Search Results (121)

The regulation of calcium (Ca²⁺) homeostasis is a critical process in both plant and animal systems, involving complex interplay between various organelles and a diverse network of channels, pumps, and transporters. This review provides a concise overview of inter-organellar Ca²⁺ homeostasis, highlighting key regulators and mechanisms in plant and animal cells. We discuss the roles of key Ca²⁺ channels and transporters, including IP₃Rs, RyRs, TPCs, MCUs, TRPMLs, and P2XRs in animals, as well as their plant counterparts. Here, we explore recent innovations in structural biology and advanced microscopic techniques that have enhanced our understanding of these proteins’ structure, functions, and regulations. We examine the importance of membrane contact sites in facilitating Ca²⁺ transfer between organelles and the specific expression patterns of Ca²⁺ channels and transporters. Furthermore, we address the physiological implications of inter-organellar Ca²⁺ homeostasis and its relevance in various pathological conditions. For extended comparability, a brief excursus into bacterial intracellular Ca²⁺ homeostasis is also made. This meta-analysis aims to bridge the gap between plant and animal Ca²⁺ signaling research, identifying common themes and unique adaptations in these diverse biological systems. Full article

Protein–polyelectrolyte nanostructures assembled via electrostatic interactions offer versatile applications in biomedicine, tissue engineering, and food science. However, several open questions remain regarding their intermolecular interactions and the influence of external conditions—such as temperature and pH—on their assembly, stability, and responsiveness. This study explores the formation and stability of networks between poly(acrylic acid) (PAA) and lysozyme (LYZ) at the nanoscale upon thermal treatment, using a combination of experimental and simulation measures. Experimental techniques of static and dynamic light scattering (SLS and DLS), Fourier transform infrared spectroscopy (FTIR), and circular dichroism (CD) are combined with all-atom molecular dynamics simulations. Model systems consisting of multiple PAA and LYZ molecules explore collective assembly and complexation in aqueous solution. Experimental results indicate that electrostatic complexation occurs between PAA and LYZ at pH values below LYZ’s isoelectric point. This leads to the formation of nanoparticles (NPs) with radii ranging from 100 to 200 nm, most pronounced at a PAA/LYZ mass ratio of 0.1. These complexes disassemble at pH 12, where both LYZ and PAA are negatively charged. However, when complexes are thermally treated (TT), they remain stable, which is consistent with earlier findings. Atomistic simulations demonstrate that thermal treatment induces partially reversible structural changes, revealing key microscopic features involved in the stabilization of the formed network. Although electrostatic interactions dominate under all pH and temperature conditions, thermally induced conformational changes reorganize the binding pattern, resulting in an increased number of contacts between LYZ and PAA upon thermal treatment. The altered hydration associated with conformational rearrangements emerges as a key contributor to the stability of the thermally treated complexes, particularly under conditions of strong electrostatic repulsion at pH 12. Moreover, enhanced polymer chain associations within the network are observed, which play a crucial role in complex stabilization. These insights contribute to the rational design of protein–polyelectrolyte materials, revealing the origins of association under thermally induced structural rearrangements. Full article

The venom of Nemopilema nomurai jellyfish represents a promising source of bioactive compounds with potential pharmacological applications. In our previous work, we identified two novel angiotensin-converting enzyme (ACE)-inhibitory peptides—IVGRPLANG (896.48 Da) and IGDEPRHQYL (1227.65 Da)—isolated from N. nomurai venom hydrolysates via papain digestion. In this study, we conducted a detailed biochemical and computational characterization of these peptides. The IC₅₀ values were determined to be 23.81 µM for IVGRPLANG and 5.68 µM for IGDEPRHQYL. Kinetic analysis using Lineweaver–Burk plots revealed that both peptides act as competitive ACE inhibitors, with calculated inhibition constants (K_i) of 51.38 µM and 5.45 µM, respectively. To assess the structural stability of the ACE–peptide complexes, molecular dynamics simulations were performed. Root mean square deviation (RMSD) and root mean square fluctuation (RMSF) analyses provided insights into complex stability, while interaction fraction analysis elucidated key bond types and residue–ligand contacts involved in binding. Furthermore, a network pharmacology approach was employed to predict therapeutic targets within the renin–angiotensin–aldosterone system (RAAS). Eleven target proteins were identified: IVGRPLANG was associated with REN, ACE, CTSB, CTSS, and AGTR2; IGDEPRHQYL was linked to REN, AGT, AGTR1, AGTR2, KNG1, and BDKR2. Molecular docking analyses using HADDOCK software (version 2.4) were conducted for all targets to evaluate binding affinities, providing further insight into the peptides’ therapeutic potential. Full article

The solubility of soybean isolate protein (SPI) undergoes significant degradation during storage and transportation. This study investigates the formulation and assessment of SPI-stabilized medium internal phase emulsions (MIPEs) with different solubilities, namely SPI80, SPI70, SPI60, and SPI50, corresponding to solubility levels of about 80%, 70%, 60%, and 50%, respectively. The contact angles of these SPI variants ranged from 79.35 to 86.55 degrees, with SPI60 and SPI50 exhibiting significantly higher values compared to SPI80 and SPI70. All SPI samples were successfully utilized for the preparation of MIPEs. However, as SPI solubility decreases, emulsion stability progressively declines, accompanied by a reduction in the absolute value of zeta potential. Additionally, interfacial protein adsorption in emulsions decreases with decreasing SPI solubility, a trend that is similarly observed in viscosity characteristics, storage modulus (G′), and loss modulus (G″). Confocal laser scanning microscopy (CLSM) and cryo-scanning electron microscopy (Cryo-SEM) analyses revealed that emulsions exhibit reduced uniformity and a less interconnected microstructural network as SPI solubility decreases. These findings provide a theoretical foundation for utilizing low-solubility SPI in MIPEs applications. Full article

Glycosaminoglycans (GAGs) are key ligands for proteins involved in physiological and pathological processes. Specific GAG-binding patterns are rarely identified, with the heparin pentasaccharide as an Antithrombin-III ligand being the best characterized. Generating glycan-specific antibodies is difficult due to their size, pattern dispersion, and flexibility. Single-domain variable new antigen receptors (VNAR nanobodies) from nurse sharks are highly soluble, stable, and versatile. Their unique properties suggest advantages over conventional antibodies, particularly for challenging biotherapeutic targets. Here we have used VNAR semi-synthetic phage libraries to select high-affinity fondaparinux-binding VNARs that did not show cross-reactivity with other GAG species. Competition ELISA and surface plasmon resonance identified a single fondaparinux-selective VNAR clone. This VNAR exhibited an extraordinarily stable protein fold: the beta-strands are stabilized by a robust hydrophobic network, as revealed by heteronuclear NMR. Docking fondaparinux to the VNAR structure revealed a large contact surface area between the CDR3 loop of the antibody and the glycan. Fusing the VNAR with a human Fc domain resulted in a stable product with a high affinity for fondaparinux (Kd = 9.3 × 10⁻⁸ M) that could efficiently discriminate between fondaparinux and other glycosaminoglycans. This novel glycan-targeting screening technology represents a promising therapeutic strategy for addressing GAG-related diseases. Full article

BCL2 is a critical regulator of intrinsic and extrinsic pathways of apoptosis that have been implicated in cancer progression and therapeutic resistance. In this study, the protein–protein interactions (PPIs) of BCL2 with potential binding partners and their role in cancer was investigated. A comprehensive PPI network for BCL2 has been generated by using the Protein Interactions Network Analysis (PINA) platform to identify key interactors. To further investigate the network, Molecular Operating Environment (MOE), Search Tool for the Retrieval of Interacting Genes (STRING), Residue Interaction Network Generation (RING), and the gProfiler server were used. Docking and Molecular Dynamics (MD) simulations were performed by using HDOCK and Gromacs to analyze the binding dynamics and stability of protein complexes. The BCL2 interactome revealed that three key interactors (p53, RAF1, and MAPK1) are involved in cancer-related processes. Docking studies highlighted BCL2 residues such as ASP111, ASP140, ARG107, and ARG146 that were predominantly involved in multiple hydrogen bonds, ionic interactions, and van der Waals contacts, highlighting conserved binding sites that play critical roles in the stability and specificity of protein–protein interactions. MD simulations (200 ns) of the BCL2-p53 complex showed that the RMSD was increased, suggesting the suppression of BCL2’s anti-apoptotic activity by p53. The RMSD for BCL2-RAF1 was also increased, showing protein domain structural rearrangements that enhance BCL2 anti-apoptotic activity. The BCL2-MAPK1 complex revealed structural, distinct flexibility patterns and dynamic hydrogen bonding interactions. These findings provide valuable insights into the molecular dynamics by which BCL2 modulates apoptosis and its potential as a promising therapeutic in cancer and apoptosis-related diseases. Full article

This work provides a comprehensive review of experimental methods used to measure rheological properties of interfacial layers stabilized by surfactants, asphaltenes, and proteins that are relevant to systems with large interfacial areas, such as emulsions and foams. Among the shear methods presented, the deep channel viscometer, bicone rheometer, and double-wall ring rheometers are the most utilized. On the other hand, the main dilational rheology techniques discussed are surface waves, capillary pressure, oscillating Langmuir trough, oscillating pendant drop, and oscillating spinning drop. Recent developments—including machine learning and artificial intelligence (AI) models, such as artificial neural networks (ANN) and convolutional neural networks (CNN)—to calculate interfacial tension from drop shape analysis in shorter times and with higher precision are critically analyzed. Additionally, configurations involving an Atomic Force Microscopy (AFM) cantilever contacting bubble, a microtensiometer platform, rectangular and radial Langmuir troughs, and high-frequency oscillation drop setups are presented. The significance of Gibbs–Marangoni effects and interfacial rheological parameters on the (de)stabilization of emulsions is also discussed. Finally, a critical review of the recent literature on the measurement of interfacial rheology is presented. Full article

Severe acute respiratory symptom coronavirus 2 (SARS-CoV-2) infection occurs via the attachment of the spike (S) protein’s receptor binding domain (RBD) to human ACE2 (hACE2). Natural polymorphisms in hACE2, particularly at the interface, may alter RBD–hACE2 interactions, potentially affecting viral infectivity across populations. This study identified the effects of six naturally occurring hACE2 polymorphisms with high allele frequency in the African population (S19P, K26R, M82I, K341R, N546D and D597Q) on the interaction with the S protein RBD of the BA.4/5 Omicron sub-lineage through post-molecular dynamics (MD), inter-protein interaction and dynamic residue network (DRN) analyses. Inter-protein interaction analysis suggested that the K26R variation, with the highest interactions, aligns with reports of enhanced RBD binding and increased SARS-CoV-2 susceptibility. Conversely, S19P, showing the fewest interactions and largest inter-protein distances, agrees with studies indicating it hinders RBD binding. The hACE2 M82I substitution destabilized RBD–hACE2 interactions, reducing contact frequency from 92 (WT) to 27. The K341R hACE2 variant, located distally, had allosteric effects that increased RBD–hACE2 contacts compared to WThACE2. This polymorphism has been linked to enhanced affinity for Alpha, Beta and Delta lineages. DRN analyses revealed that hACE2 polymorphisms may alter the interaction networks, especially in key residues involved in enzyme activity and RBD binding. Notably, S19P may weaken hACE2–RBD interactions, while M82I showed reduced centrality of zinc and chloride-coordinating residues, hinting at impaired communication pathways. Overall, our findings show that hACE2 polymorphisms affect S BA.4/5 RBD stability and modulate spike RBD–hACE2 interactions, potentially influencing SARS-CoV-2 infectivity—key insights for vaccine and therapeutic development. Full article

Telocytes (TCs) are distinctive cells widely localized in the stromal compartment of several human organs, including the skin. By means of their peculiar prolongations named telopodes, skin TCs are organized in networks interconnected with a variety of adjacent cells, being thus supposed to take part in skin homeostasis through both cell-to-cell contacts and the release of extracellular vesicles. A disarrangement/loss of the TC network was shown in human fibrotic skin as well as in the murine model of bleomycin-induced cutaneous fibrosis, but whether such TC alterations may represent just a consequence or a trigger of the fibrotic process still remains to be clarified. Thus, we investigated the effects of skin TC secretome as conditioned medium (TC-CM) on the transition of skin fibroblasts into myofibroblasts promoted by the master profibrotic cytokine transforming growth factor β1 (TGFβ1). Primary cultures of both adult human skin TCs and fibroblasts were obtained by means of immunomagnetic cell separation. Nanoparticle tracking analysis was carried out to measure extracellular vesicles in TC-CM. The combination of multiple morphological, gene/protein expression, and functional assessments demonstrated that TC-CM was able to significantly prevent TGFβ1-induced fibroblast-to-myofibroblast transition. TC-CM did not influence cell viability, while it effectively inhibited TGFβ1-induced fibroblast proliferation, migration, and morphological changes. Indeed, TC-CM was able to reduce TGFβ1-mediated skin fibroblast phenotypic and functional differentiation into myofibroblasts, as shown by a significant decrease in FAP, ACTA2, COL1A1, COL1A2, FN1, and CTGF gene expression, α-smooth muscle actin, N-cadherin, COL1A1, and FN-EDA protein levels, and collagen gel matrix contraction. Furthermore, TC-CM significantly lowered TGFβ1-mediated ERK1/2 signaling pathway activation. This in vitro study proves for the first time that TCs may play an important role in skin homeostasis through the prevention of fibroblast-to-myofibroblast transition via paracrine mechanisms and affords the necessary basis to investigate in the future the feasibility of TC secretome as an innovative antifibrotic therapeutic tool. Full article

Phosphorylation is one of the most common protein post-translational modifications. The identification of phosphorylation sites serves as the cornerstone for protein-phosphorylation-related research. This paper proposes a protein-phosphorylation site-prediction model based on graph neural networks named GraphPhos, which combines sequence features with structure features. Sequence features are derived from manual extraction and the calculation of protein pre-trained language models, and the structure feature is the secondary structure contact map calculated from protein tertiary structure. These features are then innovatively applied to graph neural networks. By inputting the features of the entire protein sequence and its contact graph, GraphPhos achieves the goal of predicting phosphorylation sites along the entire protein. Experimental results indicate that GraphPhos improves the accuracy of serine, threonine, and tyrosine site prediction by at least 8%, 15%, and 12%, respectively, exhibiting an average 7% improvement in accuracy compared to individual amino acid category prediction models. Full article

Nestin is a type VI intermediate filament protein and a well-known neural stem cell marker. It is also expressed in high-grade cancer cells, forming copolymerized filaments with vimentin. We previously showed that nestin inhibits the binding of vimentin’s tail domain to actin filaments (AFs) by steric hindrance through its large nestin tail domain (NTD), thereby increasing three-dimensional cytoskeleton network mobility, enhancing cell flexibility, and promoting cancer progression. Further, we found that nestin itself stably binds to AFs via the NTD. We therefore hypothesized that the NTD may form a flexible cytoskeletal structure by extending with weak force. In vitro tensile tests using atomic force microscopy were performed to assess the mechanical properties of NTDs. The C-terminus of the NTD bound AFs by bringing the AFM tip modified with the NTD into contact with the AFs on the substrate. NTDs were elongated to approximately 80% of their maximum length at weak forces < 150 pN. Repeated tensile tests revealed that the NTD refolded quickly and behaved like a soft elastic material. We speculate that nestin stably binds AFs, and the NTD extends with weak force, contracting quickly upon load release. Thereby, nestin would absorb mechanical load and maintain cytoskeletal integrity. Full article

EMC1 is part of the endoplasmic reticulum (ER) membrane protein complex, whose functions include the insertion of transmembrane proteins into the ER membrane, ER–mitochondria contact, and lipid exchange. Here, we show that the Drosophila melanogaster EMC1 gene is expressed in the somatic musculature and the protein localizes to the sarcoplasmic reticulum (SR) network. Muscle-specific EMC1 RNAi led to severe motility defects and partial late pupae/early adulthood lethality, phenotypes that are rescued by co-expression with an EMC1 transgene. Motility impairment in EMC1-depleted flies was associated with aberrations in muscle morphology in embryos, larvae, and adults, including tortuous and misaligned fibers with reduced size and weakness. They were also associated with an altered SR network, cytosolic calcium overload, and mitochondrial dysfunction and dysmorphology that impaired membrane potential and oxidative phosphorylation capacity. Genes coding for ER stress sensors, mitochondrial biogenesis/dynamics, and other EMC components showed altered expression and were mostly rescued by the EMC1 transgene expression. In conclusion, EMC1 is required for the SR network’s mitochondrial integrity and influences underlying programs involved in the regulation of muscle mass and shape. We believe our data can contribute to the biology of human diseases caused by EMC1 mutations. Full article

The African swine fever virus (ASFV) is a large and complex DNA virus that causes a highly lethal disease in swine, for which no antiviral drugs or vaccines are currently available. Studying viral–host protein–protein interactions advances our understanding of the molecular mechanisms underlying viral replication and pathogenesis and can facilitate the discovery of antiviral therapeutics. In this study, we employed affinity tagging and purification mass spectrometry to characterize the interactome of VPS39, an important cellular factor during the early phase of ASFV replication. The interaction network of VPS39 revealed associations with mitochondrial proteins involved in membrane contact sites formation and cellular respiration. We show that the ASFV proteins CP204L and A137R target VPS39 by interacting with its clathrin heavy-chain functional domain. Furthermore, we elaborate on the potential mechanisms by which VPS39 may contribute to ASFV replication and prioritize interactions for further investigation into mitochondrial protein function in the context of ASFV infection. Full article

The pericyte coverage of microvessels is altered in metabolic diseases, but the mechanisms regulating pericyte–endothelial cell communication remain unclear. This study investigated the formation and function of pericyte tunneling nanotubes (TNTs) and their impact on endothelial cell metabolism. TNTs were analyzed in vitro in retinas and co-cultures of pericytes and endothelial cells. Using mass spectrometry, the influence of pericytes on endothelial cell metabolism was examined. TNTs were present in the murine retina, and although diabetes was associated with a decrease in pericyte coverage, TNTs were longer. In vitro, pericytes formed TNTs in the presence of PDGF, extending toward endothelial cells and facilitating mitochondrial transport from pericytes to endothelial cells. In experiments with mitochondria-depleted endothelial cells displaying defective TCA cycle metabolism, pericytes restored the mitochondrial network and metabolism. 19,20-Dihydroxydocosapentaenoic acid (19,20-DHDP), known to disrupt pericyte–endothelial cell junctions, prevented TNT formation and metabolic rescue in mitochondria-depleted endothelial cells. 19,20-DHDP also caused significant changes in the protein composition of pericyte-endothelial cell junctions and involved pathways related to phosphatidylinositol 3-kinase, PDGF receptor, and RhoA signaling. Pericyte TNTs contact endothelial cells and support mitochondrial transfer, influencing metabolism. This protective mechanism is disrupted by 19,20-DHDP, a fatty acid mediator linked to diabetic retinopathy. Full article

CDK6 plays a key role in the regulation of the cell cycle and is considered a crucial target for cancer therapy. In this work, conformational transitions of CDK6 were identified by using Gaussian accelerated molecular dynamics (GaMD), deep learning (DL), and free energy landscapes (FELs). DL finds that the binding pocket as well as the T-loop binding to the Vcyclin protein are involved in obvious differences of conformation contacts. This result suggests that the binding pocket of inhibitors (LQQ and AP9) and the binding interface of CDK6 to the Vcyclin protein play a key role in the function of CDK6. The analyses of FELs reveal that the binding pocket and the T-loop of CDK6 have disordered states. The results from principal component analysis (PCA) indicate that the binding of the Vcyclin protein affects the fluctuation behavior of the T-loop in CDK6. Our QM/MM-GBSA calculations suggest that the binding ability of LQQ to CDK6 is stronger than AP9 with or without the binding of the Vcyclin protein. Interaction networks of inhibitors with CDK6 were analyzed and the results reveal that LQQ contributes more hydrogen binding interactions (HBIs) and hot interaction spots with CDK6. In addition, the binding pocket endures flexibility changes from opening to closing states and the Vcyclin protein plays an important role in the stabilizing conformation of the T-loop. We anticipate that this work could provide useful information for further understanding the function of CDK6 and developing new promising inhibitors targeting CDK6. Full article