Identification of molecular target of AMP-activated protein kinase activator by affinity purification and mass spectrometry
Identification of molecular target of AMP-activated protein kinase activator by affinity purification and mass spectrometry
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DOI：
10.1021/ac0484631
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发表时间：
2005-04-01
影响因子：
7.4
通讯作者：
Ichikawa, K
中科院分区：
文献类型：
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作者：
Kosaka, T;Okuyama, R;Ichikawa, K
我们展示了一种通过亲和纯化和质谱鉴定小分子化合物的分子靶标的有效方法。使用亲和柱从靶细胞裂解物中分离结合蛋白，所述亲和柱固定活性和非活性化合物。所有结合到这些亲和柱的蛋白质通过使用胰蛋白酶消化洗脱，然后通过质谱法鉴定。通过从固定有活性化合物的亲和柱中减去固定有非活性化合物的亲和柱中鉴定的蛋白质，确定与活性化合物（分子靶标的候选物）的特异性结合蛋白质。该方法被应用于鉴定D942的分子靶点，D942是一种呋喃羧酸衍生物，其通过AMP激活的蛋白激酶（AMPK-）激活来增加L 6肌细胞中的葡萄糖摄取。为了阐明D942激活AMPK的机制，制备了固定D942及其无活性衍生物D 768的亲和柱，并从L 6细胞裂解物中纯化结合蛋白。NAD（P）H脱氢酶[醌] 1（复合物1），通过减去与D 768的结合蛋白而显示为与D942的特异性结合蛋白之一，被D942而不是D 768部分抑制。由于复合物I活性的抑制导致ATP/AMP比率的降低，并且ATP/AMP比率的变化触发AMPK活化，因此我们将复合物I鉴定为D942活化AMPK的潜在蛋白质靶标。这一结果表明，我们的方法可以提供有关小分子化合物的分子靶点，特别是尚未鉴定的靶蛋白的关键信息。
We show an efficient method to identify molecular targets of small molecular compounds by affinity purification and mass spectrometry. Binding proteins were isolated from target cell lysate using affinity columns, which immobilized the active and inactive compounds. All proteins bound to these affinity columns were eluted by digestion using trypsin and then were identified by mass spectrometry. The specific binding proteins to the active compound, a candidate for molecular targets, were determined by subtracting the identified proteins in an inactive compound-immobilized affinity column from that in an active compound-immobilized affinity column. This method was applied to identification of molecular targets of D942, a furancarboxylic acid derivative, which increases glucose uptake in L6 myocytes through AMP-activated protein kinase (AMPK-) activation. To elucidate the mechanism of AMPK activation by D942, affinity columns that immobilized D942 and its inactive derivative, D768, were prepared, and the binding proteins were purified from L6 cell lysate. NAD(P)H dehydrogenase [quinone] 1 (complex 1), which was shown as one of the specific binding proteins to D942 by subtracting the binding proteins to D768, was partially inhibited by D942, not D768. Because inhibition of complex I activity led to a decrease in the ATP/AMP ratio, and the change in the ATP/AMP ratio triggered AMPK activation, we identified complex I as a potential protein target of AMPK activation by D942. This result shows our approach can provide crucial information about the molecular targets of small molecular compounds, especially target proteins not yet identified.