Kinetic Modulation of Amyloid-ß (1-42) Aggregation and Toxicity by Structure-Based Rational Design.

Several point mutations can modulate protein structure and dynamics, leading to different natures. Especially in the case of amyloidogenic proteins closely related to neurodegenerative diseases, structural changes originating from point mutations can affect fibrillation kinetics. Herein, we rationally designed mutant candidates to inhibit the fibrillation process of amyloid-ß with its point mutants through multistep in silico analyses. Our results showed that the designed mutants induced kinetic self-assembly suppression and reduced the toxicity of the aggregate. A multidisciplinary biophysical approach with small-angle X-ray scattering, ion mobility-mass spectrometry, mass spectrometry, and additional in silico experiments was performed to reveal the structural basis associated with the inhibition of fibril formation. The structure-based design of the mutants with suppressed self-assembly performed in this study could provide a different perspective for modulating amyloid aggregation based on the structural understanding of the intrinsically disordered proteins.

Gas-phase conformations of intrinsically disordered proteins and their complexes with ligands: Kinetically trapped states during transfer from solution to the gas phase.

Flexible structures of intrinsically disordered proteins (IDPs) are crucial for versatile functions in living organisms, which involve interaction with diverse partners. Electrospray ionization ion mobility mass spectrometry (ESI-IM-MS) has been widely applied for structural characterization of apo-state and ligand-associated IDPs via two-dimensional separation in the gas phase. Gas-phase IDP structures have been regarded as kinetically trapped states originated from conformational features in solution. However, an implication of the states remains elusive in the structural characterization of IDPs, because it is unclear what structural property of IDPs is preserved. Recent studies have indicated that the conformational features of IDPs in solution are not fully reproduced in the gas phase. Nevertheless, the molecular interactions captured in the gas phase amplify the structural differences between IDP conformers. Therefore, an IDP conformational change that is not observed in solution is observable in the gas-phase structures obtained by ESI-IM-MS. Herein, we have presented up-to-date researches on the key implications of kinetically trapped states in the gas phase with a brief summary of the structural dynamics of IDPs in ESI-IM-MS.

Probing drug delivery and mechanisms of action in 3D spheroid cells by quantitative analysis.

Human tumor cells in a 3-dimensional (3D) spheroid can reflect the characteristics of solid tumors by forming cell-cell interactions and microenvironments. This makes 3D cell culture useful for preclinical stability and drug efficacy tests. In this study, the drug delivery and action mechanisms in SK-N-SH neuroblastoma cells cultured in 3D spheroids were quantitatively compared to those cultured in 2D monolayers using confocal microscopy imaging and inductively coupled plasma-mass spectrometry. In the 3D spheroids, cisplatin only accessed the surface, accumulating in the cells on the spheroid exterior. As a result, an increased cellular amount of cisplatin was required to obtain similar cytotoxicity in the 3D spheroid cells to that in 2D monolayers. The mechanisms of reduction of drug efficacy by dimethyl sulfoxide (DMSO) in the 3D spheroid cells compared to those in the 2D monolayer cells were further investigated. DMSO reduced the drug cytotoxicity by forming stable DMSO-substituted compounds that inhibited the cellular uptake of cisplatin and DNA-Pt adduct formation. The quantitative analysis used in this study is promising for understanding drug delivery and drug action mechanisms in cells in various microenvironments.

Accurate Quantification of N-Glycolylneuraminic Acid in Therapeutic Proteins Using Supramolecular Mass Spectrometry.

Practical applications of innovative host-guest systems are challenging because of unexpected guest competitors and/or subtle environmental differences. Herein, a supramolecular mass spectrometry (MS)-based method using a synthetic host, cucurbit[7]uril (CB[7]), was developed for identifying and quantifying N-glycolylneuraminic acid (Neu5Gc) in therapeutic glycoproteins, which critically reduces drug efficacy. The development of a reliable derivatization-free analytical method for Neu5Gc is highly challenging because of the interference by the abundant N-acetylneuraminic acid (Neu5Ac). CB[7] recognized the subtle structural differences between Neu5Gc and Neu5Ac. Distinct host-guest interactions between CB[7] and the two sialic acids produced a highly linear relationship between the complexation and concentration proportions of the two sialic acids in MS. Furthermore, the developed method had sub-picomolar quantification limits and a wide range of applicability for diverse glycoproteins, demonstrating the potential utility of this method as a reliable assay of Neu5Gc in therapeutic glycoproteins.

Effect of packing density of lipid vesicles on the Aß42 fibril polymorphism.

The aggregation of amyloid-ß 1-42 (Aß42) on lipid membranes is closely related to the pathology of Alzheimer's disease (AD). Herein, we demonstrated the effect of the packing density of lipid vesicles on the Aß42 fibrillation kinetics and fibril morphology. We used three distinct phosphatidylcholine (PC) lipids, containing different numbers of cis-double bonds in acyl chains, and therefore, a different packing density in the lipid vesicles. Our results showed that the fibrillation of Aß42 was greatly enhanced and the formed fibrils became shorter as the number of double bonds in lipids increased. Due to the low-density characteristics of dioleoyl phosphatidylcholine (DOPC), Aß42 monomers were able to interact with the hydrophobic acyl chain of lipids exposed to the aqueous phase, thereby inducing rapid fibrillation and short fibril morphologies. Furthermore, the effects of the anionic lipids dioleoyl phosphatidylserine (DOPS) and dioleoyl phosphatidylglycerol (DOPG), and mixed vesicles of DOPC/DOPS and DOPC/DOPG on Aß42 fibrillations were investigated. The tight binding of Aß42 to the lipid head groups via electrostatic interactions was able to suppress the modulation of Aß42 fibrillations compared to accelerated fibrillations on loosely packed membranes. Our proposed mechanism regarding the influence of lipid packing density on Aß42 fibrillations provides an advanced understanding of lipid-associated amyloid fibrillations.

Ion Mobility Mass Spectrometry Analysis of Oxygen Affinity-Associated Structural Changes in Hemoglobin.

Hemoglobin (Hb) is a major oxygen-transporting protein with allosteric properties reflected in the structural changes that accompany binding of O2. Glycated hemoglobin (GHb), which is a minor component of human red cell hemolysate, is generated by a nonenzymatic reaction between glucose and hemoglobin. Due to the long lifetime of human erythrocytes (∼120 days), GHb is widely used as a reliable biomarker for monitoring long-term glucose control in diabetic patients. Although the structure of GHb differs from that of Hb, structural changes relating to the oxygen affinity of these proteins remain incompletely understood. In this study, the oxygen-binding kinetics of Hb and GHb are evaluated, and their structural dynamics are investigated using solution small-angle X-ray scattering (SAXS), electrospray ionization mass spectrometry equipped with ion mobility spectrometry (ESI-IM-MS), and molecular dynamic (MD) simulations to understand the impact of structural alteration on their oxygen-binding properties. Our results show that the oxygen-binding kinetics of GHb are diminished relative to those of Hb. ESI-IM-MS reveals structural differences between Hb and GHb, which indicate the preference of GHb for a more compact structure in the gas phase relative to Hb. MD simulations also reveal an enhancement of intramolecular interactions upon glycation of Hb. Therefore, the more rigid structure of GHb makes the conformational changes that facilitate oxygen capture more difficult creating a delay in the oxygen-binding process. Our multiple biophysical approaches provide a better understanding of the allosteric properties of hemoglobin that are reflected in the structural alterations accompanying oxygen binding.

ATP Kinetically Modulates Pathogenic Tau Fibrillations.

Advanced understanding of Alzheimer's disease (AD) and several tauopathies over the past decades indicates the pathological importance of tau aggregation in these diseases. Herein, we demonstrated that adenosine triphosphate (ATP), a highly charged anionic molecule found abundantly in the cytosol of cells, catalyzes fibrillation of tau as well as human islet amyloid polypeptide, a representative of basic intrinsically disordered proteins. Our results showed that ATP attracts multiple lysine residues of the four-repeat domain of tau (K18) via supramolecular complexation, thereby forming dimers that are converted to nuclei and accelerate fibril elongation. However, ATP was not directly incorporated into the K18 fibrils, suggesting that ATP plays the role of a catalyst, rather than a reactant, during K18 fibrillation. We also characterized the correlation between ATP dyshomeostasis and tau aggregation in the cellular environment. Our multiple biophysical approaches, including native mass spectrometry (MS), small-angle X-ray scattering (SAXS), and molecular dynamics (MD) simulation, provided insights into the molecular-level influence of ATP on the structural changes and fibrillation of tau.

Highly active ruthenium metathesis catalysts enabling ring-opening metathesis polymerization of cyclopentadiene at low temperatures.

Development of versatile ruthenium olefin-metathesis catalysts with high activity, stability, and selectivity is a continuous challenge. Here we report highly controllable ruthenium catalysts using readily accessible and versatile N-vinylsulfonamides as carbene precursors. Catalyst initiation rates were controlled in a straightforward manner, from latent to fast initiating, through the facile modulation of the N-vinylsulfonamide ligands. Trifluoromethanesulfonamide-based catalysts initiated ultrarapidly even at temperatures as low as -60 °C and continuously propagated rapidly, enabling the enthalpically and entropically less-favored ring-opening metathesis polymerizations of low-strained functionalized cyclopentene derivatives, some of which are not accessible with previous olefin-metathesis catalysts. To our surprise, the developed catalysts facilitated the polymerization of cyclopentadiene (CPD), a feedstock that is easily and commonly obtainable through the steam cracking of naphtha, which has, to the best of our knowledge, not been previously achieved due to its low ring strain and facile dimerization even at low temperatures (below 0 °C).

Electrostatic and hydrophobic interactions of lipid-associated α-synuclein: The role of a water-limited interfaces in amyloid fibrillation.

Human α­synuclein (αSyn) is an intrinsically disordered protein (IDP) whose biological and pathological functions in brain neuronal cells have not yet been fully elucidated. αSyn intrinsically participates in aiding neurotransmitter trafficking through αSyn the association with lipid membranes. However, lipid-associated states of αSyn also induce amyloid self-assembly that is linked to the pathogenesis of various synucleinopathies. These contradicting actions arise from the limited water content near lipid-water interfaces that controls αSyn electrostatic and hydrophobic interactions. Thus, understanding the molecular interactions between αSyn and lipid membranes in the presence of water molecules is critical in elucidating the pivotal role of lipid-associated αSyn in amyloid self-assembly. In this review, we describe how the membrane interface controls electrostatic and hydrophobic interactions of lipid-associated αSyn. Moreover, membrane amyloid self-assembly of αSyn will be further discussed with regards to the structural dynamics of lipid-associated αSyn and water molecules near the interface.

Distinct Fragmentation Pathways of Anticancer Drugs Induced by Charge-Carrying Cations in the Gas Phase.

With the growth of the pharmaceutical industry, structural elucidation of drugs and derivatives using tandem mass spectrometry (MS2) has become essential for drug development and pharmacokinetics studies because of its high sensitivity and low sample requirement. Thus, research seeking to understand fundamental relationships between fragmentation patterns and precursor ion structures in the gas phase has gained attention. In this study, we investigate the fragmentation of the widely used anticancer drugs, doxorubicin (DOX), vinblastine (VBL), and vinorelbine (VRL), complexed by a singly charged proton or alkali metal ion (Li+, Na+, K+) in the gas phase. The drug-cation complexes exhibit distinct fragmentation patterns in tandem mass spectra as a function of cation size. The trends in fragmentation patterns are explicable in terms of structures derived from ion mobility mass spectrometry (IM-MS) and theoretical calculations. Graphical Abstract ᅟ.