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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 heat induced aggregation of 112-syn.(A) The chaperone-like activity of WT-syn was monitored by incubating 112-syn (0.4 mg/mL) in the presence or absence of different ratios of WT-syn as described in the “Methods” section. The time dependent increase in absorbance at 360 nm was recorded over a period of 10 min. (B) Fold change in the net absorbance values from (A) before and after 10 min of incubation at 65°C recorded at 360 nm.*p<0.01 as compared to 112-syn before heating.
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pone-0098657-g003: Effect of WT-syn on heat induced aggregation of 112-syn.(A) The chaperone-like activity of WT-syn was monitored by incubating 112-syn (0.4 mg/mL) in the presence or absence of different ratios of WT-syn as described in the “Methods” section. The time dependent increase in absorbance at 360 nm was recorded over a period of 10 min. (B) Fold change in the net absorbance values from (A) before and after 10 min of incubation at 65°C recorded at 360 nm.*p<0.01 as compared to 112-syn before heating.

Mentions: Based on the above observations on distinctly different property of 112-syn as compared to WT-syn, we surmised whether the chaperone-like activity of WT-syn confers any protection towards the heat-induced aggregation of 112-syn. To examine this, we have exposed 112-syn to 65°C for 10 min in the presence or absence of WT-syn (in the ratios of 1∶0.5, 1∶1 and1∶2). Results demonstrate that WT-syn dose-dependently inhibited the heat-induced aggregation of 112-syn. At 1∶0.5, 1∶1 and 1∶2 ratios of 112-syn: WT-syn, the percent inhibition was found to be nearly 46, 62 and 72% respectively [Fig. 3A and B]. Under similar experimental conditions no gross changes in the absorbance were noticed in incubation mixtures consisting of WT-syn alone [Fig. 3B].


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 heat induced aggregation of 112-syn.(A) The chaperone-like activity of WT-syn was monitored by incubating 112-syn (0.4 mg/mL) in the presence or absence of different ratios of WT-syn as described in the “Methods” section. The time dependent increase in absorbance at 360 nm was recorded over a period of 10 min. (B) Fold change in the net absorbance values from (A) before and after 10 min of incubation at 65°C recorded at 360 nm.*p<0.01 as compared to 112-syn before heating.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098657-g003: Effect of WT-syn on heat induced aggregation of 112-syn.(A) The chaperone-like activity of WT-syn was monitored by incubating 112-syn (0.4 mg/mL) in the presence or absence of different ratios of WT-syn as described in the “Methods” section. The time dependent increase in absorbance at 360 nm was recorded over a period of 10 min. (B) Fold change in the net absorbance values from (A) before and after 10 min of incubation at 65°C recorded at 360 nm.*p<0.01 as compared to 112-syn before heating.
Mentions: Based on the above observations on distinctly different property of 112-syn as compared to WT-syn, we surmised whether the chaperone-like activity of WT-syn confers any protection towards the heat-induced aggregation of 112-syn. To examine this, we have exposed 112-syn to 65°C for 10 min in the presence or absence of WT-syn (in the ratios of 1∶0.5, 1∶1 and1∶2). Results demonstrate that WT-syn dose-dependently inhibited the heat-induced aggregation of 112-syn. At 1∶0.5, 1∶1 and 1∶2 ratios of 112-syn: WT-syn, the percent inhibition was found to be nearly 46, 62 and 72% respectively [Fig. 3A and B]. Under similar experimental conditions no gross changes in the absorbance were noticed in incubation mixtures consisting of WT-syn alone [Fig. 3B].

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