Michels A A, Nguyen V T, Konings A W, Kampinga H H, Bensaude O
Department of Radiobiology, Faculty of Medicine, University of Groningen, The Netherlands.
Eur J Biochem. 1995 Dec 1;234(2):382-9. doi: 10.1111/j.1432-1033.1995.382_b.x.
Protein denaturation and aggregation are most likely the cause for the noxious effects of heat shock. There are some indications that the nucleus is one of the most sensitive cellular compartments. To test the possibility that the intranuclear microenvironment might be detrimental to the heat stability of proteins, we compared the in situ thermal stability of a reporter protein localized in the nucleus or in the cytoplasm. A recombinant firefly (Photynus pyralis) luciferase carrying a point mutation in the C-terminal domain remains in the cytoplasm (cyt-luciferase). A nuclear localization sequence was fused to the N-terminal domain of cyt-luciferase; the resulting nuc-luciferase was efficiently targeted to the cell nucleus. In both cases, decreased luciferase activity and solubility were found in lysates from heat-shocked cells. These characteristics were taken as an indication of thermal denaturation in situ. The heat-inactivated luciferases were partially reactivated during recovery after stress, indicating the capacity of both the cytoplasmic and nuclear compartments to reassemble proteins from an aggregated state. Although both the nuc- and the cyt-luciferases were heat inactivated at similar rates in vitro, nuc-luciferase was more susceptible to thermal denaturation in situ compared to cyt-luciferase. This observation suggests that the microenvironment of an intracellular compartment may modulate the thermal stability of proteins. The local concentration might be one element of this microenvironment affecting the heat-stability of proteins. In cells made thermotolerant by a priming shock, the thermal inactivation of the recombinant luciferases occurred at a slower rate during a second challenging stress. However, this decreased thermal sensitivity was less pronounced for the nuc-luciferase (threefold) than for the cyt-luciferase (sevenfold). The nuclear luciferase might become a useful tool to investigate the action of molecular chaperones in the nucleus.
蛋白质变性和聚集很可能是热休克产生有害影响的原因。有一些迹象表明细胞核是最敏感的细胞区室之一。为了测试核内微环境可能对蛋白质热稳定性有害的可能性，我们比较了定位于细胞核或细胞质中的报告蛋白的原位热稳定性。一种在C末端结构域携带点突变的重组萤火虫（Photynus pyralis）荧光素酶保留在细胞质中（胞质荧光素酶）。将一个核定位序列融合到胞质荧光素酶的N末端结构域；由此产生的核荧光素酶被有效地靶向到细胞核。在这两种情况下，热休克细胞的裂解物中都发现荧光素酶活性和溶解度降低。这些特征被视为原位热变性的指标。热失活的荧光素酶在应激后的恢复过程中部分重新激活，这表明细胞质和细胞核区室都有能力从聚集状态重新组装蛋白质。尽管核荧光素酶和胞质荧光素酶在体外以相似的速率被热失活，但与胞质荧光素酶相比，核荧光素酶在原位更易受热变性影响。这一观察结果表明细胞内区室的微环境可能调节蛋白质的热稳定性。局部浓度可能是影响蛋白质热稳定性的这种微环境的一个因素。在通过引发性休克产生耐热性的细胞中，重组荧光素酶在第二次挑战性应激期间热失活的速率较慢。然而，这种热敏感性降低对于核荧光素酶（三倍）不如对胞质荧光素酶（七倍）明显。核荧光素酶可能成为研究分子伴侣在细胞核中作用的有用工具。
