Infections of many viruses induce caspase activation to regulate multiple cellular pathways, including programmed cell death, immune signaling and etc. Characterizations of caspase cleavage sites and substrates are important for understanding the regulation mechanisms of caspase activation. Here, we identified and analyzed a novel caspase cleavage motif AEAD, and confirmed its caspase dependent cleavage activity in natural substrate, such as nitric oxide-associated protein 1 (NOA1). Fusing the enhanced green fluorescent protein (EGFP) with the mitochondrial marker protein Tom20 through the AEAD motif peptide localized EGFP to the mitochondria. Upon the activation of caspase triggered by Sendai virus (SeV) or herpes simplex virus type 1 (HSV-1) infection, EGFP diffusely localized to the cell due to the caspase-mediated cleavage, thus allowing visual detection of the virus-induced caspase activation. An AEAD peptide-derived inhibitor Z-AEAD-FMK were developed, which significantly inhibited the activities of caspases-1, -3, -6, -7, -8 and -9, exhibiting a broad caspase inhibition effect. The inhibitor further prevented caspases-mediated cleavage of downstream substrates, including BID, PARP1, LMNA, pro-IL-1β, pro-IL-18, GSDMD and GSDME, protecting cells from virus-induced apoptotic and pyroptotic cell death. Together, our findings provide a new perspective for the identification of novel caspase cleavage motifs and the development of new caspase inhibitors and anti-inflammatory drugs.

中文翻译：

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新型半胱天冬酶切割基序 AEAD 的鉴定


许多病毒的感染都会诱导Caspase激活来调节多种细胞途径，包括程序性细胞死亡、免疫信号传导等。Caspase裂解位点和底物的表征对于理解Caspase激活的调节机制非常重要。在这里，我们鉴定并分析了一个新的 caspase 切割基序 AEAD，并证实了其在天然底物（如一氧化氮相关蛋白 1 (NOA1)）中具有 caspase 依赖性切割活性。通过 AEAD 基序肽将增强型绿色荧光蛋白 (EGFP) 与线粒体标记蛋白 Tom20 融合，将 EGFP 定位到线粒体。仙台病毒 (SeV) 或单纯疱疹病毒 1 型 (HSV-1) 感染触发 caspase 激活后，EGFP 由于 caspase 介导的裂解而广泛定位于细胞，从而可以目视检测病毒诱导的 caspase 激活。开发了一种AEAD肽衍生抑制剂Z-AEAD-FMK，可显着抑制caspase-1、-3、-6、-7、-8和-9的活性，表现出广泛的caspase抑制作用。该抑制剂进一步阻止 Caspases 介导的下游底物（包括 BID、PARP1、LMNA、pro-IL-1β、pro-IL-18、GSDMD 和 GSDME）裂解，保护细胞免受病毒诱导的细胞凋亡和焦亡。总之，我们的研究结果为鉴定新型半胱天冬酶裂解基序以及开发新型半胱天冬酶抑制剂和抗炎药物提供了新的视角。