【作者】 刘伟；

【导师】 王中华；

【作者基本信息】 山东农业大学 ， 畜牧学， 2024， 博士

【摘要】 亮氨酸(Leu)缺乏可通过mTORC1信号抑制蛋白质合成。在生长动物中,Leu缺乏强烈抑制肌肉蛋白合成。在HEK293等细胞中,Leu缺失对mTORC1活性的抑制程度与总氨基酸(AA)缺失类似。在奶牛等泌乳动物,Leu缺乏对乳蛋白合成的抑制作用较为温和,提示Leu缺乏对蛋白质的合成,mTORC1信号活性等的影响可能有细胞特异性,但尚无直接证据,背后的潜在机制也不清楚。针对这一科学问题,本文以MAC-T细胞为试验材料,通过观察Leu缺乏引起的细胞mTORC1信号与代谢适应变化、丝氨酸(Ser)合成和支链氨基酸(BCAA)分解代谢酶活性对Leu调控mTORC1信号的影响等,研究了MAC-T细胞mTORC1信号对Leu缺乏的响应特征及其机制,结果如下:1.MAC-T细胞mTORC1信号的Leu缺乏响应特征(1)MAC-T对Leu缺失有快速适应能力。当MAC-T细胞缺失Leu时,细胞4EBP1和RPS6蛋白磷酸化水平迅速降低,2 h后基本稳定。整合应激反应(ISR)标志蛋白ATF4水平也在缺失Leu后快速升高,2 h后基本稳定。以上结果表明,MAC-T细胞能够快速适应Leu缺失环境。(2)Leu以剂量依赖方式激活mTORC1信号。在Leu浓度分别为DMEM/F12培养基0%、5%、10%、50%、100%和300%的培养基中培养MAC-T细胞4 h,RPS6磷酸化水平随Leu浓度的升高而升高,表明Leu以剂量依赖方式激活mTORC1信号。ATF4蛋白水平在Leu浓度达10%以上基本稳定,表明ISR仅在极低Leu水平下发生。(3)总AA缺失对MAC-T细胞mTORC1信号有更强抑制作用。与在293T等细胞的研究报导不同,MAC-T细胞mTORC1信号对Leu缺失的反应远低于总AA缺失。Leu缺失4 h降低4EBP1磷酸化水平19%,总AA缺失4 h则降低了79%,葡萄糖缺失4 h使4EBP1磷酸化水平降低了43%。进一步比较了MAC-T和293T细胞mTORC1信号对Leu和总AA缺失的反应,Leu和总AA缺失分别使MAC-T细胞4EBP1磷酸化水平下降24%和70%,293T细胞则分别下降70%和86%,且向总AA缺失培养基中添加Leu后,对MAC-T细胞4EBP1磷酸化水平无影响,293T细胞处理1 h的4EBP1磷酸化水平则显著升高,表明两种细胞对Leu缺失的反应有差异。比较了Leu与总AA缺失对MAC-T细胞GCN2-e IF2α信号通路的影响,两者均显著提高了e IF2α磷酸化水平,但提高的幅度相似,未见总AA缺失对e IF2α磷酸化有更强促进作用。(4)Leu缺失对MAC-T细胞ISR有更强激活作用。将MAC-T细胞分别在缺失总AA、Leu或葡萄糖的培养基中培养,观察ATF4蛋白表达量变化,出乎意料的是,Leu而不是总AA缺失时ATF4表达量最高,葡萄糖缺失对ATF4表达量的刺激作用最小,表明Leu缺失对MAC-T细胞ISR有更强刺激作用。因AA缺乏能够引起细胞ISR,促进蛋白水解等以维持细胞内AA浓度稳定,提示ISR可能介导了部分Leu调控mTORC1信号作用。(5)ISR对MAC-T细胞mTORC1信号有调控作用。用ISR抑制剂处理MAC-T细胞显著降低了Leu缺失时ATF4蛋白表达量和4EBP1与RPS6磷酸化水平。用sh ATF4敲低ATF4基因,显著降低Leu缺失时4EBP1磷酸化水平,证实ISR减弱Leu缺失对MAC-T细胞mTORC1信号的抑制作用。(6)总AA和Leu缺失对MAC-T细胞总蛋白合成速度影响差异不显著。嘌呤霉素标记法测定了Leu或总AA缺失对MAC-T细胞总蛋白合成速度的影响,两种处理均显著抑制了细胞总蛋白合成,但抑制程度相近。综上所述,MAC-T细胞mTORC1信号对Leu缺失有响应速度快、剂量依赖和响应强度弱于全AA缺失的特征,与293T细胞mTORC1信号Leu缺失响应模式存在差异。研究还发现ISR减弱Leu缺失对MAC-T细胞mTORC1信号的抑制作用,且Leu缺失对MAC-T细胞ISR的刺激作用强于总AA缺失,Leu缺失对MAC-T细胞GCN2-e IF2α信号的刺激作用与总AA缺失相似。以上结果均对Leu缺乏对乳蛋白合成抑制作用较弱提出了解释,但需在机体水平进一步研究证实。2.MAC-T细胞对Leu缺失的适应性反应(1)Leu缺失引起细胞存活导向的适应性变化。Leu缺失升高凋亡早期细胞比例,但降低坏死与凋亡晚期细胞比例,正常细胞比例反而高于对照组。KEGG分析表明,Leu缺失引起的转录组变化显著富集到癌症相关、MAPK信号、细胞周期、细胞衰老、氨酰-t RNA合成等十条途径。细胞增殖与Leu浓度呈幂函数关系,在极低Leu浓度端剧烈变化。以上结果均表明Leu缺失引起MAC-T细胞以存活为导向的适应性变化。全AA缺失则不同,凋亡早期,坏死和凋亡晚期细胞比例均升高,正常细胞比例低于对照组。与此一致,全AA缺失引起的线粒体膜通透性的升高也远高于Leu缺失。(2)Leu缺失降低MAC-T细胞能量水平。Leu缺失升高MAC-T细胞内ADP/ATP比例,细胞能量水平降低。Leu缺失显著降低琥珀酸浓度,富马酸浓度有降低趋势,其他底物浓度升高或无显著变化。细胞相对pH值显著降低,丙酮酸脱氢酶磷酸化程度显著升高。以上结果表明,Leu缺乏可能主要通过抑制线粒体氧化降低细胞能量水平。(3)Leu缺失提高MAC-T细胞乙酰化水平。Leu缺失升高MAC-T细胞、降低293T细胞蛋白乙酰化水平。转录组分析表明,Leu缺失升高MAC-T细胞BCAA分解代谢、Leu转运、脂肪酸β氧化、蛋白乙酰化和降低脂肪酸从头合成相关基因的表达,提示Leu缺失将脂肪酸代谢导向β氧化。综上所述,Leu缺失降低MAC-T细胞能量水平,升高蛋白乙酰化水平,促进脂肪β氧化、抑制脂肪从头合成基因表达,细胞代谢呈存活导向变化。3.Ser合成-ISR介导Leu缺失对MAC-T和293T细胞mTORC1信号的差异化调控(1)Leu缺失促进MAC-T细胞Ser合成。Leu缺失升高了MAC-T细胞内包括Ser在内的多数AA浓度。KEGG等分析表明,Leu缺失引起的MAC-T细胞转录组与代谢组变化均显著富集到Ser合成相关途径,Ser合成酶基因表达升高。Leu缺失时,MAC-T细胞中Ser浓度显著升高,药物抑制Ser合成酶,Leu缺失对Ser浓度的升高作用消失,表明Leu缺失刺激Ser合成。代谢组学结果表明,Leu缺失后多种核苷酸相关代谢物浓度发生显著变化。鉴于Ser是核苷酸前体,核苷酸参与mTORC1信号调控,因此对Ser合成是否影响Leu对mTORC1信号调控进行了研究。(2)Ser合成在干扰Leu缺乏引起的ISR上有细胞特异性。Leu缺失升高MAC-T和293T细胞ATF4表达量,引起ISR。同时缺失Ser抑制MAC-T但不抑制293T细胞Leu缺失引起的ISR。药物抑制两种细胞的Ser合成,在显著激活MAC-T细胞ISR的同时抑制了293T细胞ISR。因为ATF4调控4EBP1蛋白表达量,与此相一致,药物抑制Ser合成显著降低了293T细胞4EBP1的表达量,而MAC-T细胞中表达量显著升高。(3)Ser合成介导Leu缺乏对mTORC1信号的调控。在缺失Leu的同时缺失Ser增强了Leu缺失对MAC-T细胞mTORC1信号的抑制作用,对293T细胞Leu缺失抑制mTORC1信号无显著影响。药物抑制Ser合成大幅度抑制MAC-T细胞mTORC1信号,对293T细胞mTORC1信号无显著影响。敲低Ser脱水酶,未显著影响Leu对MAC-T细胞mTORC1活性的调控作用,说明介导Leu调控mTORC1信号的主要是Ser合成而不是分解代谢过程。(4)抑制Ser合成导致MAC-T细胞代谢重编程。药物抑制MAC-T细胞Ser合成酶的同时缺失Ser,极显著地升高了7种TCA循环底物浓度,极显著降低α-KG浓度,引起的TCA循环底物浓度变化远比Leu缺失强烈,说明细胞代谢发生了剧烈重编程,提示Ser合成途径变化是Leu影响细胞代谢重要途径。综上所述,Ser合成-ISR轴介导了Leu缺失对不同细胞代谢和mTORC1信号的差异性调控作用,起介导作用的主要是Ser合成过程。4.抑制Leu分解代谢降低MAC-T细胞mTORC1信号(1)抑制BCAT升高Leu浓度但降低mTORC1信号。药物抑制MAC-T细胞BCAA转氨酶(BCAT)活性,显著升高了细胞内Leu浓度,但降低了4EBP1和RPS6磷酸化水平,mTORC1信号受到抑制。因三种BCAA共享BCAT,因此进一步敲低了Leu分解代谢途径专用MCCC1酶活性,mTORC1信号也不升反降,表明抑制Leu分解代谢抑制了mTORC1信号。(2)细胞代谢变化介导Leu分解代谢抑制的mTORC1信号调控效应。药物抑制BCAT显著改变MAC-T细胞TCA循环底物浓度,细胞丙酮酸浓度降低、乙酰-CoA浓度升高,氨基酸浓度发生广泛变化,脂肪酸分解代谢相关基因表达量普遍上调,细胞脂肪酸构成发生明显改变。试验进一步评估细胞代谢变化对Leu调控mTORC1信号的介导作用,添加乙酰-CoA显著降低了药物抑制BCAT的mTORC1信号降低作用。以上结果表明,细胞代谢变化可在很大程度上干扰Leu对mTORC1信号的调控,提示细胞代谢结构是Leu对不同类型细胞产生差异性调控作用的重要基础。综上所述,本部分研究证实了细胞代谢结构对Leu调控mTORC1信号有干扰作用。结论:(1)Leu缺失对mTORC1活性的影响具有细胞特异性;(2)Ser合成-ISR轴介导了Leu缺失对MAC-T和293T细胞mTORC1信号的差异化调控;(3)细胞代谢结构变化在Leu调控mTORC1信号活性过程中起重要作用。更多还原

【Abstract】 Leucine(Leu)deficiency inhibits protein synthesis through mTORC1 signaling.In growing animals,Leu deficiency strongly inhibits muscle protein synthesis.In HEK293 and other cells,the inhibition of mTORC1 activity by Leu deletion was similar to that of total amino acid(AA)deletion.In dairy cows and other lactation animals,the inhibition of Leu deficiency on milk protein synthesis is relatively mild,suggesting that the effects of Leu deficiency on protein synthesis and mTORC1 signal may be cell-specific,but there is no direct evidence and the underlying mechanism is not clear.In order to solve this scientific problem,MAC-T cells were used to observe the changes of mTORC1 signal and metabolic adaptation caused by Leu deficiency,the effects of serine(Ser)synthesis and branched chain amino acid(BCAA)catabolism on mTORC1 signal regulated by Leu.The response characteristics and mechanism of mTORC1 signal to Leu deficiency in MAC-T cells were studied.The results are as follows:1.Leu deletion response of mTORC1 signal in MAC-T cells(1)MAC-T can quickly adapt to Leu deficiency.When MAC-T cells lacked Leu,the phosphorylation levels of 4EBP1 and RPS6 proteins decreased rapidly and remained stable after 2 h,and the phosphorylation level of 4EBP1 increased after 10 h of culture,which was consistent with the reactivation of mTORC1 caused by cell protein decomposition in other studies.The level of ATF4,a marker of integrated stress response(ISR),also increased rapidly after the absence of Leu,and remained stable after 2 h.The above results show that MAC-T cells can quickly adapt to the environment of Leu deletion.(2)Leu activated mTORC1 signal in a dose-dependent manner.The phosphorylation level of 4EBP1 and RPS6 increased with the increase of Leu concentration in MAC-T cells,cultured in DMEM/F12 medium with 0%,5%,10%,50%,100% and 300% Leu concentration,indicating that Leu activated mTORC1 signal in a dose-dependent manner.The level of ATF4 protein is basically stable when the concentration of Leu is more than 10%,indicating that ISR occurs only at very low Leu levels.(3)Total AA deletion causes a stronger inhibitory effect on mTORC1 signal of MAC-T cells.Different from those reported in 293 T and muscle cells,the response of mTORC1 signal of MAC-T cells to Leu deletion was much lower than that of total AA deletion.Leu deletion for 4 h decreased 4EBP1 phosphorylation level by 19%,total AA deletion for 4 h decreased by 79%,and glucose deficiency for 4 h decreased by 43%.Furthermore,the responses of mTORC1 signals of MAC-T and 293 T cells to Leu and total AA deletions were compared.Leu and total AA deletion decreased the 4EBP1 phosphorylation level of MAC-T cells by24% and 70%,respectively,while that of 293 T cells decreased by 70% and 86%,respectively.The addition of Leu to the total AA deletion medium had no effect on the 4EBP1 phosphorylation level of MAC-T cells,but in 293 T cells the addition increased significantly after 1 hour treatment.The results showed that the responses of the two kinds of cells to Leu deletion were different.The effects of Leu and total AA deficiency on GCN2-e IF2α signaling pathway in MAC-T cells were compared.Both of them significantly increased the level of e IF2α phosphorylation,but the extent of increase was similar.There was no stronger effect of total AA deficiency on e IF2α phosphorylation.(4)Leu deletion has a stronger activating effect on ISR in MAC-T cells.MAC-T cells were cultured in the medium without total AA,Leu or glucose,respectively,and the changes of ATF4 protein expression were observed.Unexpectedly,the expression of ATF4 was the highest when Leu instead of total AA was deleted,and glucose deficiency had the least stimulating effect on ATF4 expression,indicating that Leu deletion had a stronger stimulating effect on ISR in MAC-T cells.The lack of AA can cause cell ISR and promote proteolysis to maintain the stability of intracellular AA concentration,suggesting that ISR may mediate part of the regulation of mTORC1 signal by Leu.(5)ISR can regulate the mTORC1 signal of MAC-T cells.Treatment of MAC-T cells with ISR inhibitors significantly decreased the expression of ATF4 protein and the level of phosphorylation of 4EBP1 and RPS6 in MAC-T cells with Leu deletion.Knockdown of ATF4 gene with sh ATF4 significantly decreased the level of 4EBP1 phosphorylation during Leu deletion,which confirmed that ISR attenuated the inhibitory effect of Leu deletion on mTORC1 signal in MAC-T cells.(6)There was no significant difference in the effect of total AA and Leu deletion on the total protein synthesis rate of MAC-T cells.Puromycin labeling method was used to determine the effect of Leu or total AA deletion on total protein synthesis in MAC-T cells.Both treatments significantly inhibited total protein synthesis,but the inhibition was similar.In summary,the response of mTORC1 signal of MAC-T cells to Leu deletion was faster,dose-dependent and weaker than that of total AA deletion,which was different from that of293 T cells.It was also found that ISR attenuated the inhibitory effect of Leu deletion on mTORC1 signal of MAC-T cells,and the stimulating effect of Leu deletion on ISR of MAC-T cells was stronger than that of total AA deletion,and the stimulating effect of Leu deletion on GCN2-e IF2 α signal of MAC-T cells was similar to that of total AA deletion.The above results provide an explanation for the weak inhibitory effect of Leu deficiency on milk protein synthesis,but it needs to be confirmed by further studies at the body level.2.Adaptive responses of MAC-T cells to Leu deficiency(1)The deletion of Leu causes the adaptive change of cell survival orientation.Leu deletion increased the proportion of early apoptotic cells,but decreased the proportion of necrotic and late apoptotic cells,and the proportion of normal cells was higher than that of the control group.KEGG analysis showed that the transcriptome changes caused by Leu deletion were significantly enriched in cancer-related pathways,MAPK signaling,cell cycle,cell senescence,aminoacyl-t RNA synthesis and so on.There was a power function relationship between cell proliferation and Leu concentration,and changed sharply at very low Leu concentration.The above results suggest that Leu deletion leads to survival-oriented adaptive changes in MAC-T cells.The percentage of normal cells in the early stage of apoptosis,necrosis and late stage of apoptosis increased,while the proportion of normal cells was lower than that in the control group.The loss of total AA was different.Consistent with this,the increase of mitochondrial membrane permeability caused by total AA deletion was much higher than that of Leu deletion.(2)The deletion of Leu reduces the energy level of MAC-T cells.Leu deletion increased the proportion of ADP/ATP in MAC-T cells and decreased the energy level of cells.Leu deletion significantly decreased succinic acid concentration,fumaric acid concentration tended to decrease,and other substrate concentrations increased or had no significant change.The relative pH value of cells decreased significantly and the degree of pyruvate dehydrogenase phosphorylation increased significantly.These results suggest that Leu deficiency may reduce cell energy level mainly by inhibiting mitochondrial oxidation.(3)Leu deletion increased the acetylation level of MAC-T cells.Leu deletion increased the level of protein acetylation in MAC-T cells and decreased in 293 T cells.Transcriptome analysis showed that Leu deletion increased BCAA catabolism,Leu transport,fatty acid βoxidation,protein acetylation and decreased the expression of genes related to de novo fatty acid synthesis in MAC-T cells,suggesting that Leu deletion leads fatty acid metabolism to βoxidation.In summary,Leu deletion decreased the energy level of MAC-T cells,increased the level of protein acetylation,promoted fat β oxidation,inhibited fatty acid synthesis gene expression,and cell metabolism showed survival-oriented changes.3.Ser synthesis-ISR mediates the differential regulation of mTORC1 signal in MAC-T and 293 T cells by Leu deletion.(1)Leu deletion promotes Ser synthesis in MAC-T cells.The deletion of Leu increased the concentration of most AA including Ser in MAC-T cells.KEGG analysis showed that the changes of MAC-T cell transcriptional group and metabolic group caused by Leu deletion were significantly enriched in the pathway related to Ser synthesis,and the expression of Ser synthase gene was increased.When Leu was deleted,the concentration of Ser in MAC-T cells increased significantly.When drugs were used to inhibit Ser synthetase,and the effect of Leu deletion on the increase of Ser concentration disappeared,indicating that Leu deletion stimulated Ser synthesis.Metabonomic results showed that the concentrations of many nucleotide-related metabolites changed significantly after Leu deletion.Since Ser is the precursor of nucleotides and nucleotides are involved in the regulation of mTORC1 signal,the effect of Ser synthesis on the regulation of mTORC1 signal by Leu was studied.(2)Ser synthesis is cell specific in ISR caused by interfering with Leu deficiency.Leu deletion increased the expression of ATF4 in MAC-T and 293 T cells,resulting in ISR.At the same time,deletion of Ser inhibits MAC-T but does not inhibit ISR caused by deletion of Leu in 293 T cells.The drug inhibited the Ser synthesis of the two kinds of cells,significantly activated ISR in MAC-T cells and inhibited ISR in 293 T cells.Because ATF4 regulates the expression of 4EBP1 protein,consistent with this,drug inhibition of Ser synthesis significantly decreased the expression of 4EBP1 in 293 T cells,while increased in MAC-T cells.(3)Ser synthesis mediates that Leu lacks the regulation of mTORC1 signal.The deletion of Leu and Ser enhanced the inhibitory effect of Leu deletion on mTORC1 signal of MAC-T cells,but had no significant effect on the inhibition of mTORC1 signal of 293 T cells by Leu deletion.Drug inhibition of Ser synthesis significantly inhibited mTORC1 signal of MAC-T cells,but had no significant effect on mTORC1 signal of 293 T cells.Knocking down the Ser dehydrase did not significantly affect the regulation of mTORC1 activity by Leu on MAC-T cells,indicating that the regulation of mTORC1 signal by Leu is mainly mediated by Ser synthesis rather than catabolism.(4)Inhibition of Ser synthesis leads to metabolic reprogramming of MAC-T cells.The drug inhibited Ser synthetase and deleted Ser in MAC-T cells,which significantly increased the concentration of seven kinds of TCA cycle substrate and decreased the concentration ofα-KG.The change of TCA cycle substrates concentrations were much stronger than that of Leu deletion,indicating that severe reprogramming occurred in cell metabolism,suggesting that the change of Ser synthesis pathway is an important pathway for Leu to affect cell metabolism.In summary,the Ser synthesis-ISR axis mediates the differential regulation of Leu deletion on different cell metabolism and mTORC1 signals,which is mainly mediated by the process of Ser synthesis.4.Inhibition of Leu catabolism can reduce mTORC1 signal activity in MAC-T cells.(1)Inhibition of BCAT increased Leu concentration but decreased mTORC1 signal.After inhibiting the activity of BCAA transaminase(BCAT)in MAC-T cells,the intracellular Leu concentration was significantly increased,but the phosphorylation levels of 4EBP1 and RPS6 were decreased.Because three kinds of BCAA share BCAT,the activity of MCCC1 enzyme specific to Leu catabolism pathway was further knocked down,and mTORC1 signal decreased,indicating that inhibition of Leu catabolism suppressed mTORC1 signal.(2)The changes of cell metabolism mediate the mTORC1 signal regulation of Leu catabolism inhibition.Drug inhibition of BCAT significantly changed the concentrations of TCA cycle substrates,the concentration of pyruvate decreased,the concentration of acetyl-CoA increased,the concentrations of AA changed widely,the expression of genes related to fatty acid catabolism was generally up-regulated,and the fatty acid composition of MAC-T cells changed significantly.The experiment further evaluated the mediating effect of cell metabolic changes on the regulation of mTORC1 signal by Leu.The addition of acetyl-CoA significantly reduced the inhibitory effect of drug on the decrease of mTORC1 signal.The above results showed that the changes of cell metabolism can interfere with the regulation of mTORC1 signal by Leu to a great extent,suggesting that the structure of cell metabolism is an important basis for the differential regulation of Leu on different types of cells.In summary,this part of the study confirmed that the cell metabolic structure interferes with the regulation of mTORC1 signal by Leu.Conclusion:(1)the effect of Leu deletion on mTORC1 activity is cell specific;(2)the Ser synthesis-ISR axis mediates the differential regulation of mTORC1 signal in MAC-T and293 T cells by Leu deletion;(3)the changes of cellular metabolic structure play an important role in the regulation of mTORC1 signal activity by Leu.更多还原