An aberrant fused in sarcoma liquid droplet of amyotrophic lateral sclerosis pathological variant, R495X, accelerates liquid-solid phase transition.

Intracellular aggregation of fused in sarcoma (FUS) is associated with the pathogenesis of familial amyotrophic lateral sclerosis (ALS). Under stress, FUS forms liquid droplets via liquid-liquid phase separation (LLPS). Two types of wild-type FUS LLPS exist in equilibrium: low-pressure LLPS (LP-LLPS) and high-pressure LLPS (HP-LLPS); the former dominates below 2 kbar and the latter over 2 kbar. Although several disease-type FUS variants have been identified, the molecular mechanism underlying accelerated cytoplasmic granule formation in ALS patients remains poorly understood. Herein, we report the reversible formation of the two LLPS states and the irreversible liquid-solid transition, namely droplet aging, of the ALS patient-type FUS variant R495X using fluorescence microscopy and ultraviolet-visible absorption spectroscopy combined with perturbations in pressure and temperature. Liquid-to-solid phase transition was accelerated in the HP-LLPS of R495X than in the wild-type variant; arginine slowed the aging of droplets at atmospheric conditions by inhibiting the formation of HP-LLPS more selectively compared to that of LP-LLPS. Our findings provide new insight into the mechanism by which R495X readily forms cytoplasmic aggregates. Targeting the aberrantly formed liquid droplets (the HP-LLPS state) of proteins with minimal impact on physiological functions could be a novel therapeutic strategy for LLPS-mediated protein diseases.

N-acetyl amino acid amide solubility in aqueous 1,6-hexanediol solutions: Insights into the protein droplet deformation mechanism.

Proteinaceous liquid droplets, generated by liquid-liquid phase separation, function as membraneless compartments that are essential for diverse biological functions. Studies addressing droplet generation have used 1,6-hexanediol (1,6-HD) as a droplet-discerning agent owing to its capacity to induce droplet deformation. Despite the empirical utility of 1,6-HD, the mechanism underlying 1,6-HD-induced droplet deformation remains unknown. In this study, the solubilities of N-acetyl amino acid amides, which correspond to proteinogenic amino acid residues, were examined in the presence of 1,6-HD at 25 °C. Other solvents included ethanol, 1-propanol, and amides. Remarkably, 1,6-HD effectively solubilized hydrophobic species (particularly aromatic species) and exhibited reduced efficacy in solubilizing hydrophilic species and peptide bond moieties. These solubilizing effects are reflected in changes in protein solubility and structure. Specifically, 1,6-HD primarily targets the hydrophobic regions of a protein, increasing protein solubility without causing substantial structural changes. This solubilization mechanism is essential for elucidating the role of 1,6-HD as a droplet-discerning agent and recognizing its potential limitations.

Solubilization of Carbon Nanobelts in Aqueous Solutions: Optical and Colloidal Properties.

Carbon nanobelts (CNBs) correspond to carbon nanotube (CNT) segments and are insoluble in most common aqueous solutions, posing challenges across diverse applications. In this study, [12] CNB, which corresponds to a (6,6) CNT segment, was solubilized by aliphatic surfactant micelles through host-guest complexation, which was confirmed by comprehensive analyses involving spectrophotometry, mass spectrometry, and molecular dynamics simulations. Through this solubilization, zero-Stokes shift emission of the CNB could occur, which could be ascribed to the symmetry-allowed transition. In contrast, CNB was insoluble in non-aliphatic surfactant solutions. The mechanism by which CNB is solubilized using aliphatic surfactants is completely distinct from that of the CNT dispersion mechanism. The present finding provides knowledge of the effectiveness of aliphatic compounds in solubilizing CNBs and their derivatives (carbon nanohoops), which show significant potential for various applications in aqueous systems, including biological applications.

Dissociation Rate Calculation via Constant-Force Steered Molecular Dynamics Simulation.

Steered molecular dynamics (SMD) simulations are used to study molecular dissociation events by applying a harmonic force to the molecules and pulling them at a constant velocity. Instead of constant-velocity pulling, we use a constant force: the constant-force SMD (CF-SMD) simulation. The CF-SMD simulation employs a constant force to reduce the activation barrier of molecular dissociation, thereby enhancing the dissociation event. Here, we present the capability of the CF-SMD simulation to estimate the dissociation time at equilibrium. We performed all-atom CF-SMD simulations for NaCl and protein-ligand systems, producing dissociation time at various forces. We extrapolated these values to the dissociation rate without a constant force using Bell's model or the Dudko-Hummer-Szabo model. We demonstrate that the CF-SMD simulations with the models predicted the dissociation time in equilibrium. A CF-SMD simulation is a powerful tool for estimating the dissociation rate in a direct and computationally efficient manner.

Fine-Tuning and Enhancement of pH-Dependent Membrane Permeation of Cyclic Peptides by Utilizing Noncanonical Amino Acids with Extended Side Chains.

The development of cyclic peptides that exhibit pH-sensitive membrane permeation is a promising strategy for tissue-selective drug delivery. We investigated the pH-dependent interactions of designed cyclic peptides bearing noncanonical amino acids of long acidic side chains with lipid membranes, including surface binding, insertion, and translocation across the membrane. As the length of the side chain of acidic amino acid increased, the binding affinity of the peptides to phosphatidylcholine bilayer surfaces decreased, while the pH for the 50% insertion of the peptides into the bilayers increased. The pH for membrane permeation of the peptides increased with the side chain length, resulting in specific membrane permeation at pH ∼6.5. The longer side chain of acidic amino acids improved the maximum rate of membrane permeation at low pH, where both entropic and enthalpic contributions affected the permeation. Our peptide also showed intracellular delivery of cargo molecules into living cells in a pH-dependent manner.

Selection of target-binding proteins from the information of weakly enriched phage display libraries by deep sequencing and machine learning.

Despite the advances in surface-display systems for directed evolution, variants with high affinity are not always enriched due to undesirable biases that increase target-unrelated variants during biopanning. Here, our goal was to design a library containing improved variants from the information of the "weakly enriched" library where functional variants were weakly enriched. Deep sequencing for the previous biopanning result, where no functional antibody mimetics were experimentally identified, revealed that weak enrichment was partly due to undesirable biases during phage infection and amplification steps. The clustering analysis of the deep sequencing data from appropriate steps revealed no distinct sequence patterns, but a Bayesian machine learning model trained with the selected deep sequencing data supplied nine clusters with distinct sequence patterns. Phage libraries were designed on the basis of the sequence patterns identified, and four improved variants with target-specific affinity (EC50 = 80-277 nM) were identified by biopanning. The selection and use of deep sequencing data without undesirable bias enabled us to extract the information on prospective variants. In summary, the use of appropriate deep sequencing data and machine learning with the sequence data has the possibility of finding sequence space where functional variants are enriched.

Coenzyme corona formation on carbon nanotubes leads to disruption of the redox balance in metabolic reactions.

Carbon nanotubes (CNTs) have adverse impacts on metabolism in biological systems. The impacts should be associated with interactions of the CNTs with coenzymes, such as nicotinamide adenine dinucleotide (NAD), because most metabolic processes are governed by coenzyme-dependent reactions. This study demonstrates that NAD molecules adsorb onto the CNT surface, leading to the formation of interfacial NAD layers-in other words, a coenzyme corona (coenzyme-based biomolecular corona). Coenzyme corona formation is accompanied by the oxidation of NAD at biological concentrations through electron transfer. Similar phenomena are observed for NAD derivatives. Molecular dynamics simulations indicate that the adsorption of NAD onto CNTs is driven by interactions between the aromaphilic groups of NAD and the CNT surfaces, leading to coenzyme corona formation. Generally, in living biological systems, the balance of NAD redox (NADH/NAD+ redox) is maintained to sustain metabolism. The present results suggest that CNTs affect coenzyme-dependent metabolic reactions by disrupting the redox balance through coenzyme corona formation and subsequent coenzyme oxidation. The proposed molecular mechanism not only advances the fundamental understanding of the biological impact of CNTs in terms of metabolism but also contributes to biological CNT applications.

Helix-forming aliphatic homo-δ-peptide foldamers based on the conformational restriction effects of cyclopropane.

Considerable effort has been directed toward developing artificial peptide-based foldamers. However, detailed structural analysis of δ-peptide foldamers consisting of only aliphatic δ-amino acids has not been reported. Herein, we rationally designed and stereoselectively synthesized aliphatic homo-δ-peptides forming a stable helical structure by using a chiral cyclopropane δ-amino acid as a monomer unit. Structural analysis of the homo-δ-peptides using circular dichroism, infrared, and NMR spectroscopy indicated that they form a stable 14-helical structure in solution. Furthermore, we successfully conducted X-ray crystallographic analysis of the homo-δ-peptides, demonstrating a right-handed 14-helical structure. This helical structure of the crystal was consistent with those predicted by theoretical calculations and those obtained based on NMR spectroscopy in solution. This stable helical structure is due to the effective restriction of the backbone conformation by the structural characteristics of cyclopropane. This work reports the first example of aliphatic homo-δ-peptide foldamers having a stable helical structure both in the solution and crystal states.

Rational peptide design for regulating liquid-liquid phase separation on the basis of residue-residue contact energy.

Since liquid-liquid phase separation (LLPS) of proteins is governed by their intrinsically disordered regions (IDRs), it can be controlled by LLPS-regulators that bind to the IDRs. The artificial design of LLPS-regulators based on this mechanism can be leveraged in biological and therapeutic applications. However, the fabrication of artificial LLPS-regulators remains challenging. Peptides are promising candidates for artificial LLPS-regulators because of their ability to potentially bind to IDRs complementarily. In this study, we provide a rational peptide design methodology for targeting IDRs based on residue-residue contact energy obtained using molecular dynamics (MD) simulations. This methodology provides rational peptide sequences that function as LLPS regulators. The peptides designed with the MD-based contact energy showed dissociation constants of 35-280 nM for the N-terminal IDR of the tumor suppressor p53, which are significantly lower than the dissociation constants of peptides designed with the conventional 3D structure-based energy, demonstrating the validity of the present peptide design methodology. Importantly, all of the designed peptides enhanced p53 droplet formation. The droplet-forming peptides were converted to droplet-deforming peptides by fusing maltose-binding protein (a soluble tag) to the designed peptides. Thus, the present peptide design methodology for targeting IDRs is useful for regulating droplet formation.

Affinity of phenolic compounds for transition metal ions immobilized on cation-exchange columns.

Immobilized metal ion affinity chromatography (IMAC) is useful in purification of histidine-tagged or histidine-rich proteins and peptides from a variety of hosts. However, phenolic compounds including polyphenols interfere with IMAC due to their high affinities for the transition metals immobilized on the column resins, which hampers the purification of proteins from plant-based host systems. In contrast to extensive knowledge of the mechanism of the interactions between phenolic compounds and transition metal ions in solution, an understanding of the interactions on the columns, where transition metal ions are immobilized on the resins, remains elusive. This study systematically investigated the affinity of phenolic compounds for transition metal ions by varying the number and position of phenolic hydroxyl groups (OH groups) and using different transition metals-Fe(II), Cu(II) and Ni(II)-on various IMACs, in which the columns were fabricated by equilibrating the cation-exchange column with transition metal solutions. It was found that the more OH groups the aromatic compounds have, the higher the affinity for transition metal ions; in particular, methyl gallate and pyrogallol were permanently bound to the IMAC column, which reflected coordinate bond formation with the transition metal ions. Importantly, the phenolic compounds showed no obvious affinity for the Ni(II)-IMAC column, in contrast to the Fe(II)- and Cu(II)-IMAC columns, whereas imidazole and histidine-tagged proteins showed evident binding to the Ni(II)-IMAC column. Ni(II)-IMAC should thus be especially effective in isolating histidine-tagged and histidine-rich species from phenolic compound-containing systems. These results indicate that the affinity between phenolic compounds and transition metal ions on the column is consistent with the results in solution. They also provide a comprehensive view for devising strategies to improve IMAC purification of target proteins and peptides from samples containing phenolic compounds.

Protein's Protein Corona: Nanoscale Size Evolution of Human Immunoglobulin G Aggregates Induced by Serum Albumin.

Nanoparticles are readily coated by proteins in biological systems. The protein layers on the nanoparticles, which are called the protein corona, influence the biological impacts of the nanoparticles, including internalization into cells and cytotoxicity. This study expands the scope of the nanoparticle's protein corona for exogenous artificial nanoparticles to that for exogenous proteinaceous nanoparticles. Specifically, this study addresses the formation of protein coronas on nanoscale human antibody aggregates with a radius of approximately 20-40 nm, where the antibody aggregates were induced by a pH shift from low to neutral pH. The size of the human immunoglobulin G (hIgG) aggregates grew to approximately 25 times the original size in the presence of human serum albumin (HSA). This size evolution was ascribed to the association of the hIgG aggregates, which was triggered by the formation of the hIgG aggregate's protein corona, i.e., protein's protein corona, consisting of the adsorbed HSA molecules. Because hIgG aggregate association was significantly reduced by the addition of 30-150 mM NaCl, it was attributed to electrostatic attraction, which was supported by molecular dynamics (MD) simulations. Currently, the use of antibodies as biopharmaceuticals is concerning because of undesired immune responses caused by antibody aggregates that are typically generated by a pH shift during the antibody purification process. The present findings suggest that nanoscale antibody aggregates form protein coronas induced by HSA and the resulting nanoscale antibody-HSA complexes are stable in blood containing approximately 150 mM salt ions, at least in terms of the size evolution. Mechanistic insights into protein corona formation on nanoscale antibody aggregates are useful for understanding the unintentional biological impacts of antibody drugs.

Kinetic Mechanism of Amyloid-ß-(16-22) Peptide Fibrillation.

The kinetic mechanism of amyloid fibril formation by a peptide fragment containing seven residues of the amyloid-ß protein Aß-(16-22) was investigated. We found that the N- and C-terminal unprotected Aß-(16-22), containing no aggregation nuclei, showed rapid fibrillation within seconds to minutes in a neutral aqueous buffer solution. The fibrillation kinetics were well described by the nucleation-elongation model, suggesting that primary nucleation was the rate-limiting step. On the basis of both experimental and theoretical analyses, the aggregated nucleus was estimated to be composed of 6-7 peptide molecules, wherein the two ß-sheets were associated with their hydrophobic surfaces. Thin fibers with widths of 10-20 nm were formed, which increased their length and thickness, attaining a width of >20 nm over several tens of minutes, probably owing to the lateral association of the fibers. Electrostatic and hydrophobic interactions play important roles in aggregation. These results provide a basis for understanding the fibrillation of short peptides.

Stabilization of CDK6 by ribosomal protein uS7, a target protein of the natural product fucoxanthinol.

Cyclins and cyclin-dependent kinases (CDKs) regulate the cell cycle, which is important for cell proliferation and development. Cyclins bind to and activate CDKs, which then drive the cell cycle. The expression of cyclins periodically changes throughout the cell cycle, while that of CDKs remains constant. To elucidate the mechanisms underlying the constant expression of CDKs, we search for compounds that alter their expression and discover that the natural product fucoxanthinol downregulates CDK2, 4, and 6 expression. We then develop a method to immobilize a compound with a hydroxyl group onto FG beads® and identify human ribosomal protein uS7 (also known as ribosomal protein S5) as the major fucoxanthinol-binding protein using the beads and mass spectrometry. The knockdown of uS7 induces G1 cell cycle arrest with the downregulation of CDK6 in colon cancer cells. CDK6, but not CDK2 or CDK4, is degraded by the depletion of uS7, and we furthermore find that uS7 directly binds to CDK6. Fucoxanthinol decreases uS7 at the protein level in colon cancer cells. By identifying the binding proteins of a natural product, the present study reveals that ribosomal protein uS7 may contribute to the constant expression of CDK6 via a direct interaction.

Adsorption Kinetics and Self-Assembled Structures of Aspergillus oryzae Hydrophobin RolA on Hydrophobic and Charged Solid Surfaces.

Hydrophobins are small secreted amphipathic proteins ubiquitous among filamentous fungi. Hydrophobin RolA produced by Aspergillus oryzae attaches to solid surfaces, recruits polyesterase CutL1, and thus promotes hydrolysis of polyesters. Because the N-terminal region of RolA is involved in the interaction with CutL1, the orientation of RolA on the solid surface is important. However, the kinetic properties of RolA adsorption to solid surfaces with various chemical properties remain unclear, and RolA structures assembled after the attachment to surfaces are unknown. Using a quartz crystal microbalance (QCM), we analyzed the kinetic properties of RolA adsorption to the surfaces of QCM electrodes that had been chemically modified to become hydrophobic or charged. We also observed the assembled RolA structures on the surfaces by atomic force microscopy and performed molecular dynamics (MD) simulations of RolA adsorption to self-assembled monolayer (SAM)-modified surfaces. The RolA-surface interaction was considerably affected by the zeta potential of RolA, which was affected by pH. The interactions of RolA with the surface seemed to be involved in the self-assembly of RolA. Three types of self-assembled structures of RolA were observed: spherical, rod-like, and mesh-like. The kinetics of RolA adsorption and the structures formed depended on the amount of RolA adsorbed, chemical properties of the electrode surface, and the pH of the buffer. Adsorption of RolA to solid surfaces seemed to depend mainly on its hydrophobic interaction with the surfaces; this was supported by MD simulations, which suggested that hydrophobic Cys-Cys loops of RolA attached to all SAM-modified surfaces at all pH values. IMPORTANCE The adsorption kinetics of hydrophobins to solid surfaces and self-assembled structures formed by hydrophobin molecules have been studied mostly independently. In this report, we combined the kinetic analysis of hydrophobin RolA adsorption onto solid surfaces and observation of RolA self-assembly on these surfaces. Since RolA, whose isoelectric point is close to pH 4.0, showed higher affinity to the solid surfaces at pH 4.0 than at pH 7.0 or 10.0, the affinity of RolA to these surfaces depends mainly on hydrophobic interactions. Our combined analyses suggest that not only the adsorbed amount of RolA but also the chemical properties of the solid surfaces and the zeta potential of RolA affect the self-assembled RolA structures formed on these surfaces.

A chemoproteoinformatics approach demonstrates that aspirin increases sensitivity to MEK inhibition by directly binding to RPS5.

MEK inhibitors are among the most successful molecularly targeted agents used as cancer therapeutics. However, to treat cancer more efficiently, resistance to MEK inhibitor-induced cell death must be overcome. Although previous genetic approaches based on comprehensive gene expression analysis or RNAi libraries led to the discovery of factors involved in intrinsic resistance to MEK inhibitors, a feasible combined treatment with the MEK inhibitor has not yet been developed. Here, we show that a chemoproteoinformatics approach identifies ligands overcoming the resistance to cell death induced by MEK inhibition as well as the target molecule conferring this resistance. First, we used natural products, perillyl alcohol and sesaminol, which induced cell death in combination with the MEK inhibitor trametinib, as chemical probes, and identified ribosomal protein S5 (RPS5) as their common target protein. Consistently, trametinib induced cell death in RPS5-depleted cancer cells via upregulation of the apoptotic proteins BIM and PUMA. Using molecular docking and molecular dynamics (MD) simulations, we then screened FDA- and EMA-approved drugs for RPS5-binding ligands and found that acetylsalicylic acid (ASA, also known as aspirin) directly bound to RPS5, resulting in upregulation of BIM and PUMA and induction of cell death in combination with trametinib. Our chemoproteoinformatics approach demonstrates that RPS5 confers resistance to MEK inhibitor-induced cell death, and that aspirin could be repurposed to sensitize cells to MEK inhibition by binding to RPS5.

Enzyme modification using mutation site prediction method for enhancing the regioselectivity of substrate reaction sites.

Enzymes with low regioselectivity of substrate reaction sites may produce multiple products from a single substrate. When a target product is produced industrially using these enzymes, the production of non-target products (byproducts) causes adverse effects such as increased processing costs for purification and the amount of raw material. Thus it is required the development of modified enzymes to reduce the amount of byproducts' production. In this paper, we report a method called mutation site prediction for enhancing the regioselectivity of substrate reaction sites (MSPER). MSPER takes conformational data for docking poses of an enzyme and a substrate as input and automatically generates a ranked list of mutation sites to destabilize docking poses for byproducts while maintaining those for target products in silico. We applied MSPER to the enzyme cytochrome P450 CYP102A1 (BM3) and the two substrates to enhance the regioselectivity for four target products with different reaction sites. The 13 of the total 14 top-ranked mutation sites predicted by MSPER for the four target products succeeded in selectively enhancing the regioselectivity up to 6.4-fold. The results indicate that MSPER can distinguish differences of substrate structures and the reaction sites, and can accurately predict mutation sites to enhance regioselectivity without selection by directed evolution screening.

Pressure and Temperature Phase Diagram for Liquid-Liquid Phase Separation of the RNA-Binding Protein Fused in Sarcoma.

Liquid-liquid phase separation (LLPS) of proteins and nucleic acids to form membraneless cellular compartments is considered to be involved in various biological functions. The RNA-binding protein fused in sarcoma (FUS) undergoes LLPS in vivo and in vitro. Here, we investigated the effects of pressure and temperature on the LLPS of FUS by high-pressure microscopy and high-pressure UV/vis spectroscopy. The phase-separated condensate of FUS was obliterated with increasing pressure but was observed again at a higher pressure. We generated a pressure-temperature phase diagram that describes the phase separation of FUS and provides a general understanding of the thermodynamic properties of self-assembly and phase separation of proteins. FUS has two types of condensed phases, observed at low pressure (LP-LLPS) and high pressure (HP-LLPS). The HP-LLPS state was more condensed and exhibited lower susceptibility to dissolution by 1,6-hexanediol and karyopherin-ß2 than the LP-LLPS state. Moreover, molecular dynamic simulations revealed that electrostatic interactions were destabilized, whereas cation-π, π-π, and hydrophobic interactions were stabilized in HP-LLPS. When cation-π, π-π, and hydrophobic interactions were transiently stabilized in the cellular environment, the phase transition to HP-LLPS occurred; this might be correlated to the aberrant enrichment of cytoplasmic ribonucleoprotein granules, leading to amyotrophic lateral sclerosis.

Comparative analysis of the relationship between translation efficiency and sequence features of endogenous proteins in multiple organisms.

The translation efficiency of protein genes is known to be affected by sequence features. Previous studies have found that various sequence features based on codon usage and mRNA secondary structure contribute to translation efficiency. However, most studies have focused on a specific organism, usually a model organism such as Escherichia coli or Saccharomyces cerevisiae. Here, we investigate whether the relationship between translation efficiency and sequence features is conserved among multiple organisms using publicly available ribosome profiling data and RNA-Seq data. We analyze nine organisms from various taxa: Staphylococcus aureus, five species of Streptomyces, two strains of E. coli, and S. cerevisiae. We reveal that the relationship between translation efficiency and sequence features differs across organisms, partly reflecting their taxonomy. The codon adaptation index shows high correlation in all analyzed organisms. Our study provides an insight into the diversity and commonality of sequence determinants of protein expression in these organisms.