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Structure based aggregation studies reveal the presence of helix-rich intermediate during α-Synuclein aggregation.

Ghosh D, Singh PK, Sahay S, Jha NN, Jacob RS, Sen S, Kumar A, Riek R, Maji SK - Sci Rep (2015)

Bottom Line: Using a variety of complementary biophysical techniques monitoring entire pathway of nine different synucleins, we found that transition of unstructured conformation into β-sheet rich fibril formation involves helix-rich intermediates.A multidimensional NMR study characterizing the intermediate accompanied with site-specific fluorescence study suggests that the N-terminal and central portions mainly participate in the helix-rich intermediate formation while the C-terminus remained in an extended conformation.However, significant conformational transitions occur at the middle and at the C-terminus during helix to β-sheet transition as evident from Trp fluorescence study.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosciences and Bioengineering, IIT Bombay, Powai, Mumbai, India 400076.

ABSTRACT
Mechanistic understanding of nucleation dependent polymerization by α-synuclein (α-Syn) into toxic oligomers and amyloids is important for the drug development against Parkinson's disease. However the structural and morphological characterization during nucleation and subsequent fibrillation process of α-Syn is not clearly understood. Using a variety of complementary biophysical techniques monitoring entire pathway of nine different synucleins, we found that transition of unstructured conformation into β-sheet rich fibril formation involves helix-rich intermediates. These intermediates are common for all aggregating synucleins, contain high solvent-exposed hydrophobic surfaces, are cytotoxic to SHSY-5Y cells and accelerate α-Syn aggregation efficiently. A multidimensional NMR study characterizing the intermediate accompanied with site-specific fluorescence study suggests that the N-terminal and central portions mainly participate in the helix-rich intermediate formation while the C-terminus remained in an extended conformation. However, significant conformational transitions occur at the middle and at the C-terminus during helix to β-sheet transition as evident from Trp fluorescence study. Since partial helix-rich intermediates were also observed for other amyloidogenic proteins such as Aβ and IAPP, we hypothesize that this class of intermediates may be one of the important intermediates for amyloid formation pathway by many natively unstructured protein/peptides and represent a potential target for drug development against amyloid diseases.

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Related in: MedlinePlus

Hydrophobic surface exposure and toxicity of helix-rich intermediate.(A) ANS fluorescence of different α-Syn species indicate maximum exposed hydrophobic surface for isolated helix-rich state. (B) MTT reduction by SH-SY5Y cells in presence of various α-Syn species showing higher cytotoxicity (less MTT reduction) of isolated helix and material comprising helix-rich intermediates. LMW, matured fibrils (5 days aged sample) and Aβ (25–35) fibrils were used as controls. (C) Correlation plot of ANS binding (hydrophobic surface exposure) and MTT reduction-based cytotoxicity indicates that species possessing larger hydrophobic exposed surfaces are more toxic. (D) Phase contrast images for SHSY-5Y cells treated with different species of α-Syn indicating maximum cell death for isolated helix followed by fibrils. However the morphology of cells treated with LMW remained in normal state similar to control cells (treated with buffer). Scale bar represents 100 micron. (E) The calcein and EthD-1 staining showing defective morphology with damages of cell body/neuritis of cell treated with helix-rich intermediate. Cells treated with either LMW or buffer control showed no significant of cell death/defective cell morphology. Scale bar represents 200 micron. (F) The flow cytometry analysis showing that induction of early apoptosis by cells treated with helix-rich state. The cells treated with matured fibrils and LMW samples did not show significant toxicity. Buffer was used as a control. Quadrants Q1, Q2, Q3 and Q4 represent dead cells, late apoptotic/necrotic cells, live cells and early apoptotic cells, respectively.
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f10: Hydrophobic surface exposure and toxicity of helix-rich intermediate.(A) ANS fluorescence of different α-Syn species indicate maximum exposed hydrophobic surface for isolated helix-rich state. (B) MTT reduction by SH-SY5Y cells in presence of various α-Syn species showing higher cytotoxicity (less MTT reduction) of isolated helix and material comprising helix-rich intermediates. LMW, matured fibrils (5 days aged sample) and Aβ (25–35) fibrils were used as controls. (C) Correlation plot of ANS binding (hydrophobic surface exposure) and MTT reduction-based cytotoxicity indicates that species possessing larger hydrophobic exposed surfaces are more toxic. (D) Phase contrast images for SHSY-5Y cells treated with different species of α-Syn indicating maximum cell death for isolated helix followed by fibrils. However the morphology of cells treated with LMW remained in normal state similar to control cells (treated with buffer). Scale bar represents 100 micron. (E) The calcein and EthD-1 staining showing defective morphology with damages of cell body/neuritis of cell treated with helix-rich intermediate. Cells treated with either LMW or buffer control showed no significant of cell death/defective cell morphology. Scale bar represents 200 micron. (F) The flow cytometry analysis showing that induction of early apoptosis by cells treated with helix-rich state. The cells treated with matured fibrils and LMW samples did not show significant toxicity. Buffer was used as a control. Quadrants Q1, Q2, Q3 and Q4 represent dead cells, late apoptotic/necrotic cells, live cells and early apoptotic cells, respectively.

Mentions: It has been suggested that the extent of hydrophobic surface exposure may play a significant role in cellular toxicity of protein aggregates6566. To determine the hydrophobic surface exposure of the helical intermediate and other α-Syn species, 1-anilinonaphthalene-8-sulfonate (ANS) binding study was performed with different species of α-Syn. ANS is a traditional dye frequently used for protein folding studies to detect a molten globule state67. It binds to exposed hydrophobic surfaces of proteins and, therefore, is able to monitor the relative hydrophobic surface exposure during folding and aggregation. To do the ANS binding, different species of α-Syn were isolated as described in the method section. Freshly prepared LMW was used as a negative control. The helix-rich state showed maximum ANS binding followed by isolated fibrils from the pellet purification step and fibrils collected at the end of the aggregation kinetics (Figure 10A and Supplementary Figure 12). LMW α-Syn showed least ANS binding. The increased hydrophobicity of the helix intermediate must come either from the oligomer formation or/and a structural rearrangement that brings together in closely space hydrophobic amino acid side chains. The data suggest the isolated helical intermediate contained more exposed hydrophobic surface area compared to fibrils and exposed hydrophobic surface area is least for the soluble protein (Figure 10ASupplementary Figure 12).


Structure based aggregation studies reveal the presence of helix-rich intermediate during α-Synuclein aggregation.

Ghosh D, Singh PK, Sahay S, Jha NN, Jacob RS, Sen S, Kumar A, Riek R, Maji SK - Sci Rep (2015)

Hydrophobic surface exposure and toxicity of helix-rich intermediate.(A) ANS fluorescence of different α-Syn species indicate maximum exposed hydrophobic surface for isolated helix-rich state. (B) MTT reduction by SH-SY5Y cells in presence of various α-Syn species showing higher cytotoxicity (less MTT reduction) of isolated helix and material comprising helix-rich intermediates. LMW, matured fibrils (5 days aged sample) and Aβ (25–35) fibrils were used as controls. (C) Correlation plot of ANS binding (hydrophobic surface exposure) and MTT reduction-based cytotoxicity indicates that species possessing larger hydrophobic exposed surfaces are more toxic. (D) Phase contrast images for SHSY-5Y cells treated with different species of α-Syn indicating maximum cell death for isolated helix followed by fibrils. However the morphology of cells treated with LMW remained in normal state similar to control cells (treated with buffer). Scale bar represents 100 micron. (E) The calcein and EthD-1 staining showing defective morphology with damages of cell body/neuritis of cell treated with helix-rich intermediate. Cells treated with either LMW or buffer control showed no significant of cell death/defective cell morphology. Scale bar represents 200 micron. (F) The flow cytometry analysis showing that induction of early apoptosis by cells treated with helix-rich state. The cells treated with matured fibrils and LMW samples did not show significant toxicity. Buffer was used as a control. Quadrants Q1, Q2, Q3 and Q4 represent dead cells, late apoptotic/necrotic cells, live cells and early apoptotic cells, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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f10: Hydrophobic surface exposure and toxicity of helix-rich intermediate.(A) ANS fluorescence of different α-Syn species indicate maximum exposed hydrophobic surface for isolated helix-rich state. (B) MTT reduction by SH-SY5Y cells in presence of various α-Syn species showing higher cytotoxicity (less MTT reduction) of isolated helix and material comprising helix-rich intermediates. LMW, matured fibrils (5 days aged sample) and Aβ (25–35) fibrils were used as controls. (C) Correlation plot of ANS binding (hydrophobic surface exposure) and MTT reduction-based cytotoxicity indicates that species possessing larger hydrophobic exposed surfaces are more toxic. (D) Phase contrast images for SHSY-5Y cells treated with different species of α-Syn indicating maximum cell death for isolated helix followed by fibrils. However the morphology of cells treated with LMW remained in normal state similar to control cells (treated with buffer). Scale bar represents 100 micron. (E) The calcein and EthD-1 staining showing defective morphology with damages of cell body/neuritis of cell treated with helix-rich intermediate. Cells treated with either LMW or buffer control showed no significant of cell death/defective cell morphology. Scale bar represents 200 micron. (F) The flow cytometry analysis showing that induction of early apoptosis by cells treated with helix-rich state. The cells treated with matured fibrils and LMW samples did not show significant toxicity. Buffer was used as a control. Quadrants Q1, Q2, Q3 and Q4 represent dead cells, late apoptotic/necrotic cells, live cells and early apoptotic cells, respectively.
Mentions: It has been suggested that the extent of hydrophobic surface exposure may play a significant role in cellular toxicity of protein aggregates6566. To determine the hydrophobic surface exposure of the helical intermediate and other α-Syn species, 1-anilinonaphthalene-8-sulfonate (ANS) binding study was performed with different species of α-Syn. ANS is a traditional dye frequently used for protein folding studies to detect a molten globule state67. It binds to exposed hydrophobic surfaces of proteins and, therefore, is able to monitor the relative hydrophobic surface exposure during folding and aggregation. To do the ANS binding, different species of α-Syn were isolated as described in the method section. Freshly prepared LMW was used as a negative control. The helix-rich state showed maximum ANS binding followed by isolated fibrils from the pellet purification step and fibrils collected at the end of the aggregation kinetics (Figure 10A and Supplementary Figure 12). LMW α-Syn showed least ANS binding. The increased hydrophobicity of the helix intermediate must come either from the oligomer formation or/and a structural rearrangement that brings together in closely space hydrophobic amino acid side chains. The data suggest the isolated helical intermediate contained more exposed hydrophobic surface area compared to fibrils and exposed hydrophobic surface area is least for the soluble protein (Figure 10ASupplementary Figure 12).

Bottom Line: Using a variety of complementary biophysical techniques monitoring entire pathway of nine different synucleins, we found that transition of unstructured conformation into β-sheet rich fibril formation involves helix-rich intermediates.A multidimensional NMR study characterizing the intermediate accompanied with site-specific fluorescence study suggests that the N-terminal and central portions mainly participate in the helix-rich intermediate formation while the C-terminus remained in an extended conformation.However, significant conformational transitions occur at the middle and at the C-terminus during helix to β-sheet transition as evident from Trp fluorescence study.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosciences and Bioengineering, IIT Bombay, Powai, Mumbai, India 400076.

ABSTRACT
Mechanistic understanding of nucleation dependent polymerization by α-synuclein (α-Syn) into toxic oligomers and amyloids is important for the drug development against Parkinson's disease. However the structural and morphological characterization during nucleation and subsequent fibrillation process of α-Syn is not clearly understood. Using a variety of complementary biophysical techniques monitoring entire pathway of nine different synucleins, we found that transition of unstructured conformation into β-sheet rich fibril formation involves helix-rich intermediates. These intermediates are common for all aggregating synucleins, contain high solvent-exposed hydrophobic surfaces, are cytotoxic to SHSY-5Y cells and accelerate α-Syn aggregation efficiently. A multidimensional NMR study characterizing the intermediate accompanied with site-specific fluorescence study suggests that the N-terminal and central portions mainly participate in the helix-rich intermediate formation while the C-terminus remained in an extended conformation. However, significant conformational transitions occur at the middle and at the C-terminus during helix to β-sheet transition as evident from Trp fluorescence study. Since partial helix-rich intermediates were also observed for other amyloidogenic proteins such as Aβ and IAPP, we hypothesize that this class of intermediates may be one of the important intermediates for amyloid formation pathway by many natively unstructured protein/peptides and represent a potential target for drug development against amyloid diseases.

Show MeSH
Related in: MedlinePlus