| Iron play important roles in the functioning of normal cells.Many important proteins contain iron,such as DNA helicase,some heme-containing proteins(hemoglobin,cytochrome P450 and coenzyme Q-cytochrome c reductase,succinate-coenzyme Q reductase in the mitochondrial electron transport chain)and ferrochelatase,nucleotide reductase,DNA primerase,that are involved in DNA synthesis,replication,repair and transcription etc.In cell metabolism,iron has a central position in almost all life forms.Most of these proteins are highly conserved in both prokaryotes and eukaryotes.Therefore,insufficient cellular iron level will hinder the normal functions of cell.However,extra iron ions in cells will increase the possibility of Fenton reaction occurance,generating reactive oxygen radicals(ROS),which is toxic to cells.These two factors make the maintenance of iron homeostasis very important.There are various mechanisms for maintaining iron homeostasis in cells.The main regulator during this process is Fur(Ferric uptake regulation protein).However,more and more studies have shown that a small bacterial RNA(sRNA)molecule RyhB also plays an important role in maintaining the homeostasis of iron.Some studies have even shown that,instead of being regulated by Fur,RyhB can play a role alone in controlling the level of intracellular iron ions.When RyhB is activated and expressed,it can pair with the mRNA of various iron-containing protein genes or operons(such as fumA and acnA and sdhCDAB operon,etc.)in the cell.Then the formed RyhB-mRNA complex will be degraded with the help of RNase E or RNase ? and RNA chaperone Hfq to inhibit the expression of target genes to save intracellular iron ions.Many of the targets of RyhB regulation are related to central carbon metabolic pathways.In this sense,RyhB has the potential to disturb cell metabolism.This possibility has been described in some research papers,but most of these studies failed to obtain the detailed metabolic pertubaions of RyhB regulation.There is neither systematic biological research on RyhB nor methodologies of studing the cell metabolism variations brought by RNA regulation of genes expression in our country.Therefore,by combining various systeru biology technologies,such as flux ratio analysis(METAFoR)based on 13C stable isotope labeling and mass isotopomer analysis of intracellular metabolites(fragments),we attempted to systematically resolve the central carbon metabolism pertubations brought by RyhB regulaion.Escherichia coli is the most used model bacteria in the study of bacterial sRNA.At present,a variety of sRNAs related to its pathogenic regulation have been found.We selected E.coli W3100 as the starting strain.This strain is not only suitable as a model bacterium for sRNA research,but also contains the least nutritional deficiencies,makes it most suitable for MFA analysis and METAFoR analysis.By using[1-13C]-glucose-based METAFoR technology and compairing the metabolism of multiple groups of cells,we found for the first time that there is a positive correlation between the activation of RyhB and the activity of the pentose phosphate(PP)pathway in the central carbon metabolism of the cell,that is,when RyhB is activated in the cell and exerted its biological regulation,the activity of the PP pathway will be enhanced.Therefore,it will provide more NADPH supply to the cells.Cell metabolism studies based on[U-13C]-labeled glucose in our work showed that through metabolic flux redistribution,the reversible exchange activity between serine and glycine is also enhanced,which further provides additional supply for the production of NADPH.Through the application of METAFoR,we also found that the activity of the Entnere-Doudoroff(ED)pathway was weakened in ryhB-deficient bacteria.Compared to in the ryhB-induced and the wild-type W3100 cells,the ED reaction pathway activity can hardly be observed in ryhB-deficient bacteria.We also observed that ryhB deletion will affect the growth of cells in the lag phase.In ryhB-deficient cells,the lag phase was significantly extended,but RyhB has little effect on the growth rate of the exponential growth period.Part of the reason for this may be due to the correlation between the level of RyhB and the activity of PP pathway.Because one of the important functions of the PP pathway is to produce ribose-5-phosphate,and ribose-5-phosphate is an important precursor for the synthesis of purine and pyrimidine ribonucleotides,so PP pathway activity will eventually affect the speed of synthesizing DNA and other genetic materials and one of the main functions of the lag phase is to synthesize a large amount of biomass such as nucleic acids,enzymes,coenzymes,and metabolites to prepare for cell proliferation.Quantitative studies on W3100 wild-type E.coli and other cells showed that the induction of RyhB would rapidly increase intracellular reactive oxygen species(ROS)significantly.This may due to the combined effect of RyhB modulating the degradation of non-essential iron-containing proteins in the cell to release more iron ions(increas ing the production of ROS in the cell)and the inhibition of sodB gene expression.Previous studies have shown that NADPH and NADH can be used directly as antioxidants by scavenging free radicals and repairing free radicals derived from biomolecules in cells.In our study,we found that in order to cope with the increase of ROS,the relative activity of typical NADPH-producing metabolic pathways such as PP pathway activity and serine-glycine exchange reaction activity increased.At the same time,the significantly reduced intracellular NADH/NAD+and NADPH/NADP+ratios further confirmed the higher demand for reducing power like NADPH.In our work,we also observed that the mass isotopomer distribution vector(MDV)of some key metabolite molecules(fragments),such as the MDV of OAA1-2,PEP1-3,PYR1-2,AcCoA1-2,that can be used for METAFoR calculations changed between different cells indicating that the activity of metabolic pathways around these metabolite molecules may also change accordingly.Our work,to some extent,provided a methodological reference for learning the perturbation of sRNA molecules on cell metabolism and also provided a new perspective for a more comprehensive understanding of the role of RyhB in cells.We proved that in addition to affecting the expression of various target genes at the post-transcriptional level as a regulator,this small RNA can also significantly affect cell metabolism and the redox state of the cell. |
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