Protein folding is the most fundamental and universal example of biomolecular self-organization and is characterized as an intramolecular process. In contrast, amyloidogenic proteins can interact ...with one another, leading to protein aggregation. The energy landscape of amyloid fibril formation is characterized by many minima for different competing low-energy structures and, therefore, is much more enigmatic than that of multiple folding pathways. Thus, to understand the entire energy landscape of protein aggregation, it is important to elucidate the full picture of conformational changes and polymorphisms of amyloidogenic proteins. This review provides an overview of the conformational diversity of amyloid-β (Aβ) characterized from experimental and theoretical approaches. Aβ exhibits a high degree of conformational variability upon transiently interacting with various binding molecules in an unstructured conformation in a solution, forming an α-helical intermediate conformation on the membrane and undergoing a structural transition to the β-conformation of amyloid fibrils. This review also outlines the structural polymorphism of Aβ amyloid fibrils depending on environmental factors. A comprehensive understanding of the energy landscape of amyloid formation considering various environmental factors will promote drug discovery and therapeutic strategies by controlling the fibril formation pathway and targeting the consequent morphology of aggregated structures.
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•Molecular dynamics simulations of protein–ligand complexes were performed.•Appropriate poses can be chosen from the many poses by MD with explicit solvents.•MD with implicit solvents ...cannot evaluate reasonable docking poses.•5-ns and 10-ns MD simulations worked effectively.
Computational docking programs generally predict many candidates for ligand poses, and only one or few poses should be selected for later drug design. In this study, we performed molecular dynamics (MD) simulations on 81 test sets of protein–ligand complexes and determined how well MD simulations evaluated docking poses. Our results suggest that appropriate candidate poses can be chosen from the many docking poses by using MD simulations with explicit solvents. To distinguish the appropriate pose from other candidates, 5-ns and 10-ns MD simulations were effective.
Pyruvate functions as a key molecule in energy production and as an antioxidant. The efficacy of pyruvate supplementation in diabetic retinopathy and nephropathy has been shown in animal models; ...however, its significance in the functional maintenance of neurons and Schwann cells under diabetic conditions remains unknown. We observed rapid and extensive cell death under high-glucose (> 10 mM) and pyruvate-starved conditions. Exposure of Schwann cells to these conditions led to a significant decrease in glycolytic flux, mitochondrial respiration and ATP production, accompanied by enhanced collateral glycolysis pathways (e.g., polyol pathway). Cell death could be prevented by supplementation with 2-oxoglutarate (a TCA cycle intermediate), benfotiamine (the vitamin B1 derivative that suppresses the collateral pathways), or the poly (ADP-ribose) polymerase (PARP) inhibitor, rucaparib. Our findings suggest that exogenous pyruvate plays a pivotal role in maintaining glycolysis-TCA cycle flux and ATP production under high-glucose conditions by suppressing PARP activity.
Anhydrobiosis, one of the most extensively studied forms of cryptobiosis, is induced in certain organisms as a response to desiccation. Anhydrobiotic species has been hypothesized to produce ...substances that can protect their biological components and/or cell membranes without water. In extremotolerant tardigrades, highly hydrophilic and heat-soluble protein families, cytosolic abundant heat-soluble (CAHS) proteins, have been identified, which are postulated to be integral parts of the tardigrades' response to desiccation. In this study, to elucidate these protein functions, we performed in vitro and in vivo characterizations of the reversible self-assembling property of CAHS1 protein, a major isoform of CAHS proteins from Ramazzottius varieornatus, using a series of spectroscopic and microscopic techniques. We found that CAHS1 proteins homo-oligomerized via the C-terminal α-helical region and formed a hydrogel as their concentration increased. We also demonstrated that the overexpressed CAHS1 proteins formed condensates under desiccation-mimicking conditions. These data strongly suggested that, upon drying, the CAHS1 proteins form oligomers and eventually underwent sol-gel transition in tardigrade cytosols. Thus, it is proposed that the CAHS1 proteins form the cytosolic fibrous condensates, which presumably have variable mechanisms for the desiccation tolerance of tardigrades. These findings provide insights into molecular strategies of organisms to adapt to extreme environments.
Deamidation of asparagine (Asn) residues is a nonenzymatic post-translational modification of proteins. Asn deamidation is associated with pathogenesis of age-related diseases and hypofunction of ...monoclonal antibodies. Deamidation rate is known to be affected by the residue following Asn on the carboxyl side and by secondary structure. Information about main-chain conformation of Asn residues is necessary to accurately predict deamidation rate. In this study, the effect of main-chain conformation of Asn residues on deamidation rate was computationally investigated using molecular dynamics (MD) simulations and quantum chemical calculations. The results of MD simulations for γS-crystallin suggested that frequently deamidated Asn residues have common main-chain conformations on the N-terminal side. Based on the simulated structure, initial structures for the quantum chemical calculations were constructed and optimized geometries were obtained using the B3LYP density functional method. Structures that were frequently deamidated had a lower activation energy barrier than that of the little deamidated structure. We also showed that dihydrogen phosphate and bicarbonate ions are important catalysts for deamidation of Asn residues.
Isomerized aspartic acid (Asp) residues have previously been identified in various aging tissues, and are suspected to contribute to age-related diseases. Asp-residue isomerization occurs ...nonenzymatically under physiological conditions, resulting in the formation of three types of isomerized Asp (i.e., L-isoAsp, D-Asp, and D-isoAsp) residues. Asp-residue isomerization often accelerates protein aggregation and insolubilization, making structural biology analyses difficult. Recently, Sakaue et al. reported the synthesis of a ribonuclease A (RNase A) in which Asp121 was artificially replaced with different isomerized Asp residues, and experimentally demonstrated that the enzymatic activities of these artificial mutants were completely lost. However, their structural features have not yet been elucidated. In the present study, the three-dimensional (3D) structures of these artificial-mutant RNases A were predicted using molecular dynamics (MD) simulations. The 3D structures of wild-type and artificial-mutant RNases A were converged by 3000-ns MD simulations. Our computational data show that the structures of the active site and the formation frequencies of the appropriate catalytic dyad structures in the artificial-mutant RNases A were quite different from wild-type RNase A. These computational findings may provide an explanation for the experimental data which show that artificial-mutant RNases A lack enzymatic activity. Herein, MD simulations have been used to evaluate the influences of isomerized Asp residues on the 3D structures of proteins.
•Recurrent short-term hypo- and hyper-glycemia reduced Schwann cells viabilities.•Cleaved caspase-3 and CHOP levels increased and Bcl-2 decreased in hypo- and hyper-glycemic groups.•TBARS levels ...increased by hypo- and hyperglycemia.•Recurrent short-term hypoglycemia induced apoptosis and oxidative stress in Schwann cells.•4-PBA treatment ameliorated cell death and oxidative stress in both groups.
Hypoglycemia and fluctuating high or low glucose conditions are under-appreciated sources of oxidative stress contributing to diabetic neuropathy. We investigated the effects of recurrent short-term hypoglycemia and hyperglycemia, on apoptosis and oxidative stress in Schwann cells. Immortalized adult mouse Schwann (IMS32) cells were exposed to five different glucose treatments over 3 days: 1) normal glucose (NG), 2) constant low glucose (LG), 3) constant high glucose (HG), 4) intermittent low glucose (ILG; 1 h three times per day), 5) intermittent high glucose (IHG; 1 h three times per day). Cell viability was decreased by all treatment variants, in comparison to NG. Thiobarbituric acid reactive substance (TBARS) levels were increased by HG, LG, IHG, and ILG. High glucose (HG and IHG) and low glucose (LG and ILG) increased the expression of cleaved caspase-3 and reduced that of Bcl-2. In addition, endoplasmic reticulum (ER) stress-responsive transcription factor C/EBP homologous protein (CHOP) expression was increased under low and high glucose conditions. Cell death and oxidative stress induced by HG, LG, IHG, and ILG were significantly reduced by 4-phenyl butyric acid (4-PBA), an ER stress inhibitor. These findings indicate that recurrent short-term hypoglycemia and hyperglycemia induce apoptosis and oxidative stress via the ER stress response in Schwann cells.
Ensitrelvir is a noncovalent inhibitor of the main protease (Mpro) of severe acute respiratory syndrome coronavirus 2. Acquisition of drug resistance in virus-derived proteins is a serious ...therapeutic concern, and drug resistance occurs due to amino acid mutations. In this study, we computationally constructed 24 mutants, in which one residue around the active site was replaced with alanine and performed molecular dynamics simulations to the complex of Mpro and ensitrelvir to predict the residues involved in drug resistance. We evaluated the changes in the entire protein structure and ligand configuration in each of these mutants and estimated which residues were involved in ensitrelvir recognition. This method is called a virtual alanine scan. In nine mutants (S1A, T26A, H41A, M49A, L141A, H163A, E166A, V186A, and R188A), although the entire protein structure and catalytic dyad (cysteine (Cys)145 and histidine (His)41) were not significantly moved, the ensitrelvir configuration changed. Thus, it is considered that these mutants did not recognize ensitrelvir while maintaining Mpro enzymatic activities, and Ser1, Thr26, His41, Met49, Leu141, His163, Glu166, Val186, and Arg188 may be related to ensitrelvir resistance. The ligand shift noted in M49A was similar to that observed in M49I, which has been shown to be experimentally ensitrelvir resistant. These findings suggest that our research approach can predict mutations that incite drug resistance.
The coiled-coil domains of the putative yeast cargo receptors Emp46p and Emp47p (Emp46p
and Emp47p
) assemble into heterocomplexes at neutral pH. Upon lowering the pH, the complex dissociates and ...reassembles into homo-oligomers. A glutamate residue (E303) located on the hydrophobic surface of Emp46p
serves as the pH-sensing switch for assembly and segregation, and we have suggested that its side chains are protonated in the heterocomplex, even at neutral pH. To examine this hypothesis, we constructed two structural models in which the side chains of E303 were negatively charged or protonated and analyzed the effects of these charged states on the structure of the heterocomplex using molecular dynamics (MD) simulations. The calculated structures suggested the side chains of E303 to be protonated in the heterocomplex, even at neutral pH. Based on these computational results, the pH dependence of Emp47p
homo-oligomer assembly was experimentally modified by a glutamate mutation on its hydrophobic surface. The Q306E mutant of Emp47p
underwent a structural transition at physiological pH. Our results suggest a method for modifying pH-dependent protein-protein interactions.
Hydantoins, including the antiepileptic drug phenytoin, contain an amide nitrogen and an imide nitrogen, both of which can be alkylated. However, due to the higher acidity of its proton, N3 can be ...more easily alkylated than N1 under basic conditions. In this study, we explored methods for direct N1-selective methylation of phenytoin and found that conditions using potassium bases potassium tert-butoxide (tBuOK) and potassium hexamethyldisilazide (KHMDS) in tetrahydrofuran (THF) gave N1-monomethylated phenytoin in good yield. The applicable scope of this reaction system was found to include various hydantoins and alkyl halides. To explore the function of methylated hydantoins, the effects of a series of methylated phenytoins on P-glycoprotein were examined, but none of methylated products showed inhibitory activity toward rhodamine 123 efflux by P-glycoprotein.
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