhanced fibrillation of 112-syn. Overall, the present findings highlight a plausible cross-talk between isoforms of α-syn and the relative abundance of these isoforms may dictate the nature and fate of protein aggregates.

Show MeSH
Related in: MedlinePlus