Project description:The detachment of epithelial cells, but not cancer cells, causes anoikis due to reduced energy production. Invasive tumor cells generate three splice variants of the metastasis gene osteopontin. The cancer-specific form osteopontin-c supports anchorage-independence through inducing oxidoreductases and upregulating intermediates/enzymes in the hexose monophosphate shunt, glutathione cycle, glycolysis, glycerol phosphate shuttle, and mitochondrial respiratory chain. Osteopontin-c signaling upregulates glutathione (consistent with the induction of the enzyme GPX-4), glutamine and glutamate (which can feed into the tricarboxylic acid cycle). Consecutively, the cellular ATP levels are elevated. The elevated creatine may be synthesized from serine via glycine and also supports the energy metabolism by increasing the formation of ATP. Metabolic probing with N-acetyl-L-cysteine, L-glutamate, or glycerol identified differentially regulated pathway components, with mitochondrial activity being redox dependent and the creatine pathway depending on glutamine. The effects are consistent with a stimulation of the energy metabolism that supports anti-anoikis. Our findings imply a synergism in cancer cells between osteopontin-a, which increases the cellular glucose levels, and osteopontin-c, which utilizes this glucose to generate energy. mRNA profiles of MCF-7 cells transfected with osteopontin-a, osteopontin-c and vector control were generated by RNA-Seq, in triplicate, by Illumina HiSeq.
Project description:Motility in the Archaea domain is facilitated by a unique motility structure termed the archaellum. N-glycosylation of the major structural proteins (archaellins) is important for their subsequent incorporation into the archaellum filament. Here, we report the structure of the archaellin glycan from Methanothermococcus thermolithotrophicus, a methanogen which grows optimally at 65°C. Four archaellin genes (flaB1-4) have previously been identified. In gel digestion and LC-MS analysis revealed the identity of the upper band as FlaB1 and the lower band as FlaB3. Examination of the protein sequences for the four archaellins indicated multiple possible N-linked glycosylation sites in each. We observed using mass spectrometry that Mtc. thermolithotrophicus archaellins is posttranslationally modified at multiple sites with an N-linked branched oligosaccharide composed of 7 sugars (1414 Da). NMR analysis of the purified glycan determined the structure to be α-D-glycero-D-manno-Hep3OMe6OMe-(1-3)-[α-GalNAcA3OMe-(1-2)-]-β-Man-(1-4)-[-GalA3OMe4OAc6CMe-(1-4)--GalA-(1-2)-]-α-GalAN-(1-3)-β-GalNAc-Asn. A detailed investigation by HILIC-MS discovered the presence of several, less abundant glycan variants, related to but distinct from the main heptameric glycan. In addition, we confirmed that the S-layer protein is modified with the same heptameric glycan suggesting a common N-glycosylation pathway.