Pyroptosis and the fight against lung cancer.

Pyroptosis, a newly characterized type of inflammatory programmed cell death (PCD), is usually triggered by multiple inflammasomes which can recognize different danger or damage-associated molecular patterns (DAMPs), leading to the activation of caspase-1 and the cleavage of gasdermin D (GSDMD). Gasdermin family pore-forming proteins are the executers of pyroptosis and are normally maintained in an inactive state through auto-inhibition. Upon caspases mediated cleavage of gasdermins, the pro-pyroptotic N-terminal fragment is released from the auto-inhibition of C-terminal fragment and oligomerizes, forming pores in the plasma membrane. This results in the secretion of interleukin (IL)-1ß, IL-18, and high-mobility group box 1 (HMGB1), generating osmotic swelling and lysis. Current therapeutic approaches including chemotherapy, radiotherapy, molecularly targeted therapy and immunotherapy for lung cancer treatment efficiently force the cancer cells to undergo pyroptosis, which then generates local and systemic antitumor immunity. Thus, pyroptosis is recognized as a new therapeutic regimen for the treatment of lung cancer. In this review, we briefly describe the signaling pathways involved in pyroptosis, and endeavor to discuss the antitumor effects of pyroptosis and its potential application in lung cancer therapy, focusing on the contribution of pyroptosis to microenvironmental reprogramming and evocation of antitumor immune response.

Jolkinolide B synergistically potentiates the antitumor activity of GPX4 inhibitors via inhibiting TrxR1 in cisplatin-resistant bladder cancer cells.

Glutathione peroxidase 4 (GPX4) is a promising anticancer therapeutic target; however, the application of GPX4 inhibitors (GPX4i) is limited owing to intrinsic or acquired drug resistance. Hence, understanding the mechanisms underlying drug resistance and discovering molecules that can overcome drug resistance are crucial. Herein, we demonstrated that GPX4i killed bladder cancer cells by inducing lipid reactive oxygen species-mediated ferroptosis and apoptosis, and cisplatin-resistant bladder cancer cells were also resistant to GPX4i, representing a higher half-maximal inhibitory concentration value than that of parent bladder cancer cells. In addition, thioredoxin reductase 1 (TrxR1) overexpression was responsible for GPX4i resistance in cisplatin-resistant bladder cancer cells, and inhibiting TrxR1 restored the sensitivity of these cells to GPX4i. In vitro and in vivo studies revealed that Jolkinolide B (JB), a natural diterpenoid and previously identified as a TrxR1 inhibitor, potentiated the antiproliferative efficacy of GPX4i (RSL3 and ML162) against cisplatin-resistant bladder cancer cells. Furthermore, GPX4 knockdown and inhibition could augment JB-induced paraptosis and apoptosis. Our results suggest that inhibiting TrxR1 can effectively improve GPX4 inhibition-based anticancer therapy. A combination of JB and GPX4i, which is well-tolerated and has several anticancer mechanisms, may serve as a promising therapy for treating bladder cancer.

Therapeutic potential of CB1R activation by Qingyangshen glycoside M1 for seizure relief.

ETHNOPHARMACOLOGICAL RELEVANCE: Cynanchum otophyllum C.K.Schneid.PI.Wilson, commonly referred as ''Qingyangshen'' (QYS), is a traditional folk medicine from Yunnan, renowned for its efficacy in neurological and psychiatric disorders. Glycosides isolated from QYS have shown promise in alleviating epilepsy, however, mechanisms of action and specific molecular targets remain to be elucidated. AIM OF THE STUDY: The study aimed to evaluate the anticonvulsant effects of Qingyangshen glycosides M1 (M1), a C21 steroidal glycoside from QYS, on pentylenetetrazol (PTZ)-induced convulsions in zebrafish (Danio rerio), and its neuroprotective effect on Glutamate (Glu)-induced damage to PC12 cells, and importantly to identify its potential molecular targets. MATERIALS AND METHODS: To evaluate anticonvulsant activity of M1, 7 days-post-fertilization (7-dpf) animals were pretreated (by immersion) and then exposed to PTZ (10 mM) solution. Furthermore, Glu-induced PC12 cell damage was employed to investigate the neuroprotective and anti-apoptotic capacity. Cells were pretreated with various concentrations of M1 (0-10 µM) for 12 h and then co-treated with Glu (15 mM) for an additional 24 h. The cell viability, apoptosis rate and apoptosis-related proteins (p-PI3K, PI3K, Akt, p-Akt, CREB, p-CREB, BDNF, Bax and Bcl-2) were measured using CCK-8, annexin V/PI and Western blot assays. To model the expected interaction between M1 and candidate cannabinoid receptor type 1 (CB1R), ERK phosphorylation, molecular docking, and drug affinity responsive target stability (DARTS) techniques were employed. Finally, CB1R antagonist Rimonabant (Rim) was validated by co-administration in both zebrafish and cells to confirm the requirement of CB1R for M1 efficacy. RESULTS: At a concentration of 400 µM, M1 dramatically reversed PTZ-induced convulsive-like behaviors in zebrafish, as evidenced by a significant reduction in locomotor activity. In the context of Glu-induced cytotoxicity, M1 (10 µM) demonstrated a notable increase in cell viability and suppressed apoptosis through modulation of the Bax/Bcl-2 ratio and activation of the PI3K/Akt/CREB/BDNF signaling axis. These effects were facilitated through CB1R activation. In contrast, Rim dampened the beneficial activities of M1 as a cannabinoid agonist. CONCLUSIONS: These results demonstrated that M1 as a potential CB1R activator, exhibiting anticonvulsive effects in a PTZ-induced zebrafish model and neuroprotective properties via the PI3K/Akt/CREB/BDNF signaling axis in a Glu-induced PC12 cell injury model. Notably, the observed seizure relief attenuated by CB1R chemical antagonism.

New animal model of chronic gout reproduces pathological features of the disease in humans.

OBJECTIVES: Gout, as the most prevalent form of inflammatory arthritis, necessitates the use of animal models to investigate the molecular mechanisms involved in its development. Therefore, our objective was to develop a novel chronic mouse model of gout that more closely mimics the progression of gout in humans. METHODS: A novel chronic mouse model of gout was established by a simple method, which does not require high technical proficiency, predominantly involves daily intraperitoneal injections of potassium oxonate for approximately 4 months, combined with a high fat-diet and injections of acetic acid into the hind paws to facilitate the formation of monosodium urate (MSU). Arthritis scores and paw oedema were assessed, behavioural tests were conducted, and histopathological and imaging evaluations of the arthritic paw joints were performed. RESULTS: After 4 months of induction, mice in the model group exhibited noticeable increases in arthritis severity, joint and cartilage damage, as well as bone erosion. Gomori's methenamine silver stain revealed the presence of MSU crystal deposition or tophi in the paw joints or ankle joints of up to 37.9% of the model mice (11 out of 29 mice). Moreover, treatment with benzbromarone effectively prevented the further development of gout or tophi formation in model mice. CONCLUSIONS: Our model more accurately replicates the pathological features of gouty arthritis compared with gout induced by MSU crystal injections. Therefore, it is particularly suitable for further investigations into the pathogenesis of gout and also serves as a valuable platform for screening potential antigout agents.

SODD Promotes Lung Cancer Tumorigenesis by Activating the PDK1/AKT and RAF/MEK/ERK Signaling.

BACKGROUND: The Bcl2-associated athanogene4 (BAG4/SODD) protein could be identified as a tumor marker for several malignancies and plays a major role in the occurrence, development, and drug resistance of tumors. However, the role of Silencer of death domains (SODD) in lung carcinogenesis is still elusive. OBJECTIVE: To illuminate the effect of SODD on the proliferation, migration, invasion, and apoptosis of lung cancer cells and tumor growth in vivo and explore the corresponding mechanism. METHODS: The expression of SODD in tumor and normal tissues was determined and compared via western blot. SODD gene knockout lung cancer cells (H1299 cells) were established through a CRISPR/Cas9 gene deleting system, and a transient SODD overexpression of H1299 cells was also constructed. Then, cell proliferation and invasion were assessed through colony formation and cell counting kit-8 assays, transwell migration assays, and wound healing assays. Cell drug sensitivity is also analyzed by Cell Counting Kit-8 assay. The flow cytometer was used to perform cell circle and apoptosis analysis. The interaction of SODD and RAF-1 was confirmed by co-immunoprecipitation, and the phosphorylated level of Phosphatidylinositol 3-kinase (PI3K), Serine/threonine-protein kinase (AKT), Rapidly accelerated fibrosarcoma (RAF)-1,and extracellular signal regulated kinase (ERK) in cells was examined by western blot to evaluate the activation of PI3K/PDK1/AKT and RAF/MEK/ERK pathways. In vivo, Xenograft tumor assay of SODD knockout H1299 cells was used to evaluate further the role of SODD on the proliferation of H1299 cells. RESULTS: SODD binds to RAF-1 and is over-expressed in lung tissues, and promotes the proliferation, migration, invasion, and drug sensitivity of H1299 cells. The reduced cells in the S phase and increased cells arrested in the G2/M phase were found in SODD knockout H1299 cells, and more cells got apoptosis. The expression of 3-phosphoinositide-dependent protein kinase 1(PDK1) protein in SODD knockout H1299 cells decreases distinctively, and the phosphorylated level of AKT, RAF-1, and ERK-1 kinase in SODD knockout H1299 cells is also less than that in normal H1299 cells. In contrast, SODD overexpression significantly increases the phosphorylation of AKT. In vivo, SODD promotes the tumorigenicity of H1299 cells in nude mice. CONCLUSIONS: SODD is overexpressed in lung tissues and plays a considerable role in the development and progression of lung cancer by regulating the PI3K/PDK1/AKT and RAF/MEK/ERK pathways.

Challenges and Perspectives in Target Identification and Mechanism Illustration for Chinese Medicine.

Chinese medicine (CM) is an important resource for human life understanding and discovery of drugs. However, due to the unclear pharmacological mechanism caused by unclear target, research and international promotion of many active components have made little progress in the past decades of years. CM is mainly composed of multi-ingredients with multi-targets. The identification of targets of multiple active components and the weight analysis of multiple targets in a specific pathological environment, that is, the determination of the most important target is the main obstacle to the mechanism clarification and thus hinders its internationalization. In this review, the main approach to target identification and network pharmacology were summarized. And BIBm (Bayesian inference modeling), a powerful method for drug target identification and key pathway determination was introduced. We aim to provide a new scientific basis and ideas for the development and international promotion of new drugs based on CM.

Dictamnine, a novel c-Met inhibitor, suppresses the proliferation of lung cancer cells by downregulating the PI3K/AKT/mTOR and MAPK signaling pathways.

Dictamnine (Dic), a naturally occurring small-molecule furoquinoline alkaloid isolated from the root bark of Dictamnus dasycarpus Turcz., is reported to display anticancer properties. However, little is known about the direct target proteins and anticancer mechanisms of Dic. In the current study, Dic was found to suppress the growth of lung cancer cells in vitro and in vivo, and to attenuate the activation of PI3K/AKT/mTOR and mitogen-activated protein kinase (MAPK) signaling pathways by inhibiting the phosphorylation and activation of receptor tyrosine kinase c-Met. Moreover, the binding of Dic to c-Met was confirmed by using cellular thermal shift assay (CETSA) and drug affinity responsive target stability (DARTS) assay. Among all cancer cell lines tested, Dic inhibited the proliferation of c-Met-dependent EBC-1 cells with the greatest potency (IC50 = 2.811 µM). Notably, Dic was shown to synergistically improve the chemo-sensitivity of epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI)-resistant lung cancer cells to gefitinib and osimertinib. These results suggest that Dic is a c-Met inhibitor that can serve as a potential therapeutic agent in the treatment of lung cancer, especially against EGFR TKI-resistant and c-Met-dependent lung cancer.

A Novel 5-HT1B Receptor Agonist of Herbal Compounds and One of the Therapeutic Uses for Alzheimer's Disease.

The serotonin receptor 5-HT1B is widely expressed in the central nervous system and has been considered a drug target in a variety of cognitive and psychiatric disorders. The anti-inflammatory effects of 5-HT1B agonists may present a promising approach for Alzheimer's disease (AD) treatment. Herbal antidepressants used in the treatment of AD have shown functional overlap between the active compounds and 5-HT1B receptor stimulation. Therefore, compounds in these medicinal plants that target and stimulate 5-HT1B deserve careful study. Molecular docking, drug affinity responsive target stability, cellular thermal shift assay, fluorescence resonance energy transfer (FRET), and extracellular regulated protein kinases (ERK) 1/2 phosphorylation tests were used to identify emodin-8-O-ß-d-glucopyranoside (EG), a compound from Chinese medicinal plants with cognitive deficit attenuating and antidepressant effects, as an agonist of 5-HT1B. EG selectively targeted 5-HT1B and activated the 5-HT1B-induced signaling pathway. The activated 5-HT1B pathway suppressed tumor necrosis factor (TNF)-α levels, thereby protecting neural cells against beta-amyloid (Aß)-induced death. Moreover, the agonist activity of EG towards 5-HT1B receptor, in FRET and ERK1/2 phosphorylation, was antagonized by SB 224289, a 5-HT1B antagonist. In addition, EG relieved AD symptoms in transgenic worm models. These results suggested that 5-HT1B receptor activation by EG positively affected Aß-related inflammatory process regulation and neural death resistance, which were reversed by antagonist SB 224289. The active compounds such as EG might act as potential therapeutic agents through targeting and stimulating 5-HT1B receptor for AD and other serotonin-related disorders. This study describes methods for identification of 5-HT1B agonists from herbal compounds and for evaluating agonists with biological functions, providing preliminary information on medicinal herbal pharmacology.

Akt scaffold proteins: the key to controlling specificity of Akt signaling.

The phosphatidylinositol 3-kinase-Akt signaling pathway plays an essential role in regulating cell proliferation and apoptosis. Akt kinase is at the center of this signaling pathway and interacts with a variety of proteins. Akt is overexpressed in almost 80% of tumors. However, inhibiting Akt has serious clinical side effects so is not a suitable treatment for cancer. During recent years, Akt scaffold proteins have received increasing attention for their ability to regulate Akt signaling and have emerged as potential targets for cancer therapy. In this paper, we categorize Akt kinase scaffold proteins into four groups based on their cellular location: membrane-bound activator and inhibitor, cytoplasm, and endosome. We describe how these scaffolds interact with Akt kinase, how they affect Akt activity, and how they regulate the specificity of Akt signaling. We also discuss the clinical application of Akt scaffold proteins as targets for cancer therapy.

IRS4 promotes the progression of non-small cell lung cancer and confers resistance to EGFR-TKI through the activation of PI3K/Akt and Ras-MAPK pathways.

IRS4 is a member of the insulin receptor substrate (IRS) protein family. It acts as a cytoplasmic adaptor protein, integrating and transmitting signals from receptor protein tyrosine kinases to the intracellular environment. IRS4 can induce mammary tumorigenesis and is usually overexpressed in non-small cell lung cancer (NSCLC). However, little is known about the role of IRS4 in the development and progression of lung cancer. In this study, we show that IRS4 knockout suppresses the proliferation, colony formation, migration, and invasion of A549 lung cancer cells, as well as tumor growth in a nude mouse xenograft model. In contrast, stable expression of IRS4 showed the opposite effects. As expected, IRS4 was found to activate the PI3K/Akt and Ras-MAPK pathways, and we also showed that IRS4 depletion significantly enhanced the sensitivity of EGFR tyrosine kinase inhibitor (EGFR-TKI)-resistant cells to gefitinib. Taken together, these results show that IRS4 promotes NSCLC progression and may represent a potential therapeutic target for EGFR-TKI-resistant NSCLC.

The C-terminal of the α1b-adreneroceptor is a key determinant for its structure integrity and biological functions.

The C-terminal of G protein-coupled receptors is now recognized as being important for G protein activation and signaling function. To detect the role of C-terminal tail in receptor activation, we used the α1b-AR, which has a long C-terminal of 164 amino acids. We constructed the intramolecular FRET sensors, in which the C-terminal was truncated to 10 (∆C-10), 20 (∆C-20), 30 (∆C-30), 50 (∆C-50), 70 (∆C-70), or 90 (∆C-90). The truncated mutants of ∆C-10, ∆C-20, or ∆C-30 cannot induce FRET signal changes and downstream ERK1/2 phosphorylation. However, the truncated mutants of ∆C-50, ∆C-70, or ∆C-90 induce significant FRET signal changes and downstream ERK1/2 phosphorylation, especially ∆C-90. This is particularly true in the case of the ∆C-90, ∆C-70, or ∆C-50 which retained the potential phosphorylation sites (Ser401, Ser404, Ser408, or Ser410). The ∆C-90 showed an increase in agonist-induced FRET signal changes and ERK1/2 phosphorylation in PKC- or endocytosis-dependent and EGFR-, src-, or ß-arrestin2-independent.

Effects of ultrasonic pre-treatment on physicochemical properties of proteins extracted from cold-pressed sesame cake.

Sesame is an oil crop with high nutritional value. Protein is one of the main ingredients of sesame, however research on protein of cold-pressed sesame cake is limited. This study aimed to investigate the effects of ultrasonic pre-treatment (UPT) on physicochemical properties of proteins (yield, solubility, amino acid composition, surface properties, structural and thermal stability) extracted from the cold-pressed sesame cake, after removing lignans by ultrasonic-assisted extraction. By comparison, the extraction yield of protein was significantly (p ＜ 0.05) increased from 22.24% (without UPT) to 25.95% (with UPT), while the purity (54.08% without UPT, 55.43% with UPT), total amount of essential amino acids (22.48% without UPT, 23.10% with UPT) and non-essential amino acids (37.48% without UPT, 36.54% with UPT) were not significantly influenced. Besides, UPT slightly reduced the solubility, foaming capacity and stability (FC and FS) of protein. In addition, scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR) and thermal stability (TG) analysis demonstrated that UPT could disorder and loose protein molecular structure, resulting in the change of morphology, secondary structure and thermal stability. In conclusion, this study provides a way for the separation and future application of sesame cake protein. UPT is a good option to remove the lignans from sesame cake proteins.

HDAC1-Smad3-mSin3A complex is required for Smad3-induced transcriptional inhibition of hepatocyte growth factor receptor in human lung cancers.

c-Met hyperactivity has been observed in numerous neoplasms. Several researchers have shown that the abnormal activation of c-Met is mainly caused by transcriptional activation. However, the molecular mechanism behind this transcriptional regulation is poorly understood. Here, we suggest that Smad3 negatively regulates the expression and activation of c-Met via a transcriptional mechanism. We explore the molecular mechanisms that underlie Smad3-induced c-Met transcription inhibition. We found in contrast to the high expression of c-Met, Smad3 showed low protein and mRNA levels. Smad3 and c-Met expressions were inconsistent between lung cancer tissues and cell lines. We also found that Smad3 overexpression suppresses whereas Smad3 knockdown significantly promotes Epithelial-Mesenchymal Transition and production of the angiogenic factors VEGF, CTGF and COX-2 through the ERK1/2 pathway. In addition, Smad3 overexpression decreases whereas Smad3 knockdown significantly increases protein and mRNA levels of invasion-related ß-catenin and FAK through the PI3K/Akt pathway. Furthermore, using the chromatin immunoprecipitation analysis method, we demonstrate that a transcriptional regulatory complex consisting of HDAC1, Smad3 and mSin3A binds to the promoter of the c-Met gene. By either silencing endogenous mSin3A expression with siRNA or by pretreating cells with a specific HDAC1 inhibitor (MS-275), Smad3-induced transcriptional suppression of c-Met could be effectively attenuated. These results demonstrate that Smad3-induced inhibition of c-Met transcription depends on of a functional transcriptional regulatory complex that includes Smad3, mSin3A and HDAC1 at the c-Met promoter. Collectively, our findings reveal a new regulatory mechanism of c-Met signaling, and suggest a potential molecular target for the development of anticancer drugs.

Clinical use of vascular endothelial growth factor receptor inhibitors for the treatment of renal cell carcinoma.

In recent years, there have been increased incidences of metastatic renal cell carcinoma (RCC), which is refractory to conventional chemotherapy. Owing to the insensitivity to traditional therapy, targeted therapy becomes a possible alternative strategy. Over the past decade, the development of targeted treatments for metastatic RCC has advanced considerably. Several studies have shown that the vascular endothelial growth factor pathway is an important mediator for the occurrence and development of RCC, and tyrosine kinase inhibitors (TKIs) that target vascular endothelial growth factor receptors (VEGFRs) have been considered optimal therapeutic options for RCC. Six small molecules that inhibit VEGFR1/2/3, namely, sunitinib, sorafenib, axitinib, pazopanib, cabozantinib, and lenvatinib, have been approved by the Food and Drug Administration (FDA) for the treatment of RCC. Additionally, clinical trials assessing seven TKIs that target VEGFRs are currently in progress. To some extent, these drugs improve quality of life and prolong the survival of patients. This paper presents a review of the systemic targeted therapies against VEGFRs that have been approved so far or are undergoing trials as treatments for RCC.

The Application of the RNA Interference Technologies for KRAS: Current Status, Future Perspective and Associated Challenges.

KRAS is a member of the murine sarcoma virus oncogene-RAS gene family. It plays an important role in the prevention, diagnosis and treatment of tumors during tumor cell growth and angiogenesis. KRAS is the most commonly mutated oncogene in human cancers, such as pancreatic cancers, colon cancers, and lung cancers. Detection of KRAS gene mutation is an important indicator for tracking the status of oncogenes, highlighting the developmental prognosis of various cancers, and the efficacy of radiotherapy and chemotherapy. However, the efficacy of different patients in clinical treatment is not the same. Since RNA interference (RNAi) technologies can specifically eliminate the expression of specific genes, these technologies have been widely used in the field of gene therapy for exploring gene function, infectious diseases and malignant tumors. RNAi refers to the phenomenon of highly specific degradation of homologous mRNA induced by double-stranded RNA (dsRNA), which is highly conserved during evolution. There are three classical RNAi technologies, including siRNA, shRNA and CRISPR-Cas9 system, and a novel synthetic lethal interaction that selectively targets KRAS mutant cancers. Therefore, the implementation of individualized targeted drug therapy has become the best choice for doctors and patients. Thus, this review focuses on the current status, future perspective and associated challenges in silencing of KRAS with RNAi technology.

Comparative interactome analysis reveals distinct and overlapping properties of Raf family kinases.

The three mammalian Raf proteins (A-Raf, B-Raf, and C-Raf) are key components of the MAPK pathway. Although diverse functions have been proposed for Raf kinases, it is still not clear how interacting proteins contribute to differences in the signaling functions of the three Raf kinases. Here, we report the comparative interactomes of the three Raf kinases under serum-starved and EGF-stimulated conditions. We identified nearly 400 novel interacting proteins; some interacted with all three isoforms while others interacted exclusively with one or two. Comparing the interactomes of the three Raf kinases under different conditions revealed Raf proteins perform distinct functions through specific interactions. Our interactome data help define the differences between the three Raf kinases and may uncover new functions or regulatory mechanisms. Knowledge of Raf kinase protein-protein interactions will help us to investigate the function of specific pathways in the future.

[Research progress on pathophysiological function of SOS1 protein].

Son of sevenless homolog 1 (SOS1) protein is a ubiquitously expressed adapter. As a key protein in intracellular signaling, SOS1 plays an important role in many signal transduction pathways, such as Ras and Rac signaling pathways. The abnormal expression or mutation of SOS1 is closely related to clinical diseases. In this article, we review research progress on SOS1 functions and its roles in physiology and pathophysiology.

KAP1 inhibits the Raf-MEK-ERK pathway to promote tumorigenesis in A549 lung cancer cells.

Aberrant activation of the Raf-MEK-ERK pathway has frequently been associated with various cancers, especially lung cancer. However, the key regulators of this pathway are largely unknown. Using functional proteomics screening, we found that KAP1 interacts with c-Raf. Knocking out KAP1 decreased c-Raf phosphorylation at serine 259 and increased its phosphorylation at serine 338, which activated MEK and ERK. We detected higher KAP1 expression in lung cancer tissues than in normal peri-tumoral tissues. KAP1 knockdown arrested A549 lung cancer cells in the G0/G1 phase of the cell cycle and attenuated cell growth, metastasis, the epithelial-mesenchymal transition, angiogenesis, stemness, and colony formation. Furthermore, knocking out KAP1 remarkably increased the susceptibility of A549 cells to the anti-cancer drug 5-Fluorouracil, which correlated with increasing ERK phosphorylation. In vivo xenograft experiments suggested that KAP1 deficiency significantly decreases the tumorigenicity of A549 cells. Taken together, our findings indicate that KAP1 acts as a key module in the c-Raf-interactome complex and regulates lung cancer development through the Raf-MEK-ERK pathway. Therefore, KAP1 may represent a potential diagnosis biomarker and new treatment target for lung cancer.

Revealing A-Raf functions through its interactome.

A-Raf is a member of the Raf kinase family. Unlike B-Raf and C-Raf, the functions of A-Raf remain obscure. To gain more insight into the biological functions of A-Raf, we investigated the A-Raf interactome using proteomics. We found 132 proteins that interact with A-Raf and confirmed the interaction of 12 of these proteins with A-Raf by western blotting. Our data suggested that A-Raf regulates apoptosis, RNA catabolism, GTPase activity, and cell adhesion by interacting with proteins located in different cellular compartments. We identified all ten hallmarks of cancer in these interacting proteins, suggesting that A-Raf is involved in carcinogenesis. Our results also indicated that A-Raf may play a role in different diseases and signaling pathways. These findings have identified potential regulators of A-Raf and provide a systemic insight into its biological functions.

Visualization of the activation of the histamine H3 receptor (H3R) using novel fluorescence resonance energy transfer biosensors and their potential application to the study of H3R pharmacology.

Activation of the histamine-3 receptor (H3R) is involved in memory processes and cognitive action, while blocking H3R activation can slow the progression of neurological disorders, such as Alzheimer's disease, schizophrenia and narcolepsy. To date, however, no direct way to examine the activation of H3R has been utilized. Here, we describe a novel biosensor that can visualize the activation of H3R through an intramolecular fluorescence resonance energy transfer (FRET) signal. To achieve this, we constructed an intramolecular H3R FRET sensor with cyan fluorescent protein (CFP) attached at the C terminus and yellow fluorescent protein (YFP) inserted into the third intracellular loop. The sensor was found to internalize normally on agonist treatment. We measured FRET signals between the donor CFP and the acceptor YFP in living cells in real time, the results of which indicated that H3R agonist treatment (imetit or histamine) increases the FRET signal in a time- and concentration-dependent manner with Kon and Koff values consistent with published data and which maybe correlated with decreasing cAMP levels and the promotion of ERK1/2 phosphorylation. The FRET signal was inhibited by H3R antagonists, and the introduction of mutations at F419A, F423A, L426A and L427A, once again, the promotion of ERK1/2 phosphorylation, was diminished. Thus, we have built a H3R biosensor which can visualize the activation of receptor through real-time structure changes and which can obtain pharmacological kinetic data at the same time. The FRET signals may allow the sensor to become a useful tool for screening compounds and optimizing useful ligands.