Abstract
Amyloid fibril formation involves three steps; structural perturbation, nucleation and elongation. We have investigated amyloidogenesis using prion protein as a model system and UV-light as a structural perturbant. We find that UV-exposed prion protein fails to form amyloid fibrils. Interestingly, if provided with pre-formed fibrils as seeds, UV-exposed prion protein formed amyloid fibrils albeit with slightly different morphology. Atomic force microscopy and electron microscopic studies clearly show the formation of fibrils under these conditions. Circular dichroism study shows loss in helicity in UV-exposed protein. UV-exposed prion protein fails to form amyloid fibrils. However, it remains competent for fibril extension, suggesting that UV-exposure results in loss of nucleating capability. This work opens up possibility of segregating nucleation and elongation step of amyloidogenesis, facilitating screening of new drug candidates for specifically inhibiting either of these processes. In addition, the work also highlights the importance of light-induced structural and functional alterations which are important in protein based therapeutics.
Highlights
Transmissible Spongiform Encephalopathy is a group of diseases such as Kuru, Creutzfeldt - Jakob disease (CJD), Gerstmann-Straussler Syndrome (GSS), and Fatal Familial Insomnia (FFI) characterized by neurodegeneration and deposition of amyloid plaques
Amyloidogenesis of prion protein can be induced in 3 M urea and 1 M Guanidinium chloride (GdmCl) with prolonged incubation and /or addition of pre-formed fibrils as seeds
In 3 M urea and 1 M GdmCl did not show any aggregation during the period of experiment (Figure 1A)
Summary
Transmissible Spongiform Encephalopathy is a group of diseases such as Kuru, Creutzfeldt - Jakob disease (CJD), Gerstmann-Straussler Syndrome (GSS), and Fatal Familial Insomnia (FFI) characterized by neurodegeneration and deposition of amyloid plaques. Conformational transition of cellular prion protein (PrPc) is believed to be the major cause for these diseases [1]. Nucleation process is the rate-limiting step due to kinetically disfavored oligomerization (self assembly) of intermediates These assemblies are partially concentration-dependent [11] and show presence of hydrophobic cooperativity in the process [12]. This rate-limiting phase is reflected as the lag phase in kinetics of amyloid formation. Binding of monomer to continuously growing fiber and subsequent conformational change characterize this event [15,16] These amyloid aggregates show congo-red birefringence and cross-b-sheet structure. The organization of these fibrils remains the same amongst different types of proteins- unbranched 2–3 subprotofibrils (10–15 A ) helically arrange to form protofilaments (protofibril) (25–30 A ), which associate laterally or twisted in bundle of five to form mature fibrils [17]
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