【作者】 陈雷；

【导师】 吴嘉炜；

【作者基本信息】 清华大学 ， 生物学， 2010， 博士

【摘要】 AMPK(AMP激活的蛋白质激酶)是一种保守的异源三聚体蛋白质激酶,其酶活力能够被AMP上调。该蛋白质激酶能够通过感受细胞能量状态来维持真核细胞的ATP生成和消耗的平衡,即能量稳态。同时,AMPK在调控细胞生长和增殖、建立和稳定细胞极性、调节动物寿命、调控生理节律等方面也起着重要作用。这些重要的功能使AMPK成为治疗肥胖症、II型糖尿病和某些癌症的药物靶点。然而,AMPK的活力是如何被AMP调节的目前仍然不清楚。在本论文工作中,我们解析了裂殖酵母AMPKα亚基KD-AID片段的结构。该片段包含激酶的催化结构域(KD)和自抑制结构域(AID)。AID结合在KD背面的铰链区,与KD的N-lobe及C-lobe均有相互作用。结构分析表明,AID的结合可能限制了αC的移动,从而导致了KD-AID片段与单独的KD相比活力较低。AMP不仅能够别构激活AMPK,还可以抑制AMPKα亚基T172位点的去磷酸化。进一步的体外酶动力学研究发现:破坏KD-AID相互作用界面能够解除自抑制现象,并且含有这些突变的AMPK全酶的活力也不再受AMP调节。这些结构和生化上的研究揭示了AMPK自抑制的主要机制。据此,我们提出了AMP对AMPK调节的构象转变模型。此外,本论文工作对已发表的AMPK糖原结合结构域(GBD)位置提出了质疑,并指出了新的GBD可能的结合位置及该位置相应的生理意义。本论文工作还对AMPK全酶蛋白进行了结晶筛选,为今后进一步的结构生物学研究奠定了基础。总之,本论文工作对于理解AMPK的调节机制具有重要意义。更多还原

【Abstract】 AMPK (AMP-activated protein kinase) is a conserved heterotrimeric protein kinase. Its catalytic activity can be activated by AMP, which enables it to maintain the balance between ATP production and consumption (energy homeostasis) in eukaryotic cells by sensing the cellular energy status. It also plays important roles in the regulation of cell growth and proliferation, in the establishment and maintenance of cell polarity, in regulation of animal life span and circadian clock. These important functions have rendered AMPK a major drug target for obesity, type 2 diabetes and certain cancer treatments. However, the regulation mechanism of AMPK activity by AMP-binding remains unsolved.Here we solved the crystal structures of a fragment of AMPKα-subunit (KD-AID) from fission yeast that contains the catalytic kinase domain and an autoinhibitory domain (AID). AID binds, from the“backside”, to the hinge region of its KD, forming contacts with both amino-terminal and carboxy-terminal lobes. Structural analysis indicated that AID-binding might constrain the mobility of helixαC, hence resulting in an autoinhibited KD-AID with much lower kinase activity than that of the kinase domain alone. AMP activates AMPK both allosterically and by inhibiting dephosphorylation of T172 site onαsubunit. Our further in vitro kinetic studies demonstrated that disruption of the KD-AID interface reverses the autoinhibition and these AMPK heterotrimeric mutants no longer respond to the change of AMP concentration. The structural and biochemical data have revealed the primary mechanism of AMPK autoinhibition. Based on these data, we proposed a conformational switch model for AMPK activation by AMP.In addition, we questioned the position of glycogen binding domain (GBD) proposed by previous studies. And a new possible position of GBD has been proposed, along with the corresponding physiological significance. The crystallization conditions of AMPK holoenzyme have also been screened, which provides the basis for further structure study.Over all, these results have contributed greatly in understanding the mechanism of AMPK regulation.更多还原