Asn-glycosylation is widespread not only in eukaryotes but also in archaea and some eubacteria. Oligosaccharyltransferase (OST) catalyzes the co-translational transfer of an oligosaccharide from a lipid donor to an asparagine residue in nascent polypeptide chains. Here, we report that a thermophilic archaeon, Pyrococcus furiosus OST is composed of the STT3 protein alone, and catalyzes the transfer of a heptasaccharide, containing one hexouronate and two pentose residues, onto peptides in an Asn-X-Thr/ Ser-motif-dependent manner. We also determined the 2.7-Å resolution crystal structure of the C-terminal soluble domain of Pyrococcus STT3. The structure-based multiple sequence alignment revealed a new motif, DxxK, which is adjacent to the well-conserved WWDYG motif in the tertiary structure. The mutagenesis of the DK motif residues in yeast STT3 revealed the essential role of the motif in the catalytic activity. The function of this motif may be related to the binding of the pyrophosphate group of lipidlinked oligosaccharide donors through a transiently bound cation. Our structure provides the first structural insights into the formation of the oligosaccharide-asparagine bond.

SummaryAdvances in the understanding of the molecular basis for acute myeloid leukemia (AML) have generated new potential targets for treatment. Fms-like tyrosine kinase 3 (FLT3) is one of the most frequently mutated genes in AML and mutations in this gene are associated with poor overall survival. AXL plays a role in the activation of FLT3 and has been implicated in the pathogenesis of AML. The studies reported here evaluated the ability of a novel FLT3/AXL inhibitor, gilteritinib, to block mutated FLT3 in cellular and animal models of AML. Initial kinase studies showed that gilteritinib, a type I tyrosine kinase inhibitor, was highly selective for both FLT3 and AXL while having weak activity against c-KIT. Gilteritinib demonstrated potent inhibitory activity against the internal tandem duplication (FLT3-ITD) and FLT3-D835Y point mutations in cellular assays using MV4–11 and MOLM-13 cells as well as Ba/F3 cells expressing mutated FLT3. Gilteritinib also inhibited FLT3-F691 mutations, although to a lesser degree, in these assays. Furthermore, gilteritinib decreased the phosphorylation levels of FLT3 and its downstream targets in both cellular and animal models. In vivo, gilteritinib was distributed at high levels in xenografted tumors after oral administration. The decreased FLT3 activity and high intratumor distribution of gilteritinib translated to tumor regression and improved survival in xenograft and intra-bone marrow transplantation models of FLT3-driven AML. No overt toxicity was seen in mouse models treated with gilteritinib. These results indicate that gilteritinib may be an important next-generation FLT3 inhibitor for use in the treatment of FLT3 mutation-positive AML.Electronic supplementary materialThe online version of this article (doi:10.1007/s10637-017-0470-z) contains supplementary material, which is available to authorized users.

Macrophages play crucial roles in host defence and tissue homoeostasis, processes in which both environmental stimuli and intracellularly generated metabolites influence activation of macrophages. Activated macrophages are classified into M1 and M2 macrophages. It remains unclear how intracellular nutrition sufficiency, especially for amino acid, influences on macrophage activation. Here we show that a lysosomal adaptor protein Lamtor1, which forms an amino-acid sensing complex with lysosomal vacuolar-type H+-ATPase (v-ATPase), and is the scaffold for amino acid-activated mTORC1 (mechanistic target of rapamycin complex 1), is critically required for M2 polarization. Lamtor1 deficiency, amino-acid starvation, or inhibition of v-ATPase and mTOR result in defective M2 polarization and enhanced M1 polarization. Furthermore, we identified liver X receptor (LXR) as the downstream target of Lamtor1 and mTORC1. Production of 25-hydroxycholesterol is dependent on Lamtor1 and mTORC1. Our findings demonstrate that Lamtor1 plays an essential role in M2 polarization, coupling immunity and metabolism.

Overproduction of the Her2 oncoprotein has been found in Ϸ30% of breast tumors, and patients who have Her2 excesses typically have more aggressive disease. Here we show that the expression of the Her2 gene can be decreased by inhibiting the interaction of the two cancer-linked proteins, DRIP130͞CRSP130͞Sur-2 (a Raslinked subunit of human mediator complexes) and ESX (an epithelial-restricted transcription factor). Disruption of the interaction by a short cell-permeable peptide reduced the expression of the Her2 gene and specifically impaired the growth and viability of Her2-overexpressing breast cancer cells. The association of ESX with DRIP130 is mediated by a small hydrophobic face of an 8-aa helix in ESX, suggesting a therapeutic approach to incapacitating the Her2 gene by small organic molecules. O verexpression of the oncogene Her2 (neu͞ErbB2) has been found in Ϸ30% of breast tumors (1, 2). It is unclear why overexpression of this transmembrane receptor occurs in breast cancers, but patients who have Her2 excesses typically have more aggressive disease with enhanced metastasis and increased resistance to chemotherapy (1-6). Monoclonal antibodies against the Her2 protein have been successful in treating these Her2-positive patients (7-10). A humanized antibody called Herceptin has demonstrated tumor-inhibitory and chemosensitizing effects in clinical studies and is the only drug that the Food and Drug Administration has approved for treatment of Her2-overexpressing breast tumors (9, 10). The clinical success of the Her2-antibody therapy was an excellent example of the translation of basic cancer biology into clinical cancer treatment. However, the antibody therapy alone may not be ideal for therapeutic intervention of Her2-overexpressing breast cancers. In theory, down-regulation of Her2 may be accomplished efficiently by inhibiting the expression of the Her2 gene rather than targeting elevated levels of the Her2 proteins that are already overexpressed. By analogy with treatments for AIDS, cocktails of drugs, each with different mechanisms of action, might also be more effective at achieving complete remission of breast tumors. Thus, discovering a means to provide external control over Her2 expression, particularly through small organic molecules, remains appealing.One of the critical transcription factors that activate the Her2 gene in breast cancers is ESX (ESE-1͞ELF3͞ERT͞Jen), an Ets factor that is expressed specifically in epithelial cells including mammary glands (11-13). ESX binds and strongly activates the Her2 promoter (11), and the ESX-binding site in the Her2 promoter is absolutely required for the high-level expression of Her2 in breast cancer cells (14). The expression of ESX is boosted in Her2-overexpressing breast cancers (11); a simple correlation seems to exist between the expression levels of ESX and Her2 in breast cancer cells. Here we isolate DRIP130͞ CRSP130͞Sur-2, a Ras-linked metazoan-specific subunit of human mediator complexes, as a nuclear cofactor that binds specifically to the trans...