Structural analysis of monomeric isocitrate dehydrogenase from corynebacterium glutamicum

In this research project, structural aspects of monomeric NADP+-dependent isocitrate dehydrogenase from Corynebacterium glutamicum (CgIDH) are investigated together with site-directed mutagenesis and fluorescence spectroscopy studies. CgIDH, one of the enzymes of the Krebs cycle, catalyzes the decarboxylation of isocitrate into α-ketoglutarate, which in some bacteria and plants regulates the flow of carbon into either the Krebs cycle or the glyoxylate bypass depending on the available carbon source. The structure of CgIDH complexed with Mg2+ has been determined at 1.75 Å resolution using X-ray crystallography. In contrast to the closed conformation of published structures of monomeric NADP+-dependent IDH from Azotobactor vinelandii complexed with either isocitrate-Mn2+ or NADP+, the structure of CgIDH complexed with Mg2+ demonstrates the open conformation. The superimposed structure of CgIDH complexed with Mg2+ onto the structures of AvIDH complexes reveals that Domain II is rotated ~24° or ~35º, respectively, relative to Domain I when isocitrate-Mn2+ or NADP+ is bound, resulting in the closure of the active site between the two domains. Fluorescence spectroscopic studies support the proposal that the presence of isocitrate or NADP+ could mediate the conformational changes in CgIDH. In addition, three CgIDH mutants (S130D, K253Q, and Y416T) were created based on the structural analysis and previous mutagenesis studies of homodimeric NADP+-dependent IDH. Both the specific activities and the fluorescence spectra of these CgIDH mutants elucidate the roles of these active site residues in CgIDH catalysis. It has been suggested that the conformational changes observed in the presence of the substrate(s) may regulate enzymatic activity in CgIDH, in contrast to homodimeric NADP+-dependent IDH in Escherichia coli, where the phosphorylation cycle controls activity. It is also presumed that both Lys253 and Tyr416 may play critical roles in CgIDH activity, as do the equivalent residues in homodimeric IDH from porcine heart mitochondria. Similar structural features and conformational changes among monomeric CgIDH and homodimeric NADP+-dependent IDH enzymes suggest the phylogenetic relationships among various monomeric and homodimeric NADP+-dependent IDH from different sources

The BRAF–MAPK signaling pathway is essential for cancer-immune evasion in human melanoma cells

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