Human Lysozyme Research Articles

Cryo-EM structure of a lysozyme-derived amyloid fibril from hereditary amyloidosis

Systemic ALys amyloidosis is a debilitating protein misfolding disease that arises from the formation of amyloid fibrils from C-type lysozyme. We here present a 2.8 Å cryo-electron microscopy structure of an amyloid fibril, which was isolated from the abdominal fat tissue of a patient who expressed the D87G variant of human lysozyme. We find that the fibril possesses a stable core that is formed by all 130 residues of the fibril precursor protein. There are four disulfide bonds in each fibril protein that connect the same residues as in the globularly folded protein. As the conformation of lysozyme in the fibril is otherwise fundamentally different from native lysozyme, our data provide a structural rationale for the need of protein unfolding in the development of systemic ALys amyloidosis.

Effects of combined action of proline-rich peptides of human saliva with antimicrobial proteins

BACKGROUND: The substances that make up mixed saliva exhibit activity against bacteria, viruses and fungi. Among them are various cationic antimicrobial peptides: alpha- and beta-defensins, cathelicidins, histatins, but their concentration is relatively low. On the other hand, saliva widely contains a fraction of proline-rich proteins and products of their proteolysis — peptides, the functions of which currently remain poorly studied and unclear. The study of the antimicrobial activity of proline-rich proteins, in particular, when they act together with antimicrobial peptides, and the deciphering of the molecular and cellular basis for the implementation of the protective functions of proline-rich peptides is an urgent task in medicine and biology. The data obtained will help to understand the mechanisms of anti-infective protection of the oral cavity with the participation of the innate immune system, and in the future will allow the creation of drugs based on proline-rich proteins in humans and animals. AIM: Study of the individual antibacterial action of proline-rich salivary proteins, their combined action with the antimicrobial proteins lactoferrin and lysozyme, as well as with one of the most significant endogenous protective peptides — cathelicidin LL-37 on the formation of bacterial biofilms. MATERIALS AND METHODS: Human lactoferrin and egg lysozyme were used in the work. The minimum inhibitory concentrations of peptides against gram-negative and gram-positive bacteria were estimated using serial dilutions in a liquid nutrient medium. Using serial dilutions using the “chessboard” method, it was shown that proline-rich proteins can increase the antibacterial activity of innate immunity peptides present in the oral cavity. The ability of combinations of cationic proline-rich peptides with cathelicidin LL-37 to prevent the formation of bacterial biofilms was studied. RESULTS: In the presence of proline-rich peptides, the antimicrobial activity of some antimicrobial peptides and proteins present in saliva against planktonic bacteria increases. The combined action of proline-rich proteins with LL-37 increases the inhibition of bacterial biofilm formation. CONCLUSIONS: As a result of the study, it was shown that the analyzed cationic proline-rich peptides of human saliva exhibit anti-inflammatory effects, which suggests their significant role in the regulation of inflammatory processes in the oral cavity, as well as their participation in recovery processes.

Flavones Suppress Aggregation and Amyloid Fibril Formation of Human Lysozyme under Macromolecular Crowding Conditions.

The crowded milieu of a biological cell significantly impacts protein aggregation and interactions. Understanding the effects of macromolecular crowding on the aggregation and fibrillation of amyloidogenic proteins is crucial for the treatment of many amyloid-related disorders. Most in vitro studies of protein amyloid formation and its inhibition by small molecules are conducted in dilute buffers, which do not mimic the complexity of the cellular environment. In this study, we used PEGs to simulate macromolecular crowding and examined the inhibitory effects of flavones DHF, baicalein, and luteolin on human lysozyme (HuL) aggregation at pH 2. Naturally occurring flavones have been effective inhibitors of amyloid formation in some proteins. Our findings indicate that while flavones inhibit HuL aggregation and fibrillation in dilute buffer solutions, complete inhibition is observed with a combination of flavones and PEGs, as shown by ThT fluorescence, light scattering, TEM, and AFM studies. The species formed in the presence of PEG 8000 and flavones were less hydrophobic, less toxic, and α-helix-rich compared to control samples, which were hydrophobic and β-sheet-rich, as demonstrated by ANS hydrophobicity, MTT assay, and CD spectroscopy. Fluorescence titration studies of flavones with HuL showed a significant increase in binding constant values under crowding conditions. These findings highlight the importance of macromolecular crowding in modulating protein aggregation and amyloid inhibition. Further studies using disease-causing mutants of HuL and other amyloidogenic proteins are needed to explore the role of macromolecular crowding in small-molecule-mediated modulation and inhibition of protein aggregation and amyloid formation.

A structural rationale for reversible vs irreversible amyloid fibril formation from a single protein.

Reversible and irreversible amyloids are two diverging cases of protein (mis)folding associated with the cross-β motif in the protein folding and aggregation energy landscape. Yet, the molecular origins responsible for the formation of reversible vs irreversible amyloids have remained unknown. Here we provide evidence at the atomic level of distinct folding motifs for irreversible and reversible amyloids derived from a single protein sequence: human lysozyme. We compare the 2.8 Å structure of irreversible amyloid fibrils determined by cryo-electron microscopy helical reconstructions with molecular insights gained by solid-state NMR spectroscopy on reversible amyloids. We observe a canonical cross-β-sheet structure in irreversible amyloids, whereas in reversible amyloids, there is a less-ordered coexistence of β-sheet and helical secondary structures that originate from a partially unfolded lysozyme, thus carrying a "memory" of the original folded protein precursor. We also report the structure of hen egg-white lysozyme irreversible amyloids at 3.2 Å resolution, revealing another canonical amyloid fold, and reaffirming that irreversible amyloids undergo a complete conversion of the native protein into the cross-β structure. By combining atomic force microscopy, cryo-electron microscopy and solid-state NMR, we show that a full unfolding of the native protein precursor is a requirement for establishing irreversible amyloid fibrils.

Testing AI predictions that hyperglycemia-induced non-enzymatic glycation of immune system proteins impairs diabetic immunity

People with type 1, type 2 and gestational diabetes, and newborns of diabetic mothers, have increased risks of contracting microbial infections such as SARS-CoV-2, amplifying comorbidity and mortality risks. Hyperglycemia and its associated hyperinflammation mediate these risks but by unknown mechanisms. We hypothesize that hyperglycemia results in glycation (non-enzymatic addition of sugars to free amino groups such as lysines) of antimicrobial and anti-inflammatory proteins and peptides that mediate innate and adaptive immunity. While glycation of hemoglobin A1c is used as a benchmark for glycemic control, studies of the effects of the glycation of immune system molecules are almost non-existent. Three AI protein-glycation prediction models demonstrate that immune system proteins and peptides such as serotransferrin, lactotransferrin, lysozyme, complement proteins, immunoglobulins, Toll-like receptors, etc., probably glycate at multiple sites affecting function. However, the AI-predicted glycation sites differed significantly among the three models employed. Therefore, three tests of these predictions were carried out. Predicted glycation sites were first evaluated in terms of clinically or experimentally identified glycation sites of homologous proteins and known effects of lysine substitutions at predicted glycation sites. Second, mass spectrometry was used to identify glycation sites on lactoferrin and human lysozyme. Third, the effects on the antimicrobial activity of lactotransferrin and lysozyme glycation were tested using a Candida albicans assay. Results of tests demonstrate that protein glycation occurs and interferes with immune function but that AI glycation prediction models are very inaccurate. Suggestions for improving AI accuracy are provided.

Human milk lactoferrin and lysozyme concentrations vary in response to a dietary intervention

It is known that human milk (HM)11Human milk. antimicrobial protein composition varies during lactation. However, the impact of maternal diet on these antimicrobial proteins, particularly lactoferrin and lysozyme remains unknown. In addition, it is unclear whether daily, circadian, and between breast variations exist for lactoferrin and lysozyme concentrations. We investigated the impact of a low sugar, low fat, high fibre dietary intervention on HM lysozyme and lactoferrin concentrations. HM was sampled across a 3-week period; daily, at different times of day, and from both breasts to measure the level of intraindividual variation. The intervention significantly reduced maternal sugar, total fat, and saturated fat intake. HM lactoferrin concentration declined significantly over the course of the intervention however the effect size was relatively small. In addition, lactoferrin and lysozyme concentrations were variable over time, and differed significantly within and across the day but not between breasts.

Influence of amphiphilic chitosan-deoxycholic acid-polyacrylic acid copolymer on activity, dynamics, and structure of human lysozyme and pyrazinamidase

Cationic chitosan, a naturally degradable and biocompatible polysaccharide, has shown considerable promise as a drug delivery system. However, its limited solubility in water at neutral and alkaline pH has restricted its broader application. To overcome this challenge, various chemical modification strategies have been explored to enhance the solubility of chitosan. One such strategy involves the development of chitosan-deoxycholic acid-polyacrylic acid (CDP) copolymers, which are novel amphiphilic derivatives of chitosan that exhibit excellent water solubility and self-assembly properties. Herein, we investigated the potential of CDP as an anionic carrier for two model proteins: the positively charged Lysozyme and the negatively charged Pyrazinamidase. The objective of this investigation is to elucidate the role of electrostatic interactions between the copolymer and the proteins during the drug encapsulation process. A combined approach utilizing experimental techniques and molecular dynamics (MD) simulations was used to investigate the interactions between the polymer and the proteins, as well as any potential structural changes to the proteins. The results suggest contrasting binding mechanisms for the two proteins: Lysozyme being entrapped within the cationic polymer network and Pyrazinamidase being adsorbed onto the surface of the polymeric assemblies. Binding energy calculations revealed favorable physical interactions between CDP and Lysozyme, while Pyrazinamidase exhibited unfavorable binding. These findings demonstrated that CDP may serve as a promising carrier for positively charged drugs due to its favorable interactions and potential entrapment within the polymer network. The favorable interactions observed between CDP and Lysozyme suggest that CDP could be further explored as a delivery system for positively charged therapeutic proteins.

Tailoring the Molecular Structure of 6-Gingerol for Targeting the Phase Separation in Human Lysozyme.

Human lysozyme undergoes a phase-separation process to form insoluble amyloid-architects that cause several pathologies including systemic amyloidosis. Here we have tailored 6-gingerol by extending its molecular framework with active functional groups to specifically target lysozyme phase-transition events. Aggregation assay revealed that tailored 6-gingerol with 4-aromatic moieties (MTV4) substantially suppressed the conversion of the lysozyme low-density liquid phase (LDLP) to solid-phase structured amyloids. The data obtained from biophysical, computational, and microscopic imaging tools suggest direct intervention of MTV4 with the liquid-liquid phase separation. The CD data suggest that MTV4 was able to retain the native conformation of lysozyme. Both biomolecular and computational data reveal the interference of MTV4 with the aggregation-prone hydrophobic stretches within the lysozyme, thereby retaining the native structure and reversing the misfolded intermediates to active monomers. Also, MTV4 was able to induce rapid dissolution of preformed-toxic amyloid fibrils. These results reinforce the importance of the aromatic-aromatic interaction in preventing human lysozyme phase separation.

Multifunctional carbonized polymer dots: Synthesis and applications in retarding protein amyloid aggregation, capturing free radicals and cell imaging

In this study, a series of carbonized polymer dots were synthesized using Congo red and tryptophan as carbon source by changing their molar ratio through simple one-step method in microwave oven. The prepared carbonized polymer dots could emit red fluorescence. All prepared carbonized polymer dots could inhibit the amyloid aggregation of human lysozyme and the CPDs-3 showed the best inhibition capability. The inhibition ratio of the CPDs-3 reached 95.8% at concentration of 40 μg/mL and the IC50 was 0.41 μg/mL, which may be attributed to the highest content of Congo red on the periphery of the CPDs-3. The CPDs-3 could easily interact with β-sheet structure of human lysozyme through π-π stacking and hydrophobic interaction, which helped to retard the amyloid aggregation. The CPDs-3 also displayed good anti-oxidative property and the highest elimination efficiency for hydroxyl free radical. In addition, the CPDs-3 showed nontoxicity on bEnd.3 cell by CCK-8 assay and could be used in cell imaging.

Exploring the underlying mechanism of interaction of Sulindac with Human hemoglobin and Lysozyme: Multi-spectroscopic, molecular docking, DFT and MD simulation approaches

Contrasting behavior of Nonsteroidal anti-inflammatory drug Sulindac (SDC) towards two different proteins Human hemoglobin (HHb) and Lysozyme (LYS) has been explored by multispectroscopy, molecular docking, DFT and MD Simulation approaches. The fluorescence quenching experiments showed that SDC strongly binds with both proteins in a static manner. Alteration in the secondary structure of HHb and LYS in presence of SDC was further investigated by CD and UV–visible spectroscopy. It is observed that on addition of SDC, the % α-helix of native HHb decreases whereas it increases for LYS-SDC system substantiated the fact SDC stabilized the secondary structure of LYS and destabilized the HHb structure thus, showing the contrasting traits. Experimental results further validated by computational techniques. Molecular docking and DFT were conducted to study the binding sites, nature of interactions and HOMO-LUMO energy gap in both the systems which revealed that the binding of SDC with both proteins resulted in the considerable change in the molecular structure of the former. Additionally, we performed MD simulations to study the structural stability of Lysozyme and Hemoglobin, both unbound and bound to the Sulindac molecule. Sulindac binding showed distinct impacts on protein stability in both proteins, supported by global and essential dynamics.This study not only provides important insights into the binding of SDC with HHb and LYS but also it provides a useful direction in the pharmacology and clinical medicine fields.

Metagenomic Analysis of Rhizospheric Bacterial Community of Citrus Trees Expressing Phloem-Directed Antimicrobials

Huanglongbing, also known as citrus greening, is currently the most devastating citrus disease with limited success in prevention and mitigation. A promising strategy for Huanglongbing control is the use of antimicrobials fused to a carrier protein (phloem protein of 16 kDa or PP16) that targets vascular tissues. This study investigated the effects of genetically modified citrus trees expressing Citrus sinensis PP16 (CsPP16) fused to human lysozyme and β-defensin-2 on the soil microbiome diversity using 16S amplicon analysis. The results indicated that there were no significant alterations in alpha diversity, beta diversity, phylogenetic diversity, differential abundance, or functional prediction between the antimicrobial phloem-overexpressing plants and the control group, suggesting minimal impact on microbial community structure. However, microbiota diversity analysis revealed distinct bacterial assemblages between the rhizosphere soil and root environments. This study helps to understand the ecological implications of crops expressing phloem-targeted antimicrobials for vascular disease management, with minimal impact on soil microbiota.

Antimicrobial potential and protein binding affinity of a hybrid ternary nanocomposite matrix: Zinc oxide functionalization with poly(vinyl alcohol)-sulphonated graphene oxide-poly pyrrole

The present study focuses on the application of a ternary PVA/sGO/ZnO-NP/PP nanocomposite as an antibacterial and antifungal agent. The process involved utilizing a solution casting technique to fabricate the inorganic–organic blend material, where 4-sulfophthalic acid (SPTA) was used in the sulfonation of graphitic oxide (GO) sheets. The amalgamation of polyvinyl alcohol polymer (PVA) and sulphonated GO (sGO) was achieved through a chemical oxidation polymerization process carried out in a water-based environment. This process developed a PVA/sGO film, in which zinc oxide nanoparticles (ZnO-NP) and pyrrole monomers were introduced. The PVA/sGO film served as the initial stage for the in-situ deposition and formation of polypyrrole (PP), leading to the fabrication of PVA/sGO/ZnO-NP/PP membrane. The structure was stabilized by hydrogen bonding and electrostatic interactions between the components, while SPTA acted as a cross-linker and a sulphonating agent. The Fourier transform infrared (FTIR) spectra of the quad-component composite unraveled the presence of distinctive functional groups, including PVA, sGO, PP, and Zn-O bands. The UV–visible spectra of PVA/sGO/ZnO-NP/PP nanocomposite membrane revealed a peak in the UV-B region, while in-depth X-ray diffraction (XRD) analyses provided strong evidence of its amorphous nature. The scanning electron microscopy (SEM) image showed that the top surface of the organic film was adorned with PP polymeric chains. The membrane exhibited great water retention capabilities due to its high ion exchange capacity (IEC) value.Additionally, the minimal water loss observed underscores its extended shelf life. Due to their inherent hydrophilic properties, the ternary nanocomposite membranes exhibited enhanced hydrophilicity, porosity, zeta potential, and water uptake. The BET and BJH analyses revealed that PVA/sGO/ZnO-NP/PP nanocomposites had a substantial surface area. Remarkably, the nanocomposite membrane exhibited significantly improved antimicrobial efficacy against bacteria and fungi. The type of interactions between Human serum lysozyme (HSL) and nanocomposite at the molecular level was studied through fluorescence spectroscopy. Stern-Volmer plot revealed the occurrence of both static and dynamic quenching. However, the precise mechanisms underlying the antimicrobial and protein binding (HSL) studies of PVA/sGO/ZnO-NP/PP-based nanocomposites require further investigation.

Cytokinetic engineering enhances the secretory production of recombinant human lysozyme in Komagataella phaffii

BackgroundHuman lysozyme (hLYZ) is a natural antibacterial protein with broad applications in food and pharmaceutical industries. Recombinant production of hLYZ in Komagataella phaffii (K. phaffii) has attracted considerable attention, but there are very limited strategies for its hyper-production in yeast.ResultsHere through Atmospheric and Room Temperature Plasma (ARTP)-based mutagenesis and transcriptomic analysis, the expression of two genes MYO1 and IQG1 encoding the cytokinesis core proteins was identified downregulated along with higher hLYZ production. Deletion of either gene caused severe cytokinesis defects, but significantly enhanced hLYZ production. The highest hLYZ yield of 1,052,444 ± 23,667 U/mL bioactivity and 4.12 ± 0.11 g/L total protein concentration were obtained after high-density fed-batch fermentation in the Δmyo1 mutant, representing the best production of hLYZ in yeast. Furthermore, O-linked mannose glycans were characterized on this recombinant hLYZ.ConclusionsOur work suggests that cytokinesis-based morphology engineering is an effective way to enhance the production of hLYZ in K. phaffii.

Binding effect of tyrosine-based chiral carbon dots on human serum albumin and lysozyme

Recently, chiral carbon dots (CCDs) have generated significant interest in biological fields such as biosensing, bioimaging, and drug delivery, due to the combination of inherent chirality and nanoscale size. Here, spectroscopic methods have been used to monitor the interaction between human serum albumin (HSA) and lysozyme (LYZ) with CCDs. Long-wavelength emission CCDs (LWE-CCDs) were synthesized using L/D-tyrosine (L/D-Tyr) and o-phenylenediamine (o-PDA) through a facile and one-step solvothermal approach. Two mentioned proteins interacted with L- and D-CDs and the results of secondary structure studies revealed that proteins preserve their native structure while having a small increase in their compactness. Furthermore, studies on the tertiary structure indicated more flexibility and the possibility of disorder in the hydrophobic regions of HSA, while LYZ appeared to become more rigid. Generally, the L- and D-CDs have more effect on the secondary and tertiary structures of HSA and LYZ, respectively. In addition, the interaction of L/D-Tyr (chiral precursor) with proteins was studied using molecular docking to explain the different effects of the CCDs on HSA and LYZ, and the obtained results are almost consistent with the results of experimental tests. This research offers new perspectives on the synthesis of LWE-CCD and the impact of their chirality on protein-nanoparticle interactions. Studying the action of CCDs at the molecular level will help researchers to choose suitable isomers to apply in biological applications.

REGULATION OF ENZYME-DEPENDENT LYSIS OF MICROBIAL CELLS: IS THE EFFECTOR TARGET ENZYME OR SUBSTRATE?

In this work, an attempt was made to analyze the literature data regarding the effectors of bacterial lysis in the presence of various bacteriolytic enzymes. Despite the differences between such enzymes, it is possible to identify certain general patterns of their action on a highly complex substrate - a living bacterial cell protected by a cell wall and additional complexes of biopolymers associated with it. Chicken and human lysozymes are the best known of these enzymes. They have some structural differences, but are generally very similar in properties. Understanding the characteristics of the antibacterial action of bacteriolytic enzymes present both in medications and in the human immune system is extremely important for the development of new approaches to combating bacterial infections, including antibiotic-resistant ones. Moreover, certain logical and methodological approaches used to study bacteriolytic enzymes can be extremely useful for studying and describing other enzymes that affect complex polymer substrates in real biological situations.

Intestinal lysozyme engagement of Salmonella Typhimurium stimulates the release of barrier-impairing InvE and Lpp1

Lysozyme is a β-1,4-glycosidase that hydrolyzes the polysaccharide backbone of bacterial cell walls. With an additional bactericidal function mediated by a separate protein domain, lysozyme is considered a uniquely important antimicrobial molecule contributing to the host's innate immune response to infection. Elevated lysozyme production is found in various inflammatory conditions while patients with genetic risks for inflammatory bowel diseases demonstrate abnormal lysozyme expression, granule packaging, and secretion in Paneth cells. However, it remains unclear how a gain- or loss-of-function in host lysozyme may impact the host inflammatory responses to pathogenic infection. We challenged Lyz1-/- and ectopic Lyz1-expressing (Villin-Lyz1TG) mice with S. Typhimurium and then comprehensively assessed the inflammatory disease progression. We conducted proteomics analysis to identify molecules derived from human lysozyme-mediated processing of live Salmonella. We examined the barrier-impairing effects of these identified molecules in human intestinal epithelial cell monolayer and enteroids. Lyz1-/- mice are protected from infection in terms of morbidity, mortality, and barrier integrity, whereas Villin-Lyz1TG mice demonstrate exacerbated infection and inflammation. The growth and invasion of Salmonella invitro are not affected by human or chicken lysozyme, whereas lysozyme encountering of live Salmonella stimulates the release of barrier-disrupting factors, InvE-sipC and Lpp1, which directly or indirectly impair the tight junctions. The direct engagement of host intestinal lysozyme with an enteric pathogen such as Salmonella promotes the release of virulence factors that are barrier-impairing and pro-inflammatory. Controlling lysozyme function may help alleviate the inflammatory progression.

Characterization of the structural and molecular interactions of Ferulic acid ethyl ester with human serum albumin and Lysozyme through multi-methods

Ferulic acid ethyl ester (FAEE) is an essential raw material for the formulation of drugs for cardiovascular and cerebrovascular diseases and leukopenia. It is also used as a fixed aroma agent for food production due to its high pharmacological activity. In this study, the interaction of FAEE with Human serum albumin (HSA) and Lysozyme (LZM) was characterized by multi-spectrum and molecular dynamics simulations at four different temperatures. Additionally, the quenching mechanism of FAEE-HSA and FAEE-LZM were explored. Meanwhile, the binding constants, binding sites, thermodynamic parameters, molecular dynamics, molecular docking binding energy, and the influence of metal ions in the system were evaluated. The results of Synchronous fluorescence spectroscopy, UV–vis spectroscopy, CD, three-dimensional fluorescence spectrum, and resonance light scattering showed that the microenvironment of HSA and LZM and the protein conformation changed in the presence of FAEE. Furthermore, the effects of some common metal ions on the binding constants of FAEE-HSA and FAEE-LZM were investigated. Overall, the experimental results provide a theoretical basis for promoting the application of FAEE in the cosmetics, food, and pharmaceutical industries and significant guidance for food safety, drug design, and development.

Effect of dietary supplementation with recombinant human lysozyme on growth performance, antioxidative characteristics, and intestinal health in broiler chickens.

Lysozyme is often used as a feed additive to act as an antibacterial protein that boosts the immune system of livestock and poultry while protecting against pathogens. To investigate the effects of recombinant human lysozyme (rhLYZ) from Pichia pastoris and chlortetracycline on broiler chicken's production performance, antioxidant characteristics, and intestinal microbiota, a total of 200, 1-d-old male Arbor Acres broiler chickens (46.53 ± 0.42g) were selected for a 42-d experiment. Dietary treatments included a basal diet of corn-soybean meal supplemented with either 0mg/kg (CON), 50mg/kg aureomycin (ANT), 20mg/kg rhLYZ (LOW), 60mg/kg rhLYZ (MEDIUM), or 180mg/kg rhLYZ (HIGH). Compared with CON, MEDIUM diet increased (P < 0.05) average daily gain (67.40g) of broilers from day 22 to 42. In the early (1.29) and overall phases (1.69), MEDIUM led to a reduction (P < 0.05) in the feed conversion ratio of broiler chickens. Furthermore, in comparison to the CON and ANT, MEDIUM exhibited reduced (P < 0.05) levels of INF-γ and tumor necrosis factor-α in the serum. In the cecum, the abundance of Monoglobus and Family_XIII_AD3011_group was lower (P < 0.05) in the MEDIUM treatment compared to CON. Overall, supplementation of 60mg/kg of rhLYZ improved growth performance, nutrient utilization efficiency, and serum immune function, while also influencing the composition of intestinal microbiota. This suggests lysozyme's potential to replace antibiotic additives in feed.

A small angle X-ray scattering study comparing protic ionic liquid solvent effects on the solution behaviors of hen egg white and human lysozyme

Proteins demand customized solvents for maintaining their stability. However, the specific impact of different ions on different proteins remains unclear. This study explores the effects of protic ionic liquid (PIL)-based solvents on two structurally similar proteins, namely hen egg white and human lysozyme. By employing size exclusion chromatography small-angle X-ray scattering (SEC-SAXS) and capillary SAXS, the effects of protein concentration (3 to 60 mg/mL), IL type (five PILs) and concentration (1, 10 and 20 mol% with water) and protein type on the aggregation behavior and radius of gyration (Rg) of the two lysozymes were studied. HEWL was mostly monomeric in buffer and solubilized beyond 20 mg/mL, while HL displayed slight aggregation in buffer but had increased aggregation at over 10 mg/mL. Further capillary SAXS data show that both proteins became more aggregated at concentrations exceeding 20 mg/mL and over 1 mol% PILs. The nitrate anion generally showed the “salting in” effect, while formate and glycolate anions induced the “salting out” effect on the two proteins. Overall, anion selection and protein solution behaviors at protein concentration of over 20 mg/mL and at 20 mol% ILs can be critical for maintaining the lysozyme stability.

Inhibition of protein misfolding and aggregation by steroidal quinoxalin-2(1H)-one and their molecular docking studies

A series of D-ring fused 16-substituted steroidal quinoxalin-2(1H)-one attached to an electron-releasing (ER) or electron-withdrawing (EW) groups via steroidal oxoacetate intermediate were synthesized to investigate their protein aggregation inhibition potential using human lysozyme (HLZ). The influence of the type of substituent at the C-6 positions of the quinoxalin-2(1H)-one ring on the protein aggregation inhibition potential was observed, showing that the EW moiety improved the protein aggregation inhibition potency. Of all the evaluated compounds, NO2-substituted quinoxalin-2(1H)-one derivative 13 was the most active compound and had a maximum protein aggregation inhibition effect. Significant stabilization effects strongly support the binding of the most biologically active steroidal quinoxalin-2(1H)-one with docking studies. The predicted physicochemical and ADME properties lie within a drug-like space which shows no violation of Lipinski's rule of five except compounds 12 and 13. Combined, our results suggest that D-ring fused 16-substituted steroidal quinoxalin-2(1H)-one has the potential to modulate the protein aggregation inhibition effect.