线粒体氧化磷酸化和糖酵解课件

1IF19.679 AbstractAbstractCancer cells consume large quantities of glucose and primarily use glycolysis for ATP production,even in the presence of adequate oxygen.This metabolic signature(aerobic glycolysis or the Warburg effect)enables cancer cells to direct glucose to biosynthesis,supporting their rapid growth and proliferation.癌癌细胞胞生生长需需要要消消耗耗大大量量葡葡萄萄糖糖，主主要要是是通通过糖糖酵酵解解产生生ATP，但但这种种糖糖酵酵解解甚甚至至在在氧氧充充足足条条件件下下依依然然很很活活跃。有有氧氧酵酵解解或或瓦瓦博博格格效效应的的这种种代代谢特特征征促促进了癌了癌细胞直接将葡萄糖胞直接将葡萄糖进行生物合成，行生物合成，维持癌持癌细胞快速、无限增殖。胞快速、无限增殖。2l糖糖代代谢有有2种种途途径径：线粒粒体体氧氧化化磷磷酸酸化化和和糖糖酵酵解解。正正常常哺哺乳乳动物物细胞胞在在有有氧氧条条件件下下，糖糖酵酵解解被被抑抑制制。然然而而，1920年年，德德国国生生化化学学家家Warburg发现：肝肝癌癌细胞胞的的糖糖酵酵解解活活性性较正正常常肝肝细胞胞活活跃。提提出出：在在氧氧气气充充足足下下，恶性性肿瘤瘤细胞胞糖糖酵酵解解同同样活活跃，这种种有有氧氧糖糖酵酵解解的的代代谢特特征征称称为瓦瓦博博格格效效应，表表现为葡葡萄萄糖糖摄取取率率高高，糖酵解活糖酵解活跃，代，代谢产物乳酸含量高物乳酸含量高。l“瓦博格效应”疑问“为什么肿瘤细胞大量消耗葡萄糖却不能高效产能？”34However,both causes of the Warburg effect and its connection to biosynthesis are not well understood.Here we show that the tumour suppressor p53,the most frequently mutated gene in human tumours,inhibits the pentose phosphate pathway(PPP).然然而而，瓦瓦博博格格效效应应产产生生的的原原因因以以及及它它与与癌癌细细胞胞生生物物合合成成的的关关系系，还还不不是是十十分分清清楚楚。在在本本文文中中，介介绍绍了了一一种种抑抑癌癌基基因因p53（在在肿肿瘤瘤细细胞胞中中发发生生变变异异频频率率较较高高的的基因），基因），p53可抑制戊糖磷酸途径（可抑制戊糖磷酸途径（pentose phosphate pathway，PPP）。）。5Through the PPP,p53 suppresses glucose consumption,NADPH production and biosynthesis.The p53 protein binds to glucose-6-phosphate dehydrogenase(G6PD),the firstfirst and rate-limiting enzyme of the PPP,and prevents the formation of the active dimer.通过通过PPP，p53可抑制葡萄糖消耗、可抑制葡萄糖消耗、NADPH产生产生 及生物合成。及生物合成。p53可以与可以与戊糖戊糖磷酸途径上的第一磷酸途径上的第一 步反应的关键酶葡萄糖步反应的关键酶葡萄糖-6-磷酸脱氢酶（磷酸脱氢酶（G6PD）相结合，并抑制其活性（活性二聚体形成）。相结合，并抑制其活性（活性二聚体形成）。6Tumour-associated p53 mutants lack the G6PD-inhibitory activity.Therefore,enhanced PPP glucose fluxflux due to p53 inactivation may increase glucose consumption and direct glucose towards biosynthesis in tumour cells.在在肿肿瘤瘤细细胞胞内内，由由于于p53发发生生突突变变，无无法法抑抑制制G6PD活活性性，因因此此，由由于于被被p53抑抑制的磷酸戊糖途径被激活，大量葡萄糖被消耗，而进行生物合成。制的磷酸戊糖途径被激活，大量葡萄糖被消耗，而进行生物合成。引言7The tumour suppressor p53 invokes anti-proliferative processes,of which the best understood include cell cycle arrest,DNA repair and apoptosis.抑癌基因抑癌基因p53主要抑制癌细胞增殖过程，包括细胞周期停滞、主要抑制癌细胞增殖过程，包括细胞周期停滞、DNA修复以及细修复以及细 胞凋亡等已研究比较清楚。胞凋亡等已研究比较清楚。lp53可可使使周周期期蛋蛋白白cyclinB启启动子子区区关关闭而而下下调cyclinB的的转录水水平平。如如在在卵卵巢巢癌癌细胞胞中中，球球毛毛壳壳甲甲素素K在在p53介介导作作用用下下，发生生细胞胞G2期期阻阻滞滞(Li et al.,2015)。lDNA损伤若若被被修修复复，细胞胞周周期期恢恢复复正正常常；如如果果损伤严重重，DNA无无法法被被修修复复，细胞胞则经历凋凋亡亡。细胞胞凋凋亡亡的的起起始始阶段段的的特特征征是是Caspase被被激激活活，主主要要有有：p53p53介介介介导导的的的的线线粒粒粒粒体体体体凋凋凋凋亡亡亡亡通通通通路路路路，死死死死亡亡亡亡受受受受体体体体凋凋凋凋亡通路亡通路亡通路亡通路。89Recent studies indicated that p53 also has a role in modulating metabolism including glycolysis and oxidative phosphorylation.近近年年来来研研究究表表明明，p53在在调调节节代代谢谢方方面面有有重重要要作作用用，包包括括葡葡萄萄糖糖酵酵解解和和氧氧化化磷磷酸酸化。化。However,the role of p53 in regulating biosynthesis is less well understood.但是，关于但是，关于p53在生物合成中的调节功能研究甚少。在生物合成中的调节功能研究甚少。10The PPP is important for both glucose catabolism and biosynthesis.In an oxidative phase,the PPP generates NADPH(nicotinamide adenine dinucleotide phosphate,reduced),the principal intracellular reductant required for reductive biosynthesis such as the synthesis of lipid,and ribose 5-phosphate,an essential precursor for biosynthesis of nucleotides.This is followed by a non-oxidative interconversion of ribose 5-phosphate to the intermediates in the glycolytic pathways.Despite the vital role of the PPP in biosynthesis and its close link to glycolysis,the regulation of the PPP in tumour cells remains unclear.11To investigate whether p53 modulates the PPP,we compared the oxidative PPP flux in isogenic p53+/+and p53-/-human colon cancer HCT116 cells.Cells were cultured in medium containing 2-13Cglucose,and the glucose metabolites were measured by nuclear magnetic resonance(NMR)spectroscopy.结果与分析结果与分析12These results indicate that p53 deficiency increases glucose consumption mainly through an enhanced PPP flux.colon cancer HCT116 cellscolon cancer HCT116 cellsmouse embryonic fibroblast(EMF)cellsp53 deficiency correlates with increases in PPP flux,glucose consumption and lactate production13p53 deficiency correlates with increases in PPP flux,glucose consumption and lactate productionInhibition of G6PD in these cells increased,rather than decreased,lactate production,regardless of p53 status.14p53 regulates NADPH levelsThe PPP plays a significant role in the production of cellular NADPH.The lack of p53 led to a strong increase in the NADPH level in HCT116 cells.Similarly,knocking down p53 in U2OS cells with small hairpin RNA(shRNA)strongly increased NADPH levels.Figure 2 p53 regulates NADPH levelsp53 regulates NADPH levels15Treatment with G6PD siRNA minimized the difference in NADPH levels between p53-proficient and-deficient cells.The tissues from p53-/-mice-including heart,liver,kidney and lung-exhibited substantially elevated NADPH levels,compared with those in the correspondingtissues from p53+/+mice.The exception was found in the spleen.16In the spleen,the activity of G6PD was very low(Fig.2g),and the PPP might not contribute substantially to the overall NADPH production.In contrast to p53 downregulation,overexpression of p53 led to a strong decrease in NADPH levels(Supplementary Fig.S1b).17NADPH is required for the biosynthesis of lipidThe p53-/-MEF cells showed enhanced lipid levels,compared with p53+/+MEF cells,as evaluated by Oil Red O staining.The lack of p53 also resulted in higher levels of lipid in HCT116 cells.The difference in lipid accumulation between p53+/+and p53-/-cells diminished on treatment with G6PD siRNA or DHEA.18Histological sections of liver tissue from p53-/-and p53+/+mice were stained with haematoxylin and eosin.Arrows indicate fat droplets.We also evaluated the effect of p53 on the formation of fat droplets in the liver.The liver of p53-/-mice had a larger number of bigger fat droplets,compared with the liver of p53+/+mice.19Together,these results indicate that p53 inhibits NADPH production and lipid accumulation by lowering the glucose flux through the PPP.20To investigate the mechanism by which p53 regulates the PPP,we assayed the activity activity of of G6PDG6PD,a key regulatory point of the PPP.21MEF cellsHCT116 cellsMEF cellsThe lack of p53 correlated with a strong elevation in G6PD activity in both MEF and HCT116 cells.Similarly,when p53 was knocked down in U2OS cells with shRNA,G6PD activity nearly doubled.U2OS cells22The lack of p53 was associated with highly elevated G6PD activity.Conversely,overexpression of wild-type p53 in the p53-deficient cell lines(H1299 and p53-/-Mdm-/-MEF)caused a noticeable decrease in G6PD activity.H1299These results show that p53 suppresses G6PD activity.23In each of the cell lines and tissues that were examined,the levels of the G6PD protein remained unchanged when p53 was downregulated or overexpressed.Moreover,p53 did not change the level of G6PD transcript.24To rule out the involvement of other p53 target genes in the inhibition of G6PD,we used an inhibitor of p53 transcriptional activity,pifithrin-(PFT).25PFT impeded p53-induced expression of p21,but did not restore p53-inhibited G6PD activity.Treatment of p53+/+HCT116 cells with cycloheximide alone resulted in a lower level of p53,which was accompanied by a higher activity of G6PD.Simultaneous treatment with cycloheximide and doxorubicin led to a stabilization of p53 above the basal level in unstressed cells,and a concurrent drop of G6PD activity below its basal level.As controls,none of these treatments altered G6PD activity in p53-/-HCT116 cells.26In addition,the p53 mutant V122A,which has a transactivation activity comparable to or even higher than wild-type p53 dependent on the target gene(Inga et al.,2001),failed to inhibit G6PD.(Inga et al.,2001)27Moreover,we treated cells with the nuclear export inhibitor leptomycin B to prevent cytoplasmic accumulation of p53.Leptomycin B reversed p53-mediated inhibition of G6PD.28Together,these results show that inhibition of G6PD by p53 is independent of transcription or translation and is a cytoplasmic,not nuclear,function of p53.29We next investigated whether p53 interacts with G6PDwhether p53 interacts with G6PD.30Flag-tagged p53 specifically associated with enhanced green fluorescent protein(eGFP)G6PD in vivo.Similarly,endogenous p53 interacted with endogenous G6PD.This interaction was enhanced when cells were treated with the proteasome inhibitor MG132 doxorubicin,both of which stabilized p53.31G6PD is a cytoplasmic protein,whereas p53 is present in both the cytoplasm and the nucleus,and consistently,the p53-G6PD interaction occurred in the cytoplasm.谢 谢 聆 听32