Quambalarine B, a Secondary Metabolite from Quambalaria cyanescens with Potential Anticancer Properties.

Quambalarine B (QB) is a secondary metabolite produced by the basidiomycete Quambalaria cyanescens with potential anticancer activity. Here we report that QB at low micromolar concentration inhibits proliferation of several model leukemic cell lines (Jurkat, NALM6, and REH), whereas higher concentrations induce cell death. By contrast, the effect of QB on primary leukocytes (peripheral blood mononuclear cells) is significantly milder with lower toxicity and cytostatic activity. Moreover, QB inhibited expression of the C-MYC oncoprotein and mRNA expression of its target genes, LDHA, PKM2, and GLS. Finally, QB blocked the phosphorylation of P70S6K, a downstream effector kinase in mTOR signaling that regulates translation of C-MYC. This observation could explain the molecular mechanism behind the antiproliferative and cytotoxic effects of QB on leukemic cells. Altogether, our results establish QB as a promising molecule in anticancer treatment.

Nkrp1 family, from lectins to protein interacting molecules.

The C-type lectin-like receptors include the Nkrp1 protein family that regulates the activity of natural killer (NK) cells. Rat Nkrp1a was reported to bind monosaccharide moieties in a Ca2+-dependent manner in preference order of GalNac > GlcNAc >> Fuc >> Gal > Man. These findings established for rat Nkrp1a have been extrapolated to all additional Nkrp1 receptors and have been supported by numerous studies over the past two decades. However, since 1996 there has been controversy and another article showed lack of interactions with saccharides in 1999. Nevertheless, several high affinity saccharide ligands were synthesized in order to utilize their potential in antitumor therapy. Subsequently, protein ligands were introduced as specific binders for Nkrp1 proteins and three dimensional models of receptor/protein ligand interaction were derived from crystallographic data. Finally, for at least some members of the NK cell C-type lectin-like proteins, the "sweet story" was impaired by two reports in recent years. It has been shown that the rat Nkrp1a and CD69 do not bind saccharide ligands such as GlcNAc, GalNAc, chitotetraose and saccharide derivatives (GlcNAc-PAMAM) do not directly and specifically influence cytotoxic activity of NK cells as it was previously described.

The C-type lectin-like receptor Nkrp1b: Structural proteomics reveals features affecting protein conformation and interactions.

The cytotoxicity of mouse natural killer (NK) cells in response to pathological changes in target cells is regulated via the Nkrp1b receptor. Here, we characterized the Nkrp1b structure and structural features (stalk, loop, and oligomerization state) that affect its interactions. To study the Nkrp1b protein structure and the functional importance of its stalk, two Nkrp1b protein variants differing by the presence of the stalk were prepared. These variants were studied using a combination of structural mass spectrometry approaches with computational modeling to derive structural models. In addition, information about biological activity and localization in mammalian cells was acquired using scanning microscopy techniques and western blotting. Based on these methods, we obtained the structure of Nkrp1b ectodomain in its monomeric and dimeric conformations, identified the dimerization interface, and determined disulfide connections within the molecule. We found that Nkrp1b occurs as a mixture of monomers and homodimers, both in vitro and in vivo. SIGNIFICANCE: Despite the long-standing assumption that Nkrp1 proteins are homodimers connected by disulfide bonds in the stalk region, our data showed that both Nkrp1b protein variants form monomers and homodimers irrespective of the presence of the stalk. We demonstrated that the stalk is not crucial for protein dimerization or ligand binding and that Nkrp1b interacts with its natural ligands only in its monomeric conformation; therefore, dimers may have another regulatory function. Using a unique combination of computational, biochemical, and biological methods, we revealed the structural conformation and behavior of Nkrp1b in its native state. In addition, it is a first report utilizing the intermolecular chemical cross-linking of light- and heavy-labeled protein chains together with ion mobility-mass spectrometry to design the structural models of protein homodimers.

Reprogramming of leukemic cell metabolism through the naphthoquinonic compound Quambalarine B.

Abnormalities in cancer metabolism represent potential targets for cancer therapy. We have recently identified a natural compound Quambalarine B (QB), which inhibits proliferation of several leukemic cell lines followed by cell death. We have predicted ubiquinone binding sites of mitochondrial respiratory complexes as potential molecular targets of QB in leukemia cells. Hence, we tracked the effect of QB on leukemia metabolism by applying several omics and biochemical techniques. We have confirmed the inhibition of respiratory complexes by QB and found an increase in the intracellular AMP levels together with respiratory substrates. Inhibition of mitochondrial respiration by QB triggered reprogramming of leukemic cell metabolism involving disproportions in glycolytic flux, inhibition of proteins O-glycosylation, stimulation of glycine synthesis pathway, and pyruvate kinase activity, followed by an increase in pyruvate and a decrease in lactate levels. Inhibition of mitochondrial complex I by QB suppressed folate metabolism as determined by a decrease in formate production. We have also observed an increase in cellular levels of several amino acids except for aspartate, indicating the dependence of Jurkat (T-ALL) cells on aspartate synthesis. These results indicate blockade of mitochondrial complex I and II activity by QB and reduction in aspartate and folate metabolism as therapeutic targets in T-ALL cells. Anti-cancer activity of QB was also confirmed during in vivo studies, suggesting the therapeutic potential of this natural compound.