Search Results (195)

The normal metabolism of transient starch in leaves plays a vital role in determining photosynthesis and final crop yield. However, the molecular mechanisms linking abnormal transient starch metabolism to premature leaf senescence remain unclear. Here, we isolate a rice mutant, lses1, with leaf yellowing and premature senescence, as well as excessive accumulation of starch granules in chloroplasts. Genetic analysis revealed that this trait is controlled by a single recessive nuclear gene. Through BSA-seq preliminary gene mapping, map-based cloning, and sequencing alignment, the candidate gene was pinpointed to LOC_Os02g40860 on chromosome 2, which encodes OsCKI1, a casein kinase I family member. The identity of LSES1 was confirmed functionally: genetic complementation with the native genomic sequence rescued the wild-type phenotype, while CRISPR/Cas9 knockout of the gene in wild-type plants recapitulated the premature senescence. This confirmed that LSES1/OsCKI1 is involved in the regulation of leaf senescence. Notably, one improved knockout line, KO-2, displayed significant agronomic improvements in grain length, grain width, number of productive ears, and number of filled grains per panicle, along with a significant increase in grain yield per plant, highlighting its potential breeding value. Subcellular localization and tissue-specific expression analysis showed that LSES1 is primarily nuclear-localized and constitutively expressed. Full article

FAM20C, previously known as Golgi casein kinase (GCK), is a serine/threonine kinase localized to the Golgi apparatus and classified within the acidophilic kinase family. Its phosphorylation motif is characterized by a glutamic acid residue at the +2 position relative to the target site. Before its molecular identity was established, analysis of a limited number of phosphosites in secreted proteins showed that around 70% matched the GCK consensus sequence, suggesting that GCK is the principal kinase for secreted proteins. Following the identification of GCK as FAM20C, the generation of FAM20C knockout cell lines and phosphoproteomic data confirmed its role: approximately 80% of serine/threonine phosphosites in the secretome of two different human cell lines were shown to depend on FAM20C. In this study, comparative analysis of in vitro phosphorylation datasets from a broad panel of recombinant Ser/Thr kinases confirmed that the FAM20C consensus sequence is distinct from those of other acidophilic kinases. Examination of experimentally identified human phosphosites within the secretory pathway revealed strong conservation of the FAM20C consensus, firmly establishing this enzyme as the master Ser kinase of the entire pathway. From this dataset, we defined the putative FAM20C substratome, comprising 443 phosphosites across 256 proteins, ~77% of which had not been previously linked to FAM20C. This represents the most extensive FAM20C substratome to date and a valuable resource for functional studies. Notably, enrichment analysis highlights strong links between FAM20C and major extracellular pathways, including collagen fibril organization, complement activation, and blood coagulation, underscoring an underappreciated role for this kinase in regulating hemostasis and innate immunity. Full article

Background: β-casomorphin-10 (CM-10), a peptide fragment derived from milk casein with the sequence YPFPGPIPNS, has demonstrated notable anxiolytic activity in BALB/c mice. Yet, its cellular responses and mechanistic pathways remain largely uncharacterized. Methods: We performed RNA-seq analysis to profile gene expression changes in δ-opioid receptor-expressing HEK293 cells (DOR-HEK), comparing CM-10-treated and untreated conditions. Results: CM-10 exposure led to differential expression of 1714 genes in DOR-HEK cells, with 34 upregulated (>1.4-fold) (1.9%) and 1680 downregulated (<0.71-fold) (98.1%), based on a predicted p-value threshold of <0.05. Notably, we identified 10 clusters that were associated with reduced cyclic AMP (cAMP) in DOR-HEK cells following CM-10 treatment. These clusters particularly involved genes related to regulatory subunits of cAMP-dependent protein kinases, such as PRKAR2A, cAMP-responsive element-binding pathway, circadian rhythms, such as CLOCK, ARNT1, CRY2, PER1, and PER2, and anxiety and depression, such as NOTCH1, NOTCH2 and ANK2. A network with these selected genes was confirmed by STRING analysis. Conclusions: These findings indicate that CM-10 may activate DOR-mediated signaling by suppressing cAMP levels, implicating a distinct molecular cascade in HEK293 cells. Full article

Amyotrophic Lateral Sclerosis is a progressive neurodegenerative disease characterized by the degeneration of motor neurons and the pathological accumulation of phosphorylated TDP-43. Casein kinase one delta (CK1$\delta$) has been identified as a key regulator of this aberrant phosphorylation, making it a promising therapeutic target. In this theoretical study, 26 structurally diverse compounds were evaluated against CK1$\delta$ using molecular docking, molecular dynamics simulations, and binding free energy calculations. Among them, BZH exhibited the most stable interaction with CK1$\delta$ ($-46.53\pm 1.94$ kcal/mol). An inverse correlation was observed between theoretical affinity and experimental IC₅₀ values, supporting the predictive validity of the computational approach. Pharmacokinetic analysis indicated that IMF and BIP show good oral absorption and the ability to cross the blood–brain barrier. At the same time, the toxicological profile classified all compounds in toxicity Class IV (moderate risk). Additionally, dynamic migration toward an alternative pocket was observed during simulation, highlighting the importance of considering protein flexibility in drug design. This study proposes BZH, IMF, and BIP as promising CK1$\delta$ inhibitors for future experimental validation in the treatment of ALS. Full article

Background/Objectives: Trypanosoma evansi (T. evansi) is an etiological agent of surra, and it causes significant economic losses in livestock. Rising trypanocide resistance demands alternatives that control parasitemia while mitigating oxidative and genotoxic damage. Therefore, the present study was designed to explore both the in vivo and in silico potential of Zingiber officinale (Z. officinale) as a novel phytotherapy to counter growing resistance against conventional trypanocides. Methods: Methanolic extract of Z. officinale (MZ) was orally administered at dosages of 200 mg/kg (MZ 200), 400 mg/kg (MZ 400), and 800 mg/kg (MZ 800) on a daily basis to the experimentally infected mice and compared against treated control (TC) and untreated control (UC) groups. After the infection, different parameters such as parasitemia counts, body weight changes, and the survival of infected mice were monitored for up to 7 days post-infection, while hematobiochemical parameters, oxidative stress profiles (catalase, malondialdehyde, and superoxide dismutase), and genotoxicity in brain tissues were compared at the end of the trial. Moreover, computational tools were used to predict the affinities of key bioactive compounds with twenty-one essential proteins of T. evansi. Results: The findings showed that the administration of MZ significantly (p < 0.05) reduced parasitemia and improved the survival rates in the experimentally infected mice in a dose-dependent manner. Noteworthy, significant (p < 0.05) improvements in hematological parameters and liver enzyme profiles were also recorded in MZ-treated groups. Compared to the untreated control, MZ-treated groups showed a significant amelioration in oxidative stress and genotoxicity in brain tissue in a dose-dependent fashion. The current study’s findings suggest that MZ potentially inhibits various essential proteins of T. evansi, including adenosine transporter-1, casein kinase, leucyl-tRNA synthetase, and multidrug resistance E protein. Among its constituents, 6-Isoshogaol and 6-Gingerol showed the most stable interactions in the molecular dynamics simulation. Conclusions: MZ efficiently reduced parasitemia, oxidative stress, and genotoxicity, and increased the survival rate in infected mice, suggesting it as a promising natural trypanicidal agent. Full article

Background/Objectives: Colorectal cancer (CRC) remains a leading cause of cancer-related deaths, with notable sex-specific differences in its incidence, diagnosis, and outcomes. Our previous work identified casein kinase 2 alpha (CK2α) as being capable of impairing DNA mismatch repair (MMR) via phosphorylation of MLH1, thereby increasing the tumor mutational burden. This study aimed to investigate sex-specific differences in CK2α protein expression in CRC. Methods: Immunohistochemical (IHC) analysis was performed on 161 CRC tumors and adjacent normal tissues to quantify the CK2α protein levels. A multi-cohort meta-analysis of proteomic and clinical data was conducted to validate our findings and assess the correlations with age, sex, and relevant signaling pathways. Results: Female CRC patients exhibited significantly higher CK2α expression than male patients, which was confirmed in two independent cohorts. Additionally, CK2α expression was positively correlated with age in female but not male patients. Cross-cohort correlation analyses linked CK2α levels with key proteins involved in estrogen receptor signaling and aging, including DEAD-box helicase 5 (DDX5), histone deacetylase 1 (HDAC1), proliferating cell nuclear antigen (PCNA), prohibitin-2 (PHB2), H/ACA ribonucleoprotein complex subunit 2 (NHP2), and dual-specificity mitogen-activated protein kinase kinase 3 (MAP2K3). Conclusions: CK2α is significantly overexpressed in the tumor tissue of female CRC patients and shows a strong age-related correlation. These findings suggest a sex- and age-specific regulatory mechanism potentially influenced by estrogen signaling or menopause. Such dimorphisms underscore the need for sex-specific strategies in CRC biomarker development and therapy. Full article

Heterologous expression of caseins in non-mammalian systems offers a sustainable and scalable alternative for producing milk proteins, with potential applications in the food and biopharmaceutical industries. However, a significant challenge in these systems is achieving proper phosphorylation, a critical post-translational modification required for casein functionality and stability. This review explores the current state of research on heterologous casein production, with a particular focus on the biological and technical hurdles associated with phosphorylation. Specifically, we examine the absence of the mammalian-specific kinase Fam20C in plant and yeast systems and the broader lack of secretory kinase machinery in bacteria, which collectively contribute to impaired phosphorylation fidelity. While some endogenous kinases may partially compensate, they are typically insufficient to replicate the phosphorylation pattern required for functionality. We evaluate potential strategies to address these limitations, analyze the role of phosphorylation in casein functionality, provide insights into existing patents and experimental approaches, and highlight ongoing research efforts. By synthesizing current knowledge and proposing new avenues for innovation, this review aims to provide a roadmap for the successful production of functional heterologous caseins. Full article

The immune response triggered when plant cell surface receptors recognize pathogen-associated molecular patterns (PAMPs) is known as PAMP-triggered immunity (PTI). Several studies have demonstrated that extracellular plant ferredoxin-like protein (PFLP) can enhance PTI signaling, thereby conferring resistance to bacterial diseases in various plants. The C-terminal casein kinase II (CK2) phosphorylation region of PFLP is essential for strengthening PTI. However, whether phosphorylation at this site directly enhances PTI signaling and consequently increases plant disease resistance remains unclear. To investigate this, site-directed mutagenesis was used to generate PFLPT90A, a non-phosphorylatable mutant, and PFLPT90D, a phospho-mimetic mutant, for functional analysis. Based on the experimental results, none of the recombinant proteins were able to enhance the hypersensitive response induced by the HrpN protein or increase resistance to the soft rot pathogen Pectobacterium carotovorum subsp. carotovorum ECC17. These findings suggest that phosphorylation at the T90 residue might be essential for PFLP-mediated enhancement of plant immune responses, implying that this post-translational modification is likely required for its disease resistance function in planta. To further explore the relationship between PFLP phosphorylation and endogenous CK2, the Arabidopsis insertion mutant cka2 and the complemented line CKA2R were analyzed under treatment with flg22_Pst from Pseudomonas syringae pv. tomato. The effects of PFLP on the hypersensitive response, rapid oxidative burst, callose deposition, and susceptibility to soft rot confirmed that CK2 is required for these immune responses. Furthermore, expression analysis of PTI-related genes FRK1 and WRKY22/29 in the mitogen-activated protein kinase (MAPK) signaling pathway demonstrated that CK2 is necessary for PFLP to enhance flg22_Pst-induced immune signaling. Taken together, these findings suggest that PFLP enhances A. thaliana resistance to bacterial soft rot primarily by promoting the MAPK signaling pathway triggered by PAMP recognition, with CK2-mediated phosphorylation being essential for its function. Full article

Background: Casein kinase I-like (CKL) protein is a member of the serine/threonine kinase CKI family and plays a pivotal regulatory role in various eukaryotic cellular processes, including stress responses. Objectives: This study aims to systematically identify the CKL gene family in the tomato genome and investigate its responsiveness to abiotic stress. Methods: Members of SlCKL were identified through genome-wide bioinformatics analysis, and their physicochemical properties, chromosomal localization, gene structure, conserved domains, phylogenetic relationships, cis-acting elements, cross-species collinearity, and tissue expression profiles were comprehensively analyzed. The expression patterns of SlCKL genes under abiotic stress were validated using real-time quantitative PCR. Results: A total of 16 SlCKL genes were identified and classified into three subfamilies (I–III), which are unevenly distributed across nine chromosomes, predominantly clustered at the ends. The gene structure, motifs, and functional domains exhibit high conservation. Collinearity analysis revealed stronger synteny between tomato and Arabidopsis thaliana or pepper compared to rice, maize, or tobacco, suggesting a common ancestral origin. The tissue expression profile indicates that SlCKLs are preferentially transcribed in roots. Promoter analysis and qRT-PCR validation demonstrated differential responses of SlCKLs to various abiotic stresses, such as drought, salt, heat, cold, and ABA treatment. Conclusions: This study represents the first systematic identification of the tomato SlCKL gene family, elucidating its evolutionary relationships, structural characteristics, tissue-specific expression patterns, and differential responsiveness to abiotic stress, thereby providing a critical foundation for further investigation into the molecular mechanisms underlying CKL-mediated abiotic stress adaptation in tomatoes. Full article

Casein Kinase II (CK2) is a ubiquitously present serine/threonine kinase essential for mammalian development. CK2 holoenzyme is a tetramer with two highly related catalytic subunits (α or α’) and two regulatory ß subunits. Global deletion of the α or β subunit in mice is embryonically lethal. We and others have shown that CK2 is overexpressed in leukemia cells and plays an important role in cell cycle, survival, and resistance to the apoptosis of leukemia stem cells (LSCs). To study the role of CK2α in adult mouse hematopoiesis, we generated hematopoietic cell-specific CK2α-conditional knockout mice (Vav-iCreCK2 ^f/f). Here we report the generation and validation of a novel mouse model that lacks CK2α in the hematopoietic compartment. Vav-iCreCK2α ^f/f mice were viable without dysmorphic features and showed a mild phenotype under baseline conditions. In Vav-iCreCK2α ^f/f mice, the blood count showed a significant decrease in total red blood cells and platelets. The spleen was enlarged in Vav-iCreCK2α ^f/f mice with evidence of extramedullary hematopoiesis. HSC and early progenitor cell compartments showed expansion in CK2α-null bone marrow, suggesting that the absence of CK2α impaired their proliferation and differentiation. Given the established roles of CK2 in cell cycle regulation and the findings reported here, further functional studies are warranted to investigate the role of CK2α in HSC self-renewal and differentiation. This mouse model serves as a valuable tool for understanding the role of CK2α in normal and malignant hematopoiesis. Full article

Heart failure is associated with dysregulation in cellular Ca²⁺ that could involve sarcolemmal L-type Ca²⁺ currents (LTCCs). Building on previous observations showing that recombinant Ca_V1.2 channels are upregulated by phosphorylated calmodulin (CaM) variants, the cellular mechanism(s) underlying this posttranslational modification was investigated in cultured cardiomyocytes. Whole-cell LTCCs decreased by ≈75% after silencing the gene coding for casein kinase 2 (CK2), a constitutively active kinase in cardiomyocytes, or after its pharmacological inhibition. The overexpression of the dominant negative phosphoresistant single, double T79A/S81A, or triple T79A/S81A/S101A CaM variants resulted in a similar inhibition. In contrast, the overexpression of CaM WT or its double T79D/S81D and triple T79D/S81D/S101D phosphomimetic variants curtailed the downregulation of LTCCs caused by CK2 partial knockdown, suggesting that CK2 is responsible for the posttranslational modification of these CaM target residues. Catecholamines, triggering the protein kinase A (PKA) cascade, partially rescued LTCCs treated with siRNA without or after the overexpression of either CaM WT or stimulating CaM phosphomimetic variants. More importantly, they thwarted the negative impact of the phosphoresistant CaM variants, altogether arguing that CK2 and PKA are acting in synergy to regulate the activity of LTCCs. We conclude that CK2-mediated phosphorylation processes exacerbate the Ca²⁺ load associated with heart failure. Full article

Background: Casein kinase 1 epsilon (CK1ε) plays a critical role in cancer progression by activating oncogenic signaling pathways, making it a target for cancer therapy. However, no inhibitors are currently available for clinical use, highlighting the need for novel therapeutic candidates. Methods: This study aimed to identify potential CK1ε inhibitors. To achieve this, a modified version of a previously reported pharmacophore model was applied to an ultra-large database of over 100 million compounds for virtual screening. Hits were filtered based on drug-likeness and pH-dependent pharmacophore compliance and then grouped according to their structural core. A representative compound from each structural group underwent molecular dynamic (MD) simulations and binding free energy calculations to predict its stability and affinity, allowing extrapolation of the results to the entire set of candidates. Results: Pharmacophore matching initially identified 290 compounds. After energy minimization, and an assessment of drug-likeness and pharmacophore compliance, we selected 29 structurally related candidates. MD simulations showed that most of the compounds representative of structural groups had stable binding modes, favorable intermolecular interactions, and free energies comparable to those of previously reported CK1ε inhibitors. An analysis of additional members of the most promising structural group showed that two 2,4-diaminopyrimidine-based compounds likely inhibit CK1ε. Conclusions: These findings provide structural insights into the design of CK1ε inhibitors, supporting compound optimization and the eventual development of targeted cancer therapeutics. Full article

The effects of zearalenone (ZEA), a fungal toxin in food and feed, remain unclear on the mammary gland and lactation. This study examines ZEA-induced damage in lactating mice and bovine mammary epithelial cells (MAC-T), focusing on the role of the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway in regulating cell proliferation and apoptosis. The results demonstrated that exposure to ZEA at different doses (5 mg/kg, 10 mg/kg, and 20 mg/kg) reduced lactation in female mice and slowed weight gain in their offspring. Hematoxylin and eosin (HE) staining and CSNK immunofluorescence staining of mammary tissue confirmed ZEA-induced mammary gland damage in vivo. Further analysis using PCNA immunohistochemistry and fluorescent TUNEL staining revealed that ZEA promoted apoptosis and decreased the proliferative capacity of mammary tissues. In vitro, 20 μM ZEA decreased MAC-T cell proliferation, increased apoptosis and oxidative stress, inhibited PI3K/AKT signaling, and decreased κ-casein (CSNK) expression. Pretreatment with a reactive oxygen species (ROS) scavenger (NAC) or PI3K/AKT activator (740-Y-P) reversed these effects, with NAC specifically restoring PI3K/AKT activity inhibited by ZEA. Overall, this study concludes that ZEA induces MAC-T cell apoptosis and disrupts proliferation via the ROS-mediated PI3K/AKT pathway, ultimately impairing lactation function. These findings highlight potential targets for managing ZEA contamination in food and its impact on lactation. Full article

This study utilized MAC-T cells cultured in vitro as a model to investigate the effects of varying concentrations of valine on α-casein synthesis and its underlying regulatory mechanisms. In this experiment, MAC-T cells were subjected to a 12 h starvation period, followed by the addition of valine in a range of concentrations (a total of seven concentrations: 0.000, 1.596, 3.192, 6.384, 12.768, 25.536, and 51.072 mM, as well as in 10% Fetal Bovine Serum). The suitable range of valine concentrations was determined using enzyme-linked immunosorbent assays (ELISAs). Real-time fluorescent quantitative PCR (RT-qPCR) and Western blot analyses were employed to evaluate the expression levels and phosphorylation states of the casein alpha s1 gene (CSN1S1), casein alpha s2 gene (CSN1S2) and mTOR signaling pathway-related genes. The functionality of the mTOR signaling pathway was further validated through rapamycin (100.000 nM) inhibition experiments. Results indicated that 1× Val (6.384 mM), 2× Val (12.768 mM), 4× Val (25.536 mM), and 8× Val (51.072 mM) significantly enhanced α-casein synthesis (p < 0.01). Within this concentration range, valine significantly upregulated the expression of CSN1S1, CSN1S2, and mTOR signaling pathway-related genes including the RagA gene (RRAGA), RagB gene (RRAGB), RagC gene (RRAGC), RagD gene (RRAGD), mTOR, raptor gene (RPTOR), and 4EBP1 gene (EIF4EBP1), eukaryotic initiation factor 4E (EIF4E), and S6 Kinase 1 (S6K1) (p < 0.01). Notably, the expression of the eukaryotic elongation factor 2 (EEF2) gene peaked at 1× Val (6.384 mM), while the expression of other genes reached their maximum at 4× Val (25.536 mM). Additionally, valine significantly increased the phosphorylation levels of mTOR, S6K1, 4E-binding protein-1 (4EBP1), ribosomal protein S6 (RPS6), and eEF2 (p < 0.01), with the highest phosphorylation levels of mTOR, S6K1, and RPS6 observed at 4× Val (25.536 mM). Rapamycin treatment significantly inhibited mTOR phosphorylation and α-casein synthesis (p < 0.01); however, the addition of 4× Val (25.536 mM) partially mitigated this inhibitory effect. In conclusion, valine promotes α-casein synthesis by activating the mTOR signaling pathway, with an optimal concentration of 4× Val (25.536 mM). Full article

Background: Metformin is a widely used oral hypoglycemic agent for treating type 2 diabetes. Planarians, with their remarkable regenerative abilities, are frequently employed as model organisms in stem cell and regeneration studies. This study aimed to investigate the effects of metformin on planarian regeneration, focusing on the regeneration of eyespots after amputation. Methods: Regenerating planarians with amputated eyespots were exposed to various concentrations of metformin. The regeneration time of the eyespots was measured to assess the effects of metformin. Subsequently, a 1 mmol/L metformin treatment for 24 h was applied to the planarians, followed by transcriptome analysis to identify differentially expressed genes (DEGs). The gene expression was validated through qPCR. The full-length gene of casein kinase 1α (DjCK1α) was cloned using RACE technology. DjCK1α interference was performed to examine its role in regeneration. Results: Low concentrations of metformin significantly reduced the regeneration time of planarians. Transcriptome analysis identified 113 DEGs, including 61 upregulated and 52 downregulated genes. GO and KEGG enrichment analyses were conducted. Notably, DjCK1α, a key gene involved in regeneration, was selected for further validation. qPCR confirmed that DjCK1α was significantly upregulated. The interference of DjCK1α prolonged the regeneration time of the eyespots of planarians cultured in water, while treatment with metformin did not promote the eyespot regeneration of the DjCK1α-interfered planarians. Conclusions: The results suggest that metformin accelerates planarian eyespot regeneration, potentially through the regulation of DjCK1α. This study provides the first transcriptome-based analysis of drug effects on regeneration in planarians, highlighting the role of metformin in the regeneration process. Full article