@article {21473, title = {Modulation of the extent of structural heterogeneity in α-synuclein fibrils by the small molecule thioflavin T.}, journal = {J Biol Chem}, year = {2017}, month = {2017 Jul 31}, abstract = {

The transition of intrinsically disordered, monomeric α-synuclein into β-sheet-rich oligomers and fibrils is associated with multiple neurodegenerative diseases. Fibrillar aggregates possessing distinct structures that differ in their toxicity, have been observed in different pathological phenotypes. Understanding the mechanism of formation of various fibril polymorphs with differing cytotoxic effects, is essential for determining how the aggregation reaction could be modulated to favor non-toxic fibrils over toxic fibrils. In this study, two morphologically different α-synuclein fibrils, one helical and the other ribbon-like, are shown to form together. Surprisingly, a widely-used small molecule for probing aggregation reactions, thioflavin T (ThT), is found to tune the structural heterogeneity found in the fibrils. The ribbon-like fibrils formed in the presence of ThT are found to have a longer structural core than do the helical fibrils formed in the absence of ThT. The ribbon-like fibrils are also more toxic to cells. By facilitating the formation of ribbon-like fibrils over helical fibrils, ThT reduces the extent of fibril polymorphism. This study highlights the role of a small molecule such as ThT in selectively favoring the formation of a specific type of fibril, by binding to aggregates formed early on one of multiple pathways, thereby altering the structural core and external morphology of the fibrils formed.

}, issn = {1083-351X}, doi = {10.1074/jbc.M117.795617}, author = {Kumar, Harish and Singh, Jogender and Kumari, Pratibha and Udgaonkar, Jayant B} } @article {3540, title = {The Pathogenic A116V Mutation Enhances Ion-Selective Channel Formation by Prion Protein in Membranes.}, journal = {Biophys J}, volume = {110}, year = {2016}, month = {2016 Apr 26}, pages = {1766-76}, abstract = {

Prion diseases are a group of fatal neurodegenerative disorders that afflict mammals. Misfolded and aggregated forms of the prion protein (PrP(Sc)) have been associated with many prion diseases. A transmembrane form of PrP favored by the pathogenic mutation A116V is associated with Gerstmann-Str{\"a}ussler-Scheinker syndrome, but no accumulation of PrP(Sc) is detected. However, the role of the transmembrane form of PrP in pathological processes leading to neuronal death remains unclear. This study reports that the full-length mouse PrP (moPrP) significantly increases the permeability of living cells to K(+), and forms K(+)- and Ca(2+)-selective channels in lipid membranes. Importantly, the pathogenic mutation A116V greatly increases the channel-forming capability of moPrP. The channels thus formed are impermeable to sodium and chloride ions, and are blocked by blockers of voltage-gated ion channels. Hydrogen-deuterium exchange studies coupled with mass spectrometry (HDX-MS) show that upon interaction with lipid, the central hydrophobic region (109-132) of the protein is protected against exchange, making it a good candidate for inserting into the membrane and lining the channel. HDX-MS also shows a dramatic increase in the protein-lipid stoichiometry for A116V moPrP, providing a rationale for its increased channel-forming capability. The results suggest that ion channel formation may be a possible mechanism of PrP-mediated neurodegeneration by the transmembrane forms of PrP.

}, issn = {1542-0086}, doi = {10.1016/j.bpj.2016.03.017}, author = {Sabareesan, Ambadi Thody and Singh, Jogender and Roy, Samrat and Udgaonkar, Jayant B and Mathew, M K} } @article {1169, title = {The pathogenic mutation T182A converts the prion protein into a molten globule-like conformation whose misfolding to oligomers but not to fibrils is drastically accelerated.}, journal = {Biochemistry}, volume = {55}, year = {2016}, month = {2016}, pages = {459-69}, abstract = {

Delineation of the effects of pathogenic mutations linked with familial prion diseases, on the structure and misfolding of prion protein (PrP), will be useful in understanding the molecular mechanism of PrP misfolding. Here, it has been shown that the pathogenic mutation T182A causes a drastic reduction in the apparent cooperativity and enthalpy of unfolding of the mouse prion protein (moPrP) under misfolding-prone conditions by converting the protein into a molten globule (MG)-like conformation. Hydrogen-deuterium exchange studies in conjunction with mass spectrometry indicate that the T182A mutation disrupts the core of the protein, thereby increasing overall structural dynamics. T182A moPrP is shown to misfold to oligomers very much faster than does wt moPrP, but to misfold to fibrils at a rate similar to that of wt moPrP. This observation suggests that oligomers are unlikely to play a productive role on the direct pathway of aggregation from monomer to fibrils. The observation that fully folded T182A moPrP has a MG-like structure, and that it misfolds to oligomers much faster than does wt moPrP, suggests that a MG-like intermediate, whose structure resembles that of fully folded T182A moPrP, might be populated early on the pathway of misfolding of wt moPrP to oligomers.

}, issn = {1520-4995}, doi = {10.1021/acs.biochem.5b01266}, author = {Singh, Jogender and Udgaonkar, Jayant B} } @article {3550, title = {Unraveling the Molecular Mechanism of pH-Induced Misfolding and Oligomerization of the Prion Protein.}, journal = {J Mol Biol}, volume = {428}, year = {2016}, month = {2016 Mar 27}, pages = {1345-55}, abstract = {

The misfolding of the prion protein (PrP) to aggregated forms is linked to several neurodegenerative diseases. Misfolded oligomeric forms of PrP are associated with neurotoxicity and/or infectivity, but the molecular mechanism by which they form is still poorly understood. A reduction in pH is known to be a key factor that triggers misfolded oligomer formation by PrP, but the residues whose protonation is linked with misfolding remain unidentified. The structural consequences of the protonation of these residues also remain to be determined. In the current study, amino acid residues whose protonation is critical for PrP misfolding and oligomerization have been identified using site-directed mutagenesis and misfolding/oligomerization assays. It is shown that the protonation of either H186 or D201, which mimics the effects of pathogenic mutations (H186R and D201N) at both residue sites, is critically linked to the stability, misfolding and oligomerization of PrP. Hydrogen-deuterium exchange studies coupled with mass spectrometry show that the protonation of either H186 or D201 leads to the same common structural change: increased structural dynamics in helix 1 and that in the loop between helix 1 and β-strand 2. It is shown that the protonation of either of these residues is sufficient for accelerating misfolded oligomer formation, most likely because the protonation of either residue causes the same structural perturbation. Hence, the increased structural dynamics in helix 1 and that in the loop between helix 1 and β-strand 2 appear to play an early critical role in acid-induced misfolding of PrP.

}, issn = {1089-8638}, doi = {10.1016/j.jmb.2016.01.030}, author = {Singh, Jogender and Udgaonkar, Jayant B} } @article {999, title = {Molecular Mechanism of the Misfolding and Oligomerization of the Prion Protein: Current Understanding and Its Implications.}, journal = {Biochemistry}, volume = {54}, year = {2015}, month = {2015 Jul 28}, pages = {4431-42}, abstract = {

Prion diseases, also known as transmissible spongiform encephalopathies, make up a group of fatal neurodegenerative disorders linked with the misfolding and aggregation of the prion protein (PrP). Although it is not yet understood how the misfolding of PrP induces neurodegeneration, it is widely accepted that the formation of misfolded prion protein (termed PrP(Sc)) is both the triggering event in the disease and the main component of the infectious agent responsible for disease transmission. Despite the clear involvement of PrP(Sc) in prion diseases, the exact composition of PrP(Sc) is not yet well-known. Recent studies show that misfolded oligomers of PrP could, however, be responsible for neurotoxicity and/or infectivity in the prion diseases. Hence, understanding the molecular mechanism of formation of the misfolded oligomers of PrP is critical for developing an understanding about the prion diseases and for developing anti-prion therapeutics. This review discusses recent advances in understanding the molecular mechanism of misfolded oligomer formation by PrP and its implications for the development of anti-prion therapeutics.

}, keywords = {Animals, Humans, Models, Molecular, Prion Diseases, Protein Folding, Protein Multimerization, Protein Structure, Quaternary, Protein Structure, Secondary, PrPC Proteins, PrPSc Proteins}, issn = {1520-4995}, doi = {10.1021/acs.biochem.5b00605}, author = {Singh, Jogender and Udgaonkar, Jayant B} } @article {1014, title = {Structural effects of multiple pathogenic mutations suggest a model for the initiation of misfolding of the prion protein.}, journal = {Angew Chem Int Ed Engl}, volume = {54}, year = {2015}, month = {2015 Jun 22}, pages = {7529-33}, abstract = {

A molecular understanding of the prion diseases requires delineation of the origin of misfolding of the prion protein (PrP). An understanding of how different disease-linked mutations affect the structure and dynamics of native monomeric PrP can provide a clue about how misfolding commences. In this study, hydrogen-deuterium exchange mass spectrometry was used to show that several disease-linked mutant variants, which are thermodynamically destabilized, share a common structural perturbation in their native states: helix 1 is destabilized to an extent that correlates well with the destabilization of the native protein. The mutant variants misfold and form oligomers faster than does the wild-type protein, at rates that increase exponentially with the extent to which helix 1 is destabilized in the native protein. It appears, therefore, that the loss of helix 1 structure marks the beginning of PrP misfolding and oligomerization.

}, issn = {1521-3773}, doi = {10.1002/anie.201501011}, author = {Singh, Jogender and Udgaonkar, Jayant B} }