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人源一磷酸腺苷激活蛋白激酶催化亚基自抑制机制研究
发表日期: 2008-05-04
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    一磷酸腺苷激活蛋白激酶(AMPK)是新发现的调节体内糖代谢和脂代谢紊乱并维持其平衡的一类Ser/Thr蛋白激酶, 在体内糖代谢和脂代谢中发挥主导作用,是反映细胞内能量状态变化的感受器和代谢主开关。体内AMPK活化后在糖代谢、脂代谢中产生的有益效应,使之成为治疗II型糖尿病及肥胖症的一个新的靶点。
     AMPK是一个由α催化亚基和β、γ调节亚基共同组成的异源三聚体蛋白激酶。已有文献报道,哺乳动物细胞内AMPKα催化亚基由于含有一个自抑制区域(α1: 313-392残基序列)几乎不具有活性,但是其具体的自抑制机制目前并不是很清楚。通过对人源AMPKα1催化亚基采用截短、缺失、定点突变及结构模拟等分子生物学方法发现:一段不同种属间保守的含有α螺旋的残基片段313-335对于α1催化亚基的自抑制活性起到很重要的作用,其中该自抑制片段内的Leu328残基与自抑制区外的Val298残基通过疏水相互作用来稳定催化亚基的自抑制活性构象。在COS7细胞内过表达V298G、L328Q或313-335自抑制区缺失突变的α1催化亚基后显著性地增强了胞内AMPK的下游底物乙酰辅酶A羧化酶(ACC)的磷酸化水平;在人肝癌细胞HepG2内瞬时转染V298G、L328Q或313-335自抑制区缺失突变体后显著性地提高了葡萄糖吸收水平,这些结果表明V298G、L328Q和自抑制区缺失突变的α1催化亚基在细胞内是具有持续活性的。
    以上结果已于2007年在J Biol Chem杂志上发表,为AMPK的自抑制机制提出的重要的理论假设和实验证据。该论文在相关领域引起关注,目前已被发表于Nature、Nat Rev Mol Cell Biol、EMBO J、J Biol Chem、BBRC等6篇论文引用。

Conserved -Helix Acts as Autoinhibitory Sequence in AMP-activated Protein Kinase Subunits

Pang T, Xiong B, Li JY, Qiu BY, Jin GZ, Shen JK, Li J*.
J. Biol. Chem. (IF: 5.808)
Vol. 282, No. 1, pp. 495–506, January 5, 2007
AMP-activated protein kinase (AMPK) acts as an energy sensor, being activated by metabolic stresses and regulating cellular metabolism. AMPK is a heterotrimer consisting of a catalytic subunit and two regulatory subunits, and . It had been reported that the mammalian AMPK subunit contained an autoinhibitory domain ( 1: residues 313-392) and had little kinase activity. We have found that a conserved short segment of the subunit ( 1-(313-335)), which includes a predicted -helix, is responsible for subunit autoinhibition. The role of the residues in this segment for autoinhibition was further investigated by systematic site-directed mutation. Several hydrophobic and charged residues, in particular Leu-328, were found to be critical for 1 autoinhibition. An autoinhibitory structural model of human AMPK 1-(1-335) was constructed and revealed that Val-298 interacts with Leu-328 through hydrophobic bonding at a distance of about 4 Å and may stabilize the autoinhibitory conformation. Further mutation analysis showed that V298G mutation significantly activated the kinase activity. Moreover, the phosphorylation level of acetyl-CoA carboxylase, the AMPK downstream substrate, was significantly increased in COS7 cells overexpressing AMPK 1-(1-394) with deletion of residues 313-335 ( 394) and a V298G or L328Q mutation, and the glucose uptake was also significantly enhanced in HepG2 cells transiently transfected with 394, V298G, or L328Q mutants, which indicated that these AMPK 1 mutants are constitutively active in mammalian cells and that interaction between Leu-328 and Val-298 plays an important role in AMPK autoinhibitory function.
 

 

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