Unveiling the domain-specific and RAS isoform-specific details of BRAF kinase regulation.

BRAF is a key member in the MAPK signaling pathway essential for cell growth, proliferation, and differentiation. Mutant BRAF is often the underlying cause of various types of cancer and mutant RAS, the upstream regulator of BRAF, is a driver of up to one-third of all cancers. BRAF interacts with RAS and undergoes a conformational change from an inactive, autoinhibited monomer to an active dimer, which propagates downstream signaling. Because of BRAF's complex regulation mechanism, the exact order and magnitude of its activation steps have yet to be confirmed experimentally. By studying the inter- and intramolecular interactions of BRAF, we unveil the domain-specific and isoform-specific details of BRAF regulation through pulldown assays, open surface plasmon resonance (OpenSPR), and hydrogen-deuterium exchange mass spectrometry (HDX-MS). We demonstrate that the BRAF specific region (BSR) and cysteine rich domain (CRD) play a crucial role in regulating the activation of BRAF in a RAS isoform-specific manner. Moreover, we quantified the binding affinities between BRAF N-terminal and kinase domains (KD) to reveal their individual roles in autoinhibition. Our findings also indicate that oncogenic BRAF-KDD594G mutant has a lower affinity for the N-terminal domains, implicating that pathogenic BRAF acts through decreased propensity for autoinhibition. Collectively, our study provides valuable insight into the activation mechanism of BRAF kinase to guide the development of new therapeutic strategies for cancer treatment.

Unveiling the Domain-Specific and RAS Isoform-Specific Details of BRAF Regulation.

BRAF is a key member in the MAPK signaling pathway essential for cell growth, proliferation, and differentiation. Dysregulation or mutation of BRAF is often the underlying cause of various types of cancer. RAS, a small GTPase protein that acts upstream of BRAF, has been identified as a driver of up to one-third of all cancers. When BRAF interacts with RAS via the RAS binding domain (RBD) and membrane recruitment, BRAF undergoes a conformational change from an inactive, autoinhibited monomer to an active dimer and subsequently phosphorylates MEK to propagate the signal. Despite the central role of BRAF in cellular signaling, the exact order and magnitude of its activation steps has yet to be confirmed experimentally. By studying the inter- and intramolecular interactions of BRAF, we unveil the domain-specific and isoform-specific details of BRAF regulation. We employed pulldown assays, open surface plasmon resonance (OpenSPR), and hydrogen-deuterium exchange mass spectrometry (HDX-MS) to investigate the roles of the regulatory regions in BRAF activation and autoinhibition. Our results demonstrate that the BRAF specific region (BSR) and cysteine rich domain (CRD) play a crucial role in regulating the activity of BRAF. Moreover, we quantified the autoinhibitory binding affinities between the N-terminal domains and the kinase domain (KD) of BRAF and revealed the individual roles of the BRAF regulatory domains. Additionally, our findings provide evidence that the BSR negatively regulates BRAF activation in a RAS isoform-specific manner. Our findings also indicate that oncogenic BRAF-KDD594G mutant has a lower affinity for the regulatory domains, implicating that pathogenic BRAF acts through decreased propensity for autoinhibition. Collectively, our study provides valuable insights into the activation mechanism of BRAF kinase and may help to guide the development of new therapeutic strategies for cancer treatment.

Isoflurane and Sevoflurane Induce Cognitive Impairment in Neonatal Rats by Inhibiting Neural Stem Cell Development Through Microglial Activation, Neuroinflammation, and Suppression of VEGFR2 Signaling Pathway.

Inhaled anesthetics are known to induce neurotoxicity in the developing brains of rodents, although the mechanisms are not well understood. The aim of this study was to elucidate the molecular mechanisms underlying anesthetics-induced neurodevelopmental toxicity by VEGF receptor 2 (VEGFR2) through the interaction between microglia and neural stem cells (NSCs) in postnatal day 7 (P7) rats. Cognitive function of P7 rats exposed to isoflurane and sevoflurane were assessed using Morris Water Maze and T maze tests. We also evaluated the expression levels of NSC biomarkers (Nestin and Sox2), microglia biomarker (CD11b or or IBA1), pro-inflammatory cytokines (IL-6 and TNF-α), and VEGFR2 using western blotting and immunohistochemistry in the brains of control and anesthesia-treated rats. We found spatial learning and working memory was impaired 2 weeks after anesthetics exposure in rats. Isoflurane induced stronger and more prolonged neurotoxicity than sevoflurane. However, cognitive functions were recovered 6 weeks after anesthesia. Isoflurane and sevoflurane decreased the levels of Nestin, Sox2, and p-VEGFR2, activated microglia, decreased the number of NSCs and reduced neurogenesis and the proliferation of NSCs, and increased the levels of IL-6, TNF-α, and CD11b. Our results suggested that isoflurane and sevoflurane induced cognitive impairment in rats by inhibiting NSC development and neurogenesis via microglial activation, neuroinflammation, and suppression of VEGFR2 signaling pathway.

Mechanism of BRAF Activation through Biochemical Characterization of the Recombinant Full-Length Protein.

BRAF kinase plays an important role in mitogen-activated protein kinase (MAPK) signaling and harbors activating mutations in about half of melanomas and in a smaller percentage in many other cancers. Despite its importance, few in vitro studies have been performed to characterize the biochemical properties of full-length BRAF. Herein, a strategy to generate an active, intact form of BRAF protein suitable for in vitro enzyme kinetics is described. It is shown that purified, intact BRAF protein autophosphorylates the kinase activation loop and this can be enhanced by binding the MEK protein substrate through an allosteric mechanism. These studies provide in vitro evidence that BRAF selectively binds to active RAS and that the BRAF/CRAF heterodimer is the most active form, relative to their respective homodimers. Full-length BRAF analysis with small-molecule BRAF inhibitors shows that two drugs, dabrafenib and vemurafenib, can modestly enhance kinase activity of BRAF at low concentration. Taken together, this characterization of intact BRAF contributes to a framework for understanding its role in cell signaling.

The separation of flavonoids from Pongamia pinnata using combination columns in high-speed counter-current chromatography with a three-phase solvent system.

The mangrove plant Pongamia pinnata (Leguminosae) is well known as a plant pesticide. Previous studies have indicated that the flavonoids are responsible of the biological activities of the plant. A new high-speed counter-current chromatography (HSCCC) method for the separation of three flavonoids, karanjin (1), pinnatin (2), and pongaflavone (3), from P. pinnata was developed in the present study. The lower and intermediate phase (LP and IP) of a new three-phase solvent system, n-hexane-acetonitrile-dichloromethane-water, at a volume ratio of 5:5:1:5, were used as the stationary phases, while the upper phase (UP) was used as the mobile phase, and the volume ratio between the stationary phases in the CCC column could be tuned by varying the initial pumped volume ratio of the stationary phases. The CCC columns containing all three phases of the solvent system were considered combination columns. According to the theories of combination column, it is possible to optimize the retention time of the target compounds by varying the volume ratio of the stationary phases in the HSCCC combination columns, as well as the suitable volume ratios of the stationary phases for the separation of the target compounds were predicted from the partition coefficients of the compounds in the three-phase solvent system. Then, three HSCCC separations using the combination columns with initial pumped LP:IP volume ratios of 1:0, 0.9:0.1, and 0.7:0.3 were performed separately based on the prediction. Three target compounds were prepared with high purity when the initial pumped volume ratio of the stationary phases was 0.9:0.1. The baseline separation of compounds 2 and 3 was achieved on the combination column with an initial pumped volume ratio of 0.7:0.3. Furthermore, the three experiments clearly demonstrated that the retentions and resolutions of the target compounds increased with an increasing volume ratio of IP, which is consistent with the prediction for the retention times for the solutes on combination columns. The method proposed here reduces the need for solvent selection compared with the conventional method and may have broad potential applicability in the preparation of natural products.

Pongaflavanol: a prenylated flavonoid from Pongamia pinnata with a modified ring A.

A new prenylated flavon-4-ol with a modified ring A, which we have named pongaflavanol (1), was isolated from the stem bark of Pongamia pinnata along with the known compound tunicatachalcone (2). The structure of compound 1 was elucidated on the basis of spectroscopic data.

A new phenylethanoid glycoside from Clerodendrum inerme.

A new phenylethanoid glycoside, 2-(3-methoxy-4-hydroxylphenyl) ethyl-O-2",3"-diacetyl-alpha-L-rhamnopyranosyl-(1-->3)-4-O-(E)-feruloyl-beta-D-glucopyranoside, was isolated from the aerial parts of Clerodendrum inerme (L.) Gaertn, together with monomelittoside, melittoside, inerminoside A1, verbascoside, isoverbascoside, campneoside I. Their structures were determined by spectroscopic methods.