Deficiency in the membrane protein Tmbim3a/Grinaa initiates cold-induced ER stress and cell death by activating an intrinsic apoptotic pathway in zebrafish
Deficiency in the membrane protein Tmbim3a/Grinaa initiates cold-induced ER stress and cell death by activating an intrinsic apoptotic pathway in zebrafish
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斑马鱼膜蛋白 Tmbim3a/Grinaa 的缺陷通过激活内在的细胞凋亡途径启动冷诱导的内质网应激和细胞死亡
DOI：
10.1074/jbc.ra119.007813
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发表时间：
2019-07-26
影响因子：
4.8
通讯作者：
Cui, Zongbin
中科院分区：
文献类型：
--
作者：
Chen, Kai;Li, Xixi;Cui, Zongbin
大多数含有跨膜BAX抑制剂基序（TMBIM）的蛋白家族成员具有抗凋亡活性，但其体内功能和细胞内机制尚不清楚。在这里，我们报道了斑马鱼Tmbim3a/Grinaa在预防冷诱导的内质网（ER）应激和细胞凋亡中起作用。使用基因捕获方法，我们获得了一个突变的斑马鱼系，其中tmbim3a/grinaa基因的表达被Tol2转座子插入破坏。纯合子tmbim3a/grinaa突变体幼虫在冷暴露（16°C）下表现出时间依赖性的死亡率和凋亡增加。在机制上，利用免疫荧光、基于荧光的细胞内/线粒体Ca2+水平评估、线粒体膜电位测量和Ca2+- atp酶测定，我们发现冷暴露抑制肌浆/ER Ca2+- atp酶（SERCA）活性并诱导未折叠蛋白反应（UPR）和内质网应激。我们还发现冷诱导的内质网应激在纯合子tmbim3a/grinaa突变胚中增加。tmbim3a/grinaa突变体的冷应激超敏性与细胞内Ca2+稳态破坏密切相关，随后线粒体Ca2+超载和细胞色素c释放，导致caspase 9和caspase 3介导的内在凋亡途径的激活。细胞内Ca2+螯合剂1,2-二（2-氨基苯氧基）乙烷-N，N,N ',N ' -四乙酸乙酯-乙酰氧基甲酯（BAPTA-AM）或2-氨基乙氧基二苯硼酸盐（2-APB）（一种钙释放蛋白肌醇1,4,5-三磷酸受体（IP3R）抑制剂）处理斑马鱼幼虫，可减轻冷诱导的细胞死亡。总之，这些发现揭示了Tmbim3a/Grinaa在缓解寒冷诱导的内质网应激和保护斑马鱼发育过程中caspase 9 -和caspase 3介导的细胞凋亡中的关键作用。
Most members of the family of proteins containing a transmembrane BAX inhibitor motif (TMBIM) have anti-apoptotic activity, but their in vivo functions and intracellular mechanisms remain obscure. Here, we report that zebrafish Tmbim3a/Grinaa functions in the prevention of cold-induced endoplasmic reticulum (ER) stress and apoptosis. Using a gene-trapping approach, we obtained a mutant zebrafish line in which the expression of the tmbim3a/grinaa gene is disrupted by a Tol2 transposon insertion. Homozygous tmbim3a/grinaa mutant larvae exhibited time-dependently increased mortality and apoptosis under cold exposure (at 16 °C). Mechanistically, using immunofluorescence, fluorescence-based assessments of intracellular/mitochondrial Ca2+ levels, mitochondrial membrane potential measurements, and Ca2+-ATPase assays, we found that cold exposure suppresses sarcoplasmic/ER Ca2+-ATPase (SERCA) activity and induces the unfolded protein response (UPR) and ER stress. We also found that the cold-induced ER stress is increased in homozygous tmbim3a/grinaa mutant embryos. The cold-stress hypersensitivity of the tmbim3a/grinaa mutants was tightly associated with disrupted intracellular Ca2+ homeostasis, followed by mitochondrial Ca2+ overload and cytochrome c release, leading to the activation of caspase 9– and caspase-3–mediated intrinsic apoptotic pathways. Treatment of zebrafish larvae with the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetate-acetoxymethyl ester (BAPTA-AM) or with 2-aminoethoxydiphenyl borate (2-APB), an inhibitor of the calcium-releasing protein inositol 1,4,5-trisphosphate receptor (IP3R), alleviated cold-induced cell death. Together, these findings unveil a key role of Tmbim3a/Grinaa in relieving cold-induced ER stress and in protecting cells against caspase 9– and caspase 3–mediated apoptosis during zebrafish development.