Acylsugars are insecticidal specialized metabolites produced in the glandular trichomes of plants in the Solanaceae family. In the tomato clade of the Solanum genus, acylsugars consist of aliphatic acids of different chain lengths esterified to sucrose, or less frequently to glucose. Through liquid chromatography-mass spectrometry screening of introgression lines, we previously identified a region of chromosome 8 in the Solanum pennellii LA0716 genome (IL8-1/8-1-1) that causes the cultivated tomato Solanum lycopersicum to shift from producing acylsucroses with abundant 3-methylbutanoic acid acyl chains derived from leucine metabolism to 2-methylpropanoic acid acyl chains derived from valine metabolism. We describe multiple lines of evidence implicating a trichome-expressed gene from this region as playing a role in this shift. S. lycopersicum M82 SlIPMS3 (Solyc08g014230) encodes a functional end product inhibition-insensitive version of the committing enzyme of leucine biosynthesis, isopropylmalate synthase, missing the carboxyl-terminal 160 amino acids. In contrast, the S. pennellii LA0716 IPMS3 allele found in IL8-1/8-1-1 encodes a nonfunctional truncated IPMS protein. M82 transformed with an SlIPMS3 RNA interference construct exhibited an acylsugar profile similar to that of IL8-1-1, whereas the expression of SlIPMS3 in IL8-1-1 partially restored the M82 acylsugar phenotype. These IPMS3 alleles are polymorphic in 14 S. pennellii accessions spread throughout the geographical range of occurrence for this species and are associated with acylsugars containing varying amounts of 2-methylpropanoic acid and 3-methylbutanoic acid acyl chains.

Subcellular compartmentalization has become an important theme in cell signaling such as spatial regulation of Ras by RasGRP1 and MEK/ERK by Sef. Here, we report spatial regulation of Raf kinase by RKTG (Raf kinase trapping to Golgi). RKTG is a seven-transmembrane protein localized at the Golgi apparatus. RKTG expression inhibits EGF-stimulated ERK and RSK phosphorylation, blocks NGF-mediated PC12 cell differentiation, and antagonizes Ras-and Raf-1-stimulated Elk-1 transactivation. Through interaction with Raf-1, RKTG changes the localization of Raf-1 from cytoplasm to the Golgi apparatus, blocks EGF-stimulated Raf-1 membrane translocation, and reduces the interaction of Raf-1 with Ras and MEK1. In RKTG-null mice, the basal ERK phosphorylation level is increased in the brain and liver. In RKTG-deleted mouse embryonic fibroblasts, EGF-induced ERK phosphorylation is enhanced. Collectively, our results reveal a paradigm of spatial regulation of Raf kinase by RKTG via sequestrating Raf-1 to the Golgi apparatus and thereby inhibiting the ERK signaling pathway.R af kinase relays the signals from Ras to MEK (MAPK and ERK kinase) and ERK/MAPK (1, 2). This pathway regulates many fundamental cellular functions, including cell proliferation, apoptosis, differentiation, motility, and metabolism, and is implicated in many human diseases including cancer (3). In the Ras/Raf/MEK/ ERK signaling cascade, several players within this pathway are exquisitely regulated by subcellular compartmentalization (4, 5). Kinase suppressor of Ras (KSR) is a scaffold protein that coordinates the assembly of multiprotein MAPK complex in plasma membrane (6). ␤-Arrestin could also function as a scaffold protein to target the MAPK protein complex to early endosome (7). Different lipid anchors are able to shuttle Ras between different membrane compartments to modulate subcellular Ras signaling (8-10). In T lymphocyte, Ras could be activated in situ on the Golgi apparatus via the Ras exchange factor RasGRP1, which is activated by phospholipase C (11). Compartmentalized signaling of Ras/ MAPK in T lymphocyte on either plasma membrane or Golgi apparatus leads to distinct output of ERK activation and thereby determines the threshold of thymic selection (12). MEK could be targeted to endosome by a scaffold protein MP1 through adaptor p14 (13). It was also found that MEK/ERK can be recruited to the Golgi by Sef, and that such spatial regulation blocks the Ras signaling to the nucleus but not to the cytosol (14). However, how Raf is regulated in a spatial manner has not been characterized.The activity of Raf kinase is mainly regulated by phosphorylation events and scaffold proteins (1, 2). In addition, Raf could be negatively regulated by interaction with other proteins. Raf kinase inhibitor protein (RKIP) is able to interact with Raf-1, MEK, and ERK (15). RKIP blocks Raf-1 signaling to MEK by competitively disrupting the interaction between Raf-1 and MEK because both Raf-1 and MEK bind to overlapping sites in RKIP (15, 16). After stimulation of G protein...

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Switching macrophages from a pro-tumor type to an anti-tumor state is a promising strategy for cancer immunotherapy. Existing agents, many derived from bacterial components, have safety or specificity concerns. Here, we postulate that the structures of the bacterial signals can be mimicked by using non-toxic biomolecules of simple design. Based on bioactivity screening, we devise a glucomannan polysaccharide with acetyl modification at a degree of 1.8 (acGM-1.8), which specifically activates toll-like receptor 2 (TLR2) signaling and consequently induces macrophages into an anti-tumor phenotype. For acGM-1.8, the degree of acetyl modification, glucomannan pattern, and acetylation-induced assembly are three crucial factors for its bioactivity. In mice, intratumoral injection of acGM-1.8 suppresses the growth of two tumor models, and this polysaccharide demonstrates higher safety than four classical TLR agonists. In summary, we report the design of a new, safe, and specific TLR2 agonist that can generate macrophages with strong anti-tumor potential in mice.

Remote ischaemic preconditioning (RIPC) is well known to protect the myocardium against ischaemia/reperfusion injury (IRI). Exosomes are small extracellular vesicles that have become the key mediators of intercellular communication. Various studies have confirmed that circulating exosomes mediate RIPC. However, the underlying mechanisms for RIPC-induced exosome-mediated cardioprotection remain elusive. In our study, we found that the expression level of miR-24 was higher in exosomes derived from the plasma of rats subjected to RIPC than in exosomes derived from the plasma of control rats in vivo. The rat plasma exosomes could be taken up by H9c2 cells. In addition, miR-24 was present in RIPC-induced exosomes and played a role in reducing oxidative stress-mediated injury and decreasing apoptosis by downregulating Bim expression in H2O2-treated H9c2 cells in vitro. In vivo, miR-24 in RIPC-induced exosomes reduced cardiomyocyte apoptosis, attenuated the infarct size and improved heart function. Furthermore, the apoptosis-reducing effect of miR-24 was counteracted by miR-24 antagomirs or inhibitors both in vitro and in vivo. Therefore, we provided evidence that RIPC-induced exosomes could reduce apoptosis by transferring miR-24 in a paracrine manner and that miR-24 in the exosomes plays a central role in mediating the protective effects of RIPC.