Biochemical and biophysical investigations of the interaction between human glucokinase and pro-apoptotic BAD.
Biochemical and biophysical investigations of the interaction between human glucokinase and pro-apoptotic BAD.
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DOI：
10.1371/journal.pone.0171587
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Miller BG
中科院分区：
文献类型：
--
作者：
Rexford A;Zorio DA;Miller BG
据报道，糖酵解酶葡萄糖激酶（GCK）和促凋亡蛋白BAD位于一个五元复合物内，该复合物定位于哺乳动物肝细胞和胰腺β细胞的线粒体。使用BAD的BH 3结构域的合成类似物的光化学交联研究和体外转录/翻译实验支持BAD和GCK之间的直接相互作用。为了研究BAD：GCK相互作用的生物化学和生物物理后果，我们开发了一种生产重组人BAD的方法。与已发表的报道一致，重组BAD显示出对Bcl-xL的高亲和力（KD = 7 nM），并且BAD在BH 3结构域内S118处的磷酸化消除了这种相互作用。出乎意料的是，我们没有检测到协会的重组，全长BAD与重组人胰腺GCK在一系列的蛋白质浓度，使用各种生化方法，包括尺寸排阻色谱，化学交联，分析超离心，等温滴定量热法。此外，荧光偏振分析和等温滴定量热法检测GCK和BAD BH 3肽之间没有直接的相互作用。在高浓度重组BAD存在下GCK的动力学表征显示GCK活性适度（<15%）增加，仅在远低于K0.5值的葡萄糖浓度下可观察到。GCK活性不受BAD BH 3肽的影响。这些结果提出了关于以BAD BH 3结构域为模型的钉合肽类似物的作用机制的问题，据报道，其增强GCK的Vmax值并刺激BAD缺陷胰岛中的胰岛素释放。基于我们的研究结果，我们假设BAD：GCK相互作用，以及任何由此产生的对GCK活性的调节作用，需要线粒体复合物的其他成员的参与。
The glycolytic enzyme glucokinase (GCK) and the pro-apoptotic protein BAD reportedly reside within a five-membered complex that localizes to the mitochondria of mammalian hepatocytes and pancreatic β-cells. Photochemical crosslinking studies using a synthetic analog of BAD’s BH3 domain and in vitro transcription/translation experiments support a direct interaction between BAD and GCK. To investigate the biochemical and biophysical consequences of the BAD:GCK interaction, we developed a method for the production of recombinant human BAD. Consistent with published reports, recombinant BAD displays high affinity for Bcl-xL (KD = 7 nM), and phosphorylation of BAD at S118, within the BH3 domain, abolishes this interaction. Unexpectedly, we do not detect association of recombinant, full-length BAD with recombinant human pancreatic GCK over a range of protein concentrations using various biochemical methods including size-exclusion chromatography, chemical cross-linking, analytical ultracentrifugation, and isothermal titration calorimetry. Furthermore, fluorescence polarization assays and isothermal titration calorimetry detect no direct interaction between GCK and BAD BH3 peptides. Kinetic characterization of GCK in the presence of high concentrations of recombinant BAD show modest (<15%) increases in GCK activity, observable only at glucose concentrations well below the K0.5 value. GCK activity is unaffected by BAD BH3 peptides. These results raise questions as to the mechanism of action of stapled peptide analogs modeled after the BAD BH3 domain, which reportedly enhance the Vmax value of GCK and stimulate insulin release in BAD-deficient islets. Based on our results, we postulate that the BAD:GCK interaction, and any resultant regulatory effect(s) upon GCK activity, requires the participation of additional members of the mitochondrial complex.