Search Results (177)

Prion diseases such as chronic wasting disease involve the conformational conversion of the cellular prion protein (PrP^C) into its misfolded, β-rich isoform (PrP^Sc). While chemical denaturants such as guanidine hydrochloride (GdnHCl) and urea are commonly used to study this process in vitro, their distinct molecular effects on native and misfolded PrP conformers remain incompletely understood. In this study, we employed 500 ns all-atom molecular dynamics simulations and essential collective dynamics analysis to investigate the differential effects of GdnHCl and urea on a composite PrP^C/PrP^Sc system, where white-tailed deer PrP^C interfaces with a corresponding PrP^Sc conformer. GdnHCl was found to preserve interfacial alignment and enhance β-sheet retention in PrP^Sc, while urea promoted partial β-strand dissolution and interfacial destabilization. Both denaturants formed transient contacts with PrP, but urea displaced water hydrogen bonds more extensively. Remarkably, we also observed long-range dynamical coupling across the PrP^C/PrP^Sc interface and between transiently bound solutes and distal protein regions. These findings highlight distinct, denaturant-specific mechanisms of protein destabilization and suggest that localized interactions may propagate non-locally via mechanical or steric pathways. Our results provide molecular-scale insights relevant to prion conversion mechanisms and inform experimental strategies using GdnHCl and urea to modulate misfolding processes in vitro. Full article

Kidney disease causes the retention of uremic metabolites in blood, which is associated with many comorbidities. Hemodialysis does not properly clear many metabolites, including large, middle-sized, and small protein-bound uremic toxins (PBUTs). Adsorption strategies for metabolite removal require the development of engineered adsorbents with tailored surfaces to increase the binding of desired metabolites. Albumin is uniquely positioned for modifying blood-contacting surfaces to absorb uremic metabolites, as it (i) minimizes non-specific protein adsorption and (ii) binds a range of molecules at Sudlow Sites I and II with different affinities. It is unknown if albumin-modified surfaces retain the adsorption qualities of solution-free albumin, namely, adsorption stability or specificity. Herein, albumin was covalently attached to iron oxide nanoparticles and characterized using multiple methods. Metabolite adsorption was conducted by incubating particles in a model solution of thirty-three uremic metabolites associated with kidney failure. Adsorption efficiency, selectivity, and stability were affected by albumin concentration and incubation time. Metabolite adsorption was found to change with time, and it was more effective on albumin-modified particles than unmodified controls. The findings outlined in this paper are crucial for the design of next-generation advanced blood-contacting materials to enhance dialysis and blood purification for patients with kidney disease. Full article

Protein-bound nano-injectable solutions represent a cutting-edge advancement in nanomedicine, offering a versatile platform for precise and controlled drug delivery. By leveraging the biocompatibility and functional versatility of proteins such as albumin, gelatin, and casein, these nano systems enhance drug solubility, prolong circulation time, and improve site-specific targeting, which are particularly beneficial in cancer and inflammatory diseases. This review provides a comprehensive overview of their formulation strategies, physicochemical characteristics, and biological behavior. Emphasis is placed on therapeutic applications, regulatory considerations, fabrication techniques, and the underlying mechanisms of drug–protein interactions. This review also highlights improved pharmacokinetics and reduced systemic toxicity, while also critically addressing challenges like immunogenicity, protein instability, and production scalability. Recent FDA-approved formulations and emerging innovations in precision medicine and theranostics underscore the transformative potential of protein-based nanosuspensions in next-generation drug delivery systems. Full article

For the interaction of angiotensin II (AngII) with AngII type 1 receptors (AT₁R), two potential proton hopping pathways have been identified, each associated with distinct physiological outcomes. The octapeptide AngII (Asp¹-Arg²-Val³-Tyr⁴-Ile⁵-His⁶-Pro⁷-Phe⁸) appears to form a charge relay system (CRS) in solution in which the C-terminal carboxylate abstracts a proton from the His⁶ imidazole group, which, in turn, abstracts a proton from the Tyr⁴ hydroxyl (OH) group, creating a tyrosinate anion. When AngII binds to the AT₁R, the CRS can be reconstituted with D281 of the receptor taking up the role of the Phe⁸ carboxylate in the tripartite interaction, whilst the Phe⁸ carboxylate forms a salt bridge with K199 of the receptor. As a consequence, the Tyr⁴ OH of AngII is positioned with accessibility to either the Phe⁸ carboxylate (bound to K199) or the His⁶ imidazole (activated by D281), thereby creating a potential gating mechanism for AT₁R receptor signaling. This study summarizes evidence based on structure activity data for various analogs wherein Tyr⁴ OH interaction with His⁶ imidazole (CRS formation) leads to G protein sequestration and vasoconstriction, whereas Tyr⁴ OH interaction with Phe⁸ carboxylate (bound to K199) engenders arrestin-mediated vasodilation and receptor desensitization. These findings, combined with quantum mechanical (semiempirical) calculations of CRS proton transfer presented herein, provide insights for the therapeutic targeting of angiotensin receptor blockers (sartans) and the development of second-generation drugs (bisartans). Full article

Structural analyses of protein filaments formed by self-assembly, such as actin, tubulin, or recombinase filaments, have suffered for decades from technical issues due to difficulties in crystallization, their large size, or the dynamic behavior inherent to their cellular function. The advent of cryo-electron microscopy has finally enabled us to obtain structures at different stages of the existence of these filaments. However, these structures correspond to frozen states, and the possibility of observations in solution is still lacking, especially for filaments characterized by a high plasticity, such as the RecA protein for homologous recombination. Here, we use a combination of SAXS measurements and integrative modeling to generate the solution structure of two known forms of the RecA nucleoprotein filament, previously characterized by electron microscopy and resolved by X-ray crystallography. The two forms differ in the cofactor bound to RecA–RecA interfaces, either ATP or ADP. Cooperative transition from one form to the other has been observed during single-molecule experiments by pulling on the filament but also in solution by modifying solvent conditions. We first compare the SAXS data against known structural information. While the crystal structure of the ATP form matches well with the SAXS data, we deduce from the SAXS profiles of the ADP-form values of the pitch (72.0 Å) and the number of monomers per turn (6.4) that differ with respect to the crystal structure (respectively, 82.7 Å and 6.0). We then monitor the transition between the two states driven by the addition of magnesium, and we show this transition occurs with 0.3 mM Mg ²⁺ ions with a high cooperativity. Full article

Solute carrier family 25 member A22 (SLC25A22) is a glutamate transporter in the inner mitochondrial membrane that is known to suppress ferroptosis in pancreatic ductal adenocarcinoma (PDAC). Sirtuin 3 (SIRT3) is the main mitochondrial deacetylase, and we previously demonstrated that targeting SIRT3 sensitized glioblastoma to ferroptosis by promoting mitophagy and inhibiting SLC7A11. The purpose of this study was to analyze the effect of SIRT3-mediated deacetylation of mitochondrial SLC25A22 on RAS-selective lethal 3 (RSL3)-induced ferroptosis in lung adenocarcinoma (LUAD). We found that the expression of SLC25A22 and SIRT3 had a high positive correlation and that their expression was greater in LUAD tissues than in adjacent tissues. The RSL3-induced ferroptosis of LUAD led to upregulation of SLC25A22 and SIRT3, and SIRT3 protected RSL3-induced LUAD from ferroptosis in vitro and in vivo. At the molecular level, SIRT3 bound with SLC25A22 and deacetylated this protein. Targeting SIRT3 enhanced the acetylation of SLC25A22, decreased its ubiquitination, and promoted 26S proteasome degradation in LUAD cells. Therefore, our results demonstrated that SIRT3 protected LUAD cells from RSL3-induced ferroptosis, and this effect is at least partially due to its deacetylation of SLC25A22, revealing that the SIRT3-SLC25A22 axis has an important role in regulating the ferroptosis of LUAD cells. Full article

The bacterium Bacillus anthracis secretes three protein exotoxins: Protective Antigen 83 (PA83), Lethal Factor (LF), and Edema Factor (EF). A cleaved form of PA83 (PA63) aids LF and EF entry into the cytoplasm, which leads to anthrax-induced cell death. The Protein Data Bank (PDB) has more than 25 structures of LF: the monomer alone, bound with inhibitors, or bound to PA63. The structures are all—with only minor shifts of a few Ångströms—nearly congruent. We have measured the structure of LF at equilibrium in D₂O solution by small-angle neutron scattering (SANS). The shape is modeled well by a parallelepiped (all angles 90°) with dimensions of 12 Å × 49 Å × 129 Å. For a protein with a typical density of 1.4, the molecular weight would be between 55 and 94 kDa, which is comparable to that of the 90.2 kDa monomer. However, the LF crystal structure PDB 1pwu (a generally V-shaped molecule with equal arm lengths ≈ 70 Å) with the same model fits the dimensions 30 Å × 48 Å × 104 Å. Given the large changes in the long and short dimensions, straightforward physical modeling of the solution structure from the crystal form is unable to match the SANS results. Full article

Inductively coupled plasma mass spectrometry (ICP-MS) is important in the biological and biochemical fields as it can quantify trace elements. Confocal laser Raman microscopy (CLRM), a powerful tool for the compositional analysis of biological samples, organic materials, and inorganic materials, can be used to analyze samples in aqueous solutions. Despite their analytical strength, the quantitative evaluation of proteins bound to mesoporous silica (SiO₂) microspheres, which are promising candidates for drug delivery systems and vaccine carriers, has not been sufficiently explored. Therefore, we investigated the applicability of ICP-MS and CLRM to quantify lactoferrin (LF), a widely studied iron-containing protein bound to mesoporous SiO₂ microspheres (SBA24). The bound LF amount was measured using ICP-MS, selectively monitoring iron derived from LF as a marker element, and CLRM. The results were compared with those obtained using a conventional bulk analysis technique. The amounts and trends of the signal intensities obtained using ICP-MS and CLRM agreed with each other and with the bulk analysis results. Our findings demonstrate that ICP-MS and CLRM are applicable for the quantitative evaluation of iron-containing proteins bound to SBA24. These methods offer a reliable platform for the quantification of biomolecules on microparticles and provide valuable insights for biomedical research and quality control in related industries. Full article

Background/Objectives: Neutrophil cells’ lysis forms the extracellular traps (NETs) to counter the foreign body during insults to the body. Peptidyl arginine deiminase (PAD) participates in this process and is then released into the extracellular fluid with the lysed cell components. In some diseases, patients with abnormal function of PADs, especially PAD 4, tend to form autoantibodies against the abnormal citrullinated proteins that are the result of PAD activity on arginine side chains. Those antibodies, which are highly distinct in RA, are distinctly anti-citrullinated protein antibodies (ACPA). This study used an in-silico drug repurposing approach of FDA-approved medications to identify potential alternative medications that can inhibit this process and address solutions to the current limitations of existing therapies. Methods: We utilized Maestro Schrödinger as a computational tool for preparing and docking simulations on the PAD 4 enzyme crystal structure that is retrieved from RCSB Protein Data Bank (PDB ID: 4X8G) while the docked FDA-approved medications are obtained from the Zinc 15 database. The protein was bound to GSK 199—an investigational compound—as a positive control for the docked molecules. Preparation of the protein was performed by Schrödinger Protein Preparation Wizard tool. Binding pocket determination was performed by Glide software (Schrödinger Release 2021–3:Schrödinger, LLC., New York, NY, USA, 2021). and validation of molecular docking was carried out through the redocking of GSK 199 and superimposition. After that, standard and induced fit docking were performed. Results/Conclusions: Among the four obtained hits Pemetrexed, Leucovorin, Chlordiazepoxide, and Ioversol, which showed the highest XP scores providing favorable binding interactions. The induced-fit docking (IFD) results displayed the strong binding affinities of Ioversol, Pemetrexed, Leucovorin, Chlordiazepoxide in the order IFD values −11.617, −10.599, −10.521, −9.988, respectively. This research investigates Pemetrexed, Leucovorin, Chlordiazepoxide, and Ioversol as potential repurposing agents in the treatment of rheumatoid arthritis (RA) as they are identified as PAD4 inhibitors. Full article

Osmotic stress impacts the cell wall properties in plants. This study aimed to elucidate the mechanisms involved in cell wall remodeling in etiolated (dark-grown) rice (Oryza sativa L.) shoots grown under polyethylene glycol (PEG)-induced osmotic stress conditions. Shoot growth was inhibited by 70% by the treatment with 60 mM PEG for 2 days. However, when the stressed seedlings were transferred to a solution without PEG, their shoot growth rate increased significantly. A measurement of the cell wall mechanical properties revealed that the cell walls of the stressed shoots became looser and more extensible than those of unstressed shoots. Among the cell wall constituents, the amounts of cell wall-bound phenolic acids, such as ferulic acid (FA), p-coumaric acid (p-CA), and diferulic acid (DFA), per shoot and per unit of matrix polysaccharide content were significantly reduced in the stressed shoots compared to those in the unstressed shoots. Concerning the formation of cell wall-bound phenolic acids, the activity of cell wall-bound peroxidase (CW-PRX) per unit of cell wall content, which is responsible for the coupling reaction of FA to produce DFA, was 3.5 times higher in stressed shoots than in unstressed shoots, while the activity was reduced by 20% on a shoot basis in stressed shoots compared to that in unstressed shoots. The expression levels of the major class III peroxidase genes in stressed shoots were either comparable to or slightly lower than those in unstressed shoots. Conversely, the phenylalanine ammonia-lyase (PAL) activity, which contributes to the biosynthesis of FA and p-CA, was reduced by 55% and 30% on a shoot and unit-of-protein-content basis, respectively, in stressed shoots compared to that in unstressed shoots. The expression levels of abundantly expressed PAL genes decreased by 14–46% under osmotic stress. Moreover, the gene expression levels of specific BAHD acyltransferases, which are responsible for the addition of FA and p-CA to form ester-linked moieties on cell wall constituents, decreased by 15–33% under osmotic stress. These results suggest that the downregulation of the expression of specific PAL and BAHD acyltransferase genes in osmotically stressed rice shoots is responsible for a reduction in the formation of cell wall-bound phenolic acid monomers. This, in turn, may result in a decrease in the levels of DFAs. The reduction in the formation of DFA-mediated cross-linking structures within the cell wall may contribute to an increase in the mechanical extensibility of the cell wall. The remodeling of cell walls in an extensible and loosened state could assist in maintaining the growth capacity of etiolated rice shoots grown under osmotic stress and contribute to rapid growth recovery following the alleviation of osmotic stress. Full article

Human heat shock protein 90 (Hsp90) is one of the most important chaperones that play a role in the late stages of protein folding. Errors in the process of the chaperone cycle can lead to diseases such as cancer and neurodegenerative diseases. Therefore, the activity of Hsp90 must be carefully regulated. One of the possibilities is allosteric regulation by its natural allosteric modulators—nucleotides, co-chaperones and client proteins—and synthetic small-molecule allosteric modulators, such as those targeting the middle domain or the C-terminal domain (CTD) of Hsp90. Since no experimentally determined structure of a small-molecule allosteric modulator bound to the CTD of human Hsp90 has yet been obtained, the challenge for a structure-based design of allosteric modulators remains. Solution nuclear magnetic resonance (NMR) spectroscopy could be utilized to overcome these problems. The main aim of this review article is to discuss how solution NMR techniques, especially protein-based, and the advanced isotope labeling of proteins have been used to investigate the allosteric regulation of the cytosolic isoforms of human Hsp90 with allosteric modulators. This article provides the basis for planning future NMR experiments, with the aim of gaining insights into allosteric sites and the mechanisms of allosteric regulation. Full article

Liver failure is the 12th leading cause of death worldwide. Protein-bound toxins such as bilirubin are responsible for many complications of the disease. Binder dialysis systems use albumin or another binding molecule in dialysate and detoxifying sorbent columns to remove these toxins. Systems like the molecular adsorbent recirculating system and BioLogic-DT have existed since the 1990s, but survival benefits in randomized controlled trials have not been consistent. New binder dialysis systems, including open albumin dialysis and the Advanced Multi-Organ Replacement system, are being developed. Optimal conditions for binder dialysis have not been established. We developed and validated a computational model of bound solute dialysis. It predicted the impact of changing between two test setups using different polysulfone dialyzers (F3 and F6HPS). We then predicted the impact of varying the dialysate flow rate on toxin removal. We found that bilirubin removal declines with dialysate flow rate. This can be explained through a linear decline in free bilirubin membrane permeability. Our model quantifies this decline through a single parameter (polysulfone dialyzers). Validation for additional dialyzers and flow rates will be needed. This model will benefit clinical trials by predicting optimal dialyzer and flow rate conditions. Accounting for toxin adsorption onto the dialyzer membrane may improve results further. Full article

Carrion’s disease, caused by infection with the bacterium Bartonella bacilliformis (B. bacilliformis), is effectively treated with antibiotics, but reaches fatality rates of ~90% if untreated. Current diagnostic methods are limited, insufficiently sensitive, or require laboratory technology unavailable in endemic areas. Electrochemical aptamer-based (E-AB) biosensors provide a potential solution for this unmet need, as these biosensors are portable, sensitive, and can rapidly report the detection of small molecule targets. Here, we developed an E-AB biosensor to detect Pap31, a biomarker of Carrion’s disease and an outer membrane protein in B. bacilliformis. We identified an aptamer with Pap31-specific binding affinity using a magnetic pull-down assay with magnetic bead-bound Pap31 and an aptamer library followed by electrophoretic mobility shift assays. We incorporated the Pap31-binding aptamer into a DNA oligonucleotide that changes conformation upon binding Pap31. The resultant Pap31 E-AB biosensor produced a rapid, significant, and target-specific electrical current readout in the buffer, demonstrating an apparent K_D of 0.95 nM with a limit of detection of 0.1 nM, and no significant signal change when challenged with off-target proteins. This proof-of-concept Pap31 biosensor design is a first step toward the development of more rapid, sensitive, and portable diagnostic tools for detecting Carrion’s disease. Full article

This paper reports the development of a highly crosslinked hyper-branched polyglycerol (HPG) polymer bound to elastin-like proteins (ELPs) to create a membrane that undergoes a distinct closed-to-open permeation transition at 32 °C. The crosslinked HPG forms a robust, mesoporous structure (150–300 nm pores), suitable for selective filtration. The membranes were characterized by FTIR, UV–visible spectroscopy, SEM, and AFM, revealing their structural and morphological properties. Incorporating a synthetic polypeptide introduced thermo-responsive behavior, with the membrane transitioning from impermeable to permeable above the lower critical solution temperature (LCST) of 32 °C. Permeation studies using crystal violet (CV) demonstrated selective transport, where CV permeated only above 32 °C, while water permeated at all temperatures. This hybrid HPG-ELP membrane system, acting as a molecular switch, offers potential for applications in drug delivery, bioseparations, and smart filtration systems, where permeability can be controlled by temperature. Full article

This work reported gold nanoparticles (AuNPs)-based colorimetric immunoassay with the Cu-based metal–organic framework (MOF) to load pyrroloquinoline quinone (PQQ) for the catalytic oxidation of cysteine. In this method, both Cu²⁺ and PQQ in the MOF could promote the oxidation of inducer cysteine by redox cycling, thus limiting the cysteine-induced aggregation of AuNPs and achieving dual signal amplification. Specifically, the recombinant carcinoembryonic antigen (CEA) targets were anchored on the MOF through the metal coordination interactions between the hexahistidine (His₆) tag in CEA and the unsaturated Cu²⁺ sites in MOF. The CEA/PQQ-loaded MOF could be captured by the antibody-coated ELISA plate to catalyze the oxidation of cysteine. However, once the target CEA in the samples bound to the antibody immobilized on the plate surface, the attachment of CEA/PQQ-loaded MOF would be limited. Cysteine remaining in the solution would trigger the aggregation of AuNPs and cause a color change from red to blue. The target concentration was positively related to the aggregation and color change of AuNPs. The signal-on competitive plasmonic immunoassay exhibited a low detection limit with a linear range of 0.01–1 ng/mL. Note that most of the proteins in commercial ELISA kits are recombinant with a His₆ tag in the N- or C-terminal, so the work could provide a sensitive plasmonic platform for the detection of biomarkers. Full article