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The synthesis of indazoles from pyrazoles and internal alkynes is described. Instead of complex benzenoid compounds, readily available pyrazoles were used for the preparation of indazoles by reaction of the C-H bonds of the heterocyclic ring. Oxidative benzannulation was also applied to imidazoles, providing benzimidazoles. This convergent strategy enabled alteration of the photochemical properties of benzo-fused diazoles by varying the substituents at the benzene ring, thus leading to the development of tetraarylindazoles as new fluorophores.

Pd-catalyzed C–H annulation reactions of halo- and aryl-heteroarenes were developed using readily available o-bromobiaryls and o-dibromoaryls, respectively. A variety of five-membered heteroarenes rapidly provided the corresponding phenanthrene-fused heteroarenes, which led to the identification of phenanthro-pyrazole and thiazole as new, stable −2 V redox couples. The flexible syntheses and tunability of the redox potentials of these azole-fused phenanthrenes over a wide range are expected to facilitate their application as redox-active organic functional materials.

Elevated levels of the free fatty acid palmitate are found in the plasma of obese patients and induce insulin resistance. Skeletal muscle secretes myokines as extracellular signaling mediators in response to pathophysiological conditions. Here, we identified and characterized the skeletal muscle secretome in response to palmitate-induced insulin resistance. Using a quantitative proteomic approach, we identified 36 secretory proteins modulated by palmitate-induced insulin resistance. Bioinformatics analysis revealed that palmitate-induced insulin resistance induced cellular stress and modulated secretory events. We found that the decrease in the level of annexin A1, a secretory protein, depended on palmitate, and that annexin A1 and its receptor, formyl peptide receptor 2 agonist, played a protective role in the palmitate-induced insulin resistance of L6 myotubes through PKC-θ modulation. In mice fed with a high-fat diet, treatment with the formyl peptide receptor 2 agonist improved systemic insulin sensitivity. Thus, we identified myokine candidates modulated by palmitate-induced insulin resistance and found that the annexin A1- formyl peptide receptor 2 pathway mediated the insulin resistance of skeletal muscle, as well as systemic insulin sensitivity.

Ras homolog enriched in brain (Rheb) regulates diverse cellular functions by modulating its nucleotide-bound status. Although Rheb contains a high basal GTP level, the regulatory mechanism of Rheb is not well understood. In this study, we propose soluble αβ-tubulin acts as a constitutively active Rheb activator, which may explain the reason why Rheb has a high basal GTP levels. We found that soluble αβ-tubulin is a direct Rheb-binding protein and that its deacetylated form has a high binding affinity for Rheb. Modulation of both soluble and acetylated αβ-tubulin levels affects the level of GTP-bound Rheb. This occurs in the mitotic phase in which the level of acetylated αβ-tubulin is increased but that of GTP-bound Rheb is decreased. Constitutively active Rheb-overexpressing cells showed an abnormal mitotic progression, suggesting the deacetylated αβ-tubulin-mediated regulation of Rheb status may be important for proper mitotic progression. Taken together, we propose that deacetylated soluble αβ-tubulin is a novel type of positive regulator of Rheb and may play a role as a temporal regulator for Rheb during the cell cycle.

NHERF1/EBP50 (Na+/H+ exchanger regulating factor 1; Ezrin-binding phosphoprotein of 50 kDa) organizes stable protein complexes beneath the apical membrane of polar epithelial cells. By contrast, in cancer cells without any fixed polarity, NHERF1 often localizes in the cytoplasm. The regulation of cytoplasmic NHERF1 and its role in cancer progression remain unclear. In this study, we found that, upon lysophosphatidic acid (LPA) stimulation, cytoplasmic NHERF1 rapidly translocated to the plasma membrane, and subsequently to cortical protrusion structures, of ovarian cancer cells. This movement depended on direct binding of NHERF1 to C-terminally phosphorylated ERM proteins (cpERMs). Moreover, NHERF1 depletion downregulated cpERMs and further impaired cpERM-dependent remodeling of the cell cortex, suggesting reciprocal regulation between these proteins. The LPA-induced protein complex was highly enriched in migratory pseudopodia, whose formation was impaired by overexpression of NHERF1 truncation mutants. Consistent with this, NHERF1 depletion in various types of cancer cells abolished chemotactic cell migration toward a LPA gradient. Taken together, our findings suggest that the high dynamics of cytosolic NHERF1 provide cancer cells with a means of controlling chemotactic migration. This capacity is likely to be essential for ovarian cancer progression in tumor microenvironments containing LPA.

Phospholipases (PLC, PLD and PLA) are essential mediators of intracellular and intercellular signalling. They can function as phospholipid-hydrolysing enzymes that can generate many bioactive lipid mediators, such as diacylglycerol, phosphatidic acid, lysophosphatidic acid and arachidonic acid. Lipid mediators generated by phospholipases regulate multiple cellular processes that can promote tumorigenesis, including proliferation, migration, invasion and angiogenesis. Although many individual phospholipases have been extensively studied, how phospholipases regulate diverse cancer-associated cellular processes and the interplay between different phospholipases have yet to be fully elucidated. A thorough understanding of the cancer-associated signalling networks of phospholipases is necessary to determine whether these enzymes can be targeted therapeutically.

There is a strong possibility that skeletal muscle can respond to irregular metabolic states by secreting specific cytokines. Obesity-related chronic inflammation, mediated by pro-inflammatory cytokines, is believed to be one of the causes of insulin resistance that results in type 2 diabetes. Here, we attempted to identify and characterize the members of the skeletal muscle secretome in response to tumor necrosis factor-alpha (TNF-α)-induced insulin resistance. To conduct this study, we comparatively analyzed the media levels of proteins released from L6 skeletal muscle cells. We found 28 TNF-α modulated secretory proteins by using separate filtering methods: Gene Ontology, SignalP, and SecretomeP, as well as the normalized Spectral Index for label-free quantification. Ten of these secretory proteins were increased and 18 secretory proteins were decreased by TNF-α treatment. Using microarray analysis of Zuker diabetic rat skeletal muscle combined with bioinformatics and Q-PCR, we found a correlation between TNF-α-mediated insulin resistance and type 2 diabetes. This novel approach combining analysis of the conditioned secretome and transcriptome has identified several previously unknown, TNF-α-dependent secretory proteins, which establish a foothold for research on the different causes of insulin resistance and their relationships with each other.

mTOR complex 1 (mTORC1) is a multiprotein complex that integrates diverse signals including growth factors, nutrients, and stress to control cell growth. Raptor is an essential component of mTORC1 that functions to recruit specific substrates. Recently, Raptor was suggested to be a key target of regulation of mTORC1. Here, we show that Raptor is phosphorylated by JNK upon osmotic stress. We identified that osmotic stress induces the phosphorylation of Raptor at Ser-696, Thr-706, and Ser-863 using liquid chromatography-tandem mass spectrometry. We found that JNK is responsible for the phosphorylation. The inhibition of JNK abolishes the phosphorylation of Raptor induced by osmotic stress in cells. Furthermore, JNK physically associates with Raptor and phosphorylates Raptor in vitro, implying that JNK is responsible for the phosphorylation of Raptor. Finally, we found that osmotic stress activates mTORC1 kinase activity in a JNK-dependent manner. Our findings suggest that the molecular link between JNK and Raptor is a potential mechanism by which stress regulates the mTORC1 signaling pathway.

Phospholipase D (PLD) is a phosphatidyl choline (PC)-hydrolyzing enzyme that generates phosphatidic acid (PA), a lipid second messenger that modulates diverse intracellular signaling. Through interactions with signaling molecules, both PLD and PA can mediate a variety of cellular functions, such as, growth/proliferation, vesicle trafficking, cytoskeleton modulation, development, and morphogenesis. Therefore, systemic approaches for investigating PLD networks including interrelationship between PLD and PA and theirs binding partners, such as proteins and lipids, can enhance fundamental knowledge of roles of PLD and PA in diverse biological processes. In this review, we summarize previously reported protein-protein and protein-lipid interactions of PLD and PA and their binding partners. In addition, we describe the functional roles played by PLD and PA in these interactions, and provide PLD network that summarizes these interactions. The PLD network suggests that PLD and PA could act as a decision maker and/or as a coordinator of signal dynamics. This viewpoint provides a turning point for understanding the roles of PLD-PA as a dynamic signaling hub.