Biology subjects

Chatani, E.

Publications and source records attributed to Chatani, E..

6 recordsLinked to original sources

bioRxiv2026

Local aromatic interactions define temperature sensitivity of phase separation in an intrinsically disordered protein.

Liquid-liquid phase separation (LLPS) of intrinsically disordered proteins is highly sensitive to environmental conditions, yet the molecular basis of sharp temperature responsiveness remains poorly understood. Here, we investigate a sequence-encoded mechanism underlying the pronounced temperature sensitivity of phase separation using Sup35NM, the intrinsically disordered domain of the yeast prion Sup35. We show that a tyrosine-rich local structural region within this domain encodes strong temperature responsiveness of droplet formation. Mutational analyses reveal that tyrosine residues mediate both intramolecular interactions that stabilize local structure and intermolecular interactions required for LLPS. Substitution of the tyrosine residues with alanine disrupts local structure and weakens intermolecular interactions, thereby diminishing temperature sensitivity. In contrast, substitution with phenylalanine promotes rapid droplet gelation, abolishes internal fluidity, suppresses amyloid formation, and confers resistance to temperature-induced dissolution. Based on these findings, we propose a molecular model in which finely tuned, moderately weak aromatic interactions among tyrosines enable reversible local compaction that is sensitive to temperature, thereby generating a sharp phase transition. These results suggest that amino acid sequences encode not only phase separation propensity but also the sensitivity of condensates to environmental perturbations, providing a framework for understanding how intrinsically disordered proteins act as molecular sensors of cellular conditions.

biochemistry↗

bioRxiv2023

Mechanistic modeling of amyloid oligomer and protofibril formation

Early phase of amyloid formation, where prefibrillar aggregates such as oligomers and protofibrils are often observed, is crucial for elucidating pathogenesis. However, since oligomers and protofibrils form transiently and heterogeneously, the detailed mechanisms of their formation remain unclear. Here, we have investigated the early aggregation process of bovine and human insulin by static and dynamic light scattering in combination with thioflavin T (ThT) fluorescence and Fourier transform infrared (FTIR) spectroscopy. The time dependence of light scattering has revealed that oligomers and protofibrils form in bovine insulin, in contrast to no significant aggregation in human insulin. By focusing on bovine insulin for kinetic analysis, it has been revealed that the protofibril formation process was divided into two steps with reference to fractal dimension. When modeled the experimental data of static and dynamic light scattering based on the Smoluchowski aggregation kinetics with fractal aggregation and end-to-end association, we found the initial formation of spherical oligomers and their subsequent uniaxial docking. Furthermore, the analysis of temperature and salt concentration dependence revealed that the end-to-end association is the rate-limiting step, where structure organization occurred with dehydration. The established model for protofibril formation where oligomers are incorporated as a precursor provides insight into the molecular mechanism how protein molecules assemble during the early stage of amyloid formation. SignificanceAmyloid oligomers and protofibrils have attracted attention as critical causes of neurodegenerative diseases; however, detailed formation processes of these aggregates have been poorly understood. In this study, we established a mechanistic model of oligomer and protofibril formation of bovine insulin based on Smoluchowski aggregation kinetics in terms of static and dynamic light scattering. It has been demonstrated that early aggregation proceeds by initial fractal-like aggregation to form oligomers, and subsequent their end-to-end docking to form protofibrils. The latter step is a rate-limiting step, where structural organization occurs with dehydration. The established model is expected to broadly applicable to a variety of proteins, and thus will provides valuable insights for accelerating therapeutic development and anti-neurodegenerative drug design.

biophysics↗

bioRxiv2021

Structural development of amyloid precursors in insulin B chain and the inhibition effect by fibrinogen

Amyloid fibrils are abnormal protein aggregates that relate to a large number of amyloidoses and neurodegenerative diseases. The oligomeric precursors, or prefibrillar intermediates, which emerge prior to the amyloid fibril formation, have been known to play a crucial role for the formation. Therefore, it is essential to elucidate the mechanisms of the structural development of the prefibrillar intermediates and ways to prevent its fibril formation. An insulin-derived peptide, insulin B chain, has been known for its stable accumulation of the prefibrillar intermediates. In this study, structural development of B chain prefibrillar intermediates was monitored by transmission electron microscopy and small-angle X-ray scattering combined with size exclusion chromatography and solid-state NMR spectroscopy to elucidate the stability and secondary structure. We further tracked its inhibition process by fibrinogen (Fg), which has been known to effectively prevent the amyloid fibril formation of B chain. We demonstrated that prefibrillar intermediates are wavy structures with low {beta}-sheet content, growing in a multistep manner toward the nucleation for the amyloid fibril formation. In the presence of Fg, the formation of the prefibrillar intermediates slowed down by forming specific complexes. These observations suggest that the prefibrillar intermediates serve as reaction fields for the nucleation and its propagation for the amyloid fibril formation, whereas the inhibition of prefibrillar intermediate elongation by Fg is the significant factor to suppress the fibril formation. We propose that the obtained molecular picture could be a general inhibition mechanism of the amyloid fibril formation by the inhibitors.

biochemistry↗

bioRxiv2020

Multistep changes in amyloid structure that are induced by cross-seeding on a rugged energy landscape

Amyloid fibrils are aberrant protein aggregates associated with various amyloidoses and neurodegenerative diseases. It is recently indicated that structural diversity of amyloid fibrils often results in different pathological phenotypes including cytotoxicity and infectivity. The diverse structures are predicted to propagate by seed-dependent growth, which is one of the characteristic properties of amyloid fibrils. However, much remains unknown regarding how exactly the amyloid structures are inherited to subsequent generations by seeding reaction. Here, we investigated the behaviors of self- and cross-seeding of amyloid fibrils of human and bovine insulin in terms of thioflavin T fluorescence, morphology, secondary structure, and iodine staining. Insulin amyloid fibrils exhibited different structures depending on species, and each of which replicated in self-seeding. In contrast, gradual structural changes were observed in cross-seeding, and a new-type amyloid structure with distinct morphology and cytotoxicity was formed when human insulin was seeded with bovine insulin fibrils. Remarkably, iodine staining tracked changes in amyloid structure sensitively, and singular value decomposition (SVD) analysis of the UV-Vis absorption spectra of the fibril-bound iodine has revealed the presence of one or more intermediate metastable states during the structural changes. From these findings, we propose a propagation scheme with multistep structural changes in cross-seeding between two heterologous proteins, which is accounted for as a consequence of the rugged energy landscape of amyloid formation.

biochemistry↗

bioRxiv2020

Iodine staining as a useful probe for distinguishing amyloid fibril polymorphs

ABSTRACTIt is recently suggested that amyloid polymorphism, i.e., structural diversity of amyloid fibrils, has a deep relationship with pathology. However, its prompt recognition is almost halted due to insufficiency of analytical methods for detecting polymorphism of amyloid fibrils sensitively and quickly. Here, we propose that iodine staining, a historically known reaction that was firstly found by Virchow, can be used as a method for distinguishing amyloid polymorphs. When insulin fibrils were prepared and iodine-stained, they exhibited different colors depending on polymorphs. Each of the colors was inherited to daughter fibrils by seeding reactions. The colors were fundamentally represented as a sum of three absorption bands in visible region between 400-750 nm, and the bands showed different titration curves against iodine, suggesting that there are three specific iodine binding sites. The analysis of resonance Raman spectra and polarization microscope suggested that several polyiodide ions composed of I3− and/or I5− were formed on the grooves or the edges of β-sheets. It was concluded that the polyiodide species and conformations formed are sensitive to surface structure of amyloid fibrils, and the resultant differences in color will be useful for detecting polymorphism in a wide range of diagnostic samples.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗

bioRxiv2019

Exploration of insulin amyloid polymorphism using Raman spectroscopy and imaging

We aimed to investigate insulin amyloid fibril polymorphism caused by salt effects and heating temperature, and to visualize the structural differences of the polymorphisms in situ using Raman imaging without labeling. The time course monitoring for amyloid formation was carried out in an acidic condition without any salts and with two species of salts (NaCl and Na2SO4) by heating at 60, 70, 80, and 90 . The intensity ratio of two Raman bands at 1672 and 1657 cm-1 due to {beta}-sheet and -helix structures was revealed to be an indicator of amyloid fibril formation, and the relative proportion of the {beta}-sheet structure was higher in the case with salts, especially at a higher temperature and with Na2SO4. In conjunction with the secondary structural changes of proteins, the S-S stretching vibrational mode of a disulfide bond ([~]514 cm-1) and the ratio of the tyrosine doublet R(I850/I826) were also found to be markers distinguishing polymorphisms of insulin amyloid fibrils by principal component analysis (PCA). Especially, amyloid fibrils with Na2SO4 media formed the g-g-g conformation of disulfide bond at a higher rate and without any salts; on the contrary, the g-g-g conformation was partially transformed into the g-g-t conformation at higher temperatures. The different environments of the hydroxyl groups of the tyrosine residue were assumed to be caused by fibril polymorphism. Raman imaging using these marker bands also successfully visualized the two- and three-dimensional structural differences of amyloid polymorphisms. The present results indicate the potential of Raman imaging as a diagnostic tool for polymorphisms in tissues of amyloid-related diseases.\n\nStatement of SignificanceOur results revealed three Raman markers distinguishing amyloid fibril polymorphisms caused by salt and temperature effects; the relative proportion of protein secondary structures (-helix and {beta}-sheet), the ratio of tyrosine doublet, and the conformational differences of disulfide bonds. The lower values of tyrosine doublet in the case with salts were interpreted as the anions rob the hydration water from proteins which induced protein misfolding. Using these parameters, Raman images captured their higher order structural differences in situ without labeling. The images of hydrogen bonds strength variations due to tyrosine doublet is believed to include significant novelty. The present results imply the potential of Raman imaging for use as a diagnostic imaging tool for tissues with amyloid-induced diseases.

biophysics↗