Search Results (629)

Accessory proteins of SARS-CoV-2 play crucial roles in viral pathogenesis, yet their structural properties remain elusive. ORF7b, a small accessory protein comprising only 43 amino acids, is widely assumed to parallel the structure–function relationships of its SARS-CoV ortholog based solely on sequence homology. In this study, we challenge this paradigm through direct physicochemical and structural characterization. Sequence analysis and electrostatic profiling reveal that the SARS-CoV-2 protein is a macromolecular polyanion with a net charge of −4 at neutral pH, featuring a diffuse negative surface that is highly responsive to pH changes. Complete 3D structures generated via ab initio modeling display a helical core flanked by two highly fluctuating, disordered termini. Residue Interaction Network (RIN) topology and Normal Mode Analysis (NMA) identified specific hinges governing these flexible extremities. Furthermore, the calculated dipole moment vector is tilted outward by 24°, misaligning with the central axis. Molecular dynamics simulations suggest that while the soluble structure is highly stable in water, it undergoes severe distortions and insufficient solvation within a membrane-mimetic environment. Thermodynamic association profiles and verified interactomic data from BioGRID reveal a strong propensity for ORF7b to participate in liquid–liquid phase transitions alongside human and viral partners. Taken together, these unique properties suggest that ORF7b operates as a dynamic peripheral membrane protein rather than a sedentary transmembrane component, providing a fresh framework for future therapeutic targeting. Overall, these in silico findings shift the current paradigm on ORF7b2 topology and provide a robust, physically grounded framework that identifies specific molecular priorities for future in vitro and in vivo validation. Full article

In this work, charge characteristics of submicron heterogeneities (SMH) spontaneously formed in aqueous solutions of various biomolecules: seven amino acids of various types (nonpolar glycine, polar serine, hydrophobic valine, aromatic phenylalanine, sulfur-containing methionine, glutamic acid and basic arginine), ATP, monosaccharide glucose and disaccharide sucrose were studied. The isoelectric points of the SMH in the amino acid solutions determined turned out to be in the pH range from 2.4 to 4, being shifted to the acidic region relative to the isoelectric points of the amino acids themselves (except for glutamic acid). The zeta potential of the SMH was measured in solutions of all the biomolecules under conditions close to the intracellular environment at pH = 7 and basic K⁺ ion content 150 mM. The zeta potential appeared to be negative in all cases. Using these values of the zeta potential, the concentration of OH-anions inside the SMH was estimated, and the pH values corresponding to this concentration turned out to be in the range of 7–10. Since the cell cytosol is an aqueous solution of various biomolecules, SMH must also form inside cells. An analogy is drawn between SMH and membraneless organelles, many of which have been discovered recently. The presence of compact regions with alkaline pH inside the cell is a fundamentally new factor in cell biology, which may have important consequences. Full article

This study aimed to characterize metabolomic changes in the eye lens of senescence-accelerated OXYS rats in comparison with control Wistar rats, and to identify biochemical shifts associated with genotype, age, and cataract progression. Cataract severity was clinically graded. Rats’ lenses were analyzed using quantitative ¹H NMR spectroscopy at 3.6 and approximately 4.5 months of age. A total of 43 metabolites were quantified. We found that at 3.6 months of age, OXYS lenses exhibited a significant accumulation of 17 metabolites, primarily amino acids, compared to Wistar rats, suggesting an imbalance between amino acid uptake and crystallin biosynthesis. However, by 4.5 months, OXYS lenses exhibited rapid metabolic changes characterized by significant decreases in amino acid, glucose, and key energy/antioxidant markers, including NAD, adenylate energy charge, and hypotaurine. Clinical cataract grade (Grade 2 vs. 3) had a negligible impact on the overall metabolomic profile. Our results indicate that profound metabolic reorganization, including an initial amino acid excess followed by energy and antioxidant depletion, precedes the morphological manifestation of cataracts in OXYS rats. We suggest that a biochemical “point of no return” occurs early in cataractogenesis, while subsequent increase in lens opacification is a secondary consequence of preexisting metabolic disturbances. Full article

Dengue is an arboviral disease of global significance caused by Orthoflavivirus denguei (DENV), which has four antigenically distinct serotypes. The envelope (E) protein plays a critical role in viral entry and eliciting immune responses. This study aimed to compare the biochemical and immunological properties of the E protein across the four DENV serotypes using in silico approaches. E protein reference sequences were retrieved from RefSeq and analyzed with various bioinformatics tools. Sequence alignment revealed identities ranging from 63.08% to 77.69%. Biochemical analysis showed minimal variation in molecular weight and isoelectric point; however, the net charge of DENV-3 E protein was notably lower. Secondary structure predictions indicated a predominance of alpha-helices in DENVs-1/2, while DENVs-3/4 featured more beta-sheets. Post-translational modification analysis revealed mostly casein kinase II phosphorylation sites across all serotypes, with DENV-4 uniquely presenting also tyrosine kinase sites. Amino acids W231/D341 in DENV-1, Q86 in DENVs-2/4, and D87/D339 in DENV-3 showed maximum antigenicity scores in B cell recognition, while the human leukocyte antigen (HLA) alleles B*08:01/B*39:01 and DRB4*01:01, recognized by T cells, presented the highest number of predicted epitopes for the different DENV serotypes. Conservation analysis showed that the major antigenic regions highlighted in this study are highly conserved among contemporary DENV isolates despite the genetic variability observed within each serotype. These findings suggest that subtle structural differences in the E protein may contribute to distinct immunogenic profiles, highlighting candidate regions for future investigation. Full article

Background/Objectives: Diabetes mellitus (DM) is a critical metabolic condition with escalated blood glucose levels caused by insulin resistance, restricted insulin production, and the activity of alpha-amylase and alpha-glucosidase enzymes. Methods: This current work focuses on the synthesis and evaluation of novel Pyrimidine-derived pyrazole-based thiadiazole derivatives to target DM by inhibiting α-amylase and α-glucosidase. Results: The findings exhibited that, except for three compounds, all other synthesized derivatives inhibited α-amylase and α-glucosidase enzymes with IC₅₀ values ranging from 5.17 μM to 29.84 μM on α-amylase and 7.60 μM to 31.62 μM on α-glucosidase, in comparison to the standard drug Acarbose (α-amylase IC₅₀ = 8.25 ± 0.80 μM; α-glucosidase IC₅₀ = 10.75 ± 1.10 μM). Analogs 8g, 8k, and 8b displayed superior or comparable inhibitory activity compared to the reference drug Acarbose. The inhibition potential of the derivatives can be attributed to their stable contacts with crucial amino acid residues of targeted enzymes, as shown through molecular docking analysis. Moreover, DFT-calculated HOMO–LUMO parameters and electrostatic potential (ESP) maps were used to gain complementary insight into the electronic characteristics, charge distribution, and potential interaction behavior of the synthesized derivatives, which supported the molecular docking observations. Conclusions: Experimental outcomes and in silico support display that these derivatives serve as potential leads for anti-diabetic drug development. These potent pyrimidine-derived pyrazole-based thiadiazole derivatives were comparable to an existing diabetic mellitus inhibitor, specifying potential for further therapeutic development and optimization against diabetic mellitus. Full article

In recent years, cruor from slaughterhouse blood has garnered growing interest as a potential source of antimicrobial peptides obtained through enzymatic hydrolysis. In addition, electrodialysis with ultrafiltration membrane (EDUF) represents a strategy for valorizing peptide-rich hydrolysates, enabling the selective separation and concentration of antimicrobial peptides, according to their size and charge. Hence, this study evaluated the potential of EDUF to fractionate, for the first time, calf cruor hydrolysate and explore its use as a novel source of antimicrobial peptides. The resulting peptide fractions were characterized to investigate the selectivity of peptide migration in relation to peptide physico-chemical characteristics and membrane properties and to finally assess their antimicrobial activity. High migration rates of 12.75 ± 2.17 g/m²h and 8.94 ± 0.38 g/m²h were observed for the cationic (P⁺) and anionic (P⁻) recovery fractions, respectively. These results suggested that peptide migration from calf cruor hydrolysate to both recovery fractions during EDUF was influenced by the combined effects of molecular weight, net charge, hydrophobicity, specific amino acid residues (L, Y), and peptide–membrane interactions. Furthermore, the initial and final hydrolysates as well as P⁺ fractions exhibited antifungal activities against Paecilomyces spp. and Rhodotorula mucilaginosa with minimum inhibitory concentrations (MIC) ranging from 0.312 to 0.615 mg/mL and minimum fungicidal concentrations (MFCs) ranging from 0.312 to 1.250 mg/mL. In contrast, the P⁻ fraction did not exhibit antifungal activity, but a slight anti-Listeria activity was detected, with a MIC of 10 mg/mL. These findings highlight the potential of upcycling calf blood into functional antifungal and antibacterial agents, supporting a circular economy approach and transforming waste streams into value-added ingredients that enhance food preservation. Full article

To address the poor solubility, instability, and low oral bioavailability of quercetin (Q), Q-loaded nanoparticles (Q@DDZ) were fabricated using deamidated zein peptide (DDZ) via a pH-driven method. As a food-grade hydrophilic colloid, DDZ effectively improves the colloidal stability of the delivery system. Deamidation increased hydrophilic amino acids and surface negative charge. DDZ bound Q via static quenching with a higher binding constant (K_a = 2.25 × 10³ L/mol) and more binding sites (n = 1.7561) than zein, along with stronger hydrogen bonding and hydrophobic interactions. Q@DDZ exhibited higher encapsulation efficiency (45.36–87.32%) and loading capacity (1.82–12.27%) than Q@zein, with a smaller particle size and better dispersibility. At 50.0 μg/mL Q, Q@DDZ showed 41.06% (DPPH) and 46.62% (ABTS) higher scavenging rates than free Q. It displayed excellent stability under acidic, high ionic strength, and thermal conditions (80 °C, 180 min). In simulated digestion, Q@DDZ delayed Q release in the oral and gastric phases and prolonged intestinal release, which indicated potentially improved bioavailability. This study provides mechanistic insights into deamidation-modified plant protein delivery systems for hydrophobic bioactives, offering new perspectives for the development of functional biopolymer gel materials. Full article

Messenger RNA (mRNA) therapeutics are redefining treatment approaches in vaccines, cancer immunotherapy, protein replacement, and gene editing. Lipid nanoparticles have enabled early clinical successes, but they can be limited by liver-dominant biodistribution, long-term storage stability, and systemic tolerability. Polymeric delivery systems offer a versatile alternative, with tunable physicochemical properties enabling precise control over mRNA complexation, protection, release, and targeting. This review examines recent progress across polyethyleneimine derivatives, poly(β-amino ester)s, poly(amino acid)s, polyesters, dendrimers, charge-altering releasable transporters, and lipid-polymer hybrids. We highlight strategies such as structural modification, stimuli-responsive designs, and high-throughput polymer screening that enhance stability, reduce cytotoxicity, and enable organ- or cell-specific delivery. Addressing challenges in immunogenicity, biodistribution, and manufacturing scalability will be pivotal to translating these innovations into safe and effective mRNA therapeutics. Full article

Background: Recent work has revealed that protein-coding sequences encode regulatory information influencing mRNA stability and translation through a nascent peptide code. However, the evolutionary origin of this regulatory layer remains unclear. This study aims to determine when peptide-mediated translational control emerged during the evolution of the proteome and genetic code. Methods: Dipeptide-specific effects on mRNA stability and translation were integrated with a phylogenetic timeline of dipeptide emergence derived from dipeptide sequences across proteomes. Each of the 400 canonical dipeptides was assigned an evolutionary age, and experimentally derived regulatory effects were mapped onto this timeline, with associations assessed using rank-based correlation and regression analyses. Results: A weak but statistically significant negative association was observed between dipeptide age and mRNA stability, indicating that more recently evolved dipeptides tend to destabilize transcripts. This trend was stronger at the amino acid level, where later-emerging residues showed greater contributions to reduced mRNA levels. Destabilizing effects were associated with physicochemical properties such as positive charge, side-chain bulkiness, and β-strand propensity. Mapping these effects onto codon space revealed a non-random distribution aligned with the evolutionary and structural organization of the genetic code. Destabilizing effects were also enriched within specific codon exchange groups, indicating that regulatory signals are structured within the degeneracy and mutational neighborhoods of the code. Conclusions: These findings indicate that the nascent peptide code is a late evolutionary innovation linked to amino acid expansion and proteomic complexity, with regulation embedded within both peptide sequences and the degeneracy structure of the standard genetic code. Full article

Life demonstrates remarkable homochirality of its major building blocks: nucleic acids, amino acids, sugars, and phospholipids. Phospholipid bilayer vesicles (liposomes) are formed at the water/air interface from Langmuir layers and contain ribose, a constituent of primordial water. Although the primordial ribose was initially racemic, life, as we know it, is homochiral, with d-ribose and its derivatives as the predominant forms. The phospholipid membrane’s permeability to d-ribose, together with ribose’s interaction with the bilayer’s charged phosphate groups, leads to ribose phosphorylation, yielding d-ribose-5-phosphate. Once inside, the d-ribose-5-phosphate molecules cannot cross the membrane. A similar path also exists for l-ribose, but with a lower rate. Therefore, overall, this process is enantioselective, favoring the buildup of d-ribose over l-ribose. Through liposome fusion, fission, and self-replication, this eventually leads to the Darwinian evolution of these structures and to the conversion of d-ribose-5-phosphate into complex functional molecules, such as ribozymes and RNA, and eventually into DNA, all of which inherit d-ribose’s chirality. Full article

The present study aimed to compare the composition structure, interfacial, and antioxidant activities of flaxseed protein isolates (FPIs) in different flaxseed varieties. The results showed that apparently intact protein bodies (PBs) were manifested as densely staining cytoplasmic inclusions with distinct boundaries and varying diameter ranges among different flaxseed varieties. Through alkali extraction with isoelectric precipitation, FPIs exhibited a relatively small and irregular lamellar strip structure with varying sizes and shapes packed with spherical particles in studied flaxseed varieties. The different composition structures of FPIs among studied flaxseed varieties were also obtained, involving the protein subunits’ intrinsic fluorescence properties, secondary structures, and amino acid profiles. These structural differences also led to differential purities, aqueous solubility, dispersion properties, and surface charges. Moreover, the varying emulsifying and foaming properties of FPIs from different flaxseed varieties were also observed due to the formation of coarse lipid droplets (5~40 μm) and foams (20~100 μm) with the specific structure of the oil/gas–water interface and bulk aqueous phase. The retention of phenolic compounds into FPIs still displayed evident variety specificity from 323 to 478 mg/100 g and 210 to 347 mg/100 g, which definitely led to escalated antioxidant activities. Thus, FPIs from Longya 13# and Neiya 9# flaxseed varieties were screened for favorable emulsifying and foaming properties due to the balanced molecular rigidity/unfolding and interfacial adsorption/stabilization behavior. Full article

Fungal and bacterial pathogens significantly impact global crop yields, causing substantial economic losses and food insecurity. While chemical pesticides are effective, their excessive and improper use poses risks to the environment and human health. Antimicrobial peptides (AMPs)—components of innate immunity in plants and animals—are promising candidates for the development of novel, eco-friendly antimicrobials for agriculture and medicine. This study explores the antimicrobial activity of several γ-core peptides derived from defensins of Thinopyrum elongatum, a wild plant species known for its stress resistance. All peptides carried a net positive charge. 3D structural modeling indicated that most peptides adopted an α-helical conformation, with one predicted to form an anti-parallel β-hairpin structure. The conservation of the γ-core peptide sequences across Poaceae defensins was demonstrated, underscoring the importance of these peptide regions in biological functions of defensins. Antimicrobial assays demonstrated that all peptides exhibited broad-spectrum activity, with efficacy depending on the peptide’s amino acid sequence, 3D structure, and the pathogen tested. Notably, the peptide with the highest positive charge and β-hairpin structure showed the strongest pathogen inhibition. Additionally, synergistic interactions between some peptides against Fusarium oxysporum, which enhanced their antimicrobial effects, were shown. Our findings highlight the potential of wheatgrass γ-core peptides as templates for developing new peptide-based antimicrobials for agricultural and medical applications. Full article

Ascidians are a group of marine invertebrates, most of which are sessile and soft-bodied. Their lack of an adaptive immune system makes them rely on innate immune responses to detect and eliminate invading microbes. Antimicrobial peptides (AMPs) play an essential part in this process. In this paper, we present the isolation, structure elucidation, and bioactivities of two new cysteine-rich peptides (CRPs) from the Arctic marine ascidian Synoicum turgens. The sequences and structures of the peptides were determined by Edman degradation sequencing, mass spectrometry, and NMR analysis. This revealed two novel 2 kDa peptides, St-CRP-1 and St-CRP-2, with neutral net charge and C-terminal amidation. St-CRP-1 consisted of 18 amino acids and displayed selective and moderate growth inhibition of two Gram-positive bacterial strains (Bacillus subtilis and Corynebacterium glutamicum) at 24.6 µM, whereas St-CRP-2 consisted of 19 amino acids and inhibited the growth of B. subtilis at 49.2 µM. St-CRP-1 had no effect on two mammalian cell lines or the brine shrimp Artemia salina at the highest concentration tested. Structural analysis of the St-CRPs indicated a Cys1–Cys6, Cys2–Cys4, and Cys3–Cys5 disulfide connectivity, which is also found in alpha-defensins. The results from this study show that Arctic marine ascidians are a rich source of novel bioactive peptides. Full article

Background/Objectives: Longer oligoarginines are very effective cell-penetrating peptides. It has been shown that a minimal number of positively charged side chains is necessary for efficient cellular uptake. But a highly positively charged peptide may interact with its cargo molecule, thereby reducing its efficiency. Several chemical modifications were tested to improve the internalization of short tetraarginine derivatives. Aromatic groups, such as Dabcyl at the N-terminus, Trp in the sequence, and AMBA or PABA in the backbone, were used to improve internalization. The other useful modification was the aza-glycine substitution in the case of penetratin. Methods: In this study, the effect of aza-glycine insertion into the peptide Dabcyl-RRRRK(Cf) on internalization was studied and compared with that of the Trp-modified peptide Dabcyl-RRWRRK(Cf). To explain the noticed difference in the biological activity of peptides, DFT calculations and the prediction of membrane-binding free energy (ΔΔF) from a peptide sequence were performed. Results: It turned out that the position of the aza-glycine moiety does not have an influence on the cellular uptake. The aza-glycine-containing peptide showed higher internalization than the Dabcyl-RRRRK(Cf) peptide. Besides this, these peptides have similar or higher cellular uptake than that of octaarginine at lower concentrations (c < 2 µM). The aza-glycine affected not only cellular uptake but also the entry mechanism. The structure of peptides depended on the amino acids (Trp, Gly, or azaGly) in their sequences and their positions. Conclusions: These may result in the different amphiphilicity of peptides, and thus changes in the hydrophobic moment and in the binding affinity of peptides to the negatively charged membrane surface. Full article

Background: Arginine and related amino acids are widely used to suppress protein aggregation, thereby affecting stability, manufacturability, and therapeutic performance. However, their effectiveness remains limited, necessitating the exploration of alternative strategies. Previous studies have shown that N-acetyl-L-arginine (NA-Arg) can improve protein stability; however, the potential of other N-acetylated amino acids has not been fully explored. Methods: This study aimed to investigate the effects of multiple N-acetylated amino acids as alternative excipients on aggregation, colloidal stability, and viscosity in intravenous immunoglobulin (IVIG) formulations. Dynamic light scattering (DLS) was used to evaluate diffusion behavior and aggregation tendencies, while complementary analyses were performed using size-exclusion chromatography (SEC) and flow-imaging microscopy (FI). Results: Overall, N-acetylation of amino acids improved colloidal stability, shifting the kD values from −5.87 to 6.83 mL/g for arginine and from −8.17 to 16.22 mL/g for histidine, and increased the aggregation onset temperature (Tagg) to above 60 °C. Among the tested compounds, N-acetyl-L-histidine (NA-His) showed the most favorable results, increasing the monomer proportion by approximately 4%, reducing high-molecular-weight species to below 2%, and producing a greater than 10-fold decrease in subvisible particles relative to histidine hydrochloride after 5 days of agitation. At 50 mM, both NA-His and NA-Arg reduced the viscosity of highly concentrated 200 mg/mL IVIG formulations, with NA-His exhibiting the lowest viscosity (7.24 ± 0.12 mPa·s). Protein–protein interaction and surface charge analyses indicated improved colloidal stability relative to parent amino acids, attributable to the presence of the acetyl group. Conclusions: These findings support the potential of N-acetylation as a strategy to modulate interaction-driven instability and suggest NA-His as a promising candidate excipient for stabilizing highly concentrated therapeutic proteins at acidic pH. Full article