Dynamic architecture of the purinosome involved in human de novo purine biosynthesis.
Dynamic architecture of the purinosome involved in human de novo purine biosynthesis.
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DOI：
10.1021/bi501480d
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发表时间：
2015-01-27
期刊：
影响因子：
2.9
通讯作者：
An S
中科院分区：
文献类型：
--
作者：
Kyoung M;Russell SJ;Kohnhorst CL;Esemoto NN;An S
人类从头合成嘌呤的酶已被证明在嘌呤饥饿时形成一个可逆的、瞬时的多酶复合体--嘌呤小体。然而，对嘌呤小体的表征一直局限于HeLa细胞，并且在很大程度上依赖于对其在广域荧光显微镜下的亚细胞定位和可逆性的定性检查。与人类疾病相关的细胞系特别相容的定量方法对于明确了解活细胞中的嘌呤小体是必要的。在这项工作中，人乳腺癌Hs578T细胞已经被用来证明在嘌呤耗尽的条件下嘌呤小体的优先利用。此外，我们使用了一种基于共聚焦显微镜的生物物理技术，即光漂白后的荧光恢复，来表征Hs578T活细胞中嘌呤小体的动力学特性。所有从头合成的嘌呤生物合成酶的扩散系数的定量表征表明，它们在嘌呤小体形成时的运动动力学显著降低，每种酶在嘌呤小体中的动态分配，以及在嘌呤饥饿下嘌呤小体组装中存在三个中间物种。我们还证明了嘌呤回收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，这表明从嘌呤小体中排除了回收途径。此外，我们对非代谢酶的生物物理特征表明，在Hs578T和HeLa细胞中，嘌呤小体是不同于应激颗粒和细胞质蛋白聚集体的时空不同的细胞体。总而言之，对Hs578T细胞中的嘌呤小体的定量分析使我们对嘌呤小体组装的动态结构提供了新的见解。
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.
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影响因子：
7.8
作者：
Kenworthy, Anne K;Nichols, Benjamin J;Remmert, Catha L;Hendrix, Glenn M;Kumar, Mukesh;Zimmerberg, Joshua;Lippincott-Schwartz, Jennifer
通讯作者：
Lippincott-Schwartz, Jennifer
影响因子：
4.5
作者：
Owen, Dylan M.;Williamson, David;Gaus, Katharina
通讯作者：
Gaus, Katharina
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56.9
作者：
ELION, GB
通讯作者：
ELION, GB
影响因子：
48
作者：
Schnell, Ulrike;Dijk, Freark;Giepmans, Ben N. G.
通讯作者：
Giepmans, Ben N. G.
影响因子：
3.5
作者：
Baresova, Veronika;Skopova, Vaclava;Kmoch, Stanislav
通讯作者：
Kmoch, Stanislav