@article {110446, title = {Elongation of Fibrils Formed by a Tau Fragment is Inhibited by a Transient Dimeric Intermediate.}, journal = {J Phys Chem B}, year = {2022}, month = {2022 May 03}, abstract = {

The formation and propagation of aggregates of the tau protein in the brain are associated with the tauopathy group of neurodegenerative diseases. Different tauopathies have been shown to be associated with structurally distinct aggregates of tau. However, the mechanism by which different structural folds arise remains poorly understood. In this study of fibril formation by the fragment tau-K18 of tau, it is shown that the Lys 280 {\textrightarrow} Glu mutation in the variant tau-K18 K280E forms fibrils that are morphologically distinct from those formed by wild-type (wt) tau-K18. The mutant fibrils appear to have two protofilaments twisted around each other, whereas the wt fibrils are straight and appear to have a single protofilament. Modeling the kinetics of seeded aggregation, using a simple Michaelis-Menten-like mechanism, reveals that the two morphologically distinct fibrils are elongated with different catalytic efficiencies. Surprisingly, when the elongation of monomeric tau-K18 is seeded with tau-K18 K280E fibrils, it is seen to be inhibited at high monomer concentrations. Such inhibition is not seen when elongation is seeded with tau-K18 fibrils. The mechanism of inhibition is shown to be describable as uncompetitive inhibition, in which a transient dimeric form of tau-K18 acts as an uncompetitive inhibitor. Importantly, a dimeric form of tau-K18 is seen to be populated to a detectable extent early during aggregation. A covalently linked tau dimer, with an inter-molecular disulphide linkage, is shown to be capable of acting as an inhibitor. In summary, a quantitative kinetic approach has provided an understanding of how the formation of distinct structural folds of tau fibrils can be modulated by mutation and how the elongation of one fibril type, but not the other, is inhibited by a transiently formed dimer.

}, issn = {1520-5207}, doi = {10.1021/acs.jpcb.1c10752}, author = {Kumar, Harish and Udgaonkar, Jayant B} } @article {115046, title = {α-Synuclein aggregation intermediates form fibril polymorphs with distinct prion-like properties.}, journal = {J Mol Biol}, year = {2022}, month = {2022 Jul 27}, pages = {167761}, abstract = {

α-Synuclein (α-Syn) amyloids in synucleinopathies are suggested to be structurally and functionally diverse, reminiscent of prion-like strains. But how the aggregation of the same precursor protein results in the formation of fibril polymorphs remains elusive. Here, we demonstrate the structure-function relationship of two polymorphs, pre-matured fibrils (PMFs) and helix-matured fibrils (HMFs), based on α-Syn aggregation intermediates. These polymorphs display the structural differences as demonstrated by solid-state NMR and mass spectrometry studies and also possess different cellular activities such as seeding, internalization, and cell-to-cell transfer of aggregates. HMFs with a compact core structure exhibit low seeding potency but readily internalize and transfer from one cell to another. The less structured PMFs lack transcellular transfer ability but induce abundant α-Syn pathology and trigger the formation of aggresomes in cells. Overall, the study highlights that the conformational heterogeneity in the aggregation pathway may lead to fibril polymorphs with distinct prion-like behavior.

}, issn = {1089-8638}, doi = {10.1016/j.jmb.2022.167761}, author = {Mehra, Surabhi and Ahlawat, Sahil and Kumar, Harish and Datta, Debalina and Navalkar, Ambuja and Singh, Nitu and Patel, Komal and Gadhe, Laxmikant and Kadu, Pradeep and Kumar, Rakesh and Jha, Narendra N and Sakunthala, Arunima and Sawner, Ajay S and Padinhateeri, Ranjith and Udgaonkar, Jayant B and Agarwal, Vipin and Maji, Samir K} } @article {88406, title = {The Lys 280 ➔ Gln mutation mimicking disease-linked acetylation of Lys 280 in tau extends the structural core of fibrils and modulates their catalytic properties.}, journal = {Protein Sci}, year = {2021}, month = {2021 Jan 25}, abstract = {

Amyloid fibrillar aggregates isolated from the brains of patients with neurodegenerative diseases invariably have post-translational modifications (PTMs). The roles that PTMs play in modulating the structures and polymorphism of amyloid aggregates, and hence their ability to catalyze the conversion of monomeric protein to their fibrillar structure is, however, poorly understood. This is particularly true in the case of tau aggregates, where specific folds of fibrillar tau have been implicated in specific tauopathies. Several PTMs, including acetylation at Lys 280, increase aggregation of tau in the brain, and increase neurodegeneration. In this study, tau-K18 K280Q, in which the Lys 280 ➔ Gln mutation is used to mimic acetylation at Lys 280, is shown, using HX-MS measurements, to form fibrils with a structural core that is longer than that of tau-K18 fibrils. Measurements of critical concentrations show that the binding affinity of monomeric tau-K18 for its fibrillar counterpart is only marginally more than that of monomeric tau-K18 K280Q for its fibrillar counterpart. Quantitative analysis of the kinetics of seeded aggregation, using a simple Michaelis-Menten-like model, in which the monomer first binds and then undergoes conformational conversion to β-strand, shows that the fibrils of tau-K18 K280Q convert monomeric protein more slowly than do fibrils of tau-K18. In contrast, monomeric tau-K18 K280Q is converted faster to fibrils than is monomeric tau-K18. Thus, the effect of Lys 280 acetylation on tau aggregate propagation in brain cells is expected to depend on the amount of acetylated tau present, and on whether the propagating seed is acetylated at Lys 280 or not. This article is protected by copyright. All rights reserved.

}, issn = {1469-896X}, doi = {10.1002/pro.4030}, author = {Kumar, Harish and Udgaonkar, Jayant B} } @article {99931, title = {Microsecond dynamics during the binding-induced folding of an intrinsically disordered protein.}, journal = {J Mol Biol}, year = {2021}, month = {2021 Sep 16}, pages = {167254}, abstract = {

Tau is an intrinsically disordered protein implicated in many neurodegenerative diseases. The repeat domain fragment of tau, tau-K18, is known to undergo a disorder to order transition in the presence of lipid micelles and vesicles, in which helices form in each of the repeat domains. Here, the mechanism of helical structure formation, induced by a phospholipid mimetic, sodium dodecyl sulfate (SDS) at sub-micellar concentrations, has been studied using multiple biophysical probes. A study of the conformational dynamics of the disordered state, using photoinduced electron transfer coupled to fluorescence correlation spectroscopy (PET-FCS) has indicated the presence of an intermediate state, I, in equilibrium with the unfolded state, U. The cooperative binding of the ligand (L), SDS, to I has been shown to induce the formation of a compact, helical intermediate (IL) within the dead time (\~{}37 {\textmu}s) of a continuous flow mixer. Quantitative analysis of the PET-FCS data and the ensemble microsecond kinetic data, suggests that the mechanism of induction of helical structure can be described by a U <-> I <-> IL <-> FL mechanism, in which the final helical state, FL, forms from IL with a time constant of 50-200 {\textmu}s. Finally, it has been shown that the helical conformation is an aggregation-competent state that can directly form amyloid fibrils.

}, issn = {1089-8638}, doi = {10.1016/j.jmb.2021.167254}, author = {Sen, Sreemantee and Kumar, Harish and Udgaonkar, Jayant B} } @article {56128, title = {Mechanistic approaches to understand the prion-like propagation of aggregates of the human tau protein.}, journal = {Biochim Biophys Acta Proteins Proteom}, volume = {1867}, year = {2019}, month = {2019 Apr 12}, pages = {922-932}, abstract = {

The dynamic nature of the tau protein under physiological conditions is likely to be critical for it to perform its diverse functions inside a cell. Under some conditions, this intrinsically disordered protein assembles into pathogenic aggregates that are self-perpetuating, toxic and infectious in nature. The role of liquid-liquid phase separation in the initiation of the aggregation reaction remains to be delineated. Depending on the nature of the aggregate, its structure, and its localization, neurodegenerative disorders with diverse clinical features are manifested. The prion-like mechanism by which these aggregates propagate and spread across the brain is not well understood. Various factors (PTMs, mutations) have been strongly associated with the pathological aggregates of tau. However, little is known about how these factors modulate the pathological properties linked to aggregation. This review describes the current progress towards understanding the mechanism of propagation of tau aggregates.

}, issn = {1878-1454}, doi = {10.1016/j.bbapap.2019.04.004}, author = {Kumar, Harish and Udgaonkar, Jayant B} } @article {43838, title = {Mechanistic and Structural Origins of the Asymmetric Barrier to Prion-like Cross-Seeding between Tau-3R and Tau-4R.}, journal = {J Mol Biol}, year = {2018}, month = {2018 Sep 26}, abstract = {

The spread and deposition of infectious fibrillar protein aggregates in the brain via a prion-like mechanism, is a critical component in the patho-physiology of various neurodegenerative diseases, including the tauopathies. In tauopathies, two isoforms of tau, containing three and four microtubule binding repeats (MTBRs), are found to aggregate, and the type of isoform present in aggregates, determines the type of tauopathy. Cross-seeding between the two tau isoforms is limited by an asymmetric barrier, similar to the species barrier that restricts prion transmission across species, whose origin has remained unclear. In this study, the growth of the tau fibrils is shown to be describable by a two-step Michaelis-Menten-like model. Delineation of the mechanism as a Michaelis-Menten-like mechanism has enabled a quantitative understanding of the asymmetric seeding barrier that exists between two isoforms of tau, tau-K18 and tau-K19 (which differ in containing four and three MTBRs, respectively), wherein tau-K18 fibrils cannot seed tau-K19 monomer. Furthermore, high-resolution structural analysis of the two isoforms shows that the structural core is more ordered in tau-K19 than in tau-K18. Hence, the current work provides kinetic and structural rationales for asymmetric seeding barriers in general, and for the two tau isoforms in particular.

}, issn = {1089-8638}, doi = {10.1016/j.jmb.2018.09.010}, author = {Kumar, Harish and Udgaonkar, Jayant B} } @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} }