The mitogen-activated protein kinase (MAPK) pathway is frequently activated in human cancers, leading to malignant phenotypes such as autonomous cellular proliferation. Here, we demonstrate a novel role of the activated MAPK pathway in immune evasion by melanoma cells with the mutation of BRAF, which encodes a MAPKKs, (BRAFV600E). MEK inhibitor U0126 or RNA interference (RNAi) for BRAFV600E decreased production of the immunosuppressive soluble factors interleukin (IL)-10, VEGF, or IL-6 from melanoma cells to levels comparable to those after signal transducer and activator of transcription (STAT)3 inactivation. The suppressive activity of the culture supernatants from the melanoma cells on the production of inflammatory cytokines IL-12 and tumor necrosis factor α by dendritic cells upon lipopolysaccharide stimulation was markedly reduced after transduction with BRAFV600E RNAi, comparable to the effects observed with STAT3 RNAi transduction. No additive or synergistic effects were observed by the simultaneous transduction of RNAi for both BRAFV600E and STAT3. Furthermore, specific DNA binding and transcriptional activity of STAT3 were not affected by down-regulation of the MAPK signaling with the BRAF RNAi. These results indicate that the MAPK signal, along with the STAT3 signal, is essential for immune evasion by human melanomas that have constitutively active MAPK signaling and is a potential molecular target for overcoming melanoma cell evasion of the immune system.

Both monomeric and dimeric NADP+-dependent isocitrate dehydrogenase (IDH) belong to the metal-dependent beta-decarboxylating dehydrogenase family and catalyze the oxidative decarboxylation from 2R,3S-isocitrate to yield 2-oxoglutarate, CO2, and NADPH. It is important to solve the structures of IDHs from various species to correlate with its function and evolutionary significance. So far, only two crystal structures of substrate/cofactor-bound (isocitrate/NADP) NADP+-dependent monomeric IDH from Azotobacter vinelandii (AvIDH) have been solved. Herein, we report for the first time the substrate/cofactor-free structure of a monomeric NADP+-dependent IDH from Corynebacterium glutamicum (CgIDH) in the presence of Mg2+. The 1.75 A structure of CgIDH-Mg2+ showed a distinct open conformation in contrast to the closed conformation of AvIDH-isocitrate/NADP+ complexes. Fluorescence studies on CgIDH in the presence of isocitrate/or NADP+ suggest the presence of low energy barrier conformers. In CgIDH, the amino acid residues corresponding to the Escherichia coli IDH phosphorylation-loop are alpha-helical compared with the more flexible random-coil region in the E. coli protein where IDH activation is controlled by phosphorylation. This more structured region supports the idea that activation of CgIDH is not controlled by phosphorylation. Monomeric NADP+-specific IDHs have been identified from about 50 different bacterial species, such as proteobacteria, actinobacteria, and planctomycetes, whereas, dimeric NADP+-dependent IDHs are diversified in both prokaryotes and eukaryotes. We have constructed a phylogenetic tree based on amino acid sequences of all bacterial monomeric NADP+-dependent IDHs and also another one with specifically chosen species which either contains both monomeric and dimeric NADP+-dependent IDHs or have monomeric NADP+-dependent, as well as NAD+-dependent IDHs. This is done to examine evolutionary relationships.

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Both monomeric and dimeric NADP-dependent isocitrate dehydrogenase (IDH) catalyze the oxidative decarboxylation from 2R,3S-isocitrate to yield 2-oxaloglutarate. Monomeric NADP-specific IDHs have been identified from about 50 different bacteria, whereas, dimeric NADP-dependent IDHs are diversified in both prokaryotes and eukaryotes. We have constructed the phylogenetic tree based on amino acid sequences of all bacterial monomeric NADP-IDHs. This is done to get an idea of evolutionary relationship. It is important to solve the structures of IDH from various species to correlate with its function and evolutionary significance. So far, only two crystal structures of substrate-bound (NADP or isocitrate) NADP-dependent monomeric IDH from Azotobacter Vinelandii (AvIDH) have been solved. Here, we are reporting for the first time the substrate free structure of monomeric IDH from Corynebacterium glutamicum (CgIDH) in the presence of Mg 2+. The 1.75 structure of CgIDH-Mg 2+ showed distinctly an open conformation in contrast to the closed conformation of Av-IDH-isocitrate/NADP complexes. The changes in fluorescence intensities of CgIDH in the presence of isocitrate or NADP indicate the conformational change. The conformational changes observed in fluorescence studies agree with the structural observation. Fluorescence results also suggest a low energy barrier between CgIDH with isocitrate or NADP resulting into easy access of the other substrate to perform the catalytic reaction. In CgIDH, the amino acids corresponding to the E. coli IDH phosphorylation-loop is alpha-helical compared to the more flexible random-coil loop in E. coli. This more structured region supports the idea that activation of CgIDH is not controlled by phosphorylation. This research is funded by the NSERC and SSI.