实验海洋生物学重点实验室知识库

在脊椎动物中，Gasdermin（GSDM）家族是一类执行细胞焦亡的蛋白。但是无脊椎动物中GSDM介导的细胞焦亡研究极少。最新的基因组测序分析表明，软体动物中具有大量的GSDM同源基因，但是对于它们的功能却并没有相关的研究。

在本研究中，我们鉴定了11种软体动物的18个GSDM。通过将这些GSDM的N terminal (NT)结构域在人类细胞中表达，发现只有皱纹盘鲍（Haliotis discus）的GSDM（命名为HdGSDME）具有诱导细胞焦亡的潜力，而其它10种软体动物的GSDM没有展现出诱导细胞焦亡的能力。通过一系列实验发现并验证了HdGSDME的激活机制。HdGSDME可以被鲍Caspase-3（HdCASP3）在两个不同的酶切位点特异性剪切，产生两种不同的N-terminal （NT）片段，二者皆具有细胞焦亡诱导活性以及抑菌活性。序列分析表明，HdGSDME拥有保守的氨基酸功能位点，这些关键的氨基酸功能位点对于HdGSDME-NT的成孔活性以及HdGSDME C-terminal（CT）的自抑制活性来说是至关重要的。病原菌侵染激活鲍中HdCASP3-HdGSDME通路，并且会引发细胞焦亡以及细胞外陷阱（extracellular trap，ET）的产生。阻断HdCASP3-HdGSDME通路会促进病原菌在鲍组织内的增殖，并且会导致鲍致死率上升。比较蛋白质组学研究表明，鲍细胞焦亡改变了大量分泌蛋白和非分泌细胞蛋白的表达，暗示细胞焦亡可能诱发复杂的下游反应。

综上所述，我们首次在软体动物中发现了功能性GSDME，揭示了以皱纹盘鲍为代表的贝类的细胞焦亡激活机制及抗感染免疫作用，研究成果填补了贝类GSDM功能研究的空白，为无脊椎动物细胞程序性死亡提供了新认知。

Gasdermin (GSDM) is a family of proteins that execute pyroptosis in vertebrate. In invertebrate, the studies on GSDM-mediated pyroptosis are scarce. Recent studies identified abundant GSDM structural homologs in Mollusca, but their functions are unknown.

Herein, we screened a total of 18 GSDMs from 11 species of mollusk. By heterologous expression of the N-terminal (NT) domains of these GSDMs in mammalian cells, we found that only the Pacific abalone Haliotis discus GSDM (named HdGSDME) was able to induce pyroptosis, whereas the GSDMs of all the other 10 mollusc were unable to induce pyroptosis. We discovered and verified the activation mechanism of HdGSDME through a series of experiments. HdGSDME was specifically activated by abalone caspase-3 (HdCASP3) cleavage at two distinct sites, generating two active isoforms with pyroptotic and bacteriostatic activities at different rates. Sequence analysis showed that HdGSDME possessed conserved residues that proved to be essential to the N-terminal pore-formation and C-terminal auto-inhibition capacities. Bacterial challenge activated the HdCASP3-HdGSDME pathway and induced pyroptosis and extracellular trap in abalone. Blockage of the HdCASP3-HdGSDME axis promoted bacterial dissemination in abalone tissues and increased host mortality. Comparative proteomic analyses showed that pyroptosis changed the expressions of a large amount of secreted and non-secreted cellular proteins, suggesting that pyroptosis likely induced complex downstream reactions.

In conclusion, our study showed for the first time that GSDME with pyroptotic function existed in mollusc, and revealed the activation mechanism and antibacterial effect of mollusc GSDME represented by HdGSDME. These results added new insights into the mechanisms of programmed cell death in invertebrate.