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Background: NITRATE TRANSPORTER 1.1 (NRT1.1) functions as a dual affinity nitrate transceptor regulated by phosphorylation at threonine residue 101 (T101). Previous studies have suggested that NRT1.1 is involved in stomatal opening and contributes to drought susceptibility. However, the precise mechanism of how the phosphorylation status of NRT1.1 affects stomatal movement and drought tolerance remains unclear. Results: In this study, we observed that seedlings expressing the phosphorylated form of NRT1.1 (NRT1.1T101D, T101D) exhibited increased drought tolerance compared to dephosphorylated NRT1.1 (NRT1.1T101A, T101A) mutants under low nitrate (LN) condition, characterized by decreased stomatal aperture and water loss. Moreover, we found that the drought-induced depolarization of membrane potential was diminished in T101D mutants in comparison to T101A seedlings. Furthermore, we revealed that the reduced stomatal opening in T101D seedlings was related with depressed nitrate and potassium influx, along with the down-regulation of NRT1.1, POTASSIUM CHANNEL IN ARABIDOPSIS THALIANA 1, and ARABIDOPSIS H + ATPase 1 in comparison with that of T101A. Conclusions: Our study provides several lines of evidence to demonstrate that the phosphorylation of NRT1.1 at T101 contributes to the drought tolerance under LN condition by reducing the influx of nitrate and potassium into the cytoplasm, attenuating membrane depolarization and thereby inducing stomatal closure. This finding identified a novel drought resistance mechanism enabled by post-transcriptional regulation of plasma membrane transporter. [ABSTRACT FROM AUTHOR]

Cyclin-dependent kinase 5 (CDK5), a unique member of the CDK family, is a proline-directed serine/threonine protein kinase with critical roles in various physiological and pathological processes. Widely expressed in the central nervous system, CDK5 is strongly implicated in neurological diseases. Beyond its neurological roles, CDK5 is involved in metabolic disorders, psychiatric conditions, and tumor progression, contributing to processes such as proliferation, migration, immune evasion, genomic stability, and angiogenesis. This review explores the structure and biological functions of CDK5, highlighting its regulatory roles in disease development through the phosphorylation of diverse substrate proteins. Additionally, we examine the therapeutic potential of CDK5 inhibition, offering novel perspectives for disease diagnosis and treatment. [ABSTRACT FROM AUTHOR]

Structural maintenance of chromosome-1A (SMC1A) is overexpressed in various malignancies including triple-negative breast cancer (TNBC). As a core component of the cohesin complex, SMC1A was initially recognized for its involvement in chromosomal cohesion and DNA-repair pathways. However, recent studies have unveiled its pivotal role in epithelial–mesenchymal transition (EMT), metastasis, and chemo- and radio-resistance in cancer cells. In hepatocellular carcinoma, aberrant phosphorylation of SMC1A has been associated with enhanced cell proliferation and migration. Despite these insights, the precise role of SMC1A phosphorylation in breast cancer remains largely unexplored. This study represents the first investigation to test the phosphorylation status and subcellular localization of SMC1A (p-SMC1A) in breast cancer and normal breast tissues. Immunohistochemical (IHC) staining was conducted using previously validated phospho-SMC1A antibodies on a histological section and tissue microarray (TMA) comprising samples from primary, invasive, and metastatic breast cancer and normal breast tissues. Our results revealed that p-SMC1A staining intensity was lower in normal breast tissues compared to invasive or metastatic breast cancer tissues (p < 0.001). Approximately 40% of breast cancer tissue exhibited cytoplasmic/membranous localization of p-SMC1A, whereas nuclear expression was observed in normal breast tissues. Moreover, elevated phosphorylation levels were significantly associated with higher tumor grade and metastasis. [ABSTRACT FROM AUTHOR]

Heat shock proteins (HSPs) are essential molecular chaperones that protect cells by aiding in protein folding and preventing aggregation under stress conditions. Small heat shock proteins (sHSPs), which include members from HSPB1 to HSPB10, are particularly important for cellular stress responses. These proteins share a conserved α-crystallin domain (ACD) critical for their chaperone function, with flexible N- and C-terminal extensions that facilitate oligomer formation. Phosphorylation, a key post-translational modification (PTM), plays a dynamic role in regulating sHSP structure, oligomeric state, stability, and chaperone function. Unlike other PTMs such as deamidation, oxidation, and glycation—which are often linked to protein destabilization—phosphorylation generally induces structural transitions that enhance sHSP activity. Specifically, phosphorylation promotes the disaggregation of sHSP oligomers into smaller, more active complexes, thereby increasing their efficiency. This disaggregation mechanism is crucial for protecting cells from stress-induced damage, including apoptosis, inflammation, and other forms of cellular dysfunction. This review explores the role of phosphorylation in modulating the function of sHSPs, particularly HSPB1, HSPB4, and HSPB5, and discusses how these modifications influence their protective functions in cellular stress responses. [ABSTRACT FROM AUTHOR]

ATR signaling is essential in sensing and responding to the replication stress; as such, any defects can impair cellular function and survival. ATR itself is activated via tightly regulated mechanisms. Here, we identify FOXP1, a forkhead-box-containing transcription factor, as a regulator coordinating ATR activation. We show that, unlike its role as a transcription factor, FOXP1 functions as a scaffold and directly binds to RPA–ssDNA and ATR–ATRIP complexes, facilitating the recruitment and activation of ATR. This process is regulated by FOXP1 O-GlcNAcylation, which represses its interaction with ATR, while CHK1-mediated phosphorylation of FOXP1 inhibits its O-GlcNAcylation upon replication stress. Supporting the physiological relevance of this loop, we find pathogenic FOXP1 mutants identified in various tumor tissues with compromised ATR activation and stalled replication fork stability. We thus conclude that FOXP1 may serve as a potential chemotherapeutic target in related tumors. Synopsis: Replication stress responses regulated by ATR are crucial in resolving genomic threats. This study identifies forkhead-box-containing transcription factor FOXP1 as a new regulator, which promotes ATR activation independent of its transcriptional activity. FOXP1 loads onto stalled replication forks by directly binding to RPA-ssDNA complexes. FOXP1 promotes chromatin loading of ATR and activation of ATR-CHK1. O-GlcNAcylation of FOXP1 represses its interaction with ATR. CHK1-mediated phosphorylation of FOXP1 antagonizes its O-GlcNAcylation. Direct binding to RPA-ssDNA complexes mediates FOXP1 loading onto stalled forks, where it aids ATR-CHK1 activation independent of its transcriptional activity. [ABSTRACT FROM AUTHOR]

Summary: Reactive oxygen species (ROS) are among the most important signaling molecules, playing a significant role in plant growth, development, and responses to various environmental stresses. Respiratory burst oxidase homologs (RBOHs) are key enzymes in ROS production. Plants tightly regulate the activation and deactivation of RBOHs through various post‐translational modifications (PTMs), including phosphorylation, ubiquitination, S‐nitrosylation, and persulfidation. These PTMs fine‐tune ROS production, ensuring normal plant growth and development while facilitating rapid responses to abiotic and biotic stresses. This review discusses the effects of different PTMs on RBOH function and their biological relevance. Additionally, we examine the evolutionary conservation of PTM sites and emphasize the complex interplay between multiple PTMs regulating RBOHs. [ABSTRACT FROM AUTHOR]

Invasiveness of pituitary adenoma is the main cause of its poor prognosis, mechanism of which remains largely unknown. In this study, the differential proteins between invasive and non-invasive pituitary tumors (IPA and NIPA) were identified by TMT labeled quantitative proteomics. The differential metabolites in venous bloods from patients with IPA and NIPA were analyzed by untargeted metabolomics. Proteomic data showed that the top five up-regulated proteins were AD021, C2orf15, PLCXD3, HIST3H2BB and POU1F1, and the top five down-regulated proteins were AIPL1, CALB2, GLUD2, SLC4A10 and GTF2I. Metabolomic data showed that phosphatidylinositol (PI) was most remarkably up-regulated and melibiose was most obviously down-regulated. Further investigation demonstrated that PI stimulation increased the expression of PITPNM1, POU1F1, C2orf15 and LDHA as well as the phosphorylation of AKT and ERK, and promoted the proliferation, migration and invasion of GH3 cells, which were blocked by PITPNM1knockdown. Inhibiting AKT phosphorylation reduced the expression of POU1F1, C2orf15 and LDHA in PI-stimulated cells while activating AKT increased their expression in PITPNM1-silencing cells, which was similar to the function of ERK. POU1F1 silence suppressed the expression of LDHA and C2orf15. Luciferase report assay and ChIP assay demonstrated that POU1F1 positively regulated the transcription of LDHA and C2orf15. In addition, PI propelled the metastasis of GH3 cells in vivo, and elevated the expression of PITPNM1, POU1F1, C2orf15 and LDHA. These results suggested that elevated serum PI might contribute to the proliferation and invasion of pituitary adenoma by regulating the expression of PITPNM1/AKT/ERK/POU1F1 axis. [ABSTRACT FROM AUTHOR]

Background: Bok is a poorly characterized Bcl-2 protein family member with roles yet to be clearly defined. It is clear, however, that Bok binds strongly to inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs), which govern the mobilization of Ca2+ from the endoplasmic reticulum, a signaling pathway required for many cellular processes. Also known is that Bok has a highly conserved phosphorylation site for cAMP-dependent protein kinase at serine-8 (Ser-8). Whether Bok, or phosphorylated Bok, has any direct impact on the Ca2+ mobilizing function of IP3Rs remains to be established. Methods: Bok Ser-8 phosphorylation was characterized using purified proteins, G-protein coupled receptor agonists that increase cAMP levels in intact cells, mass spectrometry, and immunoreactivity changes. Also, using mammalian cells that exclusively or predominately express IP3R1, to which Bok binds strongly, and a fluorescent Ca2+-sensitive dye or a genetically-encoded Ca2+ sensor, we explored how endogenous and exogenous Bok controls the Ca2+ mobilizing function of IP3R1, and whether Bok phosphorylation at Ser-8, or replacement of Ser-8 with a phosphomimetic amino acid, is regulatory. Results: Our results confirm that Ser-8 of Bok is phosphorylated by cAMP-dependent protein kinase, and remarkably that phosphorylation can be detected with Bok specific antibodies. Also, we find that Bok has suppressive effects on IP3R-mediated Ca2+ mobilization in a variety of cell types. Specifically, Bok accelerated the post-maximal decline in G-protein coupled receptor-induced cytosolic Ca2+ concentration, via a mechanism that involves suppression of IP3R-dependent Ca2+ release from the endoplasmic reticulum. These effects were dependent on the Bok-IP3R interaction, as they are only seen with IP3Rs that can bind Bok (e.g., IP3R1). Surprisingly, Bok phosphorylation at Ser-8 weakened the interaction between Bok and IP3R1 and reversed the ability of Bok to suppress IP3R1-mediated Ca2+ mobilization. Conclusions: For the first time, Bok was shown to directly suppress IP3R1 activity, which was reversed by Ser-8 phosphorylation. We hypothesize that this suppression of IP3R1 activity is due to Bok regulation of the conformational changes in IP3R1 that mediate channel opening. This study provides new insights on the role of Bok, its interaction with IP3Rs, and the impact it has on IP3R-mediated Ca2+ mobilization. [ABSTRACT FROM AUTHOR]

Background: Glioblastoma multiforme (GBM) is a highly aggressive brain tumor, characterized by its poor prognosis. Glycolipid metabolism is strongly associated with GBM development and malignant behavior. However, the precise functions of snoRNAs and ADARs in glycolipid metabolism within GBM cells remain elusive. The objective of the present study is to delve into the underlying mechanisms through which snoRNAs and ADARs exert regulatory effects on glycolipid metabolism in GBM cells. Methods: RNA immunoprecipitation and RNA pull-down experiments were conducted to verify the homodimerization of ADAR2 by SNORD113-3, and Sanger sequencing and Western blot experiments were used to detect the A-to-I RNA editing of PHKA2 mRNA by ADAR2. Furthermore, the phosphorylation of EBF1 was measured by in vitro kinase assay. Finally, in vivo studies using nude mice confirmed that SNORD113-3 and ADAR2 overexpression, along with PHKA2 knockdown, could suppress the formation of subcutaneous xenograft tumors and improve the outcome of tumor-bearing nude mice. Results: We found that PHKA2 in GBM significantly promoted glycolipid metabolism, while SNORD113-3, ADAR2, and EBF1 significantly inhibited glycolipid metabolism. SNORD113-3 promotes ADAR2 protein expression by promoting ADAR2 homodimer formation. ADAR2 mediates the A-to-I RNA editing of PHKA2 mRNA. Mass spectrometry analysis and in vitro kinase testing revealed that PHKA2 phosphorylates EBF1 on Y256, reducing the stability and expression of EBF1. Furthermore, direct binding of EBF1 to PKM2 and ACLY promoters was observed, suggesting the inhibition of their expression by EBF1. These findings suggest the existence of a SNORD113-3/ADAR2/PHKA2/EBF1 pathway that collectively regulates the metabolism of glycolipid and the growth of GBM cells. Finally, in vivo studies using nude mice confirmed that knockdown of PHKA2, along with overexpression of SNORD113-3 and ADAR2, could obviously suppress GBM subcutaneous xenograft tumor formation and improve the outcome of those tumor-bearing nude mice. Conclusions: Herein, we clarified the underlying mechanism involving the SNORD113-3/ADAR2/PHKA2/EBF1 pathway in the regulation of GBM cell growth and glycolipid metabolism. Our results provide a framework for the development of innovative therapeutic interventions to improve the prognosis of patients with GBM. [ABSTRACT FROM AUTHOR]

Non-small cell lung cancer (NSCLC) is the main histological subtype of lung cancer. For locally advanced and advanced NSCLC, epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI)-targeted therapy has been the first choice for NSCLC patients with EGFR mutations. TKIs, as targeted drugs, inhibit kinase activity and autophosphorylation by competitively binding to the ATP binding site of the EGFR tyrosine kinase domain, which blocks the signal transduction mediated by EGFR and thus inhibits the proliferation of tumor cells. However, drug resistance to TKIs is inevitable. EGFR is also a highly glycosylated receptor tyrosine kinase, and a wide range of crosstalk occurs between phosphorylation and glycosylation. Therefore, can the phosphorylation state be altered by glycosylation to improve drug resistance? In this review, we summarize phosphorylation, glycosylation and the crosstalk between these processes as well as the current research status and methods. We also summarize the autophosphorylation and glycosylation sites of the EGFR protein and their crosstalk. By exploring the relationship between EGFR glycosylation and autophosphorylation in targeted TKI therapy, we find that research on EGFR glycosylation is crucial for targeted NSCLC treatment and will become a research direction for identifying potential targets related to regulating TKI drug sensitivity. [ABSTRACT FROM AUTHOR]

Objective: Electroacupuncture has been shown to play a neuroprotective role following ischemic stroke, but the underlying mechanism remains poorly understood. Ferroptosis has been shown to play a key role in the injury process. In the present study, we wanted to explore whether electroacupuncture could inhibit ferroptosis by promoting nuclear factor erythroid-2-related factor 2 (Nrf2) nuclear translocation. Methods: The ischemic stroke model was established by middle cerebral artery occlusion/reperfusion (MCAO/R) in adult rats. These rats have been randomly divided into the EA + MCAO/R group, the MCAO/R group, the EA + MCAO/R + Brusatol group (the inhibitor of Nrf2), and the EA + MCAO/R + DMSO group, and the Sham group. The EA + MCAO/R group, EA + MCAO/R + Brusatol group, and the EA + MCAO/R + DMSO group received EA intervention 24 h after modeling for 7 consecutive days. The behavioral function was evaluated by Neurologic severity score (NSS), Garcia score, Foot-fault Test, and Rotarod Test. The infarct volume was detected by TTC staining, and the neuronal damage was observed by Nissl staining. The levels of Fe2+, reactive oxygen species (ROS), superoxide dismutase (SOD), and malondialdehyde (MDA) were measured by ELISA. The immunofluorescence and Western blotting were used to detect the expression of Total Nrf2, p-Nrf2, Nuclear Nrf2, and Cytoplasmic Nrf2, and the essential ferroptosis proteins, including glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11) and ferritin heavy chain 1 (FTH1). The mitochondria were observed by transmission electron microscopy (TEM). Results: Electroacupuncture improved neurological deficits in rats model of MCAO/R, decreased the brain infarct volume, alleviated neuronal damage, inhibited the Fe2+, ROS, and MDA accumulation, increased SOD levels, increased the expression of GPX4, SLC7A11 and FTH1, and rescued injured mitochondria. Especially, we found that the electroacupuncture up-regulated the expression of Nrf2, and promoted phosphorylation of Nrf2 and nuclear translocation, However, Nrf2 inhibitor Brusatol reversed the neuroprotective effect of electroacupuncture. Conclusion: Electroacupuncture can alleviate cerebral I/R injury-induced ferroptosis by promoting Nrf2 nuclear translocation. It is expected that these data will provide novel insights into the mechanisms of electroacupuncture protecting against cerebral I/R injury and potential targets underlying ferroptosis in the stroke. [ABSTRACT FROM AUTHOR]

This study evaluated the potential usage of phosphorylated egg white protein (P-EWP) nanogels fabricated via microwave-induced phosphorylation modification and gel process and further ultrasonic nanometrization as novel delivery systems for cinnamon bark essential oil (CBEO). Compared to EWP-CBEO nanogels without chemical phosphorylation, the obtained P-EWP-CBEO nanogels have shown smaller average hydrodynamic diameter (133.6 nm), relatively uniform size distribution (polydispersity index around 0.265), enhanced negative surface charge (−35.4 mV), and improved stability under the conditions of high temperature (up to 90 °C) and ionic strength (up to 200 mM NaCl). Moreover, P-EWP-CBEO nanogels, with hydrophobic interactions and disulfide bonds as the main intermolecular forces, exhibited a remarkable conformational change in microstructures. In addition, the results of the antibacterial experiments on Escherichia coli, Staphylococcus aureus, and Listeria monocytogenes showed that the MIC values of P-EWP-CBEO nanogels were two times lower than those of EWP-CBEO nanogels and could completely inhibit the growth of pathogenic bacteria within 108 h. Hence, we have suggested that P-EWP-CBEO nanogels are successfully fabricated with improved physicochemical properties as novel potential natural preservatives in the food industry. [ABSTRACT FROM AUTHOR]

Background and Objectives: The pathophysiology of arrhythmogenic cardiomyopathy (ACM), previously known as arrhythmogenic right ventricular cardiomyopathy (ARVC), and its specific biological features remain poorly understood. High-throughput plasma proteomic profiling, a powerful tool for gaining insights into disease pathophysiology at the systems biology level, has not been used to study ACM. This study aimed at characterizing plasmatic protein changes in patients with ACM, which were compared with those of healthy controls, and at exploring the potential role of the identified proteins as biomarkers for diagnosis and monitoring. Materials and Methods: Blood samples were collected from six ACM patients, four patients with other cardiomyopathies, and two healthy controls. Plasma was processed to remove high-abundance proteins and analyzed by two-dimensional gel electrophoresis. Differential protein expressions were assessed using PDQuest software, Bio-Rad US version 8.0.1. Results: The analysis revealed several proteins with altered expressions between ACM patients and controls, including plakophilin-2, junctional plakoglobin, desmoplakin, desmin, transmembrane protein 43, and lamin A/C. Conclusions: The plasma proteomic profiling of ACM suggests that ACM is a distinct disease entity characterized by a unique dysregulation of desmosomal proteins. The identification of plasma biomarkers associated with ACM underscores their potential to improve diagnostic accuracy and facilitate early intervention strategies. Further exploration of mutations in desmosomal proteins and their phosphorylation states may provide deeper insights into the pathophysiology of ACM. [ABSTRACT FROM AUTHOR]

The members of the fourth subgroup of R2R3-MYB (Sg4 members) are well-known inhibitors of phenylpropanoid and lignin synthesis pathways. The C2 domain is closely related to the transcriptional inhibitory activity of Sg4 members. Phosphorylation modification enhances the transcriptional inhibitory activity of Sg4 members. Here, we identified a phosphorylation site on the C2 domain of CsMYB4a from tea plants (Camellia sinensis). A mitogen-activated protein kinase (MAPK), named CsMPK3-2, phosphorylated this site on the C2 domain of CsMYB4a. Further experiments revealed that phosphorylation of CsMYB4a weakened its ability to inhibit the gene expression of PAL, C4H, and 4CL in the phenylpropanoid pathway and activated the expression of transcription factor YABBY5, maintaining the adaxialeabaxial polarity of the leaf. Knocking out NtYAB5 in CsMYB4a transgenic tobacco partially repaired the leaf wrinkling phenotype caused by CsMYB4a. The C1 domain exhibited an activation function when the C2 domain of CsMYB4a was phosphorylated by CsMPK3-2, causing this reversal phenomenon. These results enrich our understanding of the regulatory diversity of Sg4 members. [ABSTRACT FROM AUTHOR]

Apiose is a rare sugar present in cell-wall glycans of all higher plants. Apiosyltransferases use uridine diphosphate-α-d-apiofuranose (UDP-Api) to biosynthesize apiose-containing pectic polysaccharides, namely rhamnogalacturonan-II (RG-II) and apiogalacturonan. However, the study of apiosyltransferases is hampered by the limited availability of UDP-Api. Here, we report the challenging chemical synthesis of α-apiosyl 1-phosphate as the key intermediate towards UDP-Api synthesis. Various protecting group patterns and synthetic routes, including phosphoglycosylation, phosphorylation of the hemiacetal, and phosphorylation of the anomeric bromide were screened. The latter strategy enabled the synthesis of apiosyl 1-phosphate with good α-selectivity (α:β = 5:1). Additionally, we describe a pyrophosphate coupling reaction of the synthesized glycosyl 1-phosphate with commercially available uridine-monophosphate morpholidate to obtain UDP-Api. This test reaction was monitored by 31P-NMR spectroscopy, which showed formation of the desired product and its partial hydrolysis due to an unwanted intramolecular cyclisation typical of 1,2-cis UDP-sugars. [ABSTRACT FROM AUTHOR]

Objective(s): LOXL2, known as Lysyl oxidase-like 2, is classified as a lysyl oxidase (LOX) family member. However, its role and mechanism in endometrial cancer (EC) are unknown. Therefore, we aimed to investigate the potential role and mechanism of LOXL2 in EC. Materials and Methods: The levels of LOXL2 expression in EC tissues and normal adjacent tissues were evaluated by immunohistochemically (IHC) labeling. Following the dye application, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Transwell methodologies were executed to evaluate the effects of LOXL2 inhibition and up-regulation on the growth, programmed cell death, migration, and susceptibility to iron-dependent cell death of EC. Moreover, protein analysis through Western blotting and gene expression analysis using Real-time quantitative PCR (RT-qPCR) was employed to measure the levels of pertinent biomarkers. Results: LOXL2 is highly expressed in both EC tissues and serum in vivo. Silencing LOXL2 reduced EC cell proliferation and migration while increasing apoptosis in vitro. LOXL2 silencing increased the ferroptosis-related proteins Solute Carrier Family 7 Member 11 (SLC7A11) and Ferritin Heavy Chain 1 (FTH1) while decreasing Glutathione Peroxidase 4 (GPX4) (both, P<0.001). Additionally, LOXL2 silencing reduced the p-PI3K and p-Akt protein expression, while LOXL2 overexpression (OE-LOXL2) elevated the p-PI3K and p-Akt protein expression (both, P<0.001). Additionally, LOXL2 silencing increases SLC7A11 and FTH1 while decreasing GPX4 (both P<0.001). LOXL2 overexpression has the opposite effect. However, the LY294002 inhibitor restores SLC7A11 and FTH1 expression while decreasing GPX4 (P<0.001). Conclusion: Our research demonstrated that LOXL2 might protect EC via phosphorylation by activating the PI3K/AKT pathway. [ABSTRACT FROM AUTHOR]

Objective: We aim to investigate transcriptomic and immunophenotypic features of muscle specimens from patients with idiopathic inflammatory myopathy (IIM). Methods: Bulk RNA‐sequencing was performed on muscle biopsy samples from 16 patients with dermatomyositis (DM) and 9 patients with polymyositis (PM). Seven tested positive for anti‐aminoacyl transfer RNA synthetase antibodies in the patients with DM (ARS‐DM). We conducted weighted gene coexpression network analysis (WGCNA), differentially expressed gene (DEG) analysis, and gene set variation analysis to assess contributions of specific pathways. Cell proportions in muscle specimens were estimated using a deconvolution approach. Results: WGCNA revealed significant positive correlations between serum creatine kinase (CK) levels and gene modules involved in cellular respiration, phagocytosis, and oxidative phosphorylation (OXPHOS). Significant positive correlations were also observed between CK levels and proportions of CD16‐positive and negative monocytes and myeloid dendritic cells. Notably, patients with DM demonstrated enrichment of complement and interferon‐α and γ pathway genes compared with those with PM. Furthermore, ARS‐DM demonstrated a higher proportion of Th1 cells and DEGs related to OXPHOS. Additionally, serum Krebs von den Lungen‐6 levels correlated with gene modules associated with extracellular matrix and the transforming growth factor‐β signaling pathway. Conclusion: Our study highlights a significant involvement of monocytes in muscle damage and delineates pathologic differences among IIM subtypes. DM was characterized by complement and interferon‐α and γ signaling, whereas ARS‐DM was associated with OXPHOS. Distinctive gene expression variations in muscle specimens suggest that different pathologic mechanisms underlie muscle damage in each IIM phenotype. [ABSTRACT FROM AUTHOR]

In recent years, the influence of dietary-related factors on neurodegenerative diseases has received considerable attention in the academic community, notably involving the food additive sodium nitrite (NaNO2) and intermittent fasting behavior. However, the effects of NaNO2 and intermittent fasting on spatial learning and memory have not been thoroughly investigated. This study conducted a controlled experiment to explore the impact of NaNO2 and intermittent fasting on the hyperphosphorylation of hippocampal neurofilament (NF) and tau proteins, as well as spatial learning and memory in rats. Through Morris water maze experiments, the spatial learning and memory abilities of rats were assessed, while immunoblotting and immunohistochemistry techniques were employed to evaluate the phosphorylation levels and distribution of NF and tau proteins in the rat hippocampus. NaNO2 was found to induce hyperphosphorylation of hippocampal NF and tau proteins at the Ser396/404 sites, which was accompanied by a decline in spatial learning and memory abilities. Conversely, intermittent fasting ameliorated the NaNO2-induced hyperphosphorylation of hippocampal neurofilaments and the decline in learning and memory abilities, with no discernible effect on hippocampal tau protein hyperphosphorylation. [ABSTRACT FROM AUTHOR]

The N-methyl-D-aspartate (NMDA) receptors are related to the various functioning of the nervous system. It has been shown that the NR2B subunit plays an important role in neurological hypoxic/ischemic diseases by regulating NMDA receptor function. NR2B tyrosine phosphorylation is also an important regulatory mechanism for NMDA receptor function. However, the mechanism of NR2B tyrosine phosphorylation in hypoxic/ischemic injury is still unclear. Therefore, in the present study, we aimed to further clarify the changes in NR2B tyrosine phosphorylation in hypoxic/ischemic damage in the brain and its relationship with neuronal survival under hypoxic/ischemic conditions. Four types of NR2B tyrosine site mutants (Tyr → Phe at 1252, 1336, and 1472, and all three mutations together, named Y1252F, Y1336F, Y1472F, and Triple) and wild-type plasmids were transfected into HT22 cells. The cells were then exposed to oxygen–glucose deprivation and reoxygenation (OGD/R). NR2B, cell apoptosis-related molecules, and neuronal survival factor CREB-related signaling proteins (CaMKII, ERK, Akt) were measured. Cell viability was assessed using the CCK-8 assay. Cell apoptosis and cell cycle were evaluated using flow cytometry. The death ratio of HT22 cells under OGD conditions was further tested using a live cell analysis platform. The viability of HT22 cells in the Y1252F, Y1336F, Y1472F, Triple mutants, and wild-type groups was elevated. Compared to the wild-type, western blotting and real-time PCR showed that Y1252F, Y1336F, Y1472F, and Triple mutants downregulated the expression of apoptosis factors and upregulated anti-apoptosis factors in the OGD/R model. Flow cytometry and cell cycle analysis demonstrated that Y1252F, Y1336F, Y1472F, and Triple mutants reduced the apoptosis rate. The percentage of cells in the S phase decreased significantly. Live cell analysis illustrated that the Y1252F, Y1336F, Y1472F, and Triple mutants contributed to HT22 cell survival under OGD conditions. Additionally, the Y1252F, Y1336F, Y1472F, and Triple mutants activated the survival signaling pathway. Furthermore, compared to the control group (without plasmid), only the Y1336F, Y1472F, and Triple mutants groups showed significant differences in the above tests. The tyrosine phosphorylation of NR2B at Y1336 and Y1472 plays key roles in hypoxic/ischemic injury. These phosphorylation sites may be potential targets for hypoxic/ischemic neural protection. [ABSTRACT FROM AUTHOR]

Summary: Nitrate (NO3−) serves as the primary inorganic nitrogen source assimilated by most terrestrial plants. The acquisition of nitrate from the soil is facilitated by NITRATE TRANSPORTERS (NRTs), with NRT2.1 being the key high‐affinity nitrate transporter. The activity of NRT2.1, which has multiple potential phosphorylation sites, is intricately regulated under various physiological conditions. Here, we discovered that CALCIUM‐DEPENDENT PROTEIN KINASE 28 (CPK28) positively regulates nitrate uptake under nitrogen deprivation conditions.We found CPK28 as the kinase targeted by immunoprecipitation followed by mass spectrometry and examined the in‐planta phosphorylation status of NRT2.1 in cpk28 mutant plants by employing quantitative MS‐based phosphoproteomics. Through a combination of in vitro phosphorylation experiment and immunoblotting using phospho‐specific antibody, we successfully demonstrated that CPK28 specifically phosphorylates NRT2.1 at Ser21.Functional analysis conducted in Xenopus oocytes revealed that co‐expression of CPK28 significantly enhanced high‐affinity nitrate uptake of NRT2.1. Further investigation using transgenic plants showed that the phosphomimic variant NRT2.1S21E, but not the nonphosphorylatable variant NRT2.1S21A, fully restored high‐affinity 15NO3− uptake ability in both nrt2.1 and cpk28 mutant backgrounds.This study clarifies that the kinase activity of CPK28 is promoted during nitrogen deprivation conditions. These significant findings provide valuable insights into the intricate regulatory mechanisms that govern nitrate‐demand adaptation. [ABSTRACT FROM AUTHOR]

Background: The 3xTg-AD transgenic mouse model of Alzheimer's disease (AD) is an important tool to investigate the relationship between development of pathological amyloid-β (Aβ) and tau, neuroinflammation, and cognitive impairments. Traditional behavioral tasks assessing aspects of learning and memory, such as mazes requiring spatial navigation, unfortunately suffer from several shortcomings, including the stress of human handling and not probing species-typical behavior. The automated IntelliCage system was developed to circumvent such issues by testing mice in a social environment while measuring multiple aspects of cognition. Water consumption can serve as a primary motivator for task engagement. Once animals adapt to the cage and can access water, mice can be subjected to operant tasks. Each of the four corners of a cage contains doors to manipulate access to water, visual LED cues, and a valve allowing administration of an air puff. Previously, we detected significant impairments in 3xTg-AD mice in the IntelliCage, however a high failure rate and genotypical differences in water motivation were observed. Methods: Here, we implemented an IntelliCage paradigm where mice underwent progressively more difficult reaction time tasks to assess attention and impulsivity, behaviors mediated by the prefrontal cortex. Mice were placed in the IntelliCage at 11.5 months of age, which corresponds with the presence of widespread pathology. Results: As the difficulty of the reaction time tasks increased, 3xTg-AD mice exhibited lower percent Correct Responses than NonTg. When implementing varying pre-cue durations, where animals are required to wait between the initiation of the trial and the LED turning on (which then requires a nose-poke to access water), 3xTg-AD mice prematurely nose-poked on trials requiring a longer delay before a second nose poke would allow water access, demonstrating heightened impulsivity. The presence of soluble and insoluble fractions of cortical Aβ40 and 42, and phosphorylated tau epitopes threonine 181 and serine 396 confirmed the presence of neuropathological hallmarks in 3xTg-AD mice. Conclusion: Together, this study describes a novel protocol that overcomes motivational differences and detects attention and impulsivity deficits in 3xTg-AD mice utilizing the IntelliCage. [ABSTRACT FROM AUTHOR]

Background: Lovastatin, the main lipid-lowering component in red yeast rice, is a golden anti-lipid drug, but its long-term application is continuously challenged by potential skeletal muscle atrophy. Daidzein, an isoflavone derived from soybeans and many Chinese medicines, shows therapeutic potential in treating muscle-related diseases and metabolic disorders. However, whether daidzein can improve lovastatin-induced muscle atrophy and the specific mechanism needs to further study. Methods: Lovastatin-induced mice and zebrafish muscle atrophy models were used to validate the protective effect of daidzein in vivo. And the lovastatin-induced C2C12 myotube atrophy model was employed to validate the therapeutic efficacy and investigate the specific mechanism of daidzein in vitro. We combined specific siRNA targeting FOXO3a and AMPK-selective inhibitor, agonist to elucidate AMPK/FOXO3a-dependent muscle-protective mechanism of daidzein. The anti-atrophy effects of daidzein through blockage of abnormal activation of AMPK/FOXO3a was presented in Immunofluorescence, H&E staining, Western blot, qRT-PCR. Serum creatine kinase level was detected by ELISA and we used mouse muscle grip instrument to detect the strength of mouse muscles. Results: In this study, we demonstrated that daidzein could dose-dependently alleviate lovastatin-induced mice skeletal muscle atrophy, reduce serum creatine kinase, and improve muscle grip strength in mice. Mechanistically, daidzein inhibited lovastatin-induced FOXO3a phosphorylation caused by AMPK activation, thereby inhibiting FOXO3a nuclear translocation to restrain the expression of muscle-related proteins Atrogin-1 and MuRF-1. In C2C12 myotube, administration of AMPK-selective inhibitor Compound C recapitulated the therapeutic effects of daidzein against lovastatin-induced myotubes atrophy, while the anti-atrophy effects of daidzein were lost in the presence of AMPK-selective agonist MK-3903. In lovastatin-induced mice muscle atrophy models, Compound C elicited similar anti-atrophy effects as daidzein, but this effect was not potentiated when it was applied in combination with daidzein, suggesting that daidzein exerted therapeutic efficacy dependent on blockage of AMPK activity. Conclusions: Our study identified daidzein as an effective component that ameliorated lovastatin-induced skeletal muscle atrophy through blockage of abnormal activation of AMPK/FOXO3a and transcriptional activation of genes encoding downstream muscle-related proteins. Our results also highlighted the therapeutic potential by regulating the AMPK/FOXO3a axis in management of statin-induced myotoxicity. [ABSTRACT FROM AUTHOR]

A circadian clock is reconstituted in vitro by incubating three proteins, KaiA, KaiB, and KaiC from the non-nitrogen-fixing cyanobacterium Synechococcus elongatus PCC 7942 in the presence of ATP. Leptolyngbya boryana is a filamentous cyanobacterium that grows diazotrophically under microoxic conditions. Among the aforementioned proteins, KaiC is the main clock oscillator belonging to the RecA ATPase superfamily. Genomic studies have revealed the presence of many genes encoding KaiC family ATPases in archaea and bacteria; however, very few have been analyzed in detail. For example, the L. boryana genome encodes two kaiC homologs designated as LbkaiC1 (LBWT_14830) and LbkaiC2 (LBWT_17950). LbKaiC1 is highly similar to KaiC from S. elongatus PCC 7942 compared with LbKaiC2. LbKaiC1 and LbKaiC2 were purified as Strep-tag fusion proteins. LbKaiC1 formed a hexamer and exhibited autophosphorylation, autodephosphorylation, and ATPase activities. Furthermore, it exhibited circadian phosphorylation rhythm in the presence of KaiA and KaiB from S. elongatus PCC 7942, indicating that LbKaiC1 is the central oscillator of the circadian clock in L. boryana. The temporal separation of nitrogen fixation from photosynthesis may be supported by the circadian rhythm generated by LbKaiC1 in L. boryana. LbKaiC2 had low ATPase activity, which depended on temperature, and its autophosphorylation activity was not detected like a circadian oscillator KaiC. Although the function of LbKaiC2 remains unknown, this work will provide comprehensive understanding of KaiC family ATPases. [ABSTRACT FROM AUTHOR]

Equine melanocytic neoplasm (EMN) represents a form of skin tumor observed predominantly in grey horses aged over 15 years. Despite its prevalence, current therapeutic and preventive strategies for EMN have been subject to limited investigation. This study endeavors to shed light on potential phosphoproteins present in equine serum and fecal samples, potentially linked to EMN, with a specific focus on functional interactions in EMN pathogenesis. We examined 50 samples (25 serum, 25 feces), divided into three groups based on EMN severity: normal (n = 16), mild (n = 18), and severe EMN (n = 16). Equine phosphoproteome analysis identified 2,359 annotated serum phosphoproteins and 2002 annotated fecal phosphoproteins through differentially expressed proteins (DEPs). KEGG analysis emphasized the role of environmental information processing. Notably, the integrin NF-kappaB binding P-TEFb to stimulate transcriptional elongation signaling pathway, involving SNW1 protein, was implicated in early stage of EMN development in both serum and fecal samples. This highlights SNW1's potential role in mediating transcriptional processes, offering a novel marker within environmental information processing. This study enhances understanding of EMN mechanisms in horses, suggesting early detection through non-invasive methods and identifying a functional pathway involving SNW1, which could inform future treatment and prevention strategies. [ABSTRACT FROM AUTHOR]

The pathological deposition of proteins is a hallmark of several devastating neurodegenerative diseases. These pathological deposits comprise aggregates of proteins that adopt distinct structures named strains. However, the molecular factors responsible for the formation of distinct aggregate strains are unknown. Here, we show that the serine/threonine kinase GSK3ß catalyzes the aggregation of the protein tau into Alzheimer's disease (AD)-like filaments. We demonstrate that phosphorylation by GSK3ß, but not by several other kinases, promotes the aggregation of full-length tau as well as enhances phase separation into gel-like condensate structures. Cryoelectron microscopy further reveals that the fibrils formed by GSK3ß-phosphorylated tau adopt a fold comparable to that of paired helical filaments isolated from the brains of AD patients. Our results elucidate the intricate relationship between posttranslational modification and the formation of tau strains in neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Purpose of review: The thick ascending limb (TAL) of loop of Henle is essential for NaCl, calcium and magnesium homeostasis, pH balance and for urine concentration. NKCC2 is the main transporter for NaCl reabsorption in the TAL and its regulation is very complex. There have been recent advancements toward understanding how NKCC2 is regulated by protein trafficking, protein-protein interaction, and phosphorylation/dephosphorylation. Here, we update the latest molecular mechanisms and players that control NKCC2 function, which gives an increasingly complex picture of NKKC2 regulation in the apical membrane of the TAL. Recent Findings: Protein-protein interactions are required as a regulatory mechanism in many cellular processes. A handful of proteins have been recently identified as an interacting partner of NKCC2, which play major roles in regulating NKCC2 trafficking and activity. New players in NKCC2 internalization and trafficking have been identified. NKCC2 activity is also regulated by kinases and phosphatases, and there have been developments in that area as well. Summary: Here we review the current understanding of apical trafficking of NKCC2 in the thick ascending limb (TAL) which is tightly controlled by protein-protein interactions, protein turnover and by phosphorylation and dephosphorylation. We discuss new proteins and processes that regulate NKCC2 that have physiological and pathological significance. [ABSTRACT FROM AUTHOR]

The correction notice for the article "Circular RNA-encoded oncogenic PIAS1 variant blocks immunogenic ferroptosis by modulating the balance between SUMOylation and phosphorylation of STAT1" in Molecular Cancer addresses inaccuracies in Figures 2 and 3. The errors involve incorrect loading controls and a recommendation to change the vertical title in Figure 3-a for improved accuracy. The correction was made by Xin Zang, Xiao‑Yu He, Cheng‑Mei Xiao, Qing Lin, Meng‑Yue Wang, Cheng‑Yan Liu, Ling‑Yi Kong, Zhong Chen, and Yuan‑Zheng Xia. [Extracted from the article]

Racemisation without Consequence: MOM deprotection during a routine synthesis of phosphoric acid 1 promoted partial racemisation of the product BINOL 5. Surprisingly, however, after phosphorylation of the partially racemised BINOL, enantiopure acid 1 was isolated. Further inspection revealed that during phosphorylation, unhydrolysed racemic phosphorochloridate 6 precipitated, thus restoring homochirality in the product phosphoric acid 1. Moreover, MOM deprotection partial racemisation was avoided by conducting reactions at lower temperatures for no longer than the required deprotection time, and/or by application of other deprotection conditions from the literature. [ABSTRACT FROM AUTHOR]

Objective: With increased incidence, breast cancer has become the most common malignant tumor in women. Transmembrane protein 100 (TMEM100) is a key factor affecting the progression of malignant tumors. The aim of the study is to examine the molecular mechanism of TMEM100 in malignant progression. Material and Methods: TMEM100 expression was analyzed by Western blot, immunohistochemistry, and real-time quantitative polymerase chain reaction. Cell migration and invasiveness after transfection with TMEM100 were investigated by Transwell assay. 5-ethynyl-2-deoxyuridine staining and cell colony-formation assay were utilized to the exploration of cell proliferation. Flow cytometry was adopted to detect whether TMEM100 affected the effect of Docetaxel on cell apoptosis. The effects of TMEM100 on the Ras-extracellular signal-regulated kinase (RAS/ERK) pathway were explored by Western blot assay. Results: Downregulated TMEM100 expression was in breast cancer tissues (P < 0.01). TMEM100 overexpression hindered the invasion (P < 0.01), migration (P < 0.01), and proliferation (P < 0.01) of breast cancer cells. Chemotherapy sensitivity of breast cancer cells to docetaxel was enhanced by TMEM100 (P < 0.01). TMEM100 inhibited Ras expression and ERK1/2 phosphorylation (P < 0.01). Furthermore, ERK agonist TertButylhydroquinone neutralized the effects of TMEM100 (P < 0.01). Conclusion: TMEM100 blocked malignant progression of breast cancer and enhanced docetaxel chemosensitivity by suppressing RAS/ERK pathway. These data manifested that regulation of TMEM100 expression may affect the progression of breast cancer, and its prognostic value and mechanism deserve further investigation. [ABSTRACT FROM AUTHOR]

In epithelial ovarian cancer (EOC), platinum resistance, potentially mediated by cancer stem cells (CSCs), often leads to relapse and treatment failure. Here, the role of spindle pole body component 25 (SPC25) as a key determinant promoting stemness and platinum resistance in EOC cells, with its expression being correlated with adverse clinical outcomes is delineated. Mechanistically, SPC25 acts as a scaffolding platform, orchestrating the assembly of an SPC25/RIOK1/MYH9 trimeric complex, triggering RIOK1‐mediated phosphorylation of MYH9 at Ser1943. This prompts MYH9 to disengage from the cytoskeleton, augmenting its nuclear accumulation, thus potentiating CTNNB1 transcription and subsequent activation of Wnt/β‐catenin signaling. CBP1, a competitive inhibitory peptide, can disrupt the formation of the aforementioned trimeric complex, diminishing the activity of the SPC25/RIOK1/MYH9 axis–mediated Wnt/β‐catenin signaling, and thus attenuate CSC phenotypes, thereby enhancing platinum efficacy in vitro, in vivo, and in patient‐derived organoids. Therefore, targeting the SPC25/RIOK1/MYH9 axis, which mediates the maintenance of stemness and platinum resistance in EOC cells, may enhance platinum sensitivity and increase survival in patients with EOC. [ABSTRACT FROM AUTHOR]

The proneural transcription factor ASCL1 regulates neurogenesis and drives somatic cell reprogramming into neurons. However, not all cell types can be reprogrammed by ASCL1, raising the questions of what provides competence and how we can overcome barriers to enable directed differentiation. Here, we investigate how levels of ASCL1 and its phosphorylation modulate its activity over progressive lineage restriction of mouse embryonic stem cells. We find that inhibition of ASCL1 phosphorylation enhances reprogramming of both mesodermal and neuroectodermal cells, while pluripotent cells remain refractory to ASCL1-directed neuronal differentiation. By performing RNA-seq and ATAC-seq in neuroectoderm, we find that un(der)phosphorylated ASCL1 causes increased chromatin accessibility at sites proximal to neuronal genes, accompanied by their increased expression. Combined analysis of protein stability and proneural function of phosphomutant and phosphomimetic ASCL1 reveals that protein stability plays only a marginal role in regulating activity, while changes in amino acid charge cannot fully explain enhanced activity of the serine-proline mutant variants of ASCL1. Our work provides new insights into proneural factor activity and regulation, and suggests ways to optimize reprogramming protocols in cancer and regenerative medicine. [ABSTRACT FROM AUTHOR]

Here, we have reported a visible-light-mediated organic photoredox-catalyzed difunctionalization of vinyl arenes via radical–radical coupling. A stabilized benzylic radical at the α-position is generated via the regioselective addition of phosphonyl radical at the β-position of the styrene. Subsequently, benzyl or allyl radical, generated via the deaminative pathway from the Katritzky salt, combines with the α-radical of the styrene to furnish the functionalised C–P and C–C bonds in a single reaction. [ABSTRACT FROM AUTHOR]

In this study, a sustainable cellulose-based flame-retardant additive was developed, characterized, and incorporated into polypropylene (PP). Microcrystalline cellulose (Cel) was chemically modified with P2O5 using the solvent-free ball-milling mechanochemistry approach at room temperature. This modification enabled phosphorus grafting onto cellulose, significantly enhancing the cellulose charring ability and improving the thermal stability of the char as revealed by thermogravimetric analysis (TGA). The resulting product, Cel-P, containing 4.15 wt.% phosphorus, was incorporated and uniformly dispersed as a flame-retardant (FR) additive at 30 wt.% in PP through melt processing. The PP+30-Cel-P composite demonstrated improved char formation and FR properties, including reduction of both peak heat release rate (pHRR) and total heat release (THR) in mass loss cone calorimetry (MLC). Moreover, lower light absorptivity was obtained by smoke opacity tests as compared to PP filled with unmodified cellulose. [ABSTRACT FROM AUTHOR]

SUMMARY: Monomeric flavan‐3‐ols and their oligomeric forms, proanthocyanidins (PAs), are closely related to the bitterness of tea beverages. Monomeric flavan‐3‐ols are characteristic flavor compounds in tea. Increasing the content of PAs and anthocyanins enhances the resistance of tea plants to pathogen invasion but decreases the quality of tea beverages. MATE family transporters play a critical role in transferring monomeric flavan‐3‐ols and anthocyanins into vacuoles for storage or subsequent condensation into PAs. Their activities modulate the ratio of monomeric flavan‐3‐ols to PAs and increase anthocyanin content in tea plants. In this study, it was observed that the gene expression and protein phosphorylation level of the MATE transporter CsTT12, a vacuole‐localized flavonoid transporter, were notably upregulated following exogenous sucrose treatment, promoting PA synthesis in tea plants. Further analysis revealed that overexpression of CsTT12 and CsTT12S17D significantly increased the content of anthocyanins and PAs in plants, whereas CsTT12S17A did not. In CsTT12 knockdown plants, PA's accumulation decreased significantly, while monomeric catechin content increased. Moreover, phosphorylation modification enhanced the vacuolar membrane localization of CsTT12, whereas dephosphorylation weakened its vacuolar membrane localization. This study uncovers the crucial role of phosphorylation in flavonoid biosynthesis and provides insights into balancing quality improvements and resistance enhancement. Significance Statement: CsTT12 promotes flavonoid biosynthesis by functioning as a MATE transporter, facilitating the transfer of monomeric flavan‐3‐ols into the vacuole for proanthocyanidin (PA) accumulation. Phosphorylation of CsTT12 at Ser‐17 is crucial for its localization to the tonoplast, which subsequently influences its transport function in tea plants. [ABSTRACT FROM AUTHOR]

Simple Summary: This research focuses on understanding why some triple-negative breast cancers (TNBC) become resistant to chemotherapy, particularly doxorubicin, which is a major obstacle in treatment. The study, by developing models of doxorubicin-resistant TNBC cells, has uncovered that these cells enter a dormant-like state, slow their growth, and activate specific survival pathways. These resistant cells also show changes in metabolism and signaling, particularly involving cytokines, lipid metabolism, and ribosomal function. In identifying these mechanisms, the study suggests new treatment approaches that could target these survival strategies to make chemotherapy more effective and potentially improve patient outcomes. Background: Chemoresistance in triple-negative breast cancer (TNBC) presents a significant clinical hurdle, limiting the efficacy of treatments like doxorubicin. This study aimed to explore the molecular changes associated with doxorubicin resistance and identify potential therapeutic targets to overcome this resistance, thereby improving treatment outcomes for TNBC patients. Methods: Doxorubicin-resistant (DoxR) TNBC models (MDA-MB-231 and BT-549) were generated by exposing cells to increasing concentrations of doxorubicin. RNA sequencing (RNA-Seq) was performed using the Illumina platform, followed by bioinformatics analysis with CLC Genomics Workbench and iDEP. Functional assays assessed proliferation, sphere formation, migration, and cell cycle changes. Protein expression and phosphorylation were confirmed via Western blotting. Pathway and network analyses were conducted using Ingenuity Pathway Analysis (IPA) and STRING, while survival analysis was performed using Kaplan–Meier Plotter database. Results: DoxR cells exhibited reduced proliferation, sphere formation, and migration, but showed enhanced tolerance to doxorubicin. Increased CHK2 and p53 phosphorylation indicated cellular dormancy as a resistance mechanism. RNA-Seq analysis revealed upregulation of cytokine signaling and stress-response pathways, while cholesterol and lipid biosynthesis were suppressed. Activation of the IL1β cytokine network was prominent in DoxR cells, and CRISPR-Cas9 screens data identified dependencies on genes involved in rRNA biogenesis and metabolism. A 27-gene signature associated with doxorubicin resistance was linked to worse clinical outcomes in a large breast cancer cohort (HR = 1.76, FDR p < 2.0 × 10−13). Conclusions: This study uncovers potential therapeutic strategies for overcoming TNBC resistance, including dormancy reversal and targeting onco-ribosomal pathways and cytokine signaling networks, to improve the efficacy of doxorubicin-based treatments. [ABSTRACT FROM AUTHOR]

Introduction: Viral infection usually stimulates a variety of host cell factors to modulate the life cycle of the virus. PIM1, a serine/threonine protein kinase widely involved in cell proliferation, survival, differentiation and apoptosis, was recently reported to be upregulated by Zika virus (ZIKV) infection. However, how ZIKV-PIM1 interactions affect the viral life cycle are not fully understood. Methods and results: Here, we demonstrated that ZIKV replication was suppressed by the PIM1 kinase inhibitor SGI-1776 in both wt and Ifnar1-/- murine peritoneal macrophages, indicating that PIM1 functions independently of type I IFN signaling. Co-immunoprecipitation and GST pull-down assays revealed that the ZIKV structural protein precursor membrane (prM) interacted with PIM1. Moreover, we found that prM protein stability was enhanced by PIM1, which was attributed to its kinase activity. Mechanistically, we revealed that prM can undergo ubiquitin‒mediated proteolysis and the E3 ubiquitin ligase AMFR can target prM for degradation. Importantly, PIM1 catalyzed phosphorylation of prM at Ser101 and Thr107, and this phosphorylation prevented the proteasome-dependent degradation of prM by impairing its association with AMFR. Therefore, the S101/T107-D phosphorylation mimic mutant of prM was more resistant to PIM1-induced increases in cellular abundance. Discussion: These findings revealed PIM1 as a critical host factor that is advantageous to ZIKV and revealed that targeting the PIM1‒prM axis is a conducive strategy for controlling ZIKV infection. [ABSTRACT FROM AUTHOR]

Circadian clocks respond to temperature changes over the calendar year, allowing organisms to adjust their daily biological rhythms to optimize health and fitness. In Drosophila, seasonal adaptations are regulated by temperature-sensitive alternative splicing (AS) of period (per) and timeless (tim) genes that encode key transcriptional repressors of clock gene expression. Although Clock (Clk) gene encodes the critical activator of circadian gene expression, AS of its transcripts and its potential role in temperature regulation of clock function have not been explored. Here, we observed that Clk transcripts undergo temperature-sensitive AS. Specifically, cold temperature leads to the production of an alternative Clk transcript, hereinafter termed Clk-cold, which encodes a CLK isoform with an in-frame deletion of four amino acids proximal to the DNA binding domain. Notably, serine 13 (S13), which we found to be a CK1α-dependent phosphorylation site, is deleted in CLK-cold protein. We demonstrated that upon phosphorylation at CLK(S13), CLK-DNA interaction is reduced, thus decreasing transcriptional activity of CLK. This is in agreement with our findings that CLK occupancy at clock genes and transcriptional output are elevated at cold temperature likely due to higher amounts of CLK-cold isoforms that lack S13 residue. Finally, we showed that PER promotes CK1α-dependent phosphorylation of CLK(S13), supporting kinase-scaffolding role of repressor proteins as a conserved feature in the regulation of eukaryotic circadian clocks. This study provides insights into the complex collaboration between AS and phospho-regulation in shaping temperature responses of the circadian clock. [ABSTRACT FROM AUTHOR]

Ribosomal action is facilitated by the orchestrated work of trans-acting factors and ribosomal elements, which are subject to regulatory events, often involving phosphorylation. One such element is the ribosomal P-stalk, which plays a dual function: it activates translational GTPases, which support basic ribosomal functions, and interacts with the Gcn2 kinase, linking the ribosomes to the ISR pathway. We show that P-stalk proteins, which form a pentamer, exist in the cell exclusively in a phosphorylated state at five C-terminal domains (CTDs), ensuring optimal translation (speed and accuracy) and may play a role in the timely regulation of the Gcn2-dependent stress response. Phosphorylation of the CTD induces a structural transition from a collapsed to a coil-like structure, and the CTD gains conformational freedom, allowing specific but transient binding to various protein partners, optimizing the ribosome action. The report reveals a unique feature of the P-stalk proteins, indicating that, unlike most ribosomal proteins, which are regulated by phosphorylation in an on/off manner, the P-stalk proteins exist in a constantly phosphorylated state, which optimizes their interaction with auxiliary factors. Synopsis: P-stalk proteins are phosphorylated at their C-termini, optimizing translation speed, accuracy, and Gcn2-dependent stress response. Phosphorylation induces a shift from a collapsed to coil-like structure allowing transient binding to various proteins and optimizing interaction with auxiliary factors. Unlike the majority of ribosomal proteins, that undergo phosphorylation in an on/off manner, P-stalk proteins remain in a constantly phosphorylated state. P-stalk proteins are phosphorylated at their five C-termini, under native steady-state conditions, ensuring optimal translation speed, accuracy, and the Gcn2-dependent stress response. Phosphorylation of the C-terminal amino acids of P-proteins causes a structural shift of the C-terminal peptide, from a collapsed to a coil-like form, providing conformational flexibility for transient binding to various protein partners, enhancing ribosome function. P-stalk proteins are phosphorylated at their C-termini, optimizing translation speed, accuracy, and Gcn2-dependent stress response. Phosphorylation induces a shift from a collapsed to coil-like structure allowing transient binding to various proteins and optimizing interaction with auxiliary factors. [ABSTRACT FROM AUTHOR]

The co-evolution of plants and pathogens has enabled them to 'outsmart' each other by promoting their own defence responses and suppressing those of the other. While plants are reliant on their sophisticated immune signalling pathways, pathogens make use of effector proteins to achieve the objective. This entails rapid regulation of underlying molecular mechanisms for prompt induction of associated signalling events in both plants as well as pathogens. The past decade has witnessed the emergence of post-translational modification (PTM) of proteins as a key a factor in modulating cellular responses. The ability of PTMs to expand the functional diversity of the proteome and induce rapid changes at the appropriate time enables them to play crucial roles in the regulation of plant–pathogen interactions. Therefore, this review will delve into the intricate interplay of five major PTMs involved in plant defence and pathogen countermeasures. We discuss how plants employ PTMs to fortify their immune networks, and how pathogen effectors utilize/target host modification systems to gain entry into plants and cause disease. We also emphasize the need for identification of novel PTMs and propose the use of PTM pathways as potential targets for genome editing approaches. [ABSTRACT FROM AUTHOR]

Hyperphosphorylated TDP-43 aggregates in the cytoplasm of motor neurons is a neuropathological signature of amyotrophic lateral sclerosis (ALS). These aggregates have been proposed to possess a toxic disease driving role in ALS pathogenesis and progression, however, the contribution of phosphorylation to TDP-43 aggregation and ALS disease mechanisms remains poorly understood. We've previously shown that CK1δ and CK1ε phosphorylate TDP-43 at disease relevant sites, and that genetic reduction and chemical inhibition could reduce phosphorylated TDP-43 (pTDP-43) levels in cellular models. In this study, we advanced our findings into the hTDP-43-ΔNLS in vivo mouse model of ALS and TDP-43 proteinopathy. This mouse model possesses robust disease-relevant features of ALS, including TDP-43 nuclear depletion, cytoplasmic pTDP-43 accumulation, motor behavior deficits, and shortened survival. We tested the effect of homozygous genetic deletion of Csnk1e in the hTDP-43-ΔNLS mouse model and observed a delay in the formation of pTDP-43 without significant ultimate rescue of TDP-43 proteinopathy or disease progression. Homozygous genetic deletion of Csnk1d is lethal in mice, and we were unable to test the role of CK1δ alone. We then targeted both CK1δ and CK1ε kinases by way of CK1δ/ε-selective PF-05236216 inhibitor in the hTDP-43-ΔNLS mouse model, reasoning that inhibiting CK1ε alone would be insufficient as shown by our Csnk1e knockout mouse model study. Treated mice demonstrated reduced TDP-43 phosphorylation, lowered Nf-L levels, and improved survival in the intermediate stages. The soluble TDP-43 may have been more amenable to the inhibitor treatment than insoluble TDP-43. However, the treatments did not result in improved functional measurements or in overall survival. Our results demonstrate that phosphorylation contributes to neuronal toxicity and suggest CK1δ/ε inhibition in combination with other therapies targeting TDP-43 pathology could potentially provide therapeutic benefit in ALS. [ABSTRACT FROM AUTHOR]

Introduction: Apolipoprotein E4 (APOE4) is the strongest genetic risk factor for Alzheimer's disease (AD), yet it's unclear how this allele mediates risk. APOE4 carriers experience reduced mobility and faster decline in muscle strength, suggesting skeletal muscle involvement. Mitochondria are critical for muscle function and although we have reported defects in muscle mitochondrial respiration during early cognitive decline, APOE4 -mediated effects on muscle mitochondria are unknown. Methods: Here, we sought to determine the impact of APOE4 on skeletal muscle bioenergetics using young, male and female APOE3 (control) and APOE4 targeted replacement mice (n = 8 per genotype/sex combination). We examined the proteome, mitochondrial respiration, fiber size, and fiber-type distribution in skeletal muscle. Results: We found that APOE4 alters mitochondrial pathway expression in young mouse muscle in a sex-dependent manner without affecting respiration and fiber size or composition relative to APOE3. In both sexes, the expression of mitochondrial pathways involved in electron transport, ATP synthesis, and heat production by uncoupling proteins and mitochondrial dysfunction significantly differed between APOE4 and APOE3 muscle. For pathways with predicted direction of activation, electron transport and oxidative phosphorylation were upregulated while mitochondrial dysfunction and sirtuin signaling were downregulated in female APOE4 vs. APOE3 muscle. In males, sulfur amino acid metabolism was upregulated in APOE4 vs. APOE3 muscle. Discussion: This work highlights early involvement of skeletal muscle in a mouse model of APOE4 -linked AD, which may contribute to AD pathogenesis or serve as a biomarker for brain health. [ABSTRACT FROM AUTHOR]

Cetylpyridinium chloride (CPC) is a quaternary ammonium antimicrobial used in numerous personal care products, human food, cosmetic products, and cleaning solutions. Yet, there is minimal published data on CPC effects on eukaryotes, immune signaling, and human health. Previously, it was shown that low-micromolar CPC inhibits rat mast cell function by inhibiting antigen (Ag)-stimulated Ca2+ mobilization, microtubule polymerization, and degranulation. In the current study, these findings are extended to human mast cells (LAD2); this paper presents data indicating that a mechanism of action for CPC might center on its positively-charged quaternary nitrogen in its pyridinium headgroup. The inhibitory effect of CPC was independent of signaling platform receptor architecture. Tyrosine phosphorylation events are a trigger of Ca2+ mobilization necessary for degranulation. CPC inhibits global tyrosine phosphorylation in Ag-stimulated mast cells. Specifically, CPC inhibits tyrosine phosphorylation of specific key players Syk kinase and LAT, a substrate of Syk. In contrast, CPC did not affect Lyn kinase phosphorylation. Thus, a root mechanism for CPC effect might be electrostatic disruption of particular tyrosine phosphorylation events essential for signaling. This work presented here outlines biochemical mechanisms underlying the effects of CPC on immune signaling. [ABSTRACT FROM AUTHOR]

In the present study effect of 3,3'-dimethoxy-4,4'-dihydroxy-stilbene triazole (STT) on plmonary fibrosis development was investigated in vitro in primary lung fibroblasts as well as in vivo in mice model. The results demonstrated that STT treatment effectively inhibited the TGF-β1 induced increase in expression of α-SMA and collagen I proteins in PLFs. STT treatment effectively reversed the TGF-β1 induced increase in expression of LOXL2 protein and phosphorylation of Smad2/3 proteins. Treatment of PLFs with STT reversed the TGF-β1-induced increase in expression of NOX4 and suppression of p-AMPK protein. In mice model of pulmonary fibrosis STT treatment significantly inhibited the BLM-mediated decrease in body weight and survival rate. The BLM induced increase in pulmonary index in mice was also effectively inhibited on treatment with STT. Treatment of the mice with STT inhibited the BLM-induced increase in α-SMA and Col I protein expression in pulmonary tissues. The BLM-induced increase in TGF-β1 protein expression in pulmonary tissues of the mice was inhibited on treatment with STT. Treatment with STT effectively promoted the AMPK activation in lung tissues of the BLM administered mice. In summary, the present study demonstrates that STT treatment prevents TGF-β1 induced up-regulation of α-SMA, collagen I, LOXL2 protein expression and targets phosphorylation of Smad2/3 proteins in PLFs. Moreover, it inhibits TGF-β1-induced increase in expression of NOX4 and reverses TGF-β1-mediated suppression in expression of p-AMPK protein. Therefore, STT inhibits fibrosis development in vitro as well as in vivo and therefore can be investigated further as a therapeutic agent for the treatment of lung fibrosis. [ABSTRACT FROM AUTHOR]

Atherosclerosis (AS) is a major cause of mortality worldwide. Geniposide (GP) has lipolytic and anti-inflammatory effects and is widely administered for the treatment of cardiovascular disease. There is considerable evidence for the importance of autophagy in the cardiovascular system, and GP can promote autophagy and improve AS. However, the underlying mechanism is still unclear; network pharmacology and molecular docking suggest that GP may play anti-atherosclerotic roles by regulating the PI3K/Akt/mTOR pathway, which is a typical autophagy signal transduction approach. We further hypothesized that GP ameliorates AS by regulating autophagy through the PI3K/Akt/mTOR pathway. Oil Red O, Sirius Red, and Masson's trichrome staining revealed that GP can inhibit atherosclerotic lipid accumulation and stabilize plaques. Macrophages absorb lipids, form foam cells, and destabilize plaques. Immunohistochemical staining revealed that GP reduces the expression of F4/80, a major macrophage marker. We used western blotting (WB) and immunofluorescence (IF) to measure the protein levels of PI3K/Akt/mTOR, sequestosome-1, Beclin1, and long-chain base 3 (LC3). The experimental results revealed that GP can increase the expression of LC3, increase the expression of Beclin1, and decrease P62. Additionally, it inhibits the phosphorylation of PI3K/Akt/mTOR. In conclusion, GP can effectively treat AS by enhancing autophagy through the PI3K/Akt/mTOR pathway. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD), the predominant form of dementia, is a neurodegenerative disorder of the central nervous system (CNS) characterized by a subtle onset and a spectrum of cognitive and functional declines. The clinical manifestation of AD encompasses memory deficits, cognitive deterioration, and behavioral disturbances, culminating in a severe impairment of daily living skills. Despite its high prevalence, accounting for 60–70% of all dementia cases, there remains an absence of curative therapeutics. Microglia (MG), the resident immune cells of the CNS, exhibit a bifurcated role in AD pathogenesis. Functioning in a neuroprotective capacity, MGs express scavenger receptors, facilitating the clearance of β -amyloid protein (A β) and cellular debris. Conversely, aberrant activation of MGs can lead to the secretion of pro-inflammatory cytokines, thereby propagating neuroinflammatory responses that are detrimental to neuronal integrity. The dynamics of MG activation and the ensuing neuroinflammation are pivotal in the evolution of AD. Chinese medicine (CM), a treasure trove of traditional Chinese cultural practices, has demonstrated significant potential in the therapeutic management of AD. Over the past triennium, CM has garnered considerable research attention for its multifaceted approaches to AD, including the regulation of MG polarization. This review synthesizes current knowledge on the origins, polarization dynamics, and mechanistic interplay of MG with AD pathology. It further explores the nexus between MG polarization and cardinal pathological hallmarks of AD, such as A β plaque deposition, hyperphosphorylation of tau, synaptic plasticity impairments, neuroinflammation, and brain–gut-axis dysregulation. The review also encapsulates the therapeutic strategies of CM, which encompass monomers, formulae, and acupuncture. These strategies modulate MG polarization in the context of AD treatment, thereby providing a robust theoretical framework in which to conduct future investigative endeavors in both the clinical and preclinical realms. [ABSTRACT FROM AUTHOR]

Arrestins are essential proteins for the regulation of G protein-coupled receptors (GPCRs). They mediate GPCR desensitization after the activated receptor has been phosphorylated by G protein receptor kinases (GRKs). In addition, GPCR-arrestin interactions may trigger signaling pathways that are distinct and independent from G proteins. The non-visual GPCRs encompass hundreds of receptors with varying phosphorylation patterns and amino acid sequences, which are regulated by only two human non-visual arrestin isoforms. This review describes recent findings on GPCR-arrestin complexes, obtained by structural techniques, biophysical, biochemical, and cellular assays. The solved structures of complete GPCR-arrestin complexes are of limited resolution ranging from 3.2 to 4.7 Å and reveal a high variability in the relative receptor-arrestin orientation. In contrast, biophysical and functional data indicate that arrestin recruitment, activation and GPCR-arrestin complex stability depend on the receptor phosphosite sequence patterns and density. At present, there is still a manifest lack of high-resolution structural and dynamical information on the interactions of native GPCRs with both GRKs and arrestins, which could provide a detailed molecular understanding of the genesis of receptor phosphorylation patterns and the specificity GPCR-arrestin interactions. Such insights seem crucial for progress in the rational design of advanced, arrestin-specific therapeutics. [ABSTRACT FROM AUTHOR]

Branched-chain amino acids (BCAA: leucine, isoleucine and valine) are three of the nine indispensable amino acids, and are frequently consumed as a dietary supplement by athletes and recreationally active individuals alike. The popularity of BCAA supplements is largely predicated on the notion that they can stimulate rates of muscle protein synthesis (MPS) and suppress rates of muscle protein breakdown (MPB), the combination of which promotes a net anabolic response in skeletal muscle. To date, several studies have shown that BCAA (particularly leucine) increase the phosphorylation status of key proteins within the mechanistic target of rapamycin (mTOR) signalling pathway involved in the regulation of translation initiation in human muscle. Early research in humans demonstrated that BCAA provision reduced indices of whole-body protein breakdown and MPB; however, there was no stimulatory effect of BCAA on MPS. In contrast, recent work has demonstrated that BCAA intake can stimulate postprandial MPS rates at rest and can further increase MPS rates during recovery after a bout of resistance exercise. The purpose of this evidence-based narrative review is to critically appraise the available research pertaining to studies examining the effects of BCAA on MPS, MPB and associated molecular signalling responses in humans. Overall, BCAA can activate molecular pathways that regulate translation initiation, reduce indices of whole-body and MPB, and transiently stimulate MPS rates. However, the stimulatory effect of BCAA on MPS rates is less than the response observed following ingestion of a complete protein source providing the full complement of indispensable amino acids. [ABSTRACT FROM AUTHOR]

Protein kinases and phosphatases are key signaling proteins and are important drug targets. An explosion in the number of publicly available 3D structures of proteins has been seen in recent years. Three-dimensional structures of kinase and phosphatase have not been systematically investigated. This is due to the difficulty of designing structure-based descriptors that are capable of quantifying conformational changes. We have developed a triangular spatial relationship (TSR)-based algorithm that enables a unique representation of a protein's 3D structure using a vector of integers (keys). The main objective of this study is to provide structural insight into conformational changes. We also aim to link TSR-based structural descriptors to their functions. The 3D structures of 2527 kinases and 505 phosphatases are studied. This study results in several major findings as follows: (i) The clustering method yields functionally coherent clusters of kinase and phosphatase families and their superfamilies. (ii) Specific TSR keys are identified as structural signatures for different types of kinases and phosphatases. (iii) TSR keys can identify different conformations of the well-known DFG motif of kinases. (iv) A significant number of phosphatases have their own distinct DFG motifs. The TSR keys from kinases and phosphatases agree with each other. TSR keys are successfully used to represent and quantify conformational changes of CDK2 upon the binding of cyclin or phosphorylation. TSR keys are effective when used as features for unsupervised machine learning and for key searches. If discriminative TSR keys are identified, they can be mapped back to atomic details within the amino acids involved. In conclusion, this study presents an advanced computational methodology with significant advantages in not only representing and quantifying conformational changes of protein structures but also having the capability of directly linking protein structures to their functions. [ABSTRACT FROM AUTHOR]

HSPB4 and HSPB5 (α-crystallins) have shown increasing promise as neuroprotective agents, demonstrating several anti-apoptotic and protective roles in disorders such as multiple sclerosis and diabetic retinopathy. HSPs are highly regulated by post-translational modification, including deamidation, glycosylation, and phosphorylation. Among them, T148 phosphorylation has been shown to regulate the structural and functional characteristics of HSPB4 and underlie, in part, its neuroprotective capacity. We recently demonstrated that this phosphorylation is reduced in retinal tissues from patients with diabetic retinopathy, raising the question of its regulation during diseases. The kinase(s) responsible for regulating this phosphorylation, however, have yet to be identified. To this end, we employed a multi-tier strategy utilizing in vitro kinome profiling, bioinformatics, and chemoproteomics to predict and discover the kinases capable of phosphorylating T148. Several kinases were identified as being capable of specifically phosphorylating T148 in vitro, and further analysis highlighted mTORC2 as a particularly strong candidate. Altogether, our data demonstrate that the HSPB4-mTORC2 interaction is multi-faceted. Our data support the role of mTORC2 as a specific kinase phosphorylating HSPB4 at T148, but also provide evidence that the HSPB4 chaperone function further strengthens the interaction. This study addresses a critical gap in our understanding of the regulatory underpinnings of T148 phosphorylation-mediated neuroprotection. [ABSTRACT FROM AUTHOR]

As new pollutants, microplastics (MPs) have attracted much attention worldwide because they cause serious environmental pollution and pose potential health risks to humans. However, the toxic effects of MPs are still unclear. In this study, we analysed the inflammatory effects of 0.1 μm polystyrene microplastics (PS-MPs) on mouse and human liver cell lines. After 28 days of exposure to PS-MPs, the mice presented decreased liver index values and increased AST/ALT values. HL7702 and HepG2 were treated with PS-MPs for 24 h, and the cytotoxicity, the expression levels of inflammatory factors, and the phosphorylation of proteins in inflammation related pathways were confirmed. Compared with the control, the cell viability of these two cells significantly decreased after exposure to the PS-MPs at 1000 μm/cm2, and the BMD model also exhibited a similar dose. LDH leakage and AST also increased in a dose-dependent increase after PS-MPs exposure. The relative levels of chemokines such as GM-CSF, IL-6, IL-8, and IL-12p70 were significantly greater than those in the control. Furthermore, the PS-MPs can increase the expression levels of TLR4, MyD88, and NF-κB and activate the phosphorylation of NF-κB and STATs. Based on these results, exposure to PS-MPs can stimulate liver inflammation and activate the TLR4/MyD88/NF-κB and JAK-STAT pathways. [ABSTRACT FROM AUTHOR]