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首页> 美国卫生研究院文献>Springer Open Choice >Intact Protein Analysis at 21 Tesla and X-Ray Crystallography Define Structural Differences in Single Amino Acid Variants of Human Mitochondrial Branched-Chain Amino Acid Aminotransferase 2 (BCAT2)
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Intact Protein Analysis at 21 Tesla and X-Ray Crystallography Define Structural Differences in Single Amino Acid Variants of Human Mitochondrial Branched-Chain Amino Acid Aminotransferase 2 (BCAT2)

机译：在21 Tesla和X射线晶体学上的完整蛋白质分析定义了人类线粒体支链氨基酸氨基转移酶2（BCAT2）的单个氨基酸变异中的结构差异。

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Structural technologies are an essential component in the design of precision therapeutics. Precision medicine entails the development of therapeutics directed toward a designated target protein, with the goal to deliver the right drug to the right patient at the right time. In the field of oncology, protein structural variants are often associated with oncogenic potential. In a previous proteogenomic screen of patient-derived glioblastoma (GBM) tumor materials, we identified a sequence variant of human mitochondrial branched-chain amino acid aminotransferase 2 as a putative factor of resistance of GBM to standard-of-care-treatments. The enzyme generates glutamate, which is neurotoxic. To elucidate structural coordinates that may confer altered substrate binding or activity of the variant BCAT2 T186R, a ~45 kDa protein, we applied combined ETD and CID top-down mass spectrometry in a LC-FT-ICR MS at 21 T, and X-Ray crystallography in the study of both the variant and non-variant intact proteins. The combined ETD/CID fragmentation pattern allowed for not only extensive sequence coverage but also confident localization of the amino acid variant to its position in the sequence. The crystallographic experiments confirmed the hypothesis generated by in silico structural homology modeling, that the Lys59 side-chain of BCAT2 may repulse the Arg186 in the variant protein (PDB code: 5MPR), leading to destabilization of the protein dimer and altered enzyme kinetics. Taken together, the MS and novel 3D structural data give us reason to further pursue BCAT2 T186R as a precision drug target in GBM. >Graphical Abstractᅟ
机译：结构技术是精密疗法设计中必不可少的组成部分。精密医学需要针对指定目标蛋白的治疗方法的开发，目的是在正确的时间将正确的药物输送给正确的患者。在肿瘤学领域，蛋白质结构变异通常与致癌潜力有关。在以前的患者源性胶质母细胞瘤（GBM）肿瘤材料的蛋白质组学筛选中，我们鉴定了人线粒体支链氨基酸氨基转移酶2的序列变异体，作为GBM对标准治疗的耐药性的推定因素。该酶产生谷氨酸，它具有神经毒性。为了阐明可能赋予变异的BCAT2 T186R（约45 kDa蛋白质）改变的底物结合或活性的结构坐标，我们在21 T的LC-FT-ICR MS中应用了ETD和CID自上而下的质谱联用，X-射线晶体学研究变异蛋白和非变异蛋白。组合的ETD / CID片段化模式不仅可以覆盖广泛的序列，而且还可以使氨基酸变体可靠地定位于其在序列中的位置。晶体学实验证实了计算机结构同源性建模产生的假设，即BCAT2的Lys59侧链可能排斥变异蛋白（PDB代码：5MPR）中的Arg186，从而导致蛋白质二聚体不稳定和酶动力学改变。综合起来，MS和新颖的3D结构数据为我们提供了进一步追求BCAT2 T186R作为GBM中精确药物靶点的理由。 <！-fig ft0-> <！-fig @ position =“ anchor” mode =文章f4-> <！-fig mode =“ anchred” f5-> >图形摘要<！- fig / graphic | fig / alternatives / graphic mode =“ anchored” m1-> <！-标题a7->ᅟ

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