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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 and 112-syn on heat induced aggregation of aldolase (ALD).(A) In vitro protein aggregation assay employing aldolase (0.1 mg/mL in PBS) was performed at 65°C in the absence or presence of WT and 112-syn at different ratios (1∶2 and 1∶4) and the light scattering was monitored at 360 nm for a period of 10 min. (B) The net change in the absorbance of reaction mixtures of (A) before and after 10 min of incubation at 65°C. Data are the mean ± SD of three separate experiments. * indicates p<0.01 as compared to aldolase (decrease); #p<0.01 as compared to aldolase (increase) and $ p<0.01 as compared to the respective controls (before heating).
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pone-0098657-g001: Effect of WT and 112-syn on heat induced aggregation of aldolase (ALD).(A) In vitro protein aggregation assay employing aldolase (0.1 mg/mL in PBS) was performed at 65°C in the absence or presence of WT and 112-syn at different ratios (1∶2 and 1∶4) and the light scattering was monitored at 360 nm for a period of 10 min. (B) The net change in the absorbance of reaction mixtures of (A) before and after 10 min of incubation at 65°C. Data are the mean ± SD of three separate experiments. * indicates p<0.01 as compared to aldolase (decrease); #p<0.01 as compared to aldolase (increase) and $ p<0.01 as compared to the respective controls (before heating).

Mentions: The ability of α-syn to suppress the aggregation of non-native conformations of substrate proteins like aldolase has been widely employed as a measure of its chaperone like activity in vitro. Previously, it was shown that chaperone-like activity of α-syn was lost due to the lack of its acidic C-terminus [40]. Concomitant with the earlier findings, our initial studies aimed at understanding the functional consequence of deleted exon-5 in 112-syn also noticed that unlike WT-syn, 112-syn did not demonstrate any inhibitory effect on the heat-induced aggregation of aldolase [Fig. 1 A and B]. Results indicate that incubation of aldolase alone (0.1 mg/mL) at 65°C induced its aggregation and the absorbance values reached from 0 to ∼0.6 during the incubation period of 10 min [Fig. 1A and B]. Moreover, WT-syn suppressed the aggregation of aldolase in a dose dependent manner [Fig. 1A and B]; which corroborates with the earlier observations [39]. However, the absorbance of incubation mixtures, wherein, a standard amount of aldolase (0.1 mg/mL) when incubated with increasing concentrations of 112-syn demonstrated a dose dependent increase in the absorbance at 360 nm which is significantly more than values exhibited by aldolase alone [Fig. 1A and B]. Under similar conditions, incubation mixture consisting of 112-syn alone exhibited a dose dependent increase in the absorbance at 360 nm and this phenomena was not observed with WT-syn [Fig. 1A and B]. The obtained results prompted us to study the heat-induced aggregation propensity of 112-syn.


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 and 112-syn on heat induced aggregation of aldolase (ALD).(A) In vitro protein aggregation assay employing aldolase (0.1 mg/mL in PBS) was performed at 65°C in the absence or presence of WT and 112-syn at different ratios (1∶2 and 1∶4) and the light scattering was monitored at 360 nm for a period of 10 min. (B) The net change in the absorbance of reaction mixtures of (A) before and after 10 min of incubation at 65°C. Data are the mean ± SD of three separate experiments. * indicates p<0.01 as compared to aldolase (decrease); #p<0.01 as compared to aldolase (increase) and $ p<0.01 as compared to the respective controls (before heating).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098657-g001: Effect of WT and 112-syn on heat induced aggregation of aldolase (ALD).(A) In vitro protein aggregation assay employing aldolase (0.1 mg/mL in PBS) was performed at 65°C in the absence or presence of WT and 112-syn at different ratios (1∶2 and 1∶4) and the light scattering was monitored at 360 nm for a period of 10 min. (B) The net change in the absorbance of reaction mixtures of (A) before and after 10 min of incubation at 65°C. Data are the mean ± SD of three separate experiments. * indicates p<0.01 as compared to aldolase (decrease); #p<0.01 as compared to aldolase (increase) and $ p<0.01 as compared to the respective controls (before heating).
Mentions: The ability of α-syn to suppress the aggregation of non-native conformations of substrate proteins like aldolase has been widely employed as a measure of its chaperone like activity in vitro. Previously, it was shown that chaperone-like activity of α-syn was lost due to the lack of its acidic C-terminus [40]. Concomitant with the earlier findings, our initial studies aimed at understanding the functional consequence of deleted exon-5 in 112-syn also noticed that unlike WT-syn, 112-syn did not demonstrate any inhibitory effect on the heat-induced aggregation of aldolase [Fig. 1 A and B]. Results indicate that incubation of aldolase alone (0.1 mg/mL) at 65°C induced its aggregation and the absorbance values reached from 0 to ∼0.6 during the incubation period of 10 min [Fig. 1A and B]. Moreover, WT-syn suppressed the aggregation of aldolase in a dose dependent manner [Fig. 1A and B]; which corroborates with the earlier observations [39]. However, the absorbance of incubation mixtures, wherein, a standard amount of aldolase (0.1 mg/mL) when incubated with increasing concentrations of 112-syn demonstrated a dose dependent increase in the absorbance at 360 nm which is significantly more than values exhibited by aldolase alone [Fig. 1A and B]. Under similar conditions, incubation mixture consisting of 112-syn alone exhibited a dose dependent increase in the absorbance at 360 nm and this phenomena was not observed with WT-syn [Fig. 1A and B]. The obtained results prompted us to study the heat-induced aggregation propensity of 112-syn.

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