Abstract：
Firefly luciferase (Fluc) from Photinus pyralis is one of the most widely used reporter proteins in biomedical research. Despite its widespread use, Fluc\'s protein phase transition behaviors and phase separation characteristics have not received much attention. Current research uncovers Fluc\'s intrinsic property to phase separate in mammalian cells upon a simple cell culture temperature change. Specifically, Fluc spontaneously produced needle-shaped crystal-like inclusion bodies upon temperature shift to the hypothermic temperatures ranging from 25 °C to 31 °C. The crystal-like inclusion bodies were not associated with or surrounded by membranous organelles and were likely built from the cytosolic pool of Fluc. Furthermore, the crystal-like inclusion formation was suppressed when cells were cultured in the presence of D-luciferin and its synthetic analog, as well as the benzothiazole family of so-called stabilizing inhibitors. These two classes of compounds inhibited intracellular Fluc crystallization by different modes of action as they had contrasting effects on steady-state luciferase protein accumulation levels. This study suggests that, under substrate insufficient conditions, the excess Fluc phase separates into a crystal-like state that can modulate intracellular soluble enzyme availability and protein turnover rate.
摘要：
萤火虫荧光素酶（Fluc）是生物医学研究中使用最广泛的报告蛋白之一。尽管它广泛使用，Fluc的蛋白质相变行为和相分离特性尚未受到广泛关注。当前的研究揭示了Fluc的内在特性，即在简单的细胞培养温度变化时在哺乳动物细胞中发生相分离。具体来说,Fluc在温度转变到25°C至31°C的低温温度时自发产生针状晶体状包涵体。晶体状包涵体与膜细胞器无关或不被膜细胞器包围，并且可能是由Fluc的细胞溶质池构建的。此外，当细胞在D-荧光素及其合成类似物的存在下培养时，晶体样包合物的形成受到抑制，以及苯并噻唑家族的所谓稳定抑制剂。这两类化合物通过不同的作用方式抑制细胞内Fluc结晶，因为它们对稳态荧光素酶蛋白积累水平具有相反的作用。这项研究表明,在底物不足的条件下，过量的Fluc相分离成晶体状状态，可以调节细胞内可溶性酶的利用率和蛋白质周转率。
