Protein Phosphorylation Research Articles

Phosphoproteomic analysis of the adaption of epididymal epithelial cells to corticosterone challenge.

The epididymis has long been of interest owing to its role in promoting the functional maturation of the male germline. More recent evidence has also implicated the epididymis as an important sensory tissue responsible for remodeling of the sperm epigenome, both under physiological conditions and in response to diverse forms of environmental stress. Despite this knowledge, the intricacies of the molecular pathways involved in regulating the adaptation of epididymal tissue to paternal stressors remains to be fully resolved. The overall objective of this study was to investigate the direct impact of corticosterone challenge on a tractable epididymal epithelial cell line (i.e., mECap18 cells), in terms of driving adaptation of the cellular proteome and phosphoproteome signaling networks. The newly developed phosphoproteomic platform EasyPhos coupled with sequencing via an Orbitrap Exploris 480 mass spectrometer, was applied to survey global changes in the mECap18 cell (phospho)proteome resulting from sub-chronic (10-day) corticosterone challenge. The imposed corticosterone exposure regimen elicited relatively subtle modifications of the global mECap18 proteome (i.e., only 73 out of 4171 [∼1.8%] proteins displayed altered abundance). By contrast, ∼15% of the mECap18 phosphoproteome was substantially altered following corticosterone challenge. In silico analysis of the corresponding parent proteins revealed an activation of pathways linked to DNA damage repair and oxidative stress responses as well as a reciprocal inhibition of pathways associated with organismal death. Corticosterone challenge also induced the phosphorylation of several proteins linked to the biogenesis of microRNAs. Accordingly, orthogonal validation strategies confirmed an increase in DNA damage, which was ameliorated upon selective kinase inhibition, and an altered abundance profile of a subset of microRNAs in corticosterone-treated cells. Together, these data confirm that epididymal epithelial cells are reactive to corticosterone challenge, and that their response is tightly coupled to the opposing action of cellular kinases and phosphatases.

Phosphoproteomic investigation of targets of protein phosphatases in EGFR signaling.

Receptor tyrosine kinases (RTKs) initiate cellular signaling pathways, which are regulated through a delicate balance of phosphorylation and dephosphorylation events. While many studies of RTKs have focused on downstream-activated kinases catalyzing the site-specific phosphorylation, few studies have focused on the phosphatases carrying out the dephosphorylation. In this study, we analyzed six protein phosphatase networks using chemical inhibitors in context of epidermal growth factor receptor (EGFR) signaling by mass spectrometry-based phosphoproteomics. Specifically, we focused on protein phosphatase 2C (PP2C), involved in attenuating p38-dependent signaling pathways in various cellular responses, and confirmed its effect in regulating p38 activity in EGFR signaling. Furthermore, utilizing a p38 inhibitor, we classified phosphosites whose phosphorylation status depends on PP2C inhibition into p38-dependent and p38-independent sites. This study provides a large-scale dataset of phosphatase-regulation of EGF-responsive phosphorylation sites, which serves as a useful resource to deepen our understanding of EGFR signaling.

TSPAN8+ myofibroblastic cancer-associated fibroblasts promote chemoresistance in patients with breast cancer.

Cancer-associated fibroblasts (CAFs) are abundant stromal cells in the tumor microenvironment that promote cancer progression and relapse. However, the heterogeneity and regulatory roles of CAFs underlying chemoresistance remain largely unclear. Here, we performed a single-cell analysis using high-dimensional flow cytometry analysis and identified a distinct senescence-like tetraspanin-8 (TSPAN8)+ myofibroblastic CAF (myCAF) subset, which is correlated with therapeutic resistance and poor survival in multiple cohorts of patients with breast cancer (BC). TSPAN8+ myCAFs potentiate the stemness of the surrounding BC cells through secretion of senescence-associated secretory phenotype (SASP)-related factors IL-6 and IL-8 to counteract chemotherapy. NAD-dependent protein deacetylase sirtuin 6 (SIRT6) reduction was responsible for the senescence-like phenotype and tumor-promoting role of TSPAN8+ myCAFs. Mechanistically, TSPAN8 promoted the phosphorylation of ubiquitin E3 ligase retinoblastoma binding protein 6 (RBBP6) at Ser772 by recruiting MAPK11, thereby inducing SIRT6 protein destruction. In turn, SIRT6 down-regulation up-regulated GLS1 and PYCR1, which caused TSPAN8+ myCAFs to secrete aspartate and proline, and therefore proved a nutritional niche to support BC outgrowth. By demonstrating that TSPAN8+SIRT6low myCAFs were tightly associated with unfavorable disease outcomes, we proposed that the combined regimen of anti-TSPAN8 antibody and SIRT6 activator MDL-800 is a promising approach to overcome chemoresistance. These findings highlight that senescence contributes to CAF heterogeneity and chemoresistance and suggest that targeting TSPAN8+ myCAFs is a promising approach to circumvent chemoresistance.

Unveiling orphan receptor-like kinases in plants: novel client discovery using high-confidence library predictions in the Kinase-Client (KiC) assay.

Plants are remarkable in their ability to adapt to changing environments, with receptor-like kinases (RLKs) playing a pivotal role in perceiving and transmitting environmental cues into cellular responses. Despite extensive research on RLKs from the plant kingdom, the function and activity of many kinases, i.e., their substrates or "clients", remain uncharted. To validate a novel client prediction workflow and learn more about an important RLK, this study focuses on P2K1 (DORN1), which acts as a receptor for extracellular ATP (eATP), playing a crucial role in plant stress resistance and immunity. We designed a Kinase-Client (KiC) assay library of 225 synthetic peptides, incorporating previously identified P2K phosphorylated peptides and novel predictions from a deep-learning phosphorylation site prediction model (MUsite) and a trained hidden Markov model (HMM) based tool, HMMER. Screening the library against purified P2K1 cytosolic domain (CD), we identified 46 putative substrates, including 34 novel clients, 27 of which may be novel peptides, not previously identified experimentally. Gene Ontology (GO) analysis among phosphopeptide candidates revealed proteins associated with important biological processes in metabolism, structure development, and response to stress, as well as molecular functions of kinase activity, catalytic activity, and transferase activity. We offer selection criteria for efficient further in vivo experiments to confirm these discoveries. This approach not only expands our knowledge of P2K1's substrates and functions but also highlights effective prediction algorithms for identifying additional potential substrates. Overall, the results support use of the KiC assay as a valuable tool in unraveling the complexities of plant phosphorylation and provide a foundation for predicting the phosphorylation landscape of plant species based on peptide library results.

KANPHOS: Kinase-associated neural phospho-signaling database for data-driven research.

Protein phosphorylation, a key regulator of cellular processes, plays a central role in brain function and is implicated in neurological disorders. Information on protein phosphorylation is expected to be a clue for understanding various neuropsychiatric disorders and developing therapeutic strategies. Nonetheless, existing databases lack a specific focus on phosphorylation events in the brain, which are crucial for investigating the downstream pathway regulated by neurotransmitters. To overcome the gap, we have developed a web-based database named "Kinase-Associated Neural PHOspho-Signaling (KANPHOS)." This paper presents the design concept, detailed features, and a series of improvements for KANPHOS. KANPHOS is designed to support data-driven research by fulfilling three key objectives: (1) enabling the search for protein kinases and their substrates related to extracellular signals or diseases; (2) facilitating a consolidated search for information encompassing phosphorylated substrate genes, proteins, mutant mice, diseases, and more; and (3) offering integrated functionalities to support pathway and network analysis. KANPHOS is also equipped with API functionality to interact with external databases and analysis tools, enhancing its utility in data-driven investigations. Those key features represent a critical step toward unraveling the complex landscape of protein phosphorylation in the brain, with implications for elucidating the molecular mechanisms underlying neurological disorders. KANPHOS is freely accessible to all researchers at https://kanphos.jp.

Decreasing mitochondrial fission ameliorates HIF-1α-dependent pathological retinal angiogenesis.

Angiogenesis plays a critical role in many pathological processes, including irreversible blindness in eye diseases such as retinopathy of prematurity. Endothelial mitochondria are dynamic organelles that undergo constant fusion and fission and are critical signalling hubs that modulate angiogenesis by coordinating reactive oxygen species (ROS) production and calcium signalling and metabolism. In this study, we investigated the role of mitochondrial dynamics in pathological retinal angiogenesis. We showed that treatment with vascular endothelial growth factor (VEGF; 20 ng/ml) induced mitochondrial fission in HUVECs by promoting the phosphorylation of dynamin-related protein 1 (DRP1). DRP1 knockdown or pretreatment with the DRP1 inhibitor Mdivi-1 (5 μM) blocked VEGF-induced cell migration, proliferation, and tube formation in HUVECs. We demonstrated that VEGF treatment increased mitochondrial ROS production in HUVECs, which was necessary for HIF-1α-dependent glycolysis, as well as proliferation, migration, and tube formation, and the inhibition of mitochondrial fission prevented VEGF-induced mitochondrial ROS production. In an oxygen-induced retinopathy (OIR) mouse model, we found that active DRP1 was highly expressed in endothelial cells in neovascular tufts. The administration of Mdivi-1 (10 mg·kg-1·d-1, i.p.) for three days from postnatal day (P) 13 until P15 significantly alleviated pathological angiogenesis in the retina. Our results suggest that targeting mitochondrial fission may be a therapeutic strategy for proliferative retinopathies and other diseases that are dependent on pathological angiogenesis.

6-Gingerol attenuates hepatic ischemia/reperfusion injury through regulating MKP5-mediated P38/JNK pathway

6-Gingerol, the main bioactive compound of ginger, has antioxidant, anti-inflammatory, anti-cancer and neuroprotective effects. However, it is unclear whether 6-Gingerol has protective effects against hepatic ischemia/reperfusion (I/R) injury. In this study, the mouse liver I/R injury model and the mouse AML12 cell hypoxia/reoxygenation (H/R) model were established by pretreatment with 6-Gingerol at different concentrations to explore the potential effects of 6-Gingerol. Serum transaminase levels, liver necrotic area, cell viability, inflammatory response, and cell apoptosis were used to assess the effect of 6-Gingerol on hepatic I/R or cell H/R injury. Quantitative polymerase chain reaction (qPCR) and Western blotting were used to detect the mRNA and protein expression. The results show that 6-Gingerol decreased serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) levels, liver necrosis, inflammatory cytokines IL-1β, IL-6, MCP-1, TNF-α expression, Ly6g+ inflammatory cell infiltration, protein phosphorylation of NF-κB signaling pathway, Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) positive cells, cell apoptosis rate, the protein expression of pro-apoptotic protein BAX and C-Caspase3, increased cell viability, and expression of anti-apoptotic protein BCL-2. Moreover, 6-Gingerol could increase the mRNA and protein expression of mitogen activated protein kinase phosphatase 5 (MKP5) and inhibit the activation of P38/JNK signaling pathway. In MKP5 knockout (KO) mice, the protective effect of 6-gingerol and the inhibition of P38/JNK pathway were significantly weakened. Therefore, our results suggest that 6-Gingerol exerts anti-inflammatory and anti-apoptotic effects to attenuate hepatic I/R injury by regulating the MKP5-mediated P38/JNK signaling pathway.

Syringic acid attenuates acute lung injury by modulating macrophage polarization in LPS-induced mice

BackgroundAcute lung injury (ALI) is a continuum of lung changes caused by multiple lung injuries, characterized by a syndrome of uncontrolled systemic inflammation that often leads to significant morbidity and death. Anti-inflammatory is one of its treatment methods, but there is no safe and available drug therapy. Syringic acid (SA) is a natural organic compound commonly found in a variety of plants, especially in certain woody plants and fruits. In modern pharmacological studies, SA has anti-inflammatory effects and therefore may be a potentially safe and available compound for the treatment of acute lung injury. PurposeThis study attempts to reveal the protective mechanism of SA against ALI by affecting the polarization of macrophages and the activation of NF-κB signaling pathway. Trying to find a safer and more effective drug therapy for clinical use. MethodsWe constructed the ALI model using C57BL/6 mice by intratracheal instillation of LPS (10mg/kg). Histological analysis was performed with hematoxylin and eosin (H&E). The wet-dry ratio of the whole lung was measured to evaluate pulmonary edema. The effect of SA on macrophage M1-type was detected by flow cytometry. BCA protein quantification method was used to determine the total protein concentration in bronchoalveolar lavage fluid (BALF). The levels of Interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α in BALF were determined by the ELISA kits, and RT-qPCR was used to detect the expression levels of IL-6, IL-1β and TNF-α mRNA of lung tissue. Western blot was used to detect the expression levels of iNOS and COX-2 and the phosphorylation of p65 and IκBα in the NF-κB pathway in lung tissue. In vitro experiments were conducted with RAW267.4 cell inflammation model induced by 100ng/ml LPS and A549 cell inflammation model induced by 10μg/ml LPS. The effects of SA on M1-type and M2-type macrophages of RAW267.4 macrophages induced by LPS were detected by flow cytometry. The toxicity of compound SA to A549 cells was detected by MTT method which to determine the safe dose of SA. The expressions of COX-2 and the phosphorylation of p65 and IκBα protein in NF-κB pathway were detected by Western blot. ResultsWe found that the pre-treatment of SA significantly reduced the degree of lung injury, and the infiltration of neutrophils in the lung interstitium and alveolar space of the lung. The formation of transparent membrane in lung tissue and thickening of alveolar septum were significantly reduced compared with the model group, and the wet-dry ratio of the lung was also reduced. ELISA and RT-qPCR results showed that SA could significantly inhibit the production of IL-6, IL-1β, TNF-α. At the same time, SA could significantly inhibit the expression of iNOS and COX-2 proteins, and could inhibit the phosphorylation of p65 and IκBα proteins. in a dose-dependent manner. In vitro experiments, we found that flow cytometry showed that SA could significantly inhibit the polarization of macrophages from M0 type macrophages to M1-type macrophages, while SA could promote the polarization of M1-type macrophages to M2-type macrophages. The results of MTT assay showed that SA had no obvious cytotoxicity to A549 cells when the concentration was not higher than 80μM, while LPS could promote the proliferation of A549 cells. In the study of anti-inflammatory effect, SA can significantly inhibit the expression of COX-2 and the phosphorylation of p65 and IκBα proteins in LPS-induced A549 cells. ConclusionSA has possessed a crucial anti-ALI role in LPS-induced mice. The mechanism was elucidated, suggesting that the inhibition of macrophage polarization to M1-type and the promotion of macrophage polarization to M2-type, as well as the inhibition of NF-κB pathway by SA may be the reasons for its anti-ALI. This finding provides important molecular evidence for the further application of SA in the clinical treatment of ALI.

Proteomic and phosphoproteomic reveal immune-related function of milk fat globule membrane in bovine milk of different lactation periods

Information regarding protein expression and phosphorylation modifications in the bovine milk fat globule membrane is scarce, particularly throughout various lactation periods. This study employed a complete proteome and phosphoproteome between bovine colostrum and mature milk. A total of 11 proteins were seen in both protein expression and phosphorylation levels. There were 400 proteins identified in only protein expression, and 104 phosphoproteins identified in only phosphorylation levels. A total of 232 significant protein characteristics were identified within the proteome and significant phosphorylation sites within 86 phosphoproteins of the phosphoproteome. Biological activities and pathways primarily exhibited associations with the immune system. Simultaneously, a comprehensive analysis of proteins and phosphorylation sites using a multi-omics approach. Hence, the data we have obtained has the potential to expand our understanding of how the bovine milk fat globule membrane might be utilized as a beneficial component in dairy products.

Mechanisms of Dangua Fang in multi-target and multi-method regulation of glycolipid metabolism based on phosphoproteomics.

To explore the mechanism of Dangua Fang (, DGR) in multi-target and multi-method regulation of glycolipid metabolism based on phosphoproteomics. Sprague-Dawley rats with normal glucose levels were randomly divided into three groups, including a conventional diet control group (Group A), high-fat-high-sugar diet model group (Group B), and DGR group (Group C, high-fat-high-sugar diet containing 20.5 g DGR). After 10 weeks of intervention, the fasting blood glucose (FBG), 2 h blood glucose [PBG; using the oral glucose tolerance test (OGTT)], hemoglobin A1c (HbA1c), plasma total cholesterol (TC), and triglycerides (TG) were tested, and the livers of rats were removed to calculate the liver index. Then, hepatic portal TG were tested using the Gross permanent optimization-participatiory action planning enzymatic method and phosphoproteomics was performed using liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis followed by database search and bioinformatics analysis. Finally, cell experiments were used to verify the results of phosphoproteomics. Phosphorylated mitogen-activated protein kinase kinase kinase kinase 4 (MAP4k4) and phosphorylated adducin 1 (ADD1) were detected using western blotting. DGR effectively reduced PBG, TG, and the liver index (P < 0.05), and significantly decreased HbA1c, TC, and hepatic portal TG (P < 0.01), showed significant hematoxylin and eosin (HE) staining, red oil O staining, and Masson staining of liver tissue. The total spectrum was 805 334, matched spectrum was 260 471, accounting for accounting 32.3%, peptides were 19 995, modified peptides were 14 671, identified proteins were 4601, quantifiable proteins were 4417, identified sites were 15 749, and quantified sites were 14659. Based on the threshold of expression fold change ( > 1.2), DGR up-regulated the modification of 228 phosphorylation sites involving 204 corresponding function proteins, and down-regulated the modification of 358 phosphorylation sites involving 358 corresponding function proteins, which included correcting 75 phosphorylation sites involving 64 corresponding function proteins relating to glycolipid metabolism. Therefore, DGR improved biological tissue processes, including information storage and processing, cellular processes and signaling, and metabolism. The metabolic functions regulated by DGR mainly include energy production and conversion, carbohydrate transport and metabolism, lipid transport and metabolism, inorganic ion transport and metabolism, secondary metabolite biosynthesis, transport, and catabolism. In vitro phosphorylation validation based on cell experiments showed that the change trends in the phosphorylation level of MAP4k4 and ADD1 were consistent with that of previous phosphoproteomics studies. DGR extensively corrects the modification of phosphorylation sites to improve corresponding glycolipid metabolism-related protein expression in rats with glycolipid metabolism disorders, thereby regulating glycolipid metabolism through a multi-target and multi-method process.

One-step polymerization of functionalized ionic liquid as polymer microspheres for highly specific enrichment of phosphopeptides from cell lysate and serum

A Ti4+-functionalized ionic liquid polymer microsphere was proposed and prepared through free radical precipitation polymerization. The polymer microsphere demonstrates excellent performance on phosphopeptide enrichment, with a low detection limit (0.5 fmol) and good selectivity (1:2500). Through the analysis of HepG2 cell lysate using nano LC-MS/MS after material enrichment, a total of 1100 phosphopeptides and 733 phosphoproteins were identified. Additionally, in the serum of liver cancer patients, 69 phosphopeptides, 47 phosphoproteins, and 52 phosphorylation sites were identified, while healthy controls exhibited 55 phosphopeptides, 31 phosphoproteins, and 40 phosphorylation sites. Label-free quantification revealed 10 differentially expressed proteins (DEPs), with five proteins (FN1, SERPIND1, SERPINF2, IGFBP3, and MENT) being up-regulated and five proteins (SELENOP, PLG, SPP2, SERPINC1, and GP1BB) being down-regulated. These findings highlight the potential of the MBA-VImDOPA-Ti4+ polymer microsphere in specifically enriching phosphopeptides from clinical biological samples.

DGK5 phosphorylation finetunes PA homeostasis in plant immunity

Phosphatidic acid (PA) as a universal second messenger is transiently and rapidly produced upon immune activation in plants. A recent study by Kong et al. elucidated a mechanism for maintaining PA homeostasis via two uncoupled phosphorylation events of DIACYLGLYCEROL KINASE 5 (DGK5) at different phosphorylation sites by two distinct kinases.

Bound polyphenols from insoluble dietary fiber of navel orange peel alleviates cellular inflammation by inhibiting LPS-induced NF-κB/JAK-STAT pathway in RAW264.7 macrophages

In this study, we explored the anti-inflammatory effects of bound polyphenols derived from the insoluble dietary fiber of navel orange peel (BPP) on LPS-induced RAW264.7 macrophages, and investigated the underlying molecular mechanisms. The experimental findings revealed that BPP effectively suppressed COX-2 and iNOS protein expression, and concurrently decreased intracellular ROS production and NO release. RNA sequencing identified 1,894 differentially expressed genes between the model and BPP groups, with 1,009 genes upregulated and 885 downregulated. Notably, the NF-κB and JAK-STAT pathways were among the 20 most significantly altered signaling pathways, as determined by KEGG analysis. Western blot analysis further showed that BPP significantly inhibited the phosphorylation of key proteins in the MAPKs (ERK, JNK, p38), NF-κB (p65, IκBα), and JAK-STAT (JAK1, STAT3) pathways. Additionally, the use of p65 and STAT3 activators markedly elevated NO and ROS levels, whereas BPP treatment effectively reduced them. These findings suggest that BPP may attenuate LPS-induced inflammatory responses through the NF-κB and JAK-STAT pathways, highlighting its potential as a natural anti-inflammatory agent and its implications for functional food applications.

Regulating Lars2 in mitochondria: A potential Alzheimer's therapy by inhibiting tau phosphorylation

Driven by the scarcity of effective treatment options in clinical settings, the present study aimed to identify a new potential target for Alzheimer's disease (AD) treatment. We focused on Lars2, an enzyme synthesizing mitochondrial leucyl-tRNA, and its role in maintaining mitochondrial function. Bioinformatics analysis of human brain transcriptome data revealed downregulation of Lars2 in AD patients compared to healthy controls. During in vitro experiments, the knockdown of Lars2 in mouse neuroblastoma cells (neuro-2a cells) and primary cortical neurons led to morphological changes and decreased density in mouse hippocampal neurons. To explore the underlying mechanisms, we investigated how downregulated Lars2 expression could impede the phosphatidylinositol 3-kinase/protein kinase B (PI3K-AKT) pathway, thereby mitigating AKT's inhibitory effect on glycogen synthase kinase 3 beta (GSK3β). This led to the activation of GSK3β, causing excessive phosphorylation of Tau protein and subsequent neuronal degeneration. During in vivo experiments, knockout of lars2 in hippocampal neurons confirmed cognitive impairment through the Barnes maze test, the novel object recognition test, and nest-building experiments. Additionally, immunofluorescence assays indicated an increase in p-tau, atrophy in the hippocampal region, and a decrease in neurons following Lars2 knockout. Taken together, our findings indicate that Lars2 represents a promising therapeutic target for AD.

JHD205, A Novel Abemaciclib Derivative, Exerts Antitumor Effects on Breast Cancer by CDK4/6.

Efficient targeted molecular therapeutics are needed for the treatment of triple-negative breast cancer (TNBC), a highly invasive and difficult-to-treat form of breast cancer associated with a poor prognosis. This study aims to evaluate the potential of selective CDK4/6 inhibitors as a therapeutic option for TNBC by impairing the cell cycle G1 phase through the inhibition of retinoblastoma protein (Rb) phosphorylation. In this study, we synthesized a compound called JHD205, derived from the chemical structure of Abemaciclib, and examined its inhibitory effects on the malignant characteristics of TNBC cells. Our results demonstrated that JHD205 exhibited superior tumor growth inhibition compared to Abemaciclib in breast cancer xenograft chicken embryo models. Western blot analysis revealed that JHD205 could dosedependently degrade CDK4 and CDK6 while also causing abnormal changes in other proteins associated with CDK4/6, such as p-Rb, Rb, and E2F1. Moreover, JHD205 induced apoptosis and DNA damage and inhibited DNA repair by upregulating Caspase3 and p-H2AX protein levels. Collectively, our findings suggest that JHD205 holds promise as a potential treatment for breast carcinoma.

498 Bruton’s tyrosine kinase (BTK) inhibitors impede platelet aggregation but not adhesion to collagen.

OBJECTIVES/GOALS: The research objectives of this project are to elucidate the effects of Bruton’s tyrosine kinase inhibitors (BTKi) of varying target specificity on platelet function with regard to platelet aggregation, adhesion, spreading, and intracellular signaling as measured by kinase phosphorylation. METHODS/STUDY POPULATION: Blood from healthy volunteers was obtained and processed to obtain both washed platelets and platelet-rich plasma. The samples were then treated with one of the BTKi drugs or with vehicle (DMSO) at concentrations matching patient blood concentrations derived from clinical trials and pharmacokinetic studies. The incubated samples were then analyzed in an aggregometer using one of several agonists. Aggregation was stopped after five minutes with a perchloric acid-based lysis buffer. The samples were then analyzed by SDS-PAGE and immunoblotting to quantify BTK protein and BTK phosphorylation. Adhesion was assessed by incubating washed platelets treated with BTKi on microtiter wells coated with fibrinogen or collagen and quantifying adherent platelets by their endogenous acid phosphatase activity. RESULTS/ANTICIPATED RESULTS: We found that ibrutinib, zanubrutinib, and pirtobrutinib all completely inhibited collagen-induced platelet aggregation, whereas they did not inhibit aggregation induced by thrombin, ristocetin, arachidonic acid, or the PAR1 activator peptide SFLLRN (T6). Acalabrutinib inhibited collagen-induced platelet aggregation only at high concentrations (1-2 micromolar). At the lower concentration of 200 nanomolar, comparable to the concentration required for the other BTK inhibitors to completely inhibit platelet aggregation, acalabrutinib failed to inhibit aggregation but did inhibit auto-phosphorylation, indicating an impact on signaling. None of the BTKi drugs inhibited adhesion of platelets to collagen-coated surfaces. DISCUSSION/SIGNIFICANCE: Our data show the inhibitory effect of BTKi on collagen-induced platelet aggregation and signaling. However, it remains unclear whether the inhibition is due to an effect on BTK itself or other related kinases. Better insight into the mechanisms of platelet inhibition by BTKi may help guide the development of BTKi with a lower risk of hemorrhage.

Feedback regulation between AMPK and HIF-1α contributes to color stability via energy metabolism and mitochondrial efficiency regulation of yak meat

Despite the well-established individual roles of AMP-activated protein kinase (AMPK) and hypoxia-inducible factor-1α (HIF-1α) in sensing energy stress and/or hypoxia in postmortem muscle, a knowledge gap persists regarding their interplay and the consequent impact on the yak meat color stability. This study aimed to elucidate the AMPK–HIF–1α interaction and its consequential impact on mitochondrial functionality, energy metabolism, and ultimately, color attributes. Postmortem yak longissimus thoracis et lumborum (LTL) muscles were incubated with saline, AMPK inhibitor (Compound C), Compound C plus HIF-1α activator (DMOG), and Compound C plus HIF-1α inhibitor (YC-1) for 0, 6, 12, 24, 72, and 120 h, respectively. Results indicate that Compound C hindered the nuclear translocation of HIF-1α (P < 0.001), in turn, DMOG upregulated AMPK protein levels (P < 0.05) by a yet unknown mechanism. Mass spectrometry analysis identified seven AMPK-induced phosphorylation sites (Ser58, Thr63, Ser65, Tyr66, Thr677, Ser14, and Ser15) mapped onto three phosphopeptides on HIF-1α. Moreover, Compound C induced a significant reduction in energy metabolism levels, evidenced by decreased glycogen degradation, lactate accumulation, and pH decline (P < 0.05). Besides, Compound C promoted a consistently decreased mitochondrial protein yield, reduced NADH content, and OCR (P < 0.05). However, DMOG attenuated the effects of Compound C on mitochondrial efficiency, and interconversion of oxy-to deoxymyoglobin. Collectively, these findings establish the collaborative influence of AMPK and HIF-1α on postmortem energy metabolism, concurrently emphasizing their antagonistic effects on mitochondrial function.

PhosMap: An ensemble bioinformatic platform to empower interactive analysis of quantitative phosphoproteomics

BackgroundLiquid chromatography-mass spectrometry (LC-MS)-based quantitative phosphoproteomics has been widely used to detect thousands of protein phosphorylation modifications simultaneously from the biological specimens. However, the complicated procedures for analyzing phosphoproteomics data has become a bottleneck to widening its application. MethodsHere, we develop PhosMap, a versatile and scalable tool to accomplish phosphoproteomics data analysis. A standardized phosphorylation data format was created for data analyses, from data preprocessing to downstream bioinformatic analyses such as dimension reduction, differential phosphorylation analysis, kinase activity, survival analysis, and so on. For better usability, we distribute PhosMap as a Docker image for easy local deployment upon any of Windows, Linux, and Mac system. ResultsThe source code is deposited at https://github.com/BADD-XMU/PhosMap. A free PhosMap webserver (https://huggingface.co/spaces/Bio-Add/PhosMap), with easy-to-follow fashion of dashboards, is curated for interactive data analysis. ConclusionsPhosMap fills the technical gap of large-scale phosphorylation research by empowering researchers to process their own phosphoproteomics data expediently and efficiently, and facilitates better data interpretation.

Discovery of novel thiophene-3-carboxamide derivatives as potential VEGFR-2 inhibitors with anti-angiogenic properties

VEGFR-2 is an attractive target for the development of anti-tumor drugs and plays a crucial role in tumor angiogenesis. This study reports a series of novel thiophene-3-carboxamide derivatives based on PAN-90806 as VEGFR-2 inhibitors, among which compound 14d exhibits excellent anti-proliferative activity against HCT116, MCF7, PC3, and A549 cell lines, and has effective VEGFR-2 inhibitory activity with an IC50 value of 191.1 nM. Additionally, CETSA results indicated that VEGFR-2 was a relevant target of compound 14d in the cell lines, and compound 14d could also inhibit VEGFR-2 protein phosphorylation in A549 cell line. Furthermore, compound 14d inhibited colony formation, cell migration, and HUVECs tube formation in a dose-dependent manner. The mechanism by which 14d induced cancer cell death involves blocking the cell cycle, increasing ROS production, inducing apoptosis, and dose-dependently reducing the levels of phosphorylated ERK and MEK. Molecular docking and molecular dynamics simulations had shown that compound 14d could stably bind to the active site of VEGFR-2. These results confirmed that compound 14d might be a promising lead compound for anti-angiogenesis.

Multi-target mechanism of misoprostol in pregnancy termination based on network pharmacology and molecular docking.

Misoprostol is a prostaglandin analogue that contracts the uterus, prompting the expulsion of the embryo. No systematic evaluation of the mechanisms of misoprostol has previously been performed. In this study, known targets of misoprostol were obtained from the DrugBank database; potential targets of misoprostol were predicted using data from the SwissTargetPrediction and PharmMapper databases; and the main targets of pregnancy termination were obtained from the GeneCards database. The protein-protein interaction (PPI) network of the shared genes between misoprostol and pregnancy termination was constructed using data from the STRING database, and the "misoprostol-pregnancy termination-pathway" network was constructed and potential targets was verified through molecular docking. We analyzed 37 shared target genes and obtained a network diagram of 134 potential targets, which the core therapeutic targets were HSP90AA1, EGFR, and MAPK1. GO functional and KEGG pathway enrichment analyses showed that misoprostol can modulate the VEGF signaling pathway, calcium signaling pathway, and NF-κB signaling pathway in pregnancy termination and mainly interferes with protein phosphorylation, cell localization, and protein hydrolysis regulation processes. This research illustrates the mechanism underlying the pharmacological effect of misoprostol, namely pregnancy termination. However, further experimental verification is warranted for optimal use of misoprostol during clinical practice.