Kyoung Minjoung, Russell Sarah J, Kohnhorst Casey L, Esemoto Nopondo N, An Songon
Department of Chemistry and Biochemistry, University of Maryland Baltimore County , 1000 Hilltop Circle, Baltimore, Maryland 21250, United States.
Biochemistry. 2015 Jan 27;54(3):870-80. doi: 10.1021/bi501480d. Epub 2015 Jan 15.
Enzymes in human de novo purine biosynthesis have been demonstrated to form a reversible, transient multienzyme complex, the purinosome, upon purine starvation. However, characterization of purinosomes has been limited to HeLa cells and has heavily relied on qualitative examination of their subcellular localization and reversibility under wide-field fluorescence microscopy. Quantitative approaches, which are particularly compatible with human disease-relevant cell lines, are necessary to explicitly understand the purinosome in live cells. In this work, human breast carcinoma Hs578T cells have been utilized to demonstrate the preferential utilization of the purinosome under purine-depleted conditions. In addition, we have employed a confocal microscopy-based biophysical technique, fluorescence recovery after photobleaching, to characterize kinetic properties of the purinosome in live Hs578T cells. Quantitative characterization of the diffusion coefficients of all de novo purine biosynthetic enzymes reveals the significant reduction of their mobile kinetics upon purinosome formation, the dynamic partitioning of each enzyme into the purinosome, and the existence of three intermediate species in purinosome assembly under purine starvation. We also demonstrate that the diffusion coefficient of the purine salvage enzyme, hypoxanthine phosphoribosyltransferase 1, is not sensitive to purine starvation, indicating exclusion of the salvage pathway from the purinosome. Furthermore, our biophysical characterization of nonmetabolic enzymes clarifies that purinosomes are spatiotemporally different cellular bodies from stress granules and cytoplasmic protein aggregates in both Hs578T and HeLa cells. Collectively, quantitative analyses of the purinosome in Hs578T cells led us to provide novel insights for the dynamic architecture of the purinosome assembly.
人体从头嘌呤生物合成中的酶已被证明在嘌呤饥饿时会形成一种可逆的、短暂的多酶复合物——嘌呤体。然而，嘌呤体的特性研究仅限于HeLa细胞，并且在很大程度上依赖于在宽场荧光显微镜下对其亚细胞定位和可逆性的定性检查。对于与人类疾病相关的细胞系特别适用的定量方法，对于明确了解活细胞中的嘌呤体是必要的。在这项工作中，人类乳腺癌Hs578T细胞已被用于证明在嘌呤耗尽条件下嘌呤体的优先利用。此外，我们采用了基于共聚焦显微镜的生物物理技术——光漂白后荧光恢复，来表征活Hs578T细胞中嘌呤体的动力学特性。对所有从头嘌呤生物合成酶的扩散系数进行定量表征，揭示了它们在嘌呤体形成时移动动力学的显著降低、每种酶向嘌呤体的动态分配，以及在嘌呤饥饿时嘌呤体组装过程中存在三种中间物种。我们还证明，嘌呤补救酶次黄嘌呤磷酸核糖基转移酶1的扩散系数对嘌呤饥饿不敏感，这表明补救途径被排除在嘌呤体之外。此外，我们对非代谢酶的生物物理表征阐明，在Hs578T和HeLa细胞中，嘌呤体在时空上是与应激颗粒和细胞质蛋白聚集体不同的细胞体。总的来说，对Hs578T细胞中嘌呤体的定量分析使我们对嘌呤体组装的动态结构有了新的认识。
