Karageorgos Ioannis, Silin Vitalii I, Zvonok Nikolai, Marino John, Janero David R, Makriyannis Alexandros
Biomolecular Measurement Division, National Institute of Standards and Technology, Gaithersburg, MD, 20899, United States; Institute for Bioscience and Biotechnology Research, Rockville, MD, 20850, United States.
Institute for Bioscience and Biotechnology Research, Rockville, MD, 20850, United States.
Anal Biochem. 2017 Nov 1;536:90-95. doi: 10.1016/j.ab.2017.08.009. Epub 2017 Aug 17.
Human monoacylglycerol lipase (hMAGL) plays a key role in homeostatic tuning of the endocannabinoid signaling system and supports aggressive tumorogenesis, making this enzyme a promising therapeutic target. hMAGL features a membrane-associated lid domain that regulates entry of endocannabinoid lipid substrates into the hydrophobic channel accessing the active site, likely from the membrane bilayer. The present work applied simultaneous surface plasmon resonance and electrochemical impedance spectroscopy measurements to show that, in absence of the substrate, hMAGL can remove phospholipid molecules from the membrane and, thereby, disintegrate pre-formed, intact, tethered phospholipid bilayer membrane mimetics (tBLMs) composed of unsaturated phosphatidylcholines. To probe the mechanism of hMAGL-induced on tBLMs compromise, we investigated the effect of wild type and mutant hMAGLs and hMAGL rendered catalytically inactive, as a function of concentration and in the presence of chemically distinct active-site inhibitors. Our data show that hMAGL's lid domain and hydrophobic substrate-binding pocket play important roles in hMAGL-induced bilayer lipid mobilization, whereas hydrolytic activity of the enzyme does not appear to be a factor.
人单酰甘油脂肪酶（hMAGL）在内源性大麻素信号系统的稳态调节中起关键作用，并支持侵袭性肿瘤发生，使该酶成为一个有前景的治疗靶点。hMAGL具有一个与膜相关的盖子结构域，该结构域调节内源性大麻素脂质底物进入可能来自膜双层的通向活性位点的疏水通道。目前的工作应用同步表面等离子体共振和电化学阻抗谱测量表明，在没有底物的情况下，hMAGL可以从膜上去除磷脂分子，从而分解由不饱和磷脂酰胆碱组成的预先形成的完整、 tethered磷脂双层膜模拟物（tBLMs）。为了探究hMAGL诱导tBLMs受损的机制，我们研究了野生型和突变型hMAGLs以及催化失活的hMAGL的作用，作为浓度的函数，并在存在化学性质不同的活性位点抑制剂的情况下进行研究。我们的数据表明，hMAGL的盖子结构域和疏水底物结合口袋在hMAGL诱导的双层脂质动员中起重要作用，而该酶的水解活性似乎不是一个因素。
