乙酰化促进丙酮酸激酶M2通过自噬降解积累中间代谢产物
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摘要
蛋白质的乙酰化作为一种关键的翻译后修饰而被广泛的研究,但是此前的大量研究都集中在组蛋白和核内转录因子的乙酰化修饰上面,我们运用质谱的方法,以前列腺组织的细胞质组分为样品,鉴定了组织水平的乙酰化蛋白质组,发现了很多被乙酰化修饰的蛋白,包括很多代谢通路中的酶,这些主要的代谢通路包括糖酵解通路,糖异生通路,三羧酸循环通路,脂肪酸代谢通路。
为了进一步确定乙酰化修饰对代谢酶的调控作用,我们选取了糖酵解中跟肿瘤生长密切相关的一个丙酮酸激酶的剪接体(PKM2)进行了深入研究。丙酮酸激酶是糖酵解的三个关键调节酶之一,催化糖酵解的最后一步反应,将磷酸烯醇式丙酮酸(PEP)和二磷酸腺苷(ADP)生成丙酮酸和三磷酸腺苷(ATP)。丙酮酸激酶有四个剪接体：PKL, PKR, PKM1和PKM2。这些剪接体只在特定的组织中表达,其中PKL在肝脏中表达,PKR在红细胞中表达,PKM1在正常成人组织中表达,而PKM2在胚胎组织和肿瘤组织中表达。
我们通过实验确定了PKM2确实受到乙酰化的调控,确定了其中两个重要的乙酰化位点：赖氨酸305位和赖氨酸433位(正在进一步研究中),证明了K305位点的乙酰化可导致PKM2通过分子伴侣介导的自噬途径(CMA)降解,并且找到了K305位点的乙酰化酶pCAF和去乙酰化酶Sirt1。进一步的研究发现,并且该位点的乙酰化受细胞内葡萄糖浓度的调节,高葡萄糖浓度可促进PKM2的K305位点乙酰化,再通过CMA降解。通过PKM2的降解可积累糖酵解途径的中间产物和NADPH,用于细胞的合成代谢,从而促进肿瘤细胞的生长。
本论文首次报导了乙酰化对分子伴侣介导的自噬降解的促进作用,造成糖酵解代谢中间产物和NADPH的积累,用于肿瘤细胞的生物合成,从而促进肿瘤生长。本论文的工作揭示了造成Warburg效应的分子机制,而且为肿瘤治疗提供了一个潜在的药物靶点。
As a key posttranslational modification, protein acetylation was studied comprehensively, but the majority of acetylation studies have been focused on histones and nuclear transcription regulators. We use tandem liquid chromatography-tandem mass spectrometry (LC/LC-MS/MS) to analyse the affinity-purified acetylated peptides from cytoplasmic fraction of human prostate tissues and found that many acetylated proteins including metabolism enzymes in glycolysis, gluconeogenesis, tricarboxylic acid (TCA) cycle and fatty acid metabolism.
We selected Pyruvate kinase M2 (PKM2) which is important for tumor cell growth to elucidate the acetylation how to regulate metabolic enzymes'function. Pyruvate kinase is one of the three key regulators of glycolysis pathway, catalyzes the last step, changing phosphoenolpyruvate and ADP to pyruvate and ATP. Pyruvate kinase has four isoforms:PKL, PKR, PKM1 and PKM2 which are expressed in a tissue-specific manner. L-type is mostly present in liver; R-type is found exclusively in erythrocytes; Ml-type is mostly in normal adult tissue while M2-type is found in embryo and tumor tissue.
We identified that PKM2 is acetylated on lysine residues, including K305 and K433 (which is undering characterization). We have demonstrated acetylation at K305 promotes PKM2 degradation through chaperone mediated autophagy (CMA). We also defined the acetyltransferase (pCAF) and deacetylase (Sirtl) are responsible for K305 acetylation and deactylation respectively. Further more, we found that K305 acetylation is upregulated by high concentration of glucose in cells, resulting in accumulation of the intermediate metabolites as well as NADPH, which are essential material for growing tumor cells to build up tumor mass.
In the thesis, we have demonstrated that PKM2 acetylation promotes CMA for the first time, leads to accumulation of the intermediate metabolites as well as NADPH which may consequently promotes cancer development. This work has not only shed light on the molecular mechanism underlying Warburg effects but also is meaningful for the potential tumor therapy.
为了进一步确定乙酰化修饰对代谢酶的调控作用,我们选取了糖酵解中跟肿瘤生长密切相关的一个丙酮酸激酶的剪接体(PKM2)进行了深入研究。丙酮酸激酶是糖酵解的三个关键调节酶之一,催化糖酵解的最后一步反应,将磷酸烯醇式丙酮酸(PEP)和二磷酸腺苷(ADP)生成丙酮酸和三磷酸腺苷(ATP)。丙酮酸激酶有四个剪接体：PKL, PKR, PKM1和PKM2。这些剪接体只在特定的组织中表达,其中PKL在肝脏中表达,PKR在红细胞中表达,PKM1在正常成人组织中表达,而PKM2在胚胎组织和肿瘤组织中表达。
我们通过实验确定了PKM2确实受到乙酰化的调控,确定了其中两个重要的乙酰化位点：赖氨酸305位和赖氨酸433位(正在进一步研究中),证明了K305位点的乙酰化可导致PKM2通过分子伴侣介导的自噬途径(CMA)降解,并且找到了K305位点的乙酰化酶pCAF和去乙酰化酶Sirt1。进一步的研究发现,并且该位点的乙酰化受细胞内葡萄糖浓度的调节,高葡萄糖浓度可促进PKM2的K305位点乙酰化,再通过CMA降解。通过PKM2的降解可积累糖酵解途径的中间产物和NADPH,用于细胞的合成代谢,从而促进肿瘤细胞的生长。
本论文首次报导了乙酰化对分子伴侣介导的自噬降解的促进作用,造成糖酵解代谢中间产物和NADPH的积累,用于肿瘤细胞的生物合成,从而促进肿瘤生长。本论文的工作揭示了造成Warburg效应的分子机制,而且为肿瘤治疗提供了一个潜在的药物靶点。
As a key posttranslational modification, protein acetylation was studied comprehensively, but the majority of acetylation studies have been focused on histones and nuclear transcription regulators. We use tandem liquid chromatography-tandem mass spectrometry (LC/LC-MS/MS) to analyse the affinity-purified acetylated peptides from cytoplasmic fraction of human prostate tissues and found that many acetylated proteins including metabolism enzymes in glycolysis, gluconeogenesis, tricarboxylic acid (TCA) cycle and fatty acid metabolism.
We selected Pyruvate kinase M2 (PKM2) which is important for tumor cell growth to elucidate the acetylation how to regulate metabolic enzymes'function. Pyruvate kinase is one of the three key regulators of glycolysis pathway, catalyzes the last step, changing phosphoenolpyruvate and ADP to pyruvate and ATP. Pyruvate kinase has four isoforms:PKL, PKR, PKM1 and PKM2 which are expressed in a tissue-specific manner. L-type is mostly present in liver; R-type is found exclusively in erythrocytes; Ml-type is mostly in normal adult tissue while M2-type is found in embryo and tumor tissue.
We identified that PKM2 is acetylated on lysine residues, including K305 and K433 (which is undering characterization). We have demonstrated acetylation at K305 promotes PKM2 degradation through chaperone mediated autophagy (CMA). We also defined the acetyltransferase (pCAF) and deacetylase (Sirtl) are responsible for K305 acetylation and deactylation respectively. Further more, we found that K305 acetylation is upregulated by high concentration of glucose in cells, resulting in accumulation of the intermediate metabolites as well as NADPH, which are essential material for growing tumor cells to build up tumor mass.
In the thesis, we have demonstrated that PKM2 acetylation promotes CMA for the first time, leads to accumulation of the intermediate metabolites as well as NADPH which may consequently promotes cancer development. This work has not only shed light on the molecular mechanism underlying Warburg effects but also is meaningful for the potential tumor therapy.
引文
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[2]SPANGE S, WAGNER T, HEINZEL T, et al. Acetylation of non-histone proteins modulates cellular signalling at multiple levels[J]. Int J Biochem Cell Biol,2009, 41(1):185-98.
[3]GLOZAK M A, SENGUPTA N, ZHANG X H, et al. Acetylation and deacetylation of non-histone proteins[J]. Gene,2005,363(15-23).
[4]KOUZARIDES T. Acetylation:a regulatory modification to rival phosphorylation? [J]. Embo J,2000,19(6):1176-9.
[5]GEISS-FRIEDLANDER R, MELCHIOR F. Concepts in sumoylation:a decade on[J]. Nat Rev Mol Cell Biol,2007,8(12):947-56.
[6]SHALIZI A, GAUDILLIERE B, YUAN Z Q, et al. A calcium-regulated MEF2 sumoylation switch controls postsynaptic differentiation[J]. Science,2006,311(5763): 1012-7.
[7]KIRAN BATTA, CHANDRIMA DAS, SHRIKANTH GADAD, et al. Reversible acetylation of non histone role in cellular function and disease[M] [J]. Chromatin and Disease,,2007,193-212.
[8]KRUSE J P, GU W. SnapShot:p53 posttranslational modifications[J]. Cell,2008, 133(5).
[9]JEONG J W, BAE M K, AHN M Y, et al. Regulation and destabilization of HIF-1 alpha by ARD1-mediated acetylation[J]. Cell,2002,111(5):709-20.
[10]YOO Y G, KONG G, LEE M O. Metastasis-associated protein 1 enhances stability of hypoxia-inducible factor-1 alpha protein by recruiting histone deacetylase 1 [J]. Embo J,2006,25(6):1231-41.
[11]BILTON R, TROTTIER E, POUYSSEGUR J, et al. ARDent about acetylation and deacetylation in hypoxia signalling[J]. Trends Cell Biol,2006,16(12):616-21.
[12]SHIMAZU T, KOMATSU Y, NAKAYAMA K I, et al. Regulation of SV40 large T-antigen stability by reversible acetylation[J]. Oncogene,2006,25(56):7391-400.
[13]KRAMER O H, ZHU P, OSTENDORFF H P, et al. The histone deacetylase inhibitor valproic acid selectively induces proteasomal degradation of HDAC2[J]. Embo J,2003,22(13):3411-20.
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