Institut National de la Santé et de la Recherche Médicale, Orsay, France.
Biophys J. 2013 Sep 3;105(5):1268-75. doi: 10.1016/j.bpj.2013.06.048.
Hint2, one of the five members of the superfamily of the histidine triad AMP-lysine hydrolase proteins, is expressed in mitochondria of various cell types. In human adrenocarcinoma cells, Hint2 modulates Ca(2+) handling by mitochondria. As Hint2 is highly expressed in hepatocytes, we investigated if this protein affects Ca(2+) dynamics in this cell type. We found that in hepatocytes isolated from Hint2(-/-) mice, the frequency of Ca(2+) oscillations induced by 1 μM noradrenaline was 150% higher than in the wild-type. Using spectrophotometry, we analyzed the rates of Ca(2+) pumping in suspensions of mitochondria prepared from hepatocytes of either wild-type or Hint2(-/-) mice; we found that Hint2 accelerates Ca(2+) pumping into mitochondria. We then resorted to computational modeling to elucidate the possible molecular target of Hint2 that could explain both observations. On the basis of a detailed model for mitochondrial metabolism proposed in another study, we identified the respiratory chain as the most probable target of Hint2. We then used the model to predict that the absence of Hint2 leads to a premature opening of the mitochondrial permeability transition pore in response to repetitive additions of Ca(2+) in suspensions of mitochondria. This prediction was then confirmed experimentally.
Hint2 是组氨酸三联体 AMP-赖氨酸水解酶蛋白超家族的五个成员之一，在各种细胞类型的线粒体中表达。在人肾上腺皮质癌细胞中，Hint2 调节线粒体的 Ca(2+)处理。由于 Hint2 在肝细胞中高度表达，我们研究了该蛋白是否影响该细胞类型的 Ca(2+)动力学。我们发现，在 Hint2(-/-) 小鼠分离的肝细胞中，1 μM 去甲肾上腺素诱导的 Ca(2+)振荡频率比野生型高 150%。使用分光光度法，我们分析了从野生型或 Hint2(-/-) 小鼠肝细胞制备的线粒体悬浮液中的 Ca(2+)泵入率；我们发现 Hint2 加速了 Ca(2+)向线粒体的泵入。然后，我们求助于计算建模来阐明可能解释这两种观察结果的 Hint2 的可能分子靶标。基于另一项研究中提出的详细线粒体代谢模型，我们确定呼吸链是 Hint2 的最可能靶标。然后，我们使用该模型预测，在重复加入 Ca(2+)的线粒体悬浮液中，缺失 Hint2 会导致线粒体通透性转换孔过早打开。这一预测随后通过实验得到证实。
