Melatonin alleviates arsenic-induced liver injury by regulating protein RKIP and enhancing antioxidant defencse mechanisms.

Arsenic (As) is a highly toxic metal and one of the main factors in cancer development through oxidative stress and production of reactive oxygen species. Prior research has demonstrated melatonin's potential as a free radical scavenger. Raf kinase inhibitory protein (RKIP) is an important regulator of intracellular signaling pathways that has been linked to various types of cancer. The aim of this research was to explore the influence of melatonin's antioxidant properties on the expression of the protein RKIP and the antioxidant status of liver tissue in rats that were exposed to arsenic. Thirty two male Wistar rats were divided into four groups of eight, including control, melatonin-treated (20 mg/Kg of melatonin), sodium arsenite-treated (5.5 mg/Kg of sodium arsenite), and melatonin + sodium arsenite-treated groups (combination) for 4 weeks. The expression level of protein RKIP was measured by Western blot, and malondialdehyde (MDA) content of the liver as well as the activities of antioxidant enzymes were measured. The data analyzed using one-way ANOVA (significance level of p < 0.05) and GraphPad Prism (9) software. Sodium arsenite treatment led to a significant decrease in RKIP protein expression and antioxidant enzyme activity, and an increase in liver MDA levels (p < 0.001). Conversely, melatonin treatment in the combination group resulted in a significant increase in RKIP protein expression and antioxidant enzyme activity and a decrease in liver MDA levels (p < 0.05). These findings suggest that melatonin can attenuate oxidative damage caused by arsenic in liver cells by enhancing RKIP protein expression and antioxidant enzyme activity.

The phosphatidylethanolamine-binding proteins OsMFT1 and OsMFT2 regulate seed dormancy in rice.

Seed dormancy is crucial for optimal plant life-cycle timing. However, domestication has largely diminished seed dormancy in modern cereal cultivars, leading to challenges such as preharvest sprouting (PHS) and subsequent declines in yield and quality. Therefore, it is imperative to unravel the molecular mechanisms governing seed dormancy for the development of PHS-resistant varieties. In this study, we screened a mutant of BASIC HELIX-LOOP-HELIX TRANSCRIPTION FACTOR4 (OsbHLH004) with decreased seed dormancy and revealed that OsbHLH004 directly regulates the expression of 9-CIS-EPOXYCAROTENOID DIOXYGENASE3 (OsNCED3) and GIBBERELLIN 2-OXIDASE6 (OsGA2ox6) in rice (Oryza sativa). Additionally, we determined that two phosphatidylethanolamine-binding proteins, MOTHER OF FT AND TFL1 and 2 (OsMFT1 and OsMFT2; hereafter OsMFT1/2) interact with OsbHLH004 and Ideal Plant Architecture 1 (IPA1) to regulate their binding capacities on OsNCED3 and OsGA2ox6, thereby promoting seed dormancy. Intriguingly, FT-INTERACTING PROTEIN1 (OsFTIP1) interacts with OsMFT1/2 and affects their nucleocytoplasmic translocation into the nucleus, where OsMFT1/2-OsbHLH004 and OsMFT1/2-IPA1 antagonistically modulate the expression of OsNCED3 and OsGA2ox6. Our findings reveal that OsFTIP1-mediated inhibition of nuclear translocation of OsMFT1/2 and the dynamic transcriptional modulation of OsNCED3 and OsGA2ox6 by OsMFT1/2-OsbHLH004 and OsMFT1/2-IPA1 complexes in seed dormancy in rice.

Bmi-1 promotes the proliferation, migration and invasion, and inhibits cell apoptosis of human retinoblastoma cells via RKIP.

Retinoblastoma is one of the most common ocular malignancies in children. Bmi-1, a member of the Polycomb group family of transcriptional repressors, is expressed in a variety of tumors. The purpose of our study was to explore the role of Bmi-1 in retinoblastoma. RT-qPCR and western blot were used for calculating the mRNA and protein levels of Bmi-1 and RKIP. MTT, Wound healing and Transwell assays were performed to measure the proliferation, migration and invasion in retinoblastoma cells. Cell apoptosis was detected by flow cytometry. The volume and mass of transplanted tumors were detected in nude mice. Bmi-1 was over expressed, and RKIP was low expressed in retinoblastoma cells. Bmi-1 promoted cell proliferation, migration and invasion and suppressed cell apoptosis of Y79 and SO-RB50 cells. Downregulation of Bmi-1 and overexpression of RKIP inhibited cell proliferation, migration and invasion, and increased cell apoptosis. The functions of Bmi-1 knockdown on retinoblastoma cells were blocked by RKIP knockdown, but promoted by RKIP. Down-regulated Bmi-1 inhibited xenograft tumor growth, and RKIP exacerbated this inhibitory effect. Bmi-1 served as a potential therapeutic target for improving the efficacy of clinical treatment in retinoblastoma. All the findings revealed the functions of Bmi-1/RKIP axis in retinoblastoma tumorigenesis.

Cross-Talks between Raf Kinase Inhibitor Protein and Programmed Cell Death Ligand 1 Expressions in Cancer: Role in Immune Evasion and Therapeutic Implications.

Innovations in cancer immunotherapy have resulted in the development of several novel immunotherapeutic strategies that can disrupt immunosuppression. One key advancement lies in immune checkpoint inhibitors (ICIs), which have shown significant clinical efficacy and increased survival rates in patients with various therapy-resistant cancers. This immune intervention consists of monoclonal antibodies directed against inhibitory receptors (e.g., PD-1) on cytotoxic CD8 T cells or against corresponding ligands (e.g., PD-L1/PD-L2) overexpressed on cancer cells and other cells in the tumor microenvironment (TME). However, not all cancer cells respond-there are still poor clinical responses, immune-related adverse effects, adaptive resistance, and vulnerability to ICIs in a subset of patients with cancer. This challenge showcases the heterogeneity of cancer, emphasizing the existence of additional immunoregulatory mechanisms in many patients. Therefore, it is essential to investigate PD-L1's interaction with other oncogenic genes and pathways to further advance targeted therapies and address resistance mechanisms. Accordingly, our aim was to investigate the mechanisms governing PD-L1 expression in tumor cells, given its correlation with immune evasion, to uncover novel mechanisms for decreasing PD-L1 expression and restoring anti-tumor immune responses. Numerous studies have demonstrated that the upregulation of Raf Kinase Inhibitor Protein (RKIP) in many cancers contributes to the suppression of key hyperactive pathways observed in malignant cells, alongside its broadening involvement in immune responses and the modulation of the TME. We, therefore, hypothesized that the role of PD-L1 in cancer immune surveillance may be inversely correlated with the low expression level of the tumor suppressor Raf Kinase Inhibitor Protein (RKIP) expression in cancer cells. This hypothesis was investigated and we found several signaling cross-talk pathways between the regulations of both RKIP and PD-L1 expressions. These pathways and regulatory factors include the MAPK and JAK/STAT pathways, GSK3ß, cytokines IFN-γ and IL-1ß, Sox2, and transcription factors YY1 and NFκB. The pathways that upregulated PD-L1 were inhibitory for RKIP expression and vice versa. Bioinformatic analyses in various human cancers demonstrated the inverse relationship between PD-L1 and RKIP expressions and their prognostic roles. Therefore, we suspect that the direct upregulation of RKIP and/or the use of targeted RKIP inducers in combination with ICIs could result in a more targeted anti-tumor immune response-addressing the therapeutic challenges related to PD-1/PD-L1 monotherapy alone.

Evolution of FLOWERING LOCUS T-like genes in angiosperms: a core Lamiales-specific diversification.

Plant life history is determined by two transitions, germination and flowering time, in which the phosphatidylethanolamine-binding proteins (PEBPs) FLOWERING LOCUS T (FT) and TERMINAL FLOWER1 (TFL1) play key regulatory roles. Compared with the highly conserved TFL1-like genes, FT-like genes vary significantly in copy numbers in gymnosperms, and monocots within the angiosperms, while sporadic duplications can be observed in eudicots. Here, via a systematic analysis of the PEBPs in angiosperms with a special focus on 12 representative species featuring high-quality genomes in the order Lamiales, we identified a successive lineage-specific but systematic expansion of FT-like genes in the families of core Lamiales. The first expansion event generated FT1-like genes mainly via a core Lamiales-specific whole-genome duplication (cL-WGD), while a likely random duplication produced the FT2-like genes in the lineages containing Scrophulariaceae and the rest of the core Lamiales. Both FT1- and FT2-like genes were further amplified tandemly in some families. These expanded FT-like genes featured highly diverged expression patterns and structural variation, indicating functional diversification. Intriguingly, some core Lamiales contained the relict MOTHER OF FT AND TFL1 like 2 (MFT2) that probably expanded in the common ancestor of angiosperms. Our data showcase the highly dynamic lineage-specific expansion of the FT-like genes, and thus provide important and fresh evolutionary insights into the gene regulatory network underpinning flowering time diversity in Lamiales and, more generally, in angiosperms.

Lipid peroxidation-induced ferroptosis as a therapeutic target for mitigating neuronal injury and inflammation in sepsis-associated encephalopathy: insights into the hippocampal PEBP-1/15-LOX/GPX4 pathway.

BACKGROUND: Sepsis-associated encephalopathy (SAE) refers to the widespread impairment of brain function caused by noncentral nervous system infection mediated by sepsis. Lipid peroxidation-induced ferroptosis contributes to the occurrence and course of SAE. This study aimed to investigate the relationship between neuronal injury and lipid peroxidation-induced ferroptosis in SAE. METHODS: Baseline data were collected from pediatric patients upon admission, and the expression levels of various markers related to lipid peroxidation and ferroptosis were monitored in the serum and peripheral blood mononuclear cells (PBMCs) of patients with SAE as well as SAE model mice. The hippocampal phosphatidylethanolamine-binding protein (PEBP)-1/15-lysine oxidase (LOX)/ glutathione peroxidase 4 (GPX4) pathway was assessed for its role on the inhibitory effect of ferroptosis in SAE treatment. RESULTS: The results showed elevated levels of S100 calcium-binding protein beta (S-100ß), glial fibrillary acidic protein, and malondialdehyde in the serum of SAE patients, while superoxide dismutase levels were reduced. Furthermore, analysis of PBMCs revealed increased transcription levels of PEBP1, LOX, and long-chain fatty acyl-CoA synthetase family member 4 (ACSL4) in SAE patients, while the transcription levels of GPX4 and cystine/glutamate transporter xCT (SLC7A11) were decreased. In comparison to the control group, the SAE mice exhibited increased expression of S-100ß and neuron-specific enolase (NSE) in the hippocampus, whereas the expression of S-100ß and NSE were reduced in deferoxamine (DFO) mice. Additionally, iron accumulation was observed in the hippocampus of SAE mice, while the iron ion levels were reduced in the DFO mice. Inhibition of ferroptosis alleviated the mitochondrial damage (as assessed by transmission electron microscopy, hippocampal mitochondrial ATP detection, and the JC-1 polymer-to-monomer ratio in the hippocampus) and the oxidative stress response induced by SAE as well as attenuated neuroinflammatory reactions. Further investigations revealed that the mechanism underlying the inhibitory effect of ferroptosis in SAE treatment is associated with the hippocampal PEBP-1/15-LOX/GPX4 pathway. CONCLUSION: These results offer potential therapeutic targets for the management of neuronal injury in SAE and valuable insights into the potential mechanisms of ferroptosis in neurological disorders.

Raf kinase inhibitor protein expression in smooth muscle tumours of the uterus: a diagnostic marker for leiomyosarcoma?

RESEARCH QUESTION: What is the expression pattern of Raf kinase inhibitory protein (RKIP) in different subtypes of leiomyoma (usual type, cellular, apoplectic or haemorrhagic leiomyoma, leiomyoma with bizarre nuclei and lipoleiomyoma) and leiomyosarcoma specimens, and what is its biological role in leiomyosarcoma cells? DESIGN: Leiomyoma and leiomyosarcoma specimens underwent immunohistochemistry staining. Leiomyosarcoma SK-LMS-1 cell line was RKIP knocked down and RKIP overexpressed, and cell viability, wound healing migration and clonogenicity assays were carried out. RESULTS: A higher immunohistochemical expression of RKIP was observed in bizarre leiomyomas, than in usual-type leiomyomas. Decreased expression was also found in cellular leiomyoma, with generally absent staining in leiomyosarcomas. Upon RKIP expression manipulation in SK-LMS-1 cell line, no major differences were observed in cell viability and migration capacity over time. RKIP knockout, however, resulted in a significant increase in the cell's ability to form colonies (P = 0.011). CONCLUSION: RKIP distinct expression pattern among leiomyoma histotype and leiomyosarcoma, and its effect on leiomyosarcoma cells on colony formation, encourages further studies of RKIP in uterine smooth muscle disorders.

Flowering time genes branching out.

Plants are sessile by nature, and as such they have evolved to sense changes in seasonality and their surrounding environment, and adapt to these changes. One prime example of this is the regulation of flowering time in angiosperms, which is precisely timed by the coordinated action of two proteins: FLOWERING LOCUS T (FT) and TERMINAL FLOWER 1 (TFL1). Both of these regulators are members of the PHOSPHATIDYLETHANOLAMINE BINDING PROTEIN (PEBP) family of proteins. These regulatory proteins do not interact with DNA themselves, but instead interact with transcriptional regulators, such as FLOWERING LOCUS D (FD). FT and TFL1 were initially identified as key regulators of flowering time, acting through binding with FD; however, PEBP family members are also involved in shaping plant architecture and development. In addition, PEBPs can interact with TCP transcriptional regulators, such as TEOSINTE BRANCHED 1 (TB1), a well-known regulator of plant architecture, and key domestication-related genes in many crops. Here, we review the role of PEBPs in flowering time, plant architecture, and development. As these are also key yield-related traits, we highlight examples from the model plant Arabidopsis as well as important food and feed crops such as, rice, barley, wheat, tomato, and potato.

A systems-level analysis of the mutually antagonistic roles of RKIP and BACH1 in dynamics of cancer cell plasticity.

Epithelial-mesenchymal transition (EMT) is an important axis of phenotypic plasticity-a hallmark of cancer metastasis. Raf kinase-B inhibitor protein (RKIP) and BTB and CNC homology 1 (BACH1) are reported to influence EMT. In breast cancer, they act antagonistically, but the exact nature of their roles in mediating EMT and associated other axes of plasticity remains unclear. Here, analysing transcriptomic data, we reveal their antagonistic trends in a pan-cancer manner in terms of association with EMT, metabolic reprogramming and immune evasion via PD-L1. Next, we developed and simulated a mechanism-based gene regulatory network that captures how RKIP and BACH1 engage in feedback loops with drivers of EMT and stemness. We found that RKIP and BACH1 belong to two antagonistic 'teams' of players-while BACH1 belonged to the one driving pro-EMT, stem-like and therapy-resistant cell states, RKIP belonged to the one enabling pro-epithelial, less stem-like and therapy-sensitive phenotypes. Finally, we observed that low RKIP levels and upregulated BACH1 levels associated with worse clinical outcomes in many cancer types. Together, our systems-level analysis indicates that the emergent dynamics of underlying regulatory network enable the antagonistic patterns of RKIP and BACH1 with various axes of cancer cell plasticity, and with patient survival data.

Binding of berberine to PEBP1 synergizes with sorafenib to induce the ferroptosis of hepatic stellate cells.

Hepatic stellate cell (HSC) activation is the key process in hepatic fibrosis (HF) development. Targeted death of HSCs could be effective in the prevention and treatment of HF. Phosphatidylethanolamine-binding protein (PEBP)1 can trigger ferroptosis by mediating peroxide production, but how it modulates HSC ferroptosis is not known. We screened natural small molecules that could bind with PEBP1, and investigated the mechanism by which it promotes HSC ferroptosis. The maximum binding energy of berberine with PEBP1 was - 8.51 kcal/mol, indicating that berberine could bind strongly with PEBP1. Berberine binding to PEBP1 could promote HSC ferroptosis via synergy of its actions with those of sorafenib, but it could not induce ferroptosis alone. Combined administration of berberine enhanced the ferroptotic effects of low-dose sorafenib upon HSCs. Herein, we revealed that PEBP1 might be a target that could enhance the effects of sorafenib, which could provide a new therapeutic approach for HF treatment.

Membrane regulation of 15LOX-1/PEBP1 complex prompts the generation of ferroptotic signals, oxygenated PEs.

Ferroptosis is a regulated form of cell death, the mechanism of which is still to be understood. 15-lipoxygenase (15LOX) complex with phosphatidylethanolamine (PE)-binding protein 1 (PEBP1) catalyzes the generation of pro-ferroptotic cell death signals, hydroperoxy-polyunsaturated PE. We focused on gaining new insights into the molecular basis of these pro-ferroptotic interactions using computational modeling and liquid chromatography-mass spectrometry experiments. Simulations of 15LOX-1/PEBP1 complex dynamics and interactions with lipids revealed that association with the membrane triggers a conformational change in the complex. This conformational change facilitates the access of stearoyl/arachidonoyl-PE (SAPE) substrates to the catalytic site. Furthermore, the binding of SAPE promotes tight interactions within the complex and induces further conformational changes that facilitate the oxidation reaction. The reaction yields two hydroperoxides as products, 15-HpETE-PE and 12-HpETE-PE, at a ratio of 5:1. A significant effect of PEBP1 is observed only on the predominant product. Moreover, combined experiments and simulations consistently demonstrate the significance of PEBP1 P112E mutation in generating ferroptotic cell death signals.

Raf kinase inhibitor protein combined with phosphorylated extracellular signal-regulated kinase offers valuable prognosis in gastrointestinal stromal tumor.

BACKGROUND: Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. Tyrosine kinase inhibitors, such as imatinib, have been used as first-line therapy for the treatment of GISTs. Although these drugs have achieved considerable efficacy in some patients, reports of resistance and recurrence have emerged. Extracellular signal-regulated kinase 1/2 (ERK1/2) protein, as a member of the mitogen-activated protein kinase (MAPK) family, is a core molecule of this signaling pathway. Nowadays, research reports on the important clinical and prognostic value of phosphorylated-ERK (P-ERK) and phosphorylated-MAPK/ERK kinase (P-MEK) proteins closely related to raf kinase inhibitor protein (RKIP) have gradually emerged in digestive tract tumors such as gastric cancer, colon cancer, and pancreatic cancer. However, literature on the expression of these downstream proteins combined with RKIP in GIST is scarce. This study will focus on this aspect and search for answers to the problem. AIM: To detect the expression of RKIP, P-ERK, and P-MEK protein in GIST and to analyze their relationship with clinicopathological characteristics and prognosis of this disease. Try to establish a new prognosis evaluation model using RKIP and P-ERK in combination with analysis and its prognosis evaluation efficacy. METHODS: The research object of our experiment was 66 pathologically diagnosed GIST patients with complete clinical and follow-up information. These patients received surgical treatment at China Medical University Affiliated Hospital from January 2015 to January 2020. Immunohistochemical method was used to detect the expression of RKIP, P-ERK, and P-MEK proteins in GIST tissue samples from these patients. Kaplan-Meier method was used to calculate the survival rate of 63 patients with complete follow-up data. A Nomogram was used to represent the new prognostic evaluation model. The Cox multivariate regression analysis was conducted separately for each set of risk evaluation factors, based on two risk classification systems [the new risk grade model vs the modified National Institutes of Health (NIH) 2008 risk classification system]. Receiver operating characteristic (ROC) curves were used for evaluating the accuracy and efficiency of the two prognostic evaluation systems. RESULTS: In GIST tissues, RKIP protein showed positive expression in the cytoplasm and cell membrane, appearing as brownish-yellow or brown granules. The expression of RKIP was related to GIST tumor size, NIH grade, and mucosal invasion. P-ERK protein exhibited heterogeneous distribution in GIST cells, mainly in the cytoplasm, with occasional presence in the nucleus, and appeared as brownish-yellow granules, and the expression of P-ERK protein was associated with GIST tumor size, mitotic count, mucosal invasion, and NIH grade. Meanwhile, RKIP protein expression was negatively correlated with P-ERK expression. The results in COX multivariate regression analysis showed that RKIP protein expression was not an independent risk factor for tumor prognosis. However, RKIP combined with P-ERK protein expression were identified as independent risk factors for prognosis with statistical significance. Furthermore, we establish a new prognosis evaluation model using RKIP and P-ERK in combination and obtained the nomogram of the new prognosis evaluation model. ROC curve analysis also showed that the new evaluation model had better prognostic performance than the modified NIH 2008 risk classification system. CONCLUSION: Our experimental results showed that the expression of RKIP and P-ERK proteins in GIST was associated with tumor size, NIH 2008 staging, and tumor invasion, and P-ERK expression was also related to mitotic count. The expression of the two proteins had a certain negative correlation. The combined expression of RKIP and P-ERK proteins can serve as an independent risk factor for predicting the prognosis of GIST patients. The new risk assessment model incorporating RKIP and P-ERK has superior evaluation efficacy and is worth further practical application to validate.

RKIP localizes to the nucleus through a bipartite nuclear localization signal and interaction with importin α to regulate mitotic progression.

Raf kinase inhibitor protein (RKIP) is a multifunctional modulator of intracellular signal transduction. Although most of its functions have been considered cytosolic, we show here that the localization of RKIP is primarily nuclear in both growing and quiescent Madin-Darby canine kidney epithelial cells and in Cal-51 and BT-20 human breast cancer cells. We have identified a putative bipartite nuclear localization signal (NLS) in RKIP that maps to the surface of the protein surrounding a known regulatory region. Like classical NLS sequences, the putative NLS of RKIP is rich in arginine and lysine residues. Deletion of and point mutations in the putative NLS lead to decreased nuclear localization. Point mutation of all the basic residues in the putative NLS of RKIP particularly strongly reduces nuclear localization. We found consistent results in reexpression experiments with wildtype or mutant RKIP in RKIP-silenced cells. A fusion construct of the putative NLS of RKIP alone to a heterologous reporter protein leads to nuclear localization of the fusion protein, demonstrating that this sequence alone is sufficient for import into the nucleus. We found that RKIP interacts with the nuclear transport factor importin α in BT-20 and MDA-MB-231 human breast cancer cells, suggesting importin-mediated active nuclear translocation. Evaluating the biological function of nuclear localization of RKIP, we found that the presence of the putative NLS is important for the role of RKIP in mitotic checkpoint regulation in MCF-7 human breast cancer cells. Taken together, these findings suggest that a bipartite NLS in RKIP interacts with importin α for active transport of RKIP into the nucleus and that this process may be involved in the regulation of mitotic progression.

Photoaffinity labelling-based chemoproteomic strategy identifies PEBP1 as the target of ethyl gallate against macrophage activation.

Ulcerative colitis (UC) is an inflammatory disease of the colon with an unmet need for therapeutic targets. Ethyl gallate (EG) is a natural small molecule for UC treatment, but its cellular target is unknown. By labelling EG with a diazirine photocrosslinker and a click chemistry handle, we identified phosphatidyl-ethanolamine binding protein1 (PEBP1) as a direct cellular target of EG by forming hydrogen bonds with Asp70 and Tyr120. In particular, hydrogen/deuterium exchange mass spectrometry indicated that EG induced the sequence (residues 141-153) embedding to inhibit S153 phosphorylation of PEBP1. Additionally, the EG-mediated sequence (residues 108-122) exposure significantly enhanced PEBP1-Raf-1 interaction to block the downstream NF-κB inflammatory pathway in macrophages. Moreover, PEBP1 siRNA substantially reversed the EG-dependent down-regulation of the phosphorylation of IKKß, IκBα and NF-κB, demonstrating that the NF-κB signal functioned as an essential anti-inflammation mechanism of PEBP1. Collectively, we revealed PEBP1 as a previously undescribed cellular target in macrophages for UC therapy and identified a new allosteric site for PEBP1 biology study using EG as a chemical probe.

RKIP suppresses the influenza A virus­induced airway inflammatory response via the ERK/MAPK pathway.

Raf kinase inhibitor protein (RKIP) is an inflammation­inhibiting mediator that is involved in several diseases; however, the potential mechanism of action of RKIP on the inflammatory response induced by influenza A virus (IAV) remains unclear. The present study aimed to investigate whether RKIP regulated the inflammatory response via the ERK/MAPK pathway. The present study detected the expression levels of RKIP and alterations in the inflammatory response in human normal bronchial epithelial BEAS­2B cells, diseased human bronchial epithelial cells and primary human bronchial epithelial cells infected with IAV. Cells were treated with locostatin to inhibit the expression of RKIP. RKIP was overexpressed by lentivirus transduction and the small molecule inhibitor SCH772984 was applied to specifically inhibit activation of the ERK/MAPK pathway. In addition, C57BL/6 mice were infected with IAV to further confirm the role of RKIP in regulation of the inflammatory response via ERK/MAPK in vivo. Western blotting, reverse transcription­quantitative PCR, ELISA, 5­ethynyl­-2'­deoxyuridine assay, immunofluorescence staining, Cell Counting Kit­8, cell cycle assay, hematoxylin and eosin staining, and immunohistochemistry were used to detect all of the changes. Notably, RKIP attenuated the inflammatory response that was triggered by IAV infection in airway epithelial cells, which was characterized by augmented inflammatory cytokines and cell cycle arrest. Furthermore, the ERK/MAPK pathway was revealed to be activated by IAV infection and downregulation of RKIP aggravated the airway inflammatory response. By contrast, overexpression of RKIP effectively ameliorated the airway inflammatory response induced by IAV. These findings demonstrated that RKIP may serve a protective role in airway epithelial cells by combating inflammation via the ERK/MAPK pathway. Collectively, the present findings suggested that RKIP may negatively regulate airway inflammation and thus may constitute a promising therapeutic strategy for airway inflammatory­related diseases that are induced by IAV.

Vicagrel is hydrolyzed by Raf kinase inhibitor protein in human intestine.

As an analog of clopidogrel and prasugrel, vicagrel is completely hydrolyzed to intermediate thiolactone metabolite 2-oxo-clopidogrel (also the precursor of active thiol metabolite H4) in human intestine, predominantly by AADAC and CES2; however, other unknown vicagrel hydrolases remain to be identified. In this study, recombinant human Raf kinase inhibitor protein (rhRKIP) and pooled human intestinal S9 (HIS9) fractions and microsome (HIM) preparations were used as the different enzyme sources; prasugrel as a probe drug for RKIP (a positive control), vicagrel as a substrate drug of interest, and the rate of the formation of thiolactone metabolites 2-oxo-clopidogrel and R95913 as metrics of hydrolase activity examined, respectively. In addition, an IC50 value of inhibition of rhRKIP-catalyzed vicagrel hydrolysis by locostatin was measured, and five classical esterase inhibitors with distinct esterase selectivity were used to dissect the involvement of multiple hydrolases in vicagrel hydrolysis. The results showed that rhRKIP hydrolyzed vicagrel in vitro, with the values of Km , Vmax , and CLint measured as 20.04 ± 1.99 µM, 434.60 ± 12.46 nM/min/mg protein, and 21.69 ± 0.28 ml/min/mg protein, respectively, and that an IC50 value of locostatin was estimated as 1.24 ± 0.04 mM for rhRKIP. In addition to locostatin, eserine and vinblastine strongly suppressed vicagrel hydrolysis in HIM. It is concluded that RKIP can catalyze the hydrolysis of vicagrel in the human intestine, and that vicagrel can be hydrolyzed by multiple hydrolases, such as RKIP, AADAC, and CES2, concomitantly.

Dihydromyricetin Attenuates Cerebral Ischemia Reperfusion Injury by Inhibiting SPHK1/mTOR Signaling and Targeting Ferroptosis.

Background: Dihydromyricetin (DHM) exerts protective effects in various brain diseases. The aim of this research was to investigate the biological role of DHM in cerebral ischemia reperfusion (I/R) injury. Methods: We generated a rat model of cerebral I/R injury by performing middle cerebral artery occlusion/reperfusion (MCAO/R). The neurological score and brain water content of the experimental rats was then evaluated. The infarct volume and extent of apoptosis in brain tissues was then assessed by 2,3,5-triphenyltetrazolium (TTC) and TdT-mediated dUTP nick end labeling (TUNEL) staining. Hippocampal neuronal cells (HT22) were subjected to oxygen-glucose deprivation/reperfusion (OGD/R) and cell counting kit-8 (CCK-8) assays and flow cytometry were performed to detect cell viability and apoptosis. The levels of lipid reactive oxygen species (ROS) and iron were detected and the expression levels of key proteins were assessed by Western blotting. Results: DHM obviously reduced neurological deficits, brain water content, infarct volume and cell apoptosis in the brain tissues of MCAO/R rats. DHM repressed ferroptosis and inhibited the sphingosine kinase 1 (SPHK1)/mammalian target of rapamycin (mTOR) pathway in MCAO/R rats. In addition, DHM promoted cell viability and repressed apoptosis in OGD/R-treated HT22 cells. DHM also suppressed the levels of lipid ROS and intracellular iron in OGD/R-treated HT22 cells. The expression levels of glutathione peroxidase 4 (GPX4) was enhanced while the levels of acyl-CoA synthetase long-chain family member 4 (ACSL4) and phosphatidylethanolamine binding protein 1 (PEBP1) were reduced in OGD/R-treated HT22 cells in the presence of DHM. Moreover, the influence conferred by DHM was abrogated by the overexpression of SPHK1 or treatment with MHY1485 (an activator of mTOR). Conclusion: This research demonstrated that DHM repressed ferroptosis by inhibiting the SPHK1/mTOR signaling pathway, thereby alleviating cerebral I/R injury. Our findings suggest that DHM may be a candidate drug for cerebral I/R injury treatment.

PEBP4 Directs the Malignant Behavior of Hepatocellular Carcinoma Cells via Regulating mTORC1 and mTORC2.

Phosphatidylethanolamine binding protein 4 (PEBP4) is an understudied multifunctional small protein. Previous studies have shown that the expression of PEBP4 is increased in many cancer specimens, which correlates to cancer progression. The present study explored the mechanism by which PEBP4 regulates the growth and progression of hepatocellular carcinoma cells. Thus, we showed that knockdown of PEBP4 in MHCC97H cells, where its expression was relatively high, diminished activities of serine/threonine protein kinase B (PKB, also known as Akt), mammalian target of rapamycin complex 1(mTORC1), and mTORC2, events that were not restored by insulin-like growth factor 1 (IGF-1). Conversely, overexpression of PEBP4 in MHCC97L cells with the low endogenous level yielded opposite effects. Furthermore, physical association of PEBP4 with Akt, mTORC1, and mTORC2 was observed. Interestingly, introduction of AktS473D mutant, bypassing phosphorylation by mTORC2, rescued mTORC1 activity, but without effects on mTORC2 signaling. In contrast, the effect of PEBP4 overexpression on the activity of mTORC1 but not that of mTORC2 was suppressed by MK2206, a specific inhibitor of Akt. In conjunction, PEBP4 knockdown-engendered reduction of cell proliferation, migration and invasion was partially rescued by Akt S473D while increases in these parameters induced by overexpression of PEBP4 were completely abolished by MK2206, although the expression of epithelial mesenchymal transition (EMT) markers appeared to be fully regulated by the active mutant of Akt. Finally, knockdown of PEBP4 diminished the growth of tumor and metastasis, whereas they were enhanced by overexpression of PEBP4. Altogether, our study suggests that increased expression of PEBP4 exacerbates malignant behaviors of hepatocellular cancer cells through cooperative participation of mTORC1 and mTORC2.

Circular RNA circPOLR2A promotes clear cell renal cell carcinoma progression by facilitating the UBE3C-induced ubiquitination of PEBP1 and, thereby, activating the ERK signaling pathway.

BACKGROUND: Increasing evidence has demonstrated that circular RNAs (circRNAs) are implicated in cancer progression. However, the aberrant expression and biological functions of circRNAs in clear cell renal cell carcinoma (cRCC) remain largely elusive. METHOD: Differentially expressed circRNAs in cRCC were filtered via bioinformatics analysis. Aberrant circPOLR2A expression was validated in cRCC tissues and cell lines via qRT-PCR. Sanger sequencing was used to identify the backsplicing site of circPOLR2A. In vitro and in vivo functional experiments were performed to evaluate the role of circPOLR2A in cRCC malignancy. RNA pull-down, mass spectrometry, RIP, FISH and immunofluorescence assays were used to identify and validate the circPOLR2A-interacting proteins. Ubiquitination modification and interaction between proteins were detected via Co-IP and western blotting. The m6A modification in circPOLR2A was validated by the meRIP assay. RESULTS: Bioinformatics analysis revealed that circPOLR2A was highly expressed in cRCC tissues and metastatic cRCC tissues. CircPOLR2A expression was associated with tumor size and TNM stage in cRCC patients. In vitro and in vivo functional assays revealed that circPOLR2A accelerated cRCC cell proliferation, migration, invasion and angiogenesis, while inhibiting apoptosis. Further mechanistic research suggested that circPOLR2A could interact with UBE3C and PEBP1 proteins, and that UBE3C could act as a specific ubiquitin E3 ligase for the PEBP1 protein. The UBE3C/circPOLR2A/PEBP1 protein-RNA ternary complex enhanced the UBE3C-mediated ubiquitination and degradation of the PEBP1 protein which could inactivate the ERK signaling pathway. Rescue experiments revealed that the PEBP1 protein was the functional downstream target of circPOLR2A. Furthermore, m6A modification in circPOLR2A was confirmed, and the m6A reader YTHDF2 could regulate circPOLR2A expression. CONCLUSION: Our study demonstrated that circPOLR2A modulated the UBE3C-mediated ubiquitination and degradation of the PEBP1 protein, and further activated the ERK pathway during cRCC progression and metastasis. The m6A reader, YTHDF2, regulated circPOLR2A expression in cRCC. Hence, circPOLR2A could be a potential target for the diagnosis and treatment of cRCC.

Dioscorea nipponica Makino Relieves Ovalbumin-Induced Asthma in Mice through Regulating RKIP-Mediated Raf-1/MEK/MAPK/ERK Signaling Pathway.

Purpose: Dioscorea nipponica Makino (DNM) is a traditional herb with multiple medicinal functions. This study is aimed at exploring the therapeutic effects of DNM on asthma and the underlying mechanisms involving RKIP-mediated MAPK signaling pathway. Methods: An ovalbumin-induced asthma model was established in mice, which was further administrated with DNM and/or locostatin (RKIP inhibitor). ELISA was performed to detect the serum titers of OVA-IgE and OVA-IgG1, bronchoalveolar lavage fluid (BALF) levels of inflammation-related biomarkers, and tissue levels of oxidative stress-related biomarkers. The expression of RKIP was measured by quantitative real-time PCR, Western blot, immunohistochemistry, and immunofluorescence. HE staining was used to observe the pathological morphology of lung tissues. The protein expression of MAPK pathway-related proteins was detected by Western blot. Results: Compared with the controls, the model mice exhibited significantly higher serum titers of OVA-IgE and OVA-IgG1, BALF levels of IL-6, IL-8, IL-13, TGF-ß1, and MCP-1, tissue levels of MDA and ROS, lower BALF levels of IL-10 and IFN-γ, and tissue level of GSH. DNM relieved the allergic inflammatory response and oxidative stress in the model mice. DNM also recovered the downregulation of RKIP and the pathological injury of lung tissues in asthma mice. In addition, the Raf-1/MEK/MAPK/ERK pathway in the model mice was blocked by DNM. Silencing of RKIP by locostatin weakened the relieving effects of DNM on asthma through activating the Raf-1/MEK/MAPK/ERK pathway. Conclusion: DNM relieves asthma via blocking the Raf-1/MEK/MAPK/ERK pathway that mediated by RKIP upregulation.