Fiol Diego F, Sanmarti Enio, Lim Andreana H, Kültz Dietmar
Physiological Genomics Group, Department of Animal Sciences, University of California at Davis, CA 95616, USA.
Biochim Biophys Acta. 2011 Apr;1810(4):439-45. doi: 10.1016/j.bbagen.2010.11.005. Epub 2010 Nov 29.
Tilapia (Oreochromis mossambicus) are euryhaline fishes capable of tolerating large salinity changes. In a previous study aimed to identify genes involved in osmotolerance, we isolated an mRNA sequence with similarity to GRAIL (Gene Related to Anergy In Lymphocytes), which is a critical regulator of adaptive immunity and development. Tilapia GRAIL contains a PA (protease associated) domain and a C3H2C3 RING finger domain indicative of E3 ubiquitin ligase activity.
Western blots analysis was used to assess GRAIL expression pattern and responses to hyperosmotic stress. Immunohistochemistry was used to reveal the cellular localization of GRAIL in gill epithelium. Overexpression in HEK293 T-Rex cells was used to functionally characterize tilapia GRAIL. Salinity stress causes strong up-regulation of both mRNA and protein levels of tilapia GRAIL in gill epithelium. Tissue distribution of GRAIL protein is mainly confined to gill epithelium, which is the primary tissue responsible for osmoregulation of teleost fishes. Overexpression of tilapia GRAIL in HEK293 cells increases cell survival (cell viability) while decreases apoptosis during salinity challenge.
Our data indicate that tilapia GRAIL is a novel E3 ubiquitin ligase involved in osmotic stress signaling, which promotes environmental salinity tolerance by supporting gill cell function during hyperosmotic stress.
Involvement of tilapia GRAIL in the osmotic stress response suggests that GRAIL E3 ubiquitin ligases play a broader role in environmental stress responses, beyond their documented functions in adaptive immunity and development.
罗非鱼（莫桑比克罗非鱼）是广盐性鱼类，能够耐受大幅度的盐度变化。在之前一项旨在鉴定参与渗透压耐受性的基因的研究中，我们分离出了一个与GRAIL（淋巴细胞无反应相关基因）相似的mRNA序列，GRAIL是适应性免疫和发育的关键调节因子。罗非鱼GRAIL包含一个PA（蛋白酶相关）结构域和一个表明具有E3泛素连接酶活性的C3H2C3环指结构域。
使用蛋白质免疫印迹分析来评估GRAIL的表达模式以及对高渗应激的反应。使用免疫组织化学来揭示GRAIL在鳃上皮细胞中的细胞定位。在HEK293 T-Rex细胞中进行过表达以对罗非鱼GRAIL进行功能表征。盐度应激导致罗非鱼鳃上皮细胞中GRAIL的mRNA和蛋白质水平均强烈上调。GRAIL蛋白的组织分布主要局限于鳃上皮，鳃上皮是硬骨鱼类渗透压调节的主要组织。罗非鱼GRAIL在HEK293细胞中的过表达增加了细胞存活率，同时降低了盐度挑战期间的细胞凋亡。
我们的数据表明，罗非鱼GRAIL是一种参与渗透应激信号传导的新型E3泛素连接酶，它通过在高渗应激期间支持鳃细胞功能来促进对环境盐度的耐受性。
罗非鱼GRAIL参与渗透应激反应表明，GRAIL E3泛素连接酶在环境应激反应中发挥着更广泛的作用，超出了它们在适应性免疫和发育中已记录的功能。
