Search Results (1,118)

Background: Cleft palate (CP) is a prevalent craniofacial malformation, with the TGFβ pathway playing a critical role. Recent evidence links autophagy to regulating mouse embryonic palatal mesenchyme (MEPM) cells, but its interaction with TGFβ-activated phosphorylation cascades remains largely unknown. Here, we investigated the interplay between these pathways during palatogenesis. Methods: H&E and IHC analyses revealed increased expression of Beclin 1 and LC3 during the critical period of palatal shelf elevation and fusion (E13.5–E15.5). Bulk RNA sequencing (Bulk RNA-seq) further revealed enrichment of autophagy-related pathways and their interaction with TGFβ signaling. TMT-based phosphoproteomics was performed on TGFβ2-treated MEPM cells. Results: We identified 23,471 peptides and 3952 proteins, including 6339 phosphopeptides corresponding to 2195 phosphoproteins. Differential analysis found 477 phosphopeptides with increased abundance and 53 with decreased abundance, revealing the enrichment of seven serine (p-Ser) motifs (RxxS, SDxD, SDxE, SP, SxDE, SxEE, SxxxxD) and one threonine (p-Thr) motif (TP). Notably, kinase-substrate enrichment analysis identified CSNK2A as a previously unrecognized phosphorylation regulator, together with MAPKs and CDKs. Functional enrichment showed significant involvement of mTOR, MAPK, and autophagy/mitophagy pathways. Conclusions: Our findings revealed that TGFβ2 reshapes the MEPM phosphoproteome through Smad-independent pathway, expanding the palate-specific phospho-signaling atlas beyond the canonical Smad cascade. Full article

Reversible protein phosphorylation is an important regulatory mechanism in cellular signalling and disease, regulated by the opposing actions of kinases and phosphatases. Modern computer methods predict kinase–substrate or phosphatase–substrate interactions in isolation and lack specificity for biological conditions, neglecting triadic regulation. We present SPINET-KSP, a multi-modal LLM–Graph foundation model engineered for the prediction of kinase–substrate–phosphatase (KSP) triads with contextual awareness. SPINET-KSP integrates high-confidence interactomes (SIGNOR, BioGRID, STRING), structural contacts obtained from AlphaFold3, ESM-3 sequence embeddings, and a 512-dimensional cell-state manifold with 1612 quantitative phosphoproteomic conditions. A heterogeneous KSP graph is examined utilising a cross-attention Graphormer with Reversible Triad Attention to mimic kinase–phosphatase antagonism. SPINET-KSP, pre-trained on 3.41 million validated phospho-sites utilising masked phosphorylation modelling and contrastive cell-state learning, achieves an AUROC of 0.852 for kinase-family classification (sensitivity 0.821, specificity 0.834, MCC 0.655) and a Pearson correlation coefficient of 0.712 for phospho-occupancy prediction. In distinct 2025 mass spectrometry datasets, it identifies 72% of acknowledged cancer-resistance triads within the top 10 rankings and uncovers 247 supplementary triads validated using orthogonal proteomics. SPINET-KSP is the first foundational model for simulating context-dependent reversible phosphorylation, enabling the targeting of dysregulated kinase-phosphatase pathways in diseases. Full article

Listeria monocytogenes (LM) is a lethal foodborne intracellular pathogen. Internalins A and B (inlA and inlB) are critical virulence factors that promote LM’s adhesion and invasion into host cells. InlA is covalently anchored to the cell wall by LM SortaseA (SrtA), while inlB is anchored to the cell wall via non-covalent bonds. Therefore, inhibiting SrtA and inlB is expected to suppress LM’s adhesion and invasion of host cells, enabling the prevention and control of infections. This study demonstrated that Luteolin inhibited the activity of purified LM SrtA protein in vitro. Interactive mechanism analysis indicated that Luteolin generates interaction with the critical active sites of SrtA, which may affect its binding to its natural substrates, thereby reducing the anchoring of inlA on the cell wall and achieving the inhibition of bacterial adhesion and invasion. In addition, Luteolin binds to the groove at the binding interface between inlB and its host receptor. The key residues in inlB that interact with the host receptor form weak interactions (Hydrogen bonds and van der Waals interactions) with Luteolin, this binding may inhibit their binding, suppressing LM’s adhesion and invasion of host cells. At the tested concentrations, Luteolin did not affect the growth of LM, but remarkably reduced the mortality and alleviated the infection symptoms of LM-infected Galleria mellonella. These results provide additional theoretical evidence for the application of Luteolin in the prevention and control of LM infections, which is expected to accelerate its application progress. Full article

Pseudouridine (Ψ), the most abundant RNA modification, plays essential roles in shaping RNA structure, stability, and translational output. Beyond cancer, Ψ is dynamically regulated across numerous physiological and pathological contexts—including immune activation, metabolic disorders, stress responses, and pregnancy-related conditions such as preeclampsia—where elevated Ψ levels reflect intensified RNA turnover and modification activity. These broad functional roles highlight pseudouridylation as a central regulator of cellular homeostasis. Emerging evidence demonstrates that Ψ dysregulation contributes directly to the development and progression of several women’s cancers, including breast, ovarian, endometrial, and cervical malignancies. Elevated Ψ levels in tissues, blood, and urine correlate with tumor burden, metastatic potential, and therapeutic responsiveness. Aberrant activity of Ψ synthases such as PUS1, PUS7, and the H/ACA ribonucleoprotein component dyskerin alters pseudouridylation patterns across multiple RNA substrates, including rRNA, tRNA, mRNA, lncRNAs, snoRNAs, and ncRNAs. These widespread modifications reshape ribosome function, modify transcript stability and translational efficiency, reprogram RNA–protein interactions, and activate oncogenic signaling programs. Advances in high-resolution, site-specific Ψ mapping technologies have further revealed mechanistic links between pseudouridylation and malignant transformation, highlighting how modification of distinct RNA classes contributes to altered cellular identity and tumor progression. Collectively, Ψ and its modifying enzymes represent promising biomarkers and therapeutic targets across women’s cancers, while also serving as sensitive indicators of diverse non-cancer physiological and disease states. Full article

Although calpain-5/CAPN5 is widely expressed in mammals, little is known regarding its functions. Pathogenic mutations of CAPN5 are causal for a devastating autoimmune eye disease, neovascular inflammatory vitreoretinopathy (NIV). To provide insight into both the physiological and pathological roles of CAPN5, it is essential to identify candidate interaction partners and possible substrates. Human SH-SY5Y neuroblastoma cells, transfected with full-length catalytically dead (Cys81Ala) CAPN5-3×FLAG, were used for anti-FLAG co-immunoprecipitation (co-IP) and quantitative proteomics using Sequential Window Acquisition of all THeoretical mass spectra (SWATH-MS). Fifty-one proteins were enriched at least four-fold, p < 0.01, relative to cells transfected with an empty FLAG vector. A high proportion (24/51) of candidate CAPN5 interaction partners are associated with protein quality control, including components of the chaperonin, chaperone, and ubiquitin–proteasome systems. Additional candidate interactors include tubulins, kinases, phosphatases, G proteins, and mitochondrial proteins. CAPN5 interactions for 14 of the candidate proteins were confirmed by co-IP and immunoblotting. Of these 14 proteins, 11 exhibited in vitro calcium-induced proteolysis following co-IP with WT CAPN5-3×FLAG. Impaired calcium-induced proteolysis of co-IP proteins was observed for the pathogenic CAPN5 variants R243L and R289W. Further studies are needed to validate the association of candidate CAPN5 interactors with proteins and complexes suggested by the SWATH-MS and co-IP results, and the possible role of CAPN5 within such complexes. The possible involvement of CAPN5 in protein quality control is relevant to NIV, as defects in protein quality control have been implicated in inherited retinal disorders. Proteomic data are available via ProteomeXchange with identifier PXD068008. Full article

Soluble sugars are the key photo-assimilates in higher plants, playing critical roles in growth, development, and stress regulation. The transport of sugars in plants involves the coordinated action between several sugar transporter families, including the SUT, STP, pGlcT, VGT, TMT, INT, PLT, SFP, and SWEET families. Over recent decades, numerous studies have elucidated the molecular functions of major sugar transporters. Phylogenetic and evolutionary analyses support the conservation of substrate specificity and transport direction, at least to some extent. Structural analyses have provided key insights into the structural–function relationships of important transporters (e.g., OsSWEET2b and AtSTP10), which can be effectively leveraged for artificial intelligence (AI)-enabled protein structure prediction and rational design. Advances in omics technologies now enable low-cost, routine transcriptome profiling and cutting-edge techniques (e.g., single-cell multi-omics and spatiotemporal RNA-seq), providing unprecedented ways to understand how sugar transporters function coordinately at multiple levels. Here, we describe the classification of major sugar transporters in plants and summarize established functional knowledge. We emphasize that recent groundbreaking advances in AI-enabled protein analyses and multi-omics will revolutionize molecular physiology in crops. Specifically, the integration of functional knowledge, AI-based protein analyses, and multi-omics will help unravel the orchestration of different sugar transporters, thereby enhancing our understanding of how sugar transportation and source–sink interactions contribute to crop development, yield formation, and beyond, ultimately boosting carbohydrate transport- related crop improvement. Full article

Transmembrane facilitation of substrates by channels and secondary active transporters results in a defined steady-state concentration ratio across the membrane. Evidence is accumulating that asymmetry in the structural build of the transporters, or interaction with asymmetric partner proteins, can shift the position of the transmembrane equilibrium by biased transport directionality. For instance, the bacterial lactose transporter, LacY, and two amino acid transporters, i.e., the human excitatory amino acid carrier, EAAC1, and the yeast lysine permease, Lyp1, were reported to exhibit distinct transport kinetics in the inward and outward direction by protein-intrinsic properties. A recent example is transport modulation of human monocarboxylate transporters, MCT, by shedding of the extracellular domain of an ancillary protein, basigin. Loss of the domain selectively increases export of lactate from lung cancer cells by a factor of four, contributing to the Warburg effect and malignancy. Further, intrinsic properties of monocarboxylate transporters involving asymmetric affinities of substrate binding, or biased open probabilities were shown to generate preference for one transport direction. Here, we discuss molecular mechanisms and physiological contexts of asymmetric secondary active transmembrane transport. Focus is laid on experimentally established cases, and examples are given in which putative bias in transport directionality may have been overlooked. Full article

Emerging research indicates that neuronal activity is maintained by an architectural system of protons in a multi-scale fashion. Proton architecture is formed when organelles (such as mitochondria, endoplasmic reticulum, lysosomes, synaptic vesicles, etc.) are coupled together to produce dynamic energy domains. Techniques have been developed to visualize protons in neurons; recent advances include near-atomic structural imaging of organelle interfaces using cryo-tomography and nanoscale resolution imaging of organelle interfaces and proton tracking using ultra-fast spectroscopy. Results of these studies indicate that protons in neurons do not diffuse randomly throughout the neuron but instead exist in organized geometric configurations. The cristae of mitochondrial cells create oscillating proton micro-domains that are influenced by the curvature of the cristae, hydrogen bonding between molecules, and localized changes in dielectric properties that result in time-patterned proton signals that can be used to determine the metabolic load of the cell and the redox state of its mitochondria. These proton patterns also communicate to the rest of the cell via hydrated aligned proton-conductive pathways at the mitochon-dria-endoplasmic reticulum junctions, through acidic lipid regions, and through nano-tethered contact sites between mitochondria and other organelles, which are typically spaced approximately 10–25 nm apart. Other proton architectures exist in lysosomes, endosomes, and synaptic vesicles. In each of these organelles, the V-ATPase generates steep concentration gradients across their membranes, controlling the rate of cargo removal from the lumen of the organelle, recycling receptors from the surface of the membrane, and loading neurotransmitters into the vesicles. Recent super-resolution pH mapping has indicated that populations of synaptic vesicles contain significant heterogeneity in the amount of protons they contain, thereby influencing the amount of neurotransmitter released per vesicle, the probability of vesicle release, and the degree of post-synaptic receptor protonation. Additionally, proton gradients in each organelle interact with the cytoskeleton: the protonation status of actin and microtubules influences filament stiffness, protein–protein interactions, and organelle movement, resulting in the formation of localized spatial structures that may possess some type of computational significance. At multiple scales, it appears that neurons integrate the proton micro-domains with mechanical tension fields, dielectric nanodomains, and phase-state transitions to form distributed computing elements whose behavior is determined by the integration of energy flow, organelle geometry, and the organization of soft materials. Alterations to the proton landscape in neurons (e.g., due to alterations in cristae structure, drift in luminal pH, disruption in the hydration-structure of the cell, or imbalance in the protonation of cytoskeletal components) could disrupt the intracellular signaling network well before the onset of measurable electrical or biochemical pathologies. This article will summarize evidence indicating that proton–organelle interaction provides a previously unknown source of energetic substrate for neural computation. Using an integrated approach combining nanoscale proton energy, organelle interface geometry, cytoskeletal mechanics, and AI-based multiscale models, this article outlines current principles and unresolved questions related to the subject area as well as possible new approaches to early detection and precise intervention of pathological conditions related to altered intracellular energy flow. Full article

Extracellular polymeric substances (EPSs) produced by actinomycetes of the genus Rhodococcus play crucial roles in their ecological success, metabolic versatility, and biotechnological value. This review summarizes existing studies of Rhodococcus EPSs, emphasizing the biochemical composition, functional attributes, and practical significance of EPSs, as well as their importance in biomedicine, bioremediation, and other applications (food industry, biomineralization) with respect to the EPS chemical composition and biological roles. Rhodococcus species synthesize complex EPSs composed primarily of polysaccharides, proteins and lipids that, like in other bacteria, support cell adhesion, aggregation, biofilm formation, and horizontal gene transfer (and can prevent exogenous DNA binding) and are highly important for resistance against toxicants and dissolution/assimilation of hydrophobic compounds. EPSs produced by different species of Rhodococcus exhibit diverse structures (soluble EPSs, loosely bound and tightly bound fractions, capsules, linear and branched chains, amorphous coils, rigid helices, mushroom-like structures, extracellular matrix, and a fibrillar structure with a sheet-like texture), leading to variations in their properties (rheological features, viscosity, flocculation, sorption abilities, compression, DNA binding, and interaction with hydrophobic substrates). Notably, the EPSs exhibit marked emulsifying and flocculating properties, contributing to their recognized role in bioremediation. Furthermore, EPSs possess antiviral, antibiofilm, anti-inflammatory, and anti-proliferating activities and high viscosity, which are valuable in terms of biomedical and food applications. Despite extensive industrial and environmental interest, the molecular regulation, biosynthetic pathways, and structural diversity of Rhodococcus EPSs remain insufficiently characterized. Advancing our understanding of these biopolymers could expand new applications in biomedicine, bioremediation, and biotechnology. Full article

Most intracellular proteins are degraded by the ubiquitin–proteasome system (UPS), with proteasomes directly hydrolyzing protein substrates. Specific forms of proteasomes (non-constitutive proteasomes), implicated in antigen presentation, cellular homeostasis maintenance and stress response have been described. However, proteasomes were also identified outside cells, where their function remains unclear. Proteasome secretion via extracellular vesicles (EVs) have been reported, though the direct transmission of non-constitutive proteasomes between cells has not been shown. Using genetically modified cells, including a human adenocarcinoma cell line SW620B8-mCherry expressing the β5i subunit of non-constitutive proteasomes fused to the mCherry protein, and a number of techniques, such as differential centrifugation, affinity isolation, unspecific precipitation, NTA and microscopy, EVs containing non-constitutive proteasomes were obtained and characterized. Different cell lines were shown to secrete varying amounts of vesicles containing non-constitutive proteasomes. The content of these proteasomes in EVs was increased after the stimulation of cells with IFN-γ. The interaction of vesicles secreted by SW620B8-mCherry cells with recipient cells was demonstrated. The β5i-mCherry chimera was detected in lysates of different recipient cells following incubation with EVs secreted from SW620B8-mCherry cells. The obtained results highlight the transfer of non-constitutive proteasomes from one cell to another via EVs. Full article

Metabolites, once viewed mainly as energy substrates or structural precursors, are now increasingly recognized as key extracellular signaling mediators that regulate diverse physiological processes. This review synthesizes and systematizes current knowledge on metabolite-mediated signaling through G-protein-coupled receptors (GPCRs) in teleosts and, importantly, highlights new conceptual links between specific metabolite–GPCR axes and key physiological functions relevant to aquaculture. By integrating evidence across metabolite–GPCRs axes, including succinate–SUCNR1, aromatic amino acids (tryptophan and phenylalanine)–GPR142, basic amino acids (L-arginine)–GPRC6A, and lactate–GPR81. We clarify how metabolite–receptor interactions have the potential to modulate glucose homeostasis, immune responses, energy metabolism, and stress coping. A major contribution of this review is illustrating how metabolites act not only as nutrients but also as extracellular signaling molecules governing core physiological processes via GPCRs. Particularly from an evolutionary perspective, compared with peptide-activated GPCRs, metabolite-sensing GPCRs are relatively conserved across different species, suggesting that relevant findings from biomedical research could be translated to aquaculture applications. Therefore, understanding GPCR-mediated metabolite sensing provides a molecular foundation for improving nutrient formulation, developing functional feeds, and designing selective breeding strategies in precision aquaculture. Full article

The interaction between organic and inorganic nutrients, bacterial communities, and soil fertility has been well documented over time. Conventional agricultural systems heavily utilize both inorganic and organic fertilizers, each exerting distinct effects on soil microbial dynamics and plant growth. The objective of our experiments was to identify the most effective fertilization strategy for improving the biological quality of a microbiologically impoverished and low-productivity soil. To this end, four fertilization strategies were evaluated: (i) organic fertilizers characterized by a high content of organic carbon (Fertil 4-5-7—variant 1); (ii) organic fertilizers with 12% organic nitrogen from proteins (Bio Ostara N—variant 2) (iii) combined inorganic–organic fertilizers (P35 Bio—variant 3) and (iv) mineral (inorganic) fertilizers (BioAktiv—variant V4). This study aimed to assess the short-term effects of fertilizers with varying chemical compositions on the density of cultivable heterotrophic bacteria and their associated dehydrogenase (DH) activity in a petrocalcic chernozem soil containing pedogenic carbonates. Soil sampling was conducted according to a randomized block design, comprising four replicates per treatment (control plus four fertilizer types). The enumeration of cultivable bacteria was performed using Nutrient Agar and A2R Agar media, whereas dehydrogenase activity (DHA) was quantified based on the reduction of 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) to 1,3,5-triphenyl-tetrazolium formazan (TPF) by bacterial dehydrogenase enzymes. Marked differences were observed in both parameters between the plots amended with inorganic fertilizers and those treated with organic fertilizers, as well as among the organic fertilizer treatments of varying composition. The most pronounced increases in both bacterial density and dehydrogenase activity (DHA) were recorded in the plots receiving the fertilizer with a high organic nitrogen content. In this treatment, the maximum bacterial population density reached 6.25 log₁₀ CFU g⁻¹ dry soil after approximately two months (May), followed by a significant decline starting in July. In contrast, DHA exhibited a more rapid response, reaching its peak in April (42.75 µg TPF g⁻¹ soil), indicating an earlier DHA activation of microbial metabolism. This temporal lag between the two parameters suggests that enzymatic activity responded more swiftly to the nutrient inputs than did microbial biomass proliferation. For the other two organic fertilizer variants, bacterial population dynamics were broadly similar, with peak densities recorded in June, ranging from 5.98 log₁₀ CFU g⁻¹ soil (V3) to 6.03 log₁₀ CFU g⁻¹ soil (V1). A comparable trend was observed in DHA: in V3, maximum DHA was attained in June (30 µg TPF g⁻¹ soil), after which it remained relatively stable, whereas in V1, it peaked in June (24.05 µg TPF g⁻¹ soil) and subsequently declined slightly toward the end of the experimental period. Overall, the temporal dynamics of bacterial density and DHA demonstrated a strong dependence on the quality and biodegradability of the organic matter supplied by each fertilizer. Both parameters were consistently lower under inorganic fertilization compared with organic treatments, suggesting that the observed increases in microbial density and activity were primarily mediated by the enhanced availability of organic substrates. The relationship between the density of culturable heterotrophic bacteria and dehydrogenase (DH) activity was strongly positive (r = 0.79), indicating a close functional linkage between bacterial density and oxidative enzyme activity. This connection suggests that the culturable fraction of the heterotrophic microbial community plays a key role in the early stages of organic matter mineralization derived from the applied fertilizers, particularly in the decomposition of easily degradable substrates. Full article

Since 2014, a model of the individual response to ionizing radiation (IR), based on the radiation-induced nucleoshuttling of the ATM protein kinase (RIANS), has been developed by our lab: after irradiation, ATM dimers monomerize in cytoplasm and diffuse into the nucleus to trigger both recognition and repair of DNA double-strand breaks (DSB), the key-damage of IR response. Moderate radiosensitivity is generally caused by heterozygous mutations of ATM substrates (called X-proteins) that are over-expressed in cytoplasm and form complexes with ATM monomers, which reduces and/or delays the RIANS and DSB recognition. Here, we asked whether molecules, rather than X-proteins, can also influence RIANS. Caffeine was chosen as a potential “X-molecule” candidate. After incubation of cells with caffeine, cutaneous fibroblasts from an apparently healthy radioresistant donor, a patient suffering from Alzheimer’s disease (AD) and another suffering from neurofibromatosis type 1 (NF1) were exposed to X-rays. The functionality of ATM-dependent DSB repair and signaling was evaluated. We report here that caffeine molecule interaction with ATM leads to the inhibition of DSB recognition. This effect is significant in radioresistant cells. Conversely, in the AD and NF1 cells, the DSB recognition is already so low that caffeine does not provide any additional molecular effect. Full article

Research indicates that TRF improves mammalian metabolism and health via the microbiota–gut–brain axis. Previous studies showed that TRF promotes pig growth, but the intestinal mechanisms remain unclear. This study explored the impact of TRF on pig intestinal functions. Twelve male pigs were split into ad libitum feeding (FA) and TRF groups. FA pigs had free access to feed, whereas TRF pigs were fed during 07:00–08:00, 12:00–13:00, and 17:00–18:00. TRF enhanced crude protein digestibility by 18.9% (p = 0.045) and increased pancreatic chymotrypsin and lipase activities, while reducing ileal amylase, sucrase, and lipase activities. Transcriptomic analysis identified 1339 differentially expressed genes (DEGs) in the jejunum and 268 in the colon, indicating segment-specific responses. Jejunal DEGs were associated with protein digestion and absorption (e.g., SLC1A1, SLC38A2, XPNPEP2), extracellular matrix–receptor interaction, and PI3K-Akt signaling, while colonic DEGs were linked to starch and sucrose metabolism and circadian entrainment. Importantly, TRF decreased colonic starch by 24% (p = 0.02) and cellulose by 18% (p = 0.04), with low impact on nitrogenous substrates. These results suggest that TRF improves protein absorption in the upper intestine and carbohydrate metabolism in the lower intestine, providing insights for refining TRF strategies in precision nutrition. Full article

The peptidylarginine deiminase (PAD) family includes five isozymes (PAD1–4 and PAD6) with unique tissue distributions and substrate specificities. These enzymes facilitate citrullination, a post-translational modification where positively charged arginine residues are converted into neutral citrulline residues in the presence of calcium ions. This process significantly changes protein properties, affecting molecular interactions, structural stability, and biological functions. Over the past six years (2019–2025), there has been significant progress in understanding PAD activity mechanisms and their therapeutic potential. Recent discoveries include the regulated nuclear translocation of PAD2, PAD4’s specific role in forming cancer extracellular chromatin networks (CECNs), and the development of next-generation inhibitors with greatly improved pharmacological profiles. PAD4 is crucial in forming neutrophil extracellular traps (NETs). Citrullination of histones H3 and H4 by PAD4 destabilizes chromatin, helping release DNA-protein networks as an antibacterial defense. However, excessive NET formation can contribute to autoimmune diseases and thrombosis. Similarly, the bacterial peptidylarginine deiminase from Porphyromonas gingivalis (PPAD)—the only known prokaryotic citrullinating enzyme—plays a key role. Working with R-gingipains, PPAD triggers pathological citrullination of host proteins, leading to immune tolerance breakdown and linking periodontal disease with systemic autoimmune disorders such as rheumatoid arthritis, atherosclerosis, and Alzheimer’s disease. Once thought to be a rare post-translational modification, citrullination is now understood as a vital regulatory mechanism in both normal physiology and disease, involving both internal processes of homeostasis and external mechanisms of bacterial pathogenesis. Full article