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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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Time dependent fibrillation of WT and 112-syn.Time dependent fibrillation of WT-syn (A) and 112-syn (B) was monitored by the enhancement of ThT fluorescence. WT and 112-syn (0.5 mg/mL) at 37°C were incubated in 20 mM Tris buffer, pH 7.5 for indicated time intervals as described in the “Methods” section and the ThT fluorescence emission spectrum was recorded. (C) Fold change in the ThT fluorescence of WT and 112-syn incubated for different time intervals. Data presented are the mean ± SD of three separate experiments. (D) Aliquots from reaction mixtures incubated at 37°C for 24 h were adsorbed onto carbon coated grids and then stained with 1% uranyl acetate for 1–2 min. Negatively stained TEM images of WT and 112-syn are shown. Scale bars, 500 nm and data is a representative of three different fields of view. *p<0.01 as compared to 0 h readings for 112-syn.
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pone-0098657-g004: Time dependent fibrillation of WT and 112-syn.Time dependent fibrillation of WT-syn (A) and 112-syn (B) was monitored by the enhancement of ThT fluorescence. WT and 112-syn (0.5 mg/mL) at 37°C were incubated in 20 mM Tris buffer, pH 7.5 for indicated time intervals as described in the “Methods” section and the ThT fluorescence emission spectrum was recorded. (C) Fold change in the ThT fluorescence of WT and 112-syn incubated for different time intervals. Data presented are the mean ± SD of three separate experiments. (D) Aliquots from reaction mixtures incubated at 37°C for 24 h were adsorbed onto carbon coated grids and then stained with 1% uranyl acetate for 1–2 min. Negatively stained TEM images of WT and 112-syn are shown. Scale bars, 500 nm and data is a representative of three different fields of view. *p<0.01 as compared to 0 h readings for 112-syn.

Mentions: Earlier reports demonstrated that α-syn aggregates in a time dependent manner forming large network of fibrils and a significant increase in ThT staining was reported following prolonged incubation (three days) at room temperature [42]. In order to understand the observed differences in ThT reactivity, we have incubated individually WT and 112-syn (0.5 mg/mL) under shaking conditions for 24 h at 37°C and subjected to ThT staining. The fluorescence emission spectra of ThT at different time points (0, 12, 18 & 24 h) for WT-syn and 112-syn are shown in Figure 4A and B. Results indicate that incubation mixtures containing WT-syn did not show any gross changes in the ThT emission spectrum during the initial 24 h time period [Fig. 4A]. Nevertheless, a significant increase in the aggregation of WT-syn was evident following prolonged incubation as reported in earlier studies (data not shown) [42]. Under similar incubation conditions, 112-syn exhibited a time dependent increase in the fibrillation based on the ThT emission spectrum [Fig. 4B]. Nearly, three, five and ten-fold-increase in ThT fluorescence of 112-syn was observed at 12, 18 and 24 h respectively, whereas, ThT fluorescence of WT-syn remained the same during the initial 24 h incubation [Fig. 4C]. TEM images of 112-syn under the above incubation conditions showed the presence of long extended network of fibrils and this effect was not observed with WT-syn [Fig. 4D], which is in coherence with the obtained ThT signal [Fig. 4A and B]. The data demonstrates that 112-syn possess enhanced vulnerability towards fibrillation as compared to WT-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)

Time dependent fibrillation of WT and 112-syn.Time dependent fibrillation of WT-syn (A) and 112-syn (B) was monitored by the enhancement of ThT fluorescence. WT and 112-syn (0.5 mg/mL) at 37°C were incubated in 20 mM Tris buffer, pH 7.5 for indicated time intervals as described in the “Methods” section and the ThT fluorescence emission spectrum was recorded. (C) Fold change in the ThT fluorescence of WT and 112-syn incubated for different time intervals. Data presented are the mean ± SD of three separate experiments. (D) Aliquots from reaction mixtures incubated at 37°C for 24 h were adsorbed onto carbon coated grids and then stained with 1% uranyl acetate for 1–2 min. Negatively stained TEM images of WT and 112-syn are shown. Scale bars, 500 nm and data is a representative of three different fields of view. *p<0.01 as compared to 0 h readings for 112-syn.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098657-g004: Time dependent fibrillation of WT and 112-syn.Time dependent fibrillation of WT-syn (A) and 112-syn (B) was monitored by the enhancement of ThT fluorescence. WT and 112-syn (0.5 mg/mL) at 37°C were incubated in 20 mM Tris buffer, pH 7.5 for indicated time intervals as described in the “Methods” section and the ThT fluorescence emission spectrum was recorded. (C) Fold change in the ThT fluorescence of WT and 112-syn incubated for different time intervals. Data presented are the mean ± SD of three separate experiments. (D) Aliquots from reaction mixtures incubated at 37°C for 24 h were adsorbed onto carbon coated grids and then stained with 1% uranyl acetate for 1–2 min. Negatively stained TEM images of WT and 112-syn are shown. Scale bars, 500 nm and data is a representative of three different fields of view. *p<0.01 as compared to 0 h readings for 112-syn.
Mentions: Earlier reports demonstrated that α-syn aggregates in a time dependent manner forming large network of fibrils and a significant increase in ThT staining was reported following prolonged incubation (three days) at room temperature [42]. In order to understand the observed differences in ThT reactivity, we have incubated individually WT and 112-syn (0.5 mg/mL) under shaking conditions for 24 h at 37°C and subjected to ThT staining. The fluorescence emission spectra of ThT at different time points (0, 12, 18 & 24 h) for WT-syn and 112-syn are shown in Figure 4A and B. Results indicate that incubation mixtures containing WT-syn did not show any gross changes in the ThT emission spectrum during the initial 24 h time period [Fig. 4A]. Nevertheless, a significant increase in the aggregation of WT-syn was evident following prolonged incubation as reported in earlier studies (data not shown) [42]. Under similar incubation conditions, 112-syn exhibited a time dependent increase in the fibrillation based on the ThT emission spectrum [Fig. 4B]. Nearly, three, five and ten-fold-increase in ThT fluorescence of 112-syn was observed at 12, 18 and 24 h respectively, whereas, ThT fluorescence of WT-syn remained the same during the initial 24 h incubation [Fig. 4C]. TEM images of 112-syn under the above incubation conditions showed the presence of long extended network of fibrils and this effect was not observed with WT-syn [Fig. 4D], which is in coherence with the obtained ThT signal [Fig. 4A and B]. The data demonstrates that 112-syn possess enhanced vulnerability towards fibrillation as compared to WT-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