Protein Phosphorylation Research Articles

Evaluation of the Mechanism of Yishan Formula in Treating Breast Cancer Based on Network Pharmacology and Experimental Verification.

Yishan formula (YSF) has a significant effect on the treatment of breast cancer, which can improve the quality of life and prolong the survival of patients with breast cancer; however, its mechanism of action is unknown. In this study, network pharmacology and molecular docking methods have been used to explore the potential pharmacological effects of the YSF, and the predicted targets have been validated by in vitro experiments. Active components and targets of the YSF were obtained from the TCMSP and Swiss target prediction website. Four databases, namely GeneCards, OMIM, TTD, and DisGeNET, were used to search for disease targets. The Cytoscape v3.9.0 software was utilized to draw the network of drug-component-target and selected core targets. DAVID database was used to analyze the biological functions and pathways of key targets. Finally, molecular docking and in vitro experiments have been used to verify the hub genes. Through data collection from the database, 157 active components and 618 genes implicated in breast cancer were obtained and treated using the YSF. After screening, the main active components (kaempferol, quercetin, isorhamnetin, dinatin, luteolin, and tamarixetin) and key genes (AKT1, TP53, TNF, IL6, EGFR, SRC, VEGFA, STAT3, MAPK3, and JUN) were obtained. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that the YSF could affect the progression of breast cancer by regulating biological processes, such as signal transduction, cell proliferation and apoptosis, protein phosphorylation, as well as PI3K-Akt, Rap1, MAPK, FOXO, HIF-1, and other related signaling pathways. Molecular docking suggested that IL6 with isorhamnetin, MAPK3 with kaempferol, and EGFR with luteolin have strong binding energy. The experiment further verified that YSF can control the development of breast cancer by inhibiting the expression of the hub genes. This study showed that resistance to breast cancer may be achieved by the synergy of multiple active components, target genes, and signal pathways, which can provide new avenues for breast cancer-targeted therapy.

Unveiling the structural mechanisms behind high affinity and selectivity in phosphorylated epitope-specific rabbit antibodies.

Protein phosphorylation is a crucial process in various cellular functions, and its irregularities have been implicated in several diseases, including cancer. Antibodies are commonly employed to detect protein phosphorylation in research. However, unlike the extensive studies on recognition mechanisms of the phosphate group by proteins such as kinases and phosphatases, only a few studies have explored antibody mechanisms. In this study, we produced and characterized two rabbit monoclonal antibodies that recognize a mono-phosphorylated Akt peptide. Through crystallography, thermodynamic mutational analyses, and molecular dynamics simulations, we investigated the unique recognition mechanism that enables higher binding affinity and selectivity of the antibodies compared to other generic proteins with lower binding affinity to phosphorylated epitopes. Our results demonstrate that molecular dynamics simulations provide novel insights into the dynamic aspects of molecular recognition of post-translational modifications by proteins beyond static crystal structures, highlighting how specific atomic level interactions drive the exceptional affinity and selectivity of antibodies.

Bioactive fraction of Musa balbisiana seed mitigates D-galactose-induced brain aging via SIRT1/PGC-1α/FoxO3a activation and intestinal barrier dysfunction by modulating gut microbiota and core metabolites

Aging is an inevitable biological process, and emerging research has highlighted the potential of dietary and pharmacological interventions to decelerate the trajectory of age-related diseases and prolong the health span. This study evaluates the protective effects of Musa balbisiana seed on healthy aging using D-galactose-induced accelerated aging rats. The results suggested that the bioactive ethyl acetate fraction of Musa balbisiana seed extract (BF) exhibited protective effects against aging-induced oxidative stress by reducing oxidative DNA damage, advanced glycation end-product formation, and malondialdehyde levels while restoring antioxidant and glyoxalase enzyme activities. BF also ameliorated neurodegeneration by decreasing acetylcholinesterase enzyme activity and amyloid beta plaque formation. Histopathological analysis demonstrated the protective effects of BF against brain aging, liver disruption, renal damage, and intestinal barrier dysfunction. BF further restored intestinal permeability by upregulating the tight junctions (zonula occludens 1 and 2, claudin 1,2,3 and 4, and occludin) and mucin (mucin 2 and mucin 5ac) gene expression while downregulating the expression of inflammatory cytokines (IL-1β, IL-6, and TNF-α). BF significantly induced the phosphorylation of FoxO3a proteins and upregulated the gene expression of SIRT1, PGC-1α, and TFAM in the hippocampus. Next-generation sequencing (NGS) of 16s rRNA amplicons of fecal metagenomics DNA and metabolites profiling showed that BF intervention restructured the gut microbiota and altered core metabolites related to cholesterol metabolism. Overall, our findings demonstrated the multifaceted protective effects of Musa balbisiana seed against D-galactose-induced aging.

Impaired suppression of fatty acid release by insulin is a strong predictor of reduced whole-body insulin-mediated glucose uptake and skeletal muscle insulin receptor activation.

To examine factors underlying why most, but not all, adults with obesity exhibit impaired insulin-mediated glucose uptake, we compared: (1) adipose tissue fatty acid (FA) release, (2) skeletal muscle lipid droplet (LD) characteristics, and (3) insulin signalling events, in skeletal muscle of adults with obesity with relatively high versus low insulin-mediated glucose uptake. Seventeen adults with obesity (BMI: 36 ± 3 kg/m2) completed a 2 h hyperinsulinemic-euglycemic clamp with stable isotope tracer infusions to measure glucose rate of disappearance (glucose Rd) and FA rate of appearance (FA Ra). Skeletal muscle biopsies were collected at baseline and 30 min into the insulin infusion. Participants were stratified into HIGH (n = 7) and LOW (n = 10) insulin sensitivity cohorts by their glucose Rd during the hyperinsulinemic clamp (LOW< 400; HIGH >550 nmol/kgFFM/min/[μU/mL]). Insulin-mediated suppression of FA Ra was lower in LOW compared with HIGH (p < 0.01). In skeletal muscle, total intramyocellular lipid content did not differ between cohorts. However, the size of LDs in the subsarcolemmal region (SS) of type II muscle fibres was larger in LOW compared with HIGH (p = 0.01). Additionally, insulin receptor-β (IRβ) interactions with regulatory proteins CD36 and Fyn were lower in LOW versus HIGH (p < 0.01), which aligned with attenuated insulin-mediated Tyr phosphorylation of IRβ and downstream insulin-signalling proteins in LOW. Collectively, reduced ability for insulin to suppress FA mobilization, with accompanying modifications in intramyocellular LD size and distribution, and diminished IRβ interaction with key regulatory proteins may be key contributors to impaired insulin-mediated glucose uptake commonly found in adults with obesity.

PTH-dependent stabilization of RANKL mRNA is associated with increased phosphorylation of the KH-Type Splicing Regulatory Protein

Parathyroid hormone (PTH) receptor agonists promote bone formation but also increase osteoclastogenesis, in part by increasing expression of the receptor activator of nuclear factor kappa-Β ligand (RANKL). In addition to activation of transcription, regulation of mRNA stability is another important molecular mechanism controlling mRNA abundance. PTH treatment for 6 hrs. resulted in a 7.4-fold elevation in RANKL mRNA expression in UAMS-32P cells, despite prior inhibition of cellular transcription by thiophosphoryl (TPL). RANKL mRNA, like other TNF family members, contains AU-Rich Elements (AREs) in the 3’ UTR. AU-Rich Element Binding Proteins (ABPs including KSRP, TTP, AUF1 and HuR) bind to AREs and regulate mRNA stability. There was significantly more KSRP bound to RANKL mRNA than any of the other ABPs. PTH did not increase the amount of ABPs bound to the RANKL transcript. However, the level of cellular phosphorylated KSRP was significantly increased in UAMS-32P cells pre-treated with TPL followed by PTH exposure, compared to cells treated with vehicle following TPL. The extent of phosphorylation of cellular AUF1, HuR, and TTP did not increase with PTH treatment. There were no significant changes in the cellular content of total Pin1 and phospho-Pin1 protein with PTH treatment. We conclude that increases in cellular phospho-KSRP following PTH treatment, together with fact that the total amount of the KSRP bound to the RANKL mRNA did not change with PTH-treatment, may indicate that phospho-KSRP plays some role in stabilizing the RANKL transcript.

Sevoflurane Postconditioning Mitigates Neuronal Hypoxic-Ischemic Injury via Regulating Reactive Astrocytic STAT3 Protein Modification

Astrocyte activation plays a pivotal role in accelerating the cascade of neuroinflammation associated with the development of hypoxic-ischemic brain injury. This study aimed to investigate the mechanism by which sevoflurane postconditioning mitigates neuronal damage through astrocytes by regulating reactive astrocytic Signal Transducer and Activator of Transcription 3 (STAT3) modifications. A modified Rice‒Vannucci model in rats and a conditioned culture system established by subjecting primary astrocytes to oxygen glucose deprivation, followed by using the conditioned medium to culture the neuron cell line SH-SY5Y were used to simulate HI insult in vivo and in vitro, respectively. These models were followed by 30 minutes of 2.5% sevoflurane treatment. Stattic was used to inhibit STAT3 phosphorylation, and (Z)-PUGNAc or OSMI-1 was added to regulate O-linked-β-N-acetylglucosamine modification (O-GlcNAcylation) in primary astrocytes in vitro. Neurobehavioral tests, Nissl staining, CCK8 assay, and flow cytometry for apoptosis were used to assess neuronal function. Immunofluorescence staining was used to detect astrocyte reactivity and the intracellular distribution of STAT3. Immunoprecipitation combined with Western blotting was used to evaluate the O-GlcNAcylation of STAT3. Protein expression and phosphorylation levels were detected by Western blotting. ELISA was conducted to detect the detrimental cytokines IL-6 and IL-1β in astrocyte-conditioned medium. Sevoflurane postconditioning enhanced the O-GlcNAcylation of astrocytic STAT3 following HI insult via the manner of OGT. Crosstalk between O-GlcNAcylation and phosphorylation of STAT3 showed that O-GlcNAcylation inhibited STAT3 phosphorylation. The inhibitory effect on astrocytes suppressed STAT3 nuclear translocation, reduced astrocyte reactivity, decreased the release of the inflammatory cytokines IL6 and IL-1β, attenuated neuronal apoptosis following HI insult, and improved neuron viability. Sevoflurane postconditioning increased astrocytic STAT3 O-GlcNAcylation level to competitively inhibit STAT3 phosphorylation. This deactivated downstream inflammation pathways and reduced astrocyte reactivity, thereby mitigating HI insult in neurons both in vivo and in vitro.

Hemin attenuates bleomycin-induced lung fibrosis in mice by regulating the TGF-β1/MAPK and AMPK/SIRT1/PGC-1α/HO-1/NF-κB pathways.

The objective of this study was to investigate the protective effect and potential mechanism of action of hemin on bleomycin-induced pulmonary fibrosis in mice. Male C57BL/6 mice were randomly divided into control, bleomycin and bleomycin + hemin groups. Mice in the bleomycin and bleomycin + hemin groups were injected intratracheally with bleomycin to establish the pulmonary fibrosis model. The bleomycin + hemin group mice were injected intraperitoneally with hemin starting 7 days before modeling until the end of Day 21 after modeling. Pathological changes in lung tissue were assessed by HE and Masson staining. Malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT) levels were determined in lung tissue. Immunohistochemistry was performed to assess the expression of α-SMA and collagen I. The serum levels of IL-6 and TNF-α were measured via ELISA. Western blotting was used to determine the expression of TGF-β1, SIRT1, PGC-1α and HO-1 and the phosphorylation levels of p38, ERK1/2, JNK, AMPK and NF-κB p65 in lung tissue. Hemin significantly reduced lung indices, increased terminal body weight. It also significantly increased SOD and CAT activities; decreased MDA, IL-6 and TNF-α levels; reduced the levels of α-SMA and collagen I-positive cells; upregulated SIRT1, PGC-1α and HO-1 expression; promoted AMPK phosphorylation; and downregulated TGF-β1 expression and p38, ERK1/2, JNK and NF-κB p65 phosphorylation. Hemin might attenuate oxidative damage and inflammatory responses and reduces extracellular matrix deposition by regulating the expression and phosphorylation of proteins associated with the TGF-β1/MAPK and AMPK/SIRT1/PGC-1α/HO-1/NF-κB pathways, thereby alleviating bleomycin-induced pulmonary fibrosis.

The two-component system CpxA/CpxR regulates pathogenesis and stress adaptability in the poplar canker bacterium Lonsdalea populi.

Bacteria employ two-component systems (TCSs) to rapidly sense and respond to their surroundings often and during plant infection. Poplar canker caused by Lonsdalea populi is an emerging woody bacterial disease that leads to high mortality and poplar plantation losses in China. Nonetheless, the information about the underlying mechanism of pathogenesis remains scarce. Therefore, in this study, we reported the role of a TCS pair CpxA/CpxR in regulating virulence and stress responses in L. populi. The CpxA/R system is essential during infection, flagellum formation, and oxidative stress response. Specifically, the Cpx system affected flagellum formation by controlling the expression of flagellum-related genes. CpxR, which was activated by phosphorylation in the presence of CpxA, participated in the transcriptional regulation of a chaperone sctU and the type III secretion system (T3SS)-related genes, thereby influencing T3SS functions during L. populi infection. Phosphorylated CpxR directly manipulated the transcription of a membrane protein-coding gene yccA and the deletion of yccA resulted in reduced virulence and increased sensitivity to H2O2. Furthermore, we mutated the conserved phosphorylation site of CpxR and found that CpxRD51A could no longer bind to the yccA promoter but could still bind to the sctU promoter. Together, our findings elucidate the roles of the Cpx system in regulating virulence and reactive oxygen species resistance and provide further evidence that the TCS is crucial during infection and stress response.

Dephosphorylation of bZIP59 by PP2A ensures appropriate shade avoidance response in Arabidopsis.

Changes in light quality and quantity experienced by many shade-intolerant plants grown in close proximity lead to transcriptional reprogramming and shade avoidance syndrome (SAS). Despite the importance of phosphorylation-dependent signaling in cellular physiology, phosphorylation events during SAS are largely unknown. Here, we examined shade-regulated phosphorylation events in Arabidopsis using quantitative phosphoproteomics. We confirmed shade-induced dephosphorylation of bZIP59, a basic region/leucine zipper motif (bZIP) transcription factor. Shade treatment promotes the nuclear localization of bZIP59, which can be mimicked by mutation of the phosphorylation sites on bZIP59. Phenotypic analysis identified that bZIP59 negatively regulated shade-induced hypocotyl elongation. bZIP59 repressed the shade-induced activation of certain growth-related genes, while shade increased the DNA binding of bZIP59. Furthermore, the protein phosphatase 2A (PP2A) mediated dephosphorylation of bZIP59. Our study characterized a previously unidentified mechanism by which the phytochrome B (phyB)-PP2A-bZIP59 regulatory module integrates shade signals and transcriptomes, broadening our knowledge of phosphorylation strategies for rapid adaptation to shade.

Phosphoproteomics Analyses of Aedes aegypti fat body reveals blood meal-induced Signaling and Metabolic Pathways.

The mosquito fat body is the principal source of yolk protein precursors (YPP) during mosquito egg development in female Aedes aegypti. To better understand the metabolic and signaling pathways involved in mosquito reproduction, we investigated changes in the mosquito fat body phosphoproteome at multiple time points after a blood meal. Using LC/MS, we identified 3,570 phosphorylated proteins containing 14,551 individual phosphorylation sites. We observed protein phosphorylation changes in cellular pathways required for vitellogenesis, as well as proteins involved in primary cellular functions. Specifically, after a blood meal, proteins involved in ribosome synthesis, transcription, translation, and autophagy showed dynamic changes in their phosphorylation patterns. Our results provide new insight into blood meal-induced fat body dynamics and reveal potential proteins that can be targeted for interference with mosquito reproduction. Considering the devastating impact of mosquitoes on human health, worldwide, new approaches to control mosquitoes are urgently needed.

Posphoproteomics profiling reveals the regulatory role of a phosphorylated protein PvFBA1 in cadmium tolerance in seashore paspalum

Seashore paspalum (Paspalum vaginatum) is a warm-season and perennial turfgrass and is known for its cadmium (Cd)-stress tolerance. Here, a Phosphoproteomics analysis was performed to examine the key proteins relating to Cd tolerance in seashore paspalum. Fructose 1,6-biphosphate aldolase, PvFBA1, was identified for its phosphorylated state after exposure to Cd stress. Specifically, the phosphorylation of PvFBA1 was enhanced in several metabolic pathways, including pentose phosphate pathway (PPP), carbon fixation and biosynthesis of amino acids under Cd stress. By transforming PvFBA1 into Arabidopsis, the PvFBA1-OE plants exhibited longer roots, greater FBA activity and higher soluble sugar content than WT under 100µM CdCl2 treatment. By expressing the PvFBA1 in yeast, a serine 50 phosphorylation site was identified as functional site. By microscale thermophoresis experiment, we indicted that PvFBA1can bind Cd directly enhancing its phosphorylation level to alleviate the damage of Cd. This finding may provide new insights into the molecular mechanisms of plants Cd tolerance.

ARRHYTHMOGENIC ATRIAL REMODELING DURING PREGNANCY IN MICE

Pregnancy is associated with greater vulnerability to supraventricular tachyarrhythmias (SVT). As the underlying mechanisms remain to be elucidated, we investigated whether pregnancy induces atrial remodeling that might contribute to this. Atrial electrophysiological and contractile properties were examined in non-pregnant (NP) and pregnant (P) mice. Cell shortening and Ca2+ imaging were measured on atrial myocytes. Atrial action potential and ionic currents were recorded using patch-clamp technique. Atrial mRNA and protein expression were analyzed using qPCR and Western blot. The P-wave area on the ECG increased by 50% during pregnancy, suggesting atrial enlargement, confirmed by echocardiography. The atrial myocytes were longer in P mice, adding further evidence to the physiological hypertrophy associated with pregnancy. Echocardiography showed a 50% increase in atrial fractional area change during pregnancy, indicating much stronger contraction. A similar increase in cell shortening was observed in P mice and was associated with a decrease in sarcomere length and changes in myofilament protein phosphorylation. However, pregnancy did not affect L-type Ca2+ current, Ca2+ transients, and SR Ca2+ load. Myocytes from P mice showed twice as many spontaneous contractions and spontaneous diastolic Ca2+ releases. Moreover, pregnancy was associated with a 50% increase in action potential duration, linked to a reduction in the density of the transient outward K+ current Ito and the underlying KV4.3 channel. During pregnancy, atrial tissues undergo substantial remodeling, potentially contributing to the development of SVT.

Clinical outcomes of gynecologic tumor treatment correlate with ERK protein phosphorylation as measured by a CLIA-accredited phosphoprotein assay

Clinical outcomes of gynecologic tumor treatment correlate with ERK protein phosphorylation as measured by a CLIA-accredited phosphoprotein assay

Identification and quantification of unreported sialylated N-glycan isomers with α2-3 and α2-6 linkages in the egg yolk protein phosvitin

Phosvitin (PV), a highly phosphorylated protein found in chicken egg yolk, possesses multiple bioactivities (including anti-aging and anticancer) and functional properties (including emulsifier and metal-binding capacities). The carbohydrate moiety attached to PV has been reported, but its N-glycan structure is unknown. In this study, we performed structural and quantitative analyses of N-glycans from PV using liquid chromatography-tandem mass spectrometry (MS/MS). N-glycan structures were identified using observed precursor ion m/z and MS/MS fragment ions. Each quantity was obtained relative to the total N-glycans (100%). Thirty-seven N-glycans were identified, including 22 sialylations with a negative charge (a sum of the relative quantity of each, 96.4%) comprising 13 mono- (31.6%), 7 di- (57.5%), 2 tri- (7.3%) sialylations. The sialylated N-glycan isomers with α2-3 (flexible conformation) and α2-6 (rigid conformation) linkages were distinguished using α2-3- and α2-3,6 sialidase treatments and intensity ratios of the N-acetylglucosamine and sialic acid ions (Ln/Nn) with different fragmentation stabilities. The α2-6/α2-6 (53.8%), α2-6 (31.6%), α2-3/α2-6/α2-6 (6.5%), and α2-3/α2-6 (3.7%) linkages in mono-, di, or tri-antennary structures were identified. These negatively charged structures may affect the emulsification and metal-binding capacity of PV. This is the first study to identify and quantify N-glycans in PV, including predominantly 22 sialylated N-glycan isomers with more rigid α2-6 linkages than α2-3 linkages.

Eubacterium siraeum suppresses fat deposition via decreasing the tyrosine-mediated PI3K/AKT signaling pathway in high-fat diet-induced obesity.

Obesity in humans can lead to chronic diseases such as diabetes and cardiovascular disease. Similarly, subcutaneous fat (SCF) in pigs affects feed utilization, and excessive SCF can reduce the feed efficiency of pigs. Therefore, identifying factors that suppress fat deposition is particularly important. Numerous studies have implicated the gut microbiome in pigs' fat deposition, but research into its suppression remains scarce. The Lulai black pig (LL) is a hybrid breed derived from the Laiwu pig (LW) and the Yorkshire pig, with lower levels of SCF compared to the LW. In this study, we focused on these breeds to identify microbiota that regulate fat deposition. The key questions were: Which microbial populations reduce fat in LL pigs compared to LW pigs, and what is the underlying regulatory mechanism? In this study, we identified four different microbial strains, Eubacterium siraeum, Treponema bryantii, Clostridium sp. CAG:413, and Jeotgalibaca dankookensis, prevalent in both LW and LL pigs. Blood metabolome analysis revealed 49 differential metabolites, including tanshinone IIA and royal jelly acid, known for their anti-adipogenic properties. E. siraeum was strongly correlated with these metabolites, and its genes and metabolites were enriched in pathways linked to fatty acid degradation, glycerophospholipid, and glycerolipid metabolism. In vivo mouse experiments confirmed that E. siraeum metabolites curb weight gain, reduce SCF adipocyte size, increase the number of brown adipocytes, and regulate leptin, IL-6, and insulin secretion. Finally, we found that one important pathway through which E. siraeum inhibits fat deposition is by suppressing the phosphorylation of key proteins in the PI3K/AKT signaling pathway through the reduction of tyrosine. We compared LW and LL pigs using fecal metagenomics, metabolomics, and blood metabolomics, identifying E. siraeum as a strain linked to fat deposition. Oral administration experiments in mice demonstrated that E. siraeum effectively inhibits fat accumulation, primarily through the suppression of the PI3K/AKT signaling pathway, a critical regulator of lipid metabolism. These findings provide a valuable theoretical basis for improving pork quality and offer insights relevant to the study of human obesity and related chronic metabolic diseases. Video Abstract.

CK and LRRK2 Involvement in Neurodegenerative Diseases.

Neurodegenerative diseases (NDDs) are currently the most widespread neuronal pathologies in the world. Among these, the most widespread are Alzheimer's disease (AD), dementia, Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and Huntington's disease (HD)-all characterized by a progressive loss of neurons in specific regions of the brain leading to varied clinical symptoms. At the basis of neurodegenerative diseases, an emerging role is played by genetic mutations in the leucine-rich repeat kinase 2 (LRRK2) gene that cause increased LRRK2 activity with consequent alteration of neuronal autophagy pathways. LRRK2 kinase activity requires GTPase activity which functions independently of kinase activity and is required for neurotoxicity and to potentiate neuronal death. Important in the neurodegeneration process is the upregulation of casein kinase (CK), which causes the alteration of the AMPK pathway by enhancing the phosphorylation of α-synuclein and huntingtin proteins, known to be involved in PD and HD, and increasing the accumulation of the amyloid-β protein (Aβ) for AD. Recent research has identified CK of the kinases upstream of LRRK2 as a regulator of the stability of the LRRK2 protein. Based on this evidence, this review aims to understand the direct involvement of individual kinases in NDDs and how their crosstalk may impact the pathogenesis and early onset of neurodegenerative diseases.

Arabidopsis Actin-Binding Protein WLIM2A Links PAMP-Triggered Immunity and Cytoskeleton Organization.

Arabidopsis LIM proteins are named after the initials of three proteins Lin-11, Isl-1, and MEC-3, which belong to a class of transcription factors that play an important role in the developmental regulation of eukaryotes and are also involved in a variety of life processes, including gene transcription, the construction of the cytoskeleton, signal transduction, and metabolic regulation. Plant LIM proteins have been shown to regulate actin bundling in different cells, but their role in immunity remains elusive. Mitogen-activated protein kinases (MAPKs) are a family of conserved serine/threonine protein kinases that link upstream receptors to their downstream targets. Pathogens produce pathogen-associated molecular patterns (PAMPs) that trigger the activation of MAPK cascades in plants. Recently, we conducted a large-scale phosphoproteomic analysis of PAMP-induced Arabidopsis plants to identify putative MAPK targets. One of the identified phospho-proteins was WLIM2A, an Arabidopsis LIM protein. In this study, we investigated the role of WLIM2A in plant immunity. We employed a reverse-genetics approach and generated wlim2a knockout lines using CRISPR-Cas9 technology. We also generated complementation and phosphosite-mutated WLIM2A expression lines in the wlim2a background. The wlim2a lines were compromised in their response to Pseudomonas syringae Pst DC3000 but showed enhanced resistance to the necrotrophic fungus Botrytis cinereae. Transcriptome analyses of wlim2a mutants revealed the deregulation of immune hormone biosynthesis and signaling of salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) pathways. The wlim2a mutants also exhibited altered stomatal phenotypes. Analysis of plants expressing WLIM2A variants of the phospho-dead or phospho-mimicking MAPK phosphorylation site showed opposing stomatal behavior and resistance phenotypes in response to Pst DC3000 infection, proving that phosphorylation of WLIM2A plays a crucial role in plant immunity. Overall, these data demonstrate that phosphorylation of WLIM2A by MAPKs regulates Arabidopsis responses to plant pathogens.

CIGB-300 internalizes and impairs viability of NSCLC cells lacking actionable targets by inhibiting casein kinase-2 signaling

Overall response rates in advanced Non-Small Cell Lung Cancer (NSCLC) remains low. Thus, novel molecular targets, tailored drugs and/or drug combinations are needed. Casein Kinase-2 (CK2) is a constitutively active and frequently over-expressed enzyme which fosters tumor survival, proliferation and metastasis. By using a clinical-grade and Cell Penetrating Peptide-based inhibitor coined as CIGB-300, we explore the anti-neoplastic effects caused by interruption of CK2 signaling in lung cancer cells lacking EGFR, ALK and ROS mutations. CIGB-300 penetrated and impaired viability and proliferation of Lung Adenocarcinoma (LUAD) (A549, NCI-H522) and Lung Squamous Carcinoma (LUSC) (NCI-H226 and SK-MES-1) cells in a dose-response manner. The differential activity could not be explained by overall peptide uptake or its subcellular distribution, as evidenced by flow cytometry and confocal microscopy. Upon internalization, CIGB-300 interacted with CK2 catalytic subunits (ɑ1/ɑ2) and CK2 substrates, thus impairing phosphorylation of enzyme substrates (CDC37s13, NPM1s125) and downstream proteins (RPS6s325/326). CK2 inhibition induced an early Reactive Oxygen Species (ROS) and mitochondrial membrane depolarization, which predates lung cancer cell death. Finally, intravenous injection of CIGB-300 in a cell line-based xenograft corroborated CIGB-300’s anti-tumor effects and suggested concurrent in situ reductions of CSNK2ɑ subunit and downstream RPS6s235/236 phosphorylation. Overall, CIGB-300 therapeutic hypothesis and antineoplastic effects demonstrated herein, further support the evaluation of this clinical-grade CK2 inhibitor in advanced NSCLC with limited therapeutic options.

Exploring the inhibitory activity and mechanism on lipid production in 3T3-L1 cells by hot water extract derived from Acacia confusa flowers.

Acacia confusa Merr. (Fabaceae) (A. confusa) is a native tree species of Taiwan, commonly found in the low-altitude mountains and hilly areas of the Hengchun Peninsula. This evergreen, perennial, and large-sized tree was the focus of a study that employed various chromatographic and spectroscopic methods to analyze the hot water extract of its flowers. The analysis revealed that the major components of the extract were myricitrin, quercitrin, europetin-3-O-rhamnoside, and chalconaringenin-2'-xyloside, with respective concentrations of approximately 0.22, 0.02, 0.26, and 0.10mg/g of the flowers. Subsequent cell assays were conducted to assess the inhibitory effect of the extract on lipid synthesis in fat cells. Oil Red O staining results indicated that the extract significantly suppressed fatty acid accumulation in 3T3-L1 cells, with the most pronounced effect observed at a concentration of 180μg/ml. Furthermore, the hot water extract of A. confusa flowers was found to increase the phosphorylation of AMP-activated protein kinase (AMPK), decrease the phosphorylation of cAMP response element-binding protein (CREB), and reduce the expression of glucocorticoid receptor (GR) protein. This, in turn, inhibited the expression of downstream transcription factors such as CCAT/ehancer binding proteins α (C/EBPα), CCAT/ehancer binding proteins β (C/EBPβ), CCAT/ehancer binding proteins δ (C/EBPδ), peroxisome proliferation-actived receptor γ (PPARγ), and sterol regulatory element binding proteins-1 (SREBP-1). Consequently, the expression of lipid synthesis-related proteins acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), and fatty acid translocase (CD36) was reduced, ultimately inhibiting lipid generation. Therefore, the hot water extract of A. confusa flowers shows potential for development as a weight-loss tea.

Nicotine-induced CHRNA5 activation modulates CES1 expression, impacting head and neck squamous cell carcinoma recurrence and metastasis via MEK/ERK pathway

The mucosal epithelium of the head and neck region (including the oral cavity, nasal cavity, pharynx, nasopharynx, and larynx) is the primary site exposed to tobacco smoke, and its presence of nicotinic acetylcholine receptors (nAChRs) has been observed in the mucosal epithelial cells of this area. It remains unclear whether HNSC cells can migrate and invade through nAChR signaling. A model of HNSC cells exposed to nicotine is established. Cell proliferation following nicotine exposure is assessed using the CCK-8 assay, while migration and invasion are evaluated through wound healing and Transwell assays. The effects of CHRNA5 knockdown and overexpression are also investigated. Immunofluorescence staining is used to analyze CHRNA5 expression and localization, and clonogenic assays are performed to measure colony proliferation after CHRNA5 knockdown and overexpression. The interaction between CHRNA5 and CES1 is examined using molecular docking, co-immunoprecipitation, and immunofluorescence. Differentially expressed genes are subjected to pathway enrichment analysis, and MEK/ERK protein expression and phosphorylation are validated via western blot. Tumor formation assays are performed in nude mice using sh-CHRNA5 Cal27 cells, followed by western blot and immunohistochemical staining. Additionally, laryngeal and hypopharyngeal cancer tissues are analyzed through immunohistochemistry. Nicotine significantly enhanced the proliferation, migration, and invasion capabilities of head and neck tumor cells, including Cal27, Fadu, HN6, and Tu686 cells, through the expression of CHRNA5. Knockdown of CHRNA5 can reduce cell migration, invasion, and proliferation, whereas nicotine exposure can reverse this trend. Additionally, the mRNA and protein expression of CES1 decreases with the knockdown of CHRNA5, indicating a regulatory relationship between the two. Transcriptomics revealed that the knockdown of CHRNA5 is associated with the MEK/ERK signaling pathway. Further cellular- and tissue-level evidence confirmed that the levels of p-MEK/MEK, p-ERK/ERK, and CES1 decreased following knockdown of CHRNA5, a trend that nicotine can reverse. Nicotine promotes the proliferation, migration, and invasion of HNSC by upregulating CHRNA5 expression. Knockdown of CHRNA5 reduces these effects, which can be reversed by nicotine. Nicotine exposure activates CHRNA5, regulating CES1 expression via the MEK/ERK pathway, contributing to the recurrence and metastasis of head and neck squamous carcinoma.