Search Results (41)

Cold environments challenge the structural and functional integrity of membrane proteins, requiring specialized adaptations to maintain activity under low thermal energy. Here, we investigate the molecular basis of cold tolerance in the peptide transporter PepT1 from the Antarctic icefish (Chionodraco hamatus, ChPepT1) using molecular dynamics simulations, binding free energy calculations (MM/GBSA), and dynamic network analysis. We compare ChPepT1 to its human ortholog (hPepT1), a non-cold-adapted variant, to reveal key features enabling psychrophilic function. Our simulations show that ChPepT1 displays enhanced global flexibility, particularly in domains adjacent to the substrate-binding site and the C-terminal domain (CTD). While hPepT1 loses substrate binding affinity as temperature increases, ChPepT1 maintains stable peptide interactions across a broad thermal range. This thermodynamic buffering results from temperature-sensitive rearrangement of hydrogen bond networks and more dynamic lipid interactions. Importantly, we identify a temperature-responsive segment (TRS, residues 660–670) within the proximal CTD that undergoes an α-helix to coil transition, modulating long-range coupling with transmembrane helices. Dynamic cross-correlation analyses further suggest that ChPepT1, unlike hPepT1, reorganizes its interdomain communication in response to temperature shifts. Our findings suggest that cold tolerance in ChPepT1 arises from a combination of structural flexibility, resilient substrate binding, and temperature-sensitive interdomain dynamics. These results provide new mechanistic insight into thermal adaptation in membrane transporters and offer a framework for engineering proteins with enhanced functionality in extreme environments. Full article

Troponin C (TnC) is the Ca²⁺-sensing subunit of troponin that is responsible for activating thin filaments in striated muscle, and, in turn, for regulating the systolic and diastolic contractile function of cardiac muscle. The secondary structure of vertebrate TnC is mainly composed of α-helices, with nine helices named sequentially, starting from the amino terminus, from N to A–H. The N-helix is a 12-residue-long α-helix located at the extreme amino terminus of the protein and is the only helical structure that does not participate in forming Ca²⁺-binding EF-hands. Evolutionarily, the N-helix is found only in TnC from mammalian species and most other vertebrates and is not present in other Ca²⁺-binding protein members of the calmodulin (CaM) family. Furthermore, the primary sequence of the N-helix differs between the genetic isoforms of the fast skeletal TnC (sTnC) and cardiac/slow skeletal TnC (cTnC). The 3D location of the N-helix within the troponin complex is also distinct between skeletal and cardiac troponin. Physical chemistry and biophysical studies centered on the sTnC N-helix demonstrate that it is crucial to the thermal stability and Ca²⁺ sensitivity of thin filament-regulated MgATPase activity in solution and to isometric force generation in the sarcomere. Comparable studies on the cTnC N-helix have not yet been performed despite the identification of cardiomyopathy-associated genetic variants that affect the residues of cTnC’s N-helix. Here, we review the current status of the research on TnC’s N-helix and establish future directions to elucidate its functional significance. Full article

Honey has long been valued for its medicinal properties, yet the therapeutic potential of Irish monofloral honey remains largely unexplored. This study investigates the antioxidant and immunomodulatory effects of Irish ivy (Hedera helix) and heather (Calluna vulgaris) honey samples on PMA-differentiated THP-1 macrophages, a well-characterised immune model. Antioxidant capacity was assessed through free radical scavenging assays, DPPH and ORAC, while qPCR analysis examined the key inflammatory markers. Both the heather and ivy honey varieties demonstrated antioxidant activity, with heather honey exhibiting the highest total phenolic content (TPC), and ivy honey stimulating Nrf2 activation. Manuka honey showed the strongest radical scavenging capacity, as reflected in its higher ORAC and DPPH values. These findings suggest that the different honey varieties may exert antioxidant effects through distinct mechanisms. Exposure to honey reduced oxidative stress and upregulated the expression of a key antioxidant transcription regulator (Nrf2) and an associated downstream antioxidant defence enzyme, superoxide dismutase (SOD). Additionally, both the honey types exhibited immunomodulatory effects, upregulating pro-inflammatory cytokines, such as TNF-α and IL-1β, while increasing the expression of the anti-inflammatory cytokine IL-10. These findings suggest potential bioactive properties that warrant further investigation. Given the growing interest in alternative treatments for inflammation-related conditions, further research is warranted to determine whether the observed in vitro effects translate into clinically relevant outcomes. This study expands the current understanding of Irish monofloral honey, reinforcing its potential as a functional bioactive compound with relevance in antioxidant therapies, immune modulation, and wound healing. Full article

To produce products with standardized and optimal technical performance, probiotics, particularly Lactic Acid Bacteria (LAB), have long been utilized as fermentation starters in sausages, ensuring both the standardization and enhancement of product quality and safety. Microorganisms isolated from traditional meat products, due to their excellent adaptability to the fermentation environment and their ability to preserve desirable flavor, exhibit high potential as candidates for meat fermentation starters. Three indigenous LAB strains—Latilactobacillus sakei, Pediococcus pentosaceus, and Weissella cibaria, isolated from Yunnan ham—were applied in the fermentation of beef sausages to investigate the underlying factors responsible for quality changes. The results indicated that sausages fermented with L. sakei and P. pentosaceus exhibited the lowest pH (4.98) and a_w (0.79), while displaying significantly higher hardness, cohesiveness, and chewiness. Additionally, LF-NMR measurements showed that L. sakei and P. pentosaceus promoted the transfer of immobilized water to free water, facilitating the drying and maturation process. Raman spectroscopy analysis revealed a reduction in α-helix content and an increase in disordered β-sheet and β-turn structures in the secondary protein structure. These findings suggest that L. sakei and P. pentosaceus improved quality attributes by modifying the secondary protein structure to enhance water migration and accelerate the ripening process. L. sakei and P. pentosaceus demonstrated desirable technological characteristics, indicating their efficacy for use in fermented sausage production. This study provides valuable insights into improving the production of fermented sausages using specific LAB strains. Full article

Background: Soy allergy is an important nutritional and health issue that needs to be addressed. Lactic acid bacteria (LAB) fermentation is an effective approach to reduce soy protein allergy. Polysaccharides are commonly used in LAB-fermented products to enhance their textural properties. This study proposes a new strategy for developing hypoallergenic soy protein products. Methods: We prepared a soy protein isolate (SPI) through fermentation with LAB (FSPI) and with five types of polysaccharides supplementation, namely polydextrose (PDX), inulin (IN), long-chain inulin (LCIN), soluble soy polysaccharides (SSPS), and β-glucan (BG). The texture and microstructure of different samples were analyzed. Antigenicity and IgE-binding capacity were determined using ELISA. Finally, peptide sequencing was used to identify the degradation degree and frequency of allergenic epitopes. Results: Samples with added PDX (F-PDX) and IN (F-IN) exhibited lower hardness; smaller, irregular pores; and a honeycomb structure, whereas samples with SSPS (F-SSPS) and BG (F-BG) had higher hardness; large, regular pores; and strong sheet structures. The antigenicity and IgE-binding capacity of F-PDX and F-IN were lower both before and after 120 min of in vitro dynamic gastrointestinal digestion. The peptidomics results indicated that F-PDX and F-IN primarily facilitated the degradation of the glycinin G1 and G2 subunits, β-conglycinin α, and the β subunit. Moreover, it increased the frequency of destruction of allergenic epitopes, and further promoted the degradation of epitopes in the external α-helix structures of glycinin and β-conglycinin compared to FSPI. Conclusions: The addition of polysaccharides had a significant impact on the structure and allergenicity of the soy protein gel, especially PDX and IN. Full article

Background: Current influenza A vaccines primarily induce neutralizing antibodies targeting the variable hemagglutinin (HA) head domain, limiting their effectiveness against diverse or emerging influenza A virus (IAV) subtypes. The conserved HA stem domain, particularly the long α-helix (LAH) epitope, is a focus of universal vaccine research due to its cross-protective potential. Additionally, Fc-mediated functions such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) are recognized as important protective immune mechanisms. This study evaluated IgG responses to the HA head, stem, and LAH regions and assessed cross-reactive potential through neutralization, ADCC, and ADCP assays. Methods: IgG responses to the HA head, stem, and LAH regions were measured in vaccinated individuals. Functional assays were conducted for neutralization, ADCC, and ADCP to evaluate the association between antibody levels and immune function. Results: The results showed that HA head-specific IgG increased significantly after vaccination in 50 individuals, whereas stem-specific IgG increased by 72% and LAH-specific IgG by 12–14%. Among the induced antibody subclasses, IgG1 was predominantly increased. Neutralization titers were detected in viruses of the same strain as the vaccine strain, but not in classical or pandemic strains (H5N1, H7N9). HA stem-specific IgG1 antibody titers showed a significant correlation with ADCC/ADCP activity breadth, but no correlation was observed with neutralization breadth. Conclusions: These findings suggest that although current influenza vaccines can induce HA stem-targeted cross-reactive antibodies, their quantity may be insufficient for broad cross-protection, underscoring the need for improved vaccine strategies. Full article

HSP70 chaperones play pivotal roles in facilitating protein folding, refolding, and disaggregation through their binding and releasing activities. This intricate process is further supported by J-domain proteins (JDPs), also known as DNAJs or HSP40s, which can be categorized into classes A and B. In yeast, these classes are represented by Ydj1 and Sis1, respectively. While both classes stimulate the ATPase activity of Ssa1 (yeast HSP70) through the J-domain, only class B JDPs possess the unique ability to efficiently stimulate Ssa1 in disaggregation processes. The C-terminal EEVD motif of HSP70 plays a crucial role in mediating these interactions by connecting with both client proteins and JDPs. However, the removal of the EEVD motif disrupts the capacity of HSP70 to associate with class B JDPs, and the intricacies of the interaction between these two proteins remain incompletely understood. We employed NMR spectroscopy to investigate the structure and dynamics of the class B J domain protein (JDP) of S. cerevisiae (Sis1) complexed with an EEVD peptide of Ssa1. Our study is based on the extraordinary 70.5% residue assignment of the full-length (352 residues long) Sis1. Our findings revealed that EEVD binds to two distinct sites within the C-terminal domain I (CTDI) of Sis1, to the J domain and to the GF-rich loop located between the J domain and α-helix 6 (a structure identified by this work). We propose that the interaction between EEVD and Sis1 facilitates the dissociation of α-helix 6, promoting a conformational state that is more favorable for interaction with Ssa1. We also employed α-synuclein as a substrate to investigate the competitive nature between EEVD and the client protein. Our experimental findings provide evidence supporting the interaction of EEVD with the client protein at multiple sites and essential insights into the mechanistic cycle of class B JDPs. Full article

The effect of resveratrol (RESV) on α-lactalbumin (α-LA) thermal stability was evaluated using differential scanning calorimetry (DSC), circular dichroism (CD) and dynamic light scattering (DLS) measurements. Complementary information offered by molecular docking served to identify the binding site of the ligand on the native structure of protein and the type of interacting forces. DSC thermograms revealed a double-endotherm pattern with partial overlapping of the two components. The most relevant effect of RESV is manifested in the narrowing of the protein thermal fingerprint: the first process (peak temperature T₁) is shifted to higher temperatures while the second one (peak temperature T₂) to lower values. The CD data indicated partial conformational changes in the protein non-α-helix domain at T₁, resulting in a β-sheet richer intermediate (BSRI) with an unaffected, native-like α-helix backbone. The RESV influence on this process may be defined as slightly demoting, at least within DSC conditions (linear heating rate of 1 K min⁻¹). On further heating, unfolding of the α-helix domain takes place at T₂, with RESV acting as a promoter of the process. Long time incubation at 333 K produced the same type of BSRI: no significant effect of RESV on the secondary structure content was detected by CD spectroscopy. Nevertheless, the size distribution of the protein population obtained from DLS measurements revealed the free (non-bound) RESV action manifested in the developing of larger size aggregates. Full article

Eukaryotic release factor eRF1, encoded by the ETF1 gene, recognizes stop codons and induces peptide release during translation termination. ETF1 produces several different transcripts as a result of alternative splicing, from which two eRF1 isoforms can be formed. Isoform 1 codes well-studied canonical eRF1, and isoform 2 is 33 amino acid residues shorter than isoform 1 and completely unstudied. Using a reconstituted mammalian in vitro translation system, we showed that the isoform 2 of human eRF1 is also involved in translation. We showed that eRF1iso2 can interact with the ribosomal subunits and pre-termination complex. However, its codon recognition and peptide release activities have decreased. Additionally, eRF1 isoform 2 exhibits unipotency to UGA. We found that eRF1 isoform 2 interacts with eRF3a but stimulated its GTPase activity significantly worse than the main isoform eRF1. Additionally, we studied the eRF1 isoform 2 effect on stop codon readthrough and translation in a cell-free translation system. We observed that eRF1 isoform 2 suppressed stop codon readthrough of the uORFs and decreased the efficiency of translation of long coding sequences. Based on these data, we assumed that human eRF1 isoform 2 can be involved in the regulation of translation termination. Moreover, our data support previously stated hypotheses that the GTS loop is important for the multipotency of eRF1 to all stop codons. Whereas helix α1 of the N-domain eRF1 is proposed to be involved in conformational rearrangements of eRF1 in the A-site of the ribosome that occur after GTP hydrolysis by eRF3, which ensure hydrolysis of peptidyl-tRNA at the P site of the ribosome. Full article

Proteins that are amphiphilic and have low water solubility can self-assemble into nanoparticles useful in food science, pharmaceutical science, or biotechnology. However, protein nanoparticles exhibit drawbacks such as low stability unless the particles are coated. In the current study, tannic acid is the coating agent for nanoparticles synthesized from the protein zein. Tannic acid is a hydrolyzable tannin comprising a polyol esterified with galloyl residues. The nominal molecular formula of tannic acid (C₇₆H₅₂O₄₆) suggests the material is decagalloyl glucose, obscuring its complex composition as a mixture of galloyl esters of glucose. We prepared hollow zein nanoparticles and coated them with tannic acid preparations that had short or long galloyl ester chains. The % α-helix of zein in nanoparticles is lower than in native zein but there is no effect of coating the particles with tannic acid. Interactions between the tannic acid and the zein slightly perturb the IR spectrum of the protein but there is no effect of galloyl chain length. We confirmed that tannic acid-coated particles have a more negative zeta potential, suggesting greater stability compared to uncoated particles. Coating with longer chain length tannic acid reduces particle diameter and tends to decrease polydispersity but does not change particle digestibility. Coating with shorter galloyl chain length tannic acid tends not to change particle diameter, reduces polydispersity of the particles, and stabilizes particles to enzymatic digestion. Tannic acid is a naturally occurring tunable coating for nanoparticles that can be used to adjust properties such as particle size, polydispersity, and digestibility for specific purposes. Full article

We present an analysis of α-helix folding in the coarse-grained coordinate of number of formed helical hydrogen bonds (NHBs) for four alanine peptides (ALA)n, with n = 5, 8, 15, and 21 residues. Starting with multi-microsecond all-atom molecular dynamics trajectories in aqueous solution, we represent the system dynamics in a space of between four (for ALA5) and twenty (for ALA21) hydrogen-bonding microstates. In all cases, transitions changing the hydrogen bond count by 1–2 dominate and the coil formation, NHB 1 → 0, is the fastest process. The calculation of global maximum weight paths shows that, when analyzed at a sufficiently long lag time, folding in the NHB coordinate is consecutive, with direct folding, 0 → 3, for ALA5 and bottlenecks at transitions 4 → 6 for ALA8, 0 → 5 for ALA15, and 0 → 9 for ALA21. Further coarse-graining to 2–4 dimensions was performed with the optimal dimensionality reduction method, allowing the identification of crucial folding intermediates and time scales of their formation in ALA8, ALA15, and ALA21. The detailed analysis of hydrogen bonding patterns revealed that folding is initiated preferentially at both peptide termini. The kinetic model was also used to estimate diffusion and friction coefficients for helix propagation. The description of the helix formation process in the hydrogen bonding coordinate NHB was in good general agreement with the experimental data and qualitatively similar to previous kinetic models of higher dimensions based on structural clustering. Use of the low-dimensional hydrogen bonding picture thus provides a different, complementary way of describing the complex and fascinating mechanism of helix formation. Full article

Members of the phloem protein 16 (PP16) gene family are induced by elicitors in rice and the corresponding proteins from cucurbits, which display RNA binding and intercellular transport activities, are accumulated in phloem sap. These proteins facilitate the movement of protein complexes through the phloem translocation flow and may be involved in the response to water deficit, among other functions. However, there is scant information regarding their function in other plants, including the identification of paralog genes in non-vascular plants and chlorophytes. In the present work, an evolutionary and structural analysis of the PP16 family in green plants (Viridiplantae) was carried out. Data mining in different databases indicated that PP16 likely originated from a larger gene present in an ancestral lineage that gave rise to chlorophytes and multicellular plants. This gene encodes a protein related to synaptotagmin, which is involved in vesicular transport in animal systems, although other members of this family play a role in lipid turnover in endomembranes and organelles. These proteins contain a membrane-binding C2 domain shared with PP16 proteins in vascular plants. In silico analysis of the predicted structure of the PP16 protein family identified several β-sheets, one α-helix, and intrinsically disordered regions. PP16 may have been originally involved in vesicular trafficking and/or membrane maintenance but specialized in long-distance signaling during the emergence of the plant vascular system. Full article

Physiology disorders of the liver, as it is an important tissue in lipid metabolism, can cause fatty liver disease. The mechanism might be regulated by 17 circadian clock genes and 18 fat metabolism genes, together with a high-fat diet (HFD). Due to their rich nutritional and medicinal value, Chinese soft-shelled turtles (Trionyx sinensis) are very popular among the Chinese people. In the study, we aimed to investigate the influence of an HFD on the daily expression of both the core clock genes and the lipid metabolism genes in the liver tissue of the turtles. The two diets were formulated with 7.98% lipid (the CON group) and 13.86% lipid (the HFD group) to feed 180 juvenile turtles, which were randomly divided into two groups with three replicates per group and 30 turtles in each replicate for six weeks, and the diet experiment was administrated with a photophase regimen of a 24 h light/dark (12L:12D) cycle. At the end of the experiment, the liver tissue samples were collected from nine turtles per group every 3 h (zeitgeber time: ZT 0, 3, 6, 9, 12, 15, 18, 21 and 24) for 24 h to investigate the daily expression and correlation analysis of these genes. The results showed that 11 core clock genes [i.e., circadian locomotor output cycles kaput (Clock), brain and muscle arnt-like protein 1 and 2 (Bmal1/2), timeless (Tim), cryptochrome 1 (Cry2), period2 (Per2), nuclear factor IL-3 gene (Nfil3), nuclear receptor subfamily 1, treatment D, member 1 and 2 (Nr1d1/2) and retinoic acid related orphan receptor α/β/γ β and γ (Rorβ/γ)] exhibited circadian oscillation, but 6 genes did not, including neuronal PAS domain protein 2 (Npas2), Per1, Cry1, basic helix-loop-helix family, member E40 (Bhlhe40), Rorα and D-binding protein (Dbp), and 16 lipid metabolism genes including fatty acid synthase (Fas), diacylglycerol acyltransferase 1 (Dgat1), 3-hydroxy-3-methylglutaryl-CoA reductase (Hmgcr), Low-density lipoprotein receptor-related protein 1-like (Ldlr1), Lipin 1 (Lipin1), Carnitine palmitoyltransferase 1A (Cpt1a), Peroxisome proliferator activation receptor α, β and γ (Pparα/β/γ), Sirtuin 1 (Sirt1), Apoa (Apoa1), Apolipoprotein B (Apob), Pyruvate Dehydrogenase kinase 4 (Pdk4), Acyl-CoA synthase long-chain1 (Acsl1), Liver X receptors α (Lxrα) and Retinoid X receptor, α (Rxra) also demonstrated circadian oscillations, but 2 genes did not, Scd and Acaca, in the liver tissues of the CON group. However, in the HFD group, the circadian rhythms’ expressional patterns were disrupted for the eight core clock genes, Clock, Cry2, Per2, Nfil3, Nr1d1/2 and Rorβ/γ, and the peak expression of Bmal1/2 and Tim showed delayed or advanced phases. Furthermore, four genes (Cry1, Per1, Dbp and Rorα) displayed no diurnal rhythm in the CON group; instead, significant circadian rhythms appeared in the HFD group. Meanwhile, the HFD disrupted the circadian rhythm expressions of seven fat metabolism genes (Fas, Cpt1a, Sirt1, Apoa1, Apob, Pdk4 and Acsl1). Meanwhile, the other nine genes in the HFD group also showed advanced or delayed expression peaks compared to the CON group. Most importantly of all, there were remarkably positive or negative correlations between the core clock genes and the lipid metabolism genes, and their correlation relationships were altered by the HFD. To sum up, circadian rhythm alterations of the core clock genes and the lipid metabolism genes were induced by the high-fat diet (HFD) in the liver tissues of T. sinensis. This result provides experimental and theoretical data for the mass breeding and production of T. sinensis in our country. Full article

Bacterial-derived cellulose (BC) has been studied as a promising material for biomedical applications, including wound care, due to its biocompatibility, water-holding capacity, liquid/gas permeability, and handleability properties. Although BC has been studied as a dressing material for cutaneous wounds, to date, BC inherently lacks antibacterial properties. The current research utilizes bifunctional chimeric peptides containing carbohydrate binding peptides (CBP; either a short version or a long version) and an antimicrobial peptide (AMP), KR-12. The secondary structure of the chimeric peptides was evaluated and confirmed that the α-helix structure of KR-12 was retained for both chimeric peptides evaluated (Long-CBP-KR12 and Short-CBP-KR12). Chimeric peptides and their individual components were assessed for cytotoxicity, where only higher concentrations of Short-CBP and longer timepoints of Short-CBP-KR12 exposure exhibited negative effects on metabolic activity, which was attributed to solubility issues. All KR-12-containing peptides exhibited antibacterial activity in solution against Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa). The lipopolysaccharide (LPS) binding capability of the peptides was evaluated and the Short-CBP-KR12 peptide exhibited enhanced LPS-binding capabilities compared to KR-12 alone. Both chimeric peptides were able to bind to BC and were observed to be retained on the surface over a 7-day period. All functionalized materials exhibited no adverse effects on the metabolic activity of both normal human dermal fibroblasts (NHDFs) and human epidermal keratinocyte (HaCaT) epithelial cells. Additionally, the BC tethered chimeric peptides exhibited antibacterial activity against E. coli. Overall, this research outlines the design and evaluation of chimeric CBP-KR12 peptides for developing antimicrobial BC membranes with potential applications in wound care. Full article

Pentameric ligand-gated ion channels (pLGICs) are expressed throughout the central and peripheral nervous systems of vertebrates and modulate many aspects of human health and disease. Recent structural and computational data indicate that cation-selective pLGICs contain a long helical extension (MA) of one of the transmembrane helices. The MA helix has been shown to affect both the membrane expression of, and ion conductance levels through, these pLGICs. Here we probe the functional effects of 68 mutations in the MA region of the α4β2 nicotinic acetylcholine receptor (nAChR), using a voltage-sensitive membrane dye and radioligand binding to measure receptor function and expression/assembly. We found seven alanine mutations in a stretch of the MA helix that prevent correct receptor folding and/or assembly, as evidenced by the lack of both function and ligand binding. A further two alanine mutations resulted in receptors that were capable of binding ligand but showed no functional response, and we propose that, in these mutants, ligand binding is insufficient to trigger channel opening. The data clarify the effect of the MA helix, and as the effects of some of our mutations in the α4β2 nAChR differ from the effects of equivalent mutations in other cation-selective pLGICs, we suggest that residues in the MA helix may play subtly different roles in different receptors. Full article