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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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Effect of WT-syn on time dependent fibrillation of 112-syn.(A) 112-syn (0.5 mg/mL) was incubated in the presence or absence of different ratios of WT-syn at 37°C in 20 mM Tris buffer, pH 7.5 for 12 h. The increase in ThT fluorescence emission spectrum (470–600 nm) was recorded following excitation at 450 nm. (B) Fold change in the ThT fluorescence of 112-syn, incubated for 12 h in the presence or absence of different ratios of WT-syn. Data presented are the mean ± SD of three separate experiments. *p<0.01 as compared to 0 h reading for 112-syn; #p<0.01 as compared to112-syn alone.
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pone-0098657-g005: Effect of WT-syn on time dependent fibrillation of 112-syn.(A) 112-syn (0.5 mg/mL) was incubated in the presence or absence of different ratios of WT-syn at 37°C in 20 mM Tris buffer, pH 7.5 for 12 h. The increase in ThT fluorescence emission spectrum (470–600 nm) was recorded following excitation at 450 nm. (B) Fold change in the ThT fluorescence of 112-syn, incubated for 12 h in the presence or absence of different ratios of WT-syn. Data presented are the mean ± SD of three separate experiments. *p<0.01 as compared to 0 h reading for 112-syn; #p<0.01 as compared to112-syn alone.

Mentions: Since, WT-syn was found to inhibit heat-induced aggregation of 112-syn, we next analyzed whether similar effects would also be evident on the fibrillation of 112-syn. To examine this, fibrillation of 112-syn was monitored by ThT staining following 12 h incubation at 37°C in reaction mixture containing 112-syn in the presence or absence of WT-syn at different ratios. The obtained data indicates that WT-syn dose-dependently increased the fibrillation of 112-syn as measured by ThT staining [Fig. 5A]. 112-syn when incubated with WT-syn at 1∶0.5 and 1∶1 ratio resulted in the enhancement of ThT fluorescence by nearly 1.9 and 2.7 fold respectively and the values remained nearly the same even at 1∶2 ratio [Fig. 5A and B]. A similar enhancement in ThT staining was not observed when 112-syn was incubated with different concentrations of bovine serum albumin, which rules out the possibility of differences in protein concentration between samples for the observed effects.


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)

Effect of WT-syn on time dependent fibrillation of 112-syn.(A) 112-syn (0.5 mg/mL) was incubated in the presence or absence of different ratios of WT-syn at 37°C in 20 mM Tris buffer, pH 7.5 for 12 h. The increase in ThT fluorescence emission spectrum (470–600 nm) was recorded following excitation at 450 nm. (B) Fold change in the ThT fluorescence of 112-syn, incubated for 12 h in the presence or absence of different ratios of WT-syn. Data presented are the mean ± SD of three separate experiments. *p<0.01 as compared to 0 h reading for 112-syn; #p<0.01 as compared to112-syn alone.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098657-g005: Effect of WT-syn on time dependent fibrillation of 112-syn.(A) 112-syn (0.5 mg/mL) was incubated in the presence or absence of different ratios of WT-syn at 37°C in 20 mM Tris buffer, pH 7.5 for 12 h. The increase in ThT fluorescence emission spectrum (470–600 nm) was recorded following excitation at 450 nm. (B) Fold change in the ThT fluorescence of 112-syn, incubated for 12 h in the presence or absence of different ratios of WT-syn. Data presented are the mean ± SD of three separate experiments. *p<0.01 as compared to 0 h reading for 112-syn; #p<0.01 as compared to112-syn alone.
Mentions: Since, WT-syn was found to inhibit heat-induced aggregation of 112-syn, we next analyzed whether similar effects would also be evident on the fibrillation of 112-syn. To examine this, fibrillation of 112-syn was monitored by ThT staining following 12 h incubation at 37°C in reaction mixture containing 112-syn in the presence or absence of WT-syn at different ratios. The obtained data indicates that WT-syn dose-dependently increased the fibrillation of 112-syn as measured by ThT staining [Fig. 5A]. 112-syn when incubated with WT-syn at 1∶0.5 and 1∶1 ratio resulted in the enhancement of ThT fluorescence by nearly 1.9 and 2.7 fold respectively and the values remained nearly the same even at 1∶2 ratio [Fig. 5A and B]. A similar enhancement in ThT staining was not observed when 112-syn was incubated with different concentrations of bovine serum albumin, which rules out the possibility of differences in protein concentration between samples for the observed effects.

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