Search Results (7,591)

Over the past decade, the number and diversity of identified protein post-translational modifications (PTMs) have grown significantly. However, most PTMs occur at relatively low abundance, making selective enrichment of modified peptides essential. To address this, we developed a thermodynamic model describing the free beads enrichment in suspension enrichment process and derived a theoretical relationship between material dosage and analyte recovery. The model predicts a non-linear trend, with enrichment efficiency increasing up to an optimal dosage and declining thereafter—a pattern confirmed by experimental data. We validated the model using centrifugation-based enrichment for glycosylated peptides and magnetic-based enrichment for phosphorylated peptides. In both cases, the results aligned with theoretical predictions. Additionally, the optimal dosage varied among peptides with the same modification type, highlighting the importance of tailoring enrichment strategies. This study provides a solid theoretical and experimental basis for optimizing PTMs enrichment and advancing more sensitive, accurate, and efficient mass spectrometry-based proteomic workflows. Full article

The TP53 gene, frequently mutated across multiple cancer types, plays a pivotal role in regulating the cell cycle and apoptosis through its protein, p53. Missense variants of uncertain significance (VUSs) in TP53 present challenges in understanding their impact on protein function and complicate clinical interpretation. This study aims to analyze the effects of missense VUSs in p53, as reported in the gnomAD database, with a specific focus on their impact on protein stability and phosphorylation. In this study, 33 missense VUSs in TP53 reported in the gnomAD database were analyzed using in silico tools, including PhosphositePlus v6.7.4, the Kinase Library v0.0.11, and Dynamut2. Of these analyzed variants, five disrupted known phosphorylation sites, while another five created new consensus sequences for phosphorylation. Moreover, 20 variants exhibited a moderate destabilizing effect on the protein structure. At least three missense VUSs were identified as potentially affecting p53 function, which may contribute to cancer development. These findings highlight the importance of integrating in silico structural and functional analysis to assess the pathogenic potential of missense VUSs. Full article

Type 10 17β-hydroxysteroid dehydrogenase (17β-HSD10) is the HSD17B10 gene product. It plays an appreciable part in the carcinogenesis and pathogenesis of neurodegeneration, such as Alzheimer’s disease and infantile neurodegeneration. This mitochondrial, homo-tetrameric protein is a central hub in various metabolic pathways, e.g., branched-chain amino acid degradation and neurosteroid metabolism. It can bind to other proteins carrying out diverse physiological functions, e.g., tRNA maturation. It has also previously been proposed to be an Aβ-binding alcohol dehydrogenase (ABAD) or endoplasmic reticulum-associated Aβ-binding protein (ERAB), although those reports are controversial due to data analyses. For example, the reported k_m value of some substrate of ABAD/ERAB was five times higher than its natural solubility in the assay employed to measure k_m. Regarding any reported “one-site competitive inhibition” of ABAD/ERAB by Aβ, the k_ivalue estimations were likely impacted by non-physiological concentrations of 2-octanol at high concentrations of vehicle DMSO and, therefore, are likely artefactual. Certain data associated with ABAD/ERAB were found not reproducible, and multiple experimental approaches were undertaken under non-physiological conditions. In contrast, 17β-HSD10 studies prompted a conclusion that Aβ inhibited 17β-HSD10 activity, thus harming brain cells, replacing a prior supposition that “ABAD” mediates Aβ neurotoxicity. Furthermore, it is critical to find answers to the question as to why elevated levels of 17β-HSD10, in addition to Aβ and phosphorylated Tau, are present in the brains of AD patients and mouse AD models. Addressing this question will likely prompt better approaches to develop treatments for Alzheimer’s disease. Full article

Resveratrol (RSV), a polyphenol found in a variety of berries and wines, is known for its anti-inflammatory, anticancer, and antioxidant properties. It has been suggested that RSV may play a role in the regulation of metabolic disorders, including diabetes and insulin resistance. However, in recent years, it has been reported to completely inhibit Akt kinase function in liver cells. Akt is a central protein involved in the metabolic function of insulin and is regulated by the phosphatidylinositol-3-kinase (PI3K) pathway. In this study, we examined the effect of RSV on insulin-induced insulin receptor (IR) phosphorylation and proteins involved in the PI3K/Akt pathway in a hepatic cell model, clone 9 (C9), and in hepatoma cells, Hepa 1-6 (H1-6). In both cell lines, RSV inhibited tyrosine phosphorylation of IR and insulin-induced activation of Akt. We also evaluated the effect of RSV on the activation of protein tyrosine phosphatase 1B (PTP1B), which is associated with IR dephosphorylation, and found that RSV increased PTP1B-Tyr¹⁵² phosphorylation in a time- and concentration-dependent manner. Furthermore, we found that the protein kinase C (PKC) inhibitors BIM and Gö6976 prevented the inhibition of Akt phosphorylation by RSV and increased the phosphorylation of Ser/Thr residues in IR, suggesting that PKC is involved in the inhibition of the insulin pathway by RSV. Thus, classical PKC isoforms impair the PI3K/Akt pathway at the IR and GSK3 and GS downstream levels; however, IRS-Tyr⁶³² phosphorylation remains unaffected. These results suggest that RSV can lead to insulin resistance by activating PTP1B and PKC, consequently affecting glucose homeostasis in hepatic cells. Full article

The rabies virus (RABV) phosphoprotein (P protein) has multiple functions, including acting as the essential non-catalytic cofactor of the viral polymerase (L protein) for genome replication and transcription; the principal viral antagonist of the interferon (IFN)-mediated innate immune response; and the chaperone for the viral nucleoprotein (N protein). Although P protein is known to undergo phosphorylation by cellular kinases, the location and functions of the phosphorylation sites remains poorly defined. Here, we report the identification by mass-spectrometry (MS) of residues of P protein that are modified by phosphorylation in mammalian cells, including several novel sites. Analysis of P protein with phospho-mimetic and phospho-inhibitory mutations of three novel residues/clusters that were commonly identified by MS (Ser48, Ser183/187, Ser217/219/220) indicate that phosphorylation at each of these sites does not have a major influence on nuclear trafficking or antagonistic functions toward IFN signalling pathways. However, phosphorylation of Ser48 in the N-terminus of P protein impaired function in transcription/replication and in the formation of replication structures that contain complexes of P and N proteins, suggestive of altered interactions of these proteins. The crystal structure of P protein containing the S48E phospho-mimetic mutation indicates that Ser48 phosphorylation facilitates the binding of residues 41–52 of P protein into the RNA-binding groove of non-RNA-bound N protein (N⁰), primarily through the formation of a salt bridge with Arg434 of N protein. These data indicate that Ser48 modification regulates the cycling of P-N⁰ chaperone complexes that deliver N protein to RNA to enable transcription/replication, such that enhanced interaction due to S48E phospho-mimetic mutation reduces N protein delivery to the RNA, inhibiting subsequent transcription/replication processes. These data are, to our knowledge, the first to implicate phosphorylation of RABV P protein in conserved replication functions of the P gene. Full article

Glioblastoma (GBM) is a highly aggressive and heterogeneous brain tumor. Glioma stem-like cells (GSCs) play a central role in tumor progression, therapeutic resistance, and recurrence. Although immune cells are known to shape the GBM microenvironment, the impact of antigen-presenting-cell (APC) signature genes on tumor-intrinsic phenotypes remains underexplored. We analyzed both bulk- and single-cell RNA sequencing datasets of GBM to investigate the association between APC gene expression and tumor-cell states, including stemness and metabolic reprogramming. Signature scores were computed using curated gene sets related to APC activity, KEGG metabolic pathways, and cancer hallmark pathways. Protein–protein interaction (PPI) networks were constructed to examine the links between immune regulators and metabolic programs. The high expression of APC-related genes, such as HLA-DRA, CD74, CD80, CD86, and CIITA, was associated with lower stemness signatures and enhanced inflammatory signaling. These APC-high states (mean difference = –0.43, adjusted p < 0.001) also showed a shift in metabolic activity, with decreased oxidative phosphorylation and increased lipid and steroid metabolism. This pattern suggests coordinated changes in immune activity and metabolic status. Furthermore, TNF-α and other inflammatory markers were more highly expressed in the less stem-like tumor cells, indicating a possible role of inflammation in promoting differentiation. Our findings revealed that elevated APC gene signatures are associated with more differentiated and metabolically specialized GBM cell states. These transcriptional features may also reflect greater immunogenicity and inflammation sensitivity. The APC metabolic signature may serve as a useful biomarker to identify GBM subpopulations with reduced stemness and increased immune engagement, offering potential therapeutic implications. Full article

The midgut of Antheraea pernyi plays a critical role in antiviral defense. However, its transcriptional complexity remains poorly understood. Here, a full-length (FL) transcriptome atlas of A. pernyi midgut was developed by integrating PacBio Iso-Seq and RNA-seq techniques. The transcriptome sequences included 1850 novel protein-coding genes, 17,736 novel alternative isoforms, 1664 novel long non-coding RNAs (lncRNAs), and 858 transcription factors (TFs). In addition, 2471 alternative splicing (AS) events and 3070 alternative polyadenylation (APA) sites were identified. Moreover, 3426 and 4796 differentially expressed genes (DEGs) and isoforms were identified after ApNPV infection, respectively, besides the differentially expressed lncRNAs (164), TFs (171), and novel isoforms of ApRelish (1) and ApSOCS2 (4). Enrichment analyses showed that KEGG pathways related to metabolism were suppressed, whereas GO terms related to DNA synthesis and replication were induced. Furthermore, the autophagy and apoptosis pathways were significantly enriched among the upregulated genes. Protein–protein interaction network (PPI) analysis revealed the coordinated downregulation of genes involved in mitochondrial ribosomes, V-type and F-type ATPases, and oxidative phosphorylation, indicating the disruption of host energy metabolism and organelle acidification. Moreover, coordinated upregulation of genes associated with cytoplasmic ribosomes was observed, suggesting that the infection by ApNPV interferes with host translational machinery. These results show that ApNPV infection reprograms energy metabolism, biosynthetic processes, and immune response in A. pernyi midgut. Our study provides a foundation for elucidating the mechanisms of A. pernyi–virus interactions, particularly how the viruses affect host defense strategies. Full article

Background: Heat therapy (HT) and electroacupuncture (EA) are widely utilized pain relief methods, but the analgesic mechanisms of their combined application remain unclear. Methods: In acetic acid (AA)-induced writhing test and complete Freund’s adjuvant (CFA)-induced inflammatory pain tests, mice received one of three treatments: EA at bilateral ST36, HT via a 45 °C heating pad, or the combination (EA + HT). To probe underlying pathways, separate groups were pretreated with caffeine, DPCPX (a selective adenosine A₁ receptor antagonist), or naloxone (an opioid receptor antagonist). Spinal expression of glial fibrillary acidic protein (GFAP) and phosphorylated p38 (p-p38) was examined by Western blot and immunofluorescence. Results: Both EA and HT individually reduced AA-induced writhing, with the combination (EA + HT) exhibiting the greatest analgesic effect. EA’s analgesic effect was reversed by caffeine and DPCPX and partially by naloxone, while HT’s effect was reversed by caffeine and DPCPX but was unaffected by naloxone. AA injection elevated spinal p-p38 and GFAP expression, which were attenuated by either EA or HT, with the most substantial suppression observed in the EA + HT group. In the CFA model, both treatments alleviated mechanical allodynia, while the combined treatment resulted in significantly greater analgesia compared to either treatment alone. Conclusions: EA combined with HT synergistically enhances analgesia in both AA and CFA pain models, accompanied by reduced spinal inflammation and astrocyte activation. EA’s analgesic effects appear to involve adenosine A₁ receptor pathways and, to a lesser extent, opioid receptor mechanisms, whereas HT’s effects involve adenosine A₁ receptor pathways. Full article

The popularity of bioactive compounds extracted from sea cucumbers is growing due to their wide application in the pharmaceutical industry, particularly in the development of drugs for neurological disorders. Different types of compounds, such as saponins, phenolic compounds, cerebrosides, and glucocerebrosides, are being studied intensively for their efficacy in assessing the treatment of neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, and brain tumors, among others. Positive results have been observed in the upregulation in the content of p-CREB, p-PL3K, BDNF, SOD, and MDA. Furthermore, the neuroprotective mechanism of the compounds against Alzheimer’s disease revealed that suppressing the phosphorylation of tau protein by the PI3K/Akt/GSK3β pathway leads to improved synaptic plasticity and reduced nerve fiber tangles. This comprehensive review explores recent findings on the therapeutic potential of sea cucumber bioactives in the treatment of brain-related disorders. Full article

Background and objectives: Adducins are cytoskeletal proteins essential for membrane stability, actin–spectrin network organization, and cell signaling. Mutations in the genes ADD1, ADD2, and ADD3 have been linked to hypertension, neurodevelopmental disorders, and cancer. However, no comprehensive in silico saturation mutagenesis study has systematically evaluated the pathogenic potential and structural consequences of all possible missense mutations in adducins. This study aimed to identify high-risk variants and their potential impact on protein stability and function. Methods: We performed computational saturation mutagenesis for all possible single amino acid substitutions across the adducin proteins family. Pathogenicity predictions were conducted using four independent tools: AlphaMissense, Rhapsody, PolyPhen-2, and PMut. Predictions were validated against UniProt-annotated pathogenic variants. Predictive performance was assessed using Cohen’s Kappa, sensitivity, and precision. Mutations with a prediction probability ≥ 0.8 were further analyzed for structural stability using mCSM, DynaMut2, MutPred2, and Missense3D, with particular focus on functionally relevant domains such as phosphorylation and calmodulin-binding sites. Results: PMut identified the highest number of pathogenic mutations, while PolyPhen-2 yielded more conservative predictions. Several high-risk mutations clustered in known regulatory and binding regions. Substitutions involving glycine were consistently among the most destabilizing due to increased backbone flexibility. Validated variants showed strong agreement across multiple tools, supporting the robustness of the analysis. Conclusions: This study highlights the utility of multi-tool bioinformatic strategies for comprehensive mutation profiling. The results provide a prioritized list of high-impact adducin variants for future experimental validation and offer insights into potential therapeutic targets for disorders involving ADD1, ADD2, and ADD3 mutations. Full article

The C-kinase-activated protein phosphatase-1 (PP1) inhibitor of 17 kDa (CPI-17) is a specific inhibitor of the PP1 catalytic subunit (PP1c) and the myosin phosphatase (MP) holoenzyme. CPI-17 requires the phosphorylation of Thr38 in the peptide segment ³⁵ARV(P)TVKYDRREL⁴⁶ for inhibitory activity. CPI-17 regulates myosin phosphorylation in smooth muscle contraction and the tumorigenic transformation of several cell lines via the inhibition of MP. A phosphospecific antibody (anti-CPI-17^pThr38) against the phosphorylation peptide was used to determine the phosphorylation levels in cells. We found that phospho-CPI-17 and its closely related proteins are not present in HeLa and MCF7 cells after inducing phosphorylation by inhibiting phosphatases with calyculin A. In contrast, cross-reactions of proteins in the 40–220 kDa range with anti-CPI-17^pThr38 were apparent. Searching the protein database for similarities to the CPI-17 phosphorylation sequence revealed several proteins with 42–75% sequence identities. The LIMK2-1 isoform emerged as a possible PP1 inhibitor. Experiments with Flag-LIMK2-1 overexpressed in tsA201 cells proved that LIMK2-1 interacts with PP1c isoforms and is phosphorylated predominantly by protein kinase C. Phosphorylated LIMK2-1 inhibits PP1c and the MP holoenzyme with similar potencies (IC50 ~28–47 nM). In conclusion, our results suggest that LIMK2-1 is a novel phosphorylation-dependent inhibitor of PP1c and MP and may function as a CPI-17-like phosphatase inhibitor in cells where CPI-17 is present but not phosphorylated upon phosphatase inhibition. Full article

Although exercise is known to exert anti-inflammatory effects in neurodegenerative diseases, its specific impact and underlying mechanisms in Parkinson’s disease (PD) remain poorly understood. This study explores the effects of exercise on microglia-mediated neuroinflammation and apoptosis in a PD model, focusing on the role of irisin signaling in mediating these effects. Using a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model, we found that a 10-week treadmill exercise regimen significantly enhanced motor function, reduced dopaminergic neuron loss, attenuated neuronal apoptosis, and alleviated neuroinflammation. Exercise also shifted microglia from a pro-inflammatory to an anti-inflammatory phenotype. Notably, levels of irisin, phosphorylated AMP-activated protein kinase (p-AMPK), and sirtuin 1 (Sirt1), which were decreased in the PD brain, were significantly increased following exercise. These beneficial effects were abolished by blocking the irisin receptor with cyclic arginine–glycine–aspartic acid–tyrosine–lysine (cycloRGDyk). Our results indicate that exercise promotes neuroprotection in PD by modulating microglial activation and the AMPK/Sirt1 pathway through irisin signaling, offering new insights into exercise-based therapeutic approaches for PD. Full article

MOTS-c is a mitochondrial peptide that plays a crucial role in regulating energy metabolism, gene expression, and immune processes. However, current research primarily focuses on mammals like humans and mice, with no reports on avian MOTS-c. This study aimed to identify and characterize MOTS-c coding sequences across major poultry species through bioinformatics analysis and experimental validation. The alignment results showed high sequence similarity in the MOTS-c coding regions between avian and mammalian species. However, a single nucleotide deletion was identified in avian sequences at the position corresponding to the fourth amino acid residue of mammalian homologs, resulting in divergent downstream amino acid sequences. Despite this deletion, several residues were conserved across species. Phylogenetic analysis of mRNA sequences grouped pigeons with mammals, while protein sequence analysis revealed that poultry and mammals form separate branches, highlighting the divergence between avian and mammalian MOTS-c sequences. Tissue expression profiling demonstrated widespread distribution of chicken MOTS-c across multiple tissues, with the highest expression levels in the heart. Fasting significantly reduced heart MOTS-c expression, suggesting potential metabolic regulatory functions. Functional analysis of MOTS-c in primary hepatocytes revealed significant enrichment of the ribosome, oxidative phosphorylation, and key signaling pathways (PI3K-AKT and JAK-STAT) following 24 hours of treatment. Western blot validation confirmed MOTS-c-mediated activation of the AKT signaling pathway. This study represents the first comprehensive characterization of avian MOTS-c, providing critical insights into its evolutionary conservation and its potential functional roles in gene expression and cellular metabolism. Our findings establish a foundation for further investigation into the functions of mitochondrial-encoded peptides in avian species. Full article

T-cells constitute an essential component of the adaptive immune response, mount a protective response against foreign pathogens and are important regulators of anti-tumor immunotherapy. In this context, the activation of T-cells and chimeric antigen receptor (CAR)-expressing T-cells is orchestrated by various signaling pathways, involving the initiation of a protein tyrosine phosphorylation cascade. For T-cells, this involves initiation of the phosphorylation cascade via src-related protein-tyrosine kinase p56^lck, which we show to associate with the co-receptors CD4 and CD8 for the induction of a phosphorylation cascade needed for the activation of T-cells. Likewise, p56^lck phosphorylation of the antigen receptor immunoreceptor tyrosine-based activation motifs (ITAMs) and key CD28 tyrosine motifs ensures the functionality and the survival of CARs, while their phospho-targets are also inhibited by PD-1, a key component of the immune checkpoint blockade. This review covers historic and current elements of our knowledge of CD4/CD8–p56^lck-induced activation events and their importance to the development of CAR T-cell immunotherapies. Full article

Background: Protein kinase R (PKR) inhibits general mRNA translation by phosphorylating the alpha subunit of eukaryotic translation initiation factor 2 (eIF2). PKR also modulates NF-κB signaling during viral infections, but comparative studies of PKR-mediated NF-κB responses across mammalian species and their regulation by viral inhibitors remain largely unexplored. This study aimed to characterize the conserved antiviral and inflammatory roles of mammalian PKR orthologs and investigate their modulation by poxviral inhibitors. Methods: Using reporter gene assays and quantitative RT-PCR, we assessed the impact of 17 mammalian PKR orthologs on general translation inhibition, stress-responsive translation, and NF-κB-dependent induction of target genes. Congenic human and rabbit cell lines infected with a myxoma virus strain lacking PKR inhibitors were used to compare the effects of human and rabbit PKR on viral replication and inflammatory responses. Site-directed mutagenesis was employed to determine key residues responsible for differential sensitivity to the viral inhibitor M156. Results: All 17 mammalian PKR orthologs significantly inhibited general translation, strongly activated stress-responsive ATF4 translation, and robustly induced NF-κB target genes. Inhibition of these responses was specifically mediated by poxviral K3 orthologs that effectively suppressed PKR activation. Comparative analyses showed human and rabbit PKRs similarly inhibited virus replication and induced cytokine transcripts. Amino acid swaps between rabbit PKRs reversed their sensitivity to viral inhibitor M156 and NF-κB activation. Conclusions: Our data show that the tested PKR orthologs exhibit conserved dual antiviral and inflammatory regulatory roles, which can be antagonized by poxviral K3 orthologs that exploit eIF2α mimicry to modulate the PKR-NF-κB axis. Full article