Liaoning Key Laboratory of Marine Animal Immunology, Dalian Ocean University, Dalian, 116023, China; Liaoning Key Laboratory of Marine Animal Immunology and Disease Control, Dalian Ocean University, Dalian, 116023, China.
Liaoning Key Laboratory of Marine Animal Immunology, Dalian Ocean University, Dalian, 116023, China; Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266235, China; Liaoning Key Laboratory of Marine Animal Immunology and Disease Control, Dalian Ocean University, Dalian, 116023, China; Dalian Key Laboratory of Disease Prevention and Control for Aquaculture Animals, Dalian Ocean University, Dalian, 116023, China.
Fish Shellfish Immunol. 2018 Jul;78:248-258. doi: 10.1016/j.fsi.2018.04.048. Epub 2018 Apr 24.
The inositol-requiring enzyme 1 (IRE1), one of the primary endoplasmic reticulum (ER) transmembrane receptor proteins, is involved in regulating unfolded protein response (UPR) signaling pathway and plays an import role in maintaining cell homeostasis. In the present study, an IRE1 homologue was identified from Patinopecten yessoensis (designated as PyIRE1). The cDNA of PyIRE1 was of 3314 bp with a 2646 bp open reading frame (ORF) of IRE1 encoding a polypeptide of 881 amino acids. There was a signal peptide, four pyrrolo-quinoline quinine (PPQ) domains, a transmembrane helix region, a Serine/Threonine protein kinases domain (S_TKc) and a protein kinases or N-glycanases containing protein domain (PUG) in the deduced amino acid sequence of PyIRE1. The PyIRE1 mRNA was constitutively expressed in all the tested tissues, with the highest expression level in gills. PyIRE1 protein was mainly located in the ER of P. yessoensis hemocytes. The expression profiles of PyIRE1, glucose-regulated protein 94 (designated as PyGRP94) and glucose-regulated protein 78 (designated as PyGRP78) were determined by SYBR Green qRT-PCR after heat shock treatment. The mRNA expression levels of all these three genes were significantly up-regulated and reached their peak values at 2 h (3.97-fold, p < 0.05), 8 h (19.67-fold, p < 0.05) and 4 h (27.37-fold, p < 0.05) in hemocytes, 2 h (3.55-fold, p < 0.05), 12 h (8.58-fold, p < 0.05) and 8 h (35.31-fold, p < 0.05) in gills after heat shock treatment, respectively. After the injection with PyIRE1 dsRNA, the mRNA expression of pro-apoptotic B-cell lymphoma-2 (Bcl-2) family member PyBax and the activity of caspase-3 significantly decreased in comparison with the control group (p < 0.05) after heat shock treatment. These results collectively suggested that PyIRE1, as an ER stress sensor, was potentially involved in the response upon heat stress by regulating the expression of PyBax and apoptosis of hemocytes in P. yessoensis.
肌醇需求酶 1（IRE1）是内质网（ER）跨膜受体蛋白之一，参与调节未折叠蛋白反应（UPR）信号通路，在维持细胞内稳态方面发挥重要作用。本研究从中国蛤蜊（Patinopecten yessoensis）中鉴定出一个 IRE1 同源物（命名为 PyIRE1）。PyIRE1 的 cDNA 全长 3314 bp，包含一个 2646 bp 的开放阅读框（ORF），编码一个 881 个氨基酸的多肽。该多肽含有一个信号肽、四个吡咯并喹啉醌（PPQ）结构域、一个跨膜螺旋区、丝氨酸/苏氨酸蛋白激酶结构域（S_TKc）和一个蛋白激酶或 N-糖基酶结构域（PUG）。PyIRE1 的推定氨基酸序列中存在一个内质网定位信号肽，一个内质网定位信号肽，四个吡咯并喹啉醌（PPQ）结构域，一个跨膜螺旋区，一个丝氨酸/苏氨酸蛋白激酶结构域（S_TKc）和一个蛋白激酶或 N-糖基酶结构域（PUG）。PyIRE1 在所有测试组织中均呈组成型表达，在鳃中表达量最高。PyIRE1 蛋白主要定位于中国蛤蜊血细胞的内质网中。用 SYBR Green qRT-PCR 检测热休克处理后 PyIRE1、葡萄糖调节蛋白 94（PyGRP94）和葡萄糖调节蛋白 78（PyGRP78）的表达谱。这三个基因的 mRNA 表达水平均显著上调，在血细胞中分别于 2 h（3.97 倍，p < 0.05）、8 h（19.67 倍，p < 0.05）和 4 h（27.37 倍，p < 0.05）达到峰值，在鳃中分别于 2 h（3.55 倍，p < 0.05）、12 h（8.58 倍，p < 0.05）和 8 h（35.31 倍，p < 0.05）达到峰值。热休克处理后，注射 PyIRE1 dsRNA 后，与对照组相比，促凋亡 B 细胞淋巴瘤-2（Bcl-2）家族成员 PyBax 的 mRNA 表达和 caspase-3 的活性显著降低（p < 0.05）。这些结果表明，PyIRE1 作为内质网应激传感器，可能通过调节中国蛤蜊血细胞中 PyBax 的表达和细胞凋亡来参与热应激反应。
