Department of Biochemistry, Toho University School of Medicine, 5-21-16 Omori-Nishi, Ota-ku, Tokyo, 143-8540, Japan.
Hibernation Metabolism, Physiology, and Development Group, Environmental Biology Division, Institute of Low Temperature Science, Hokkaido University, Kita 19, Nishi 8, Kita-ku, Sapporo, Hokkaido, 060-0819, Japan.
Nat Commun. 2018 Oct 26;9(1):4457. doi: 10.1038/s41467-018-06985-6.
Necroptosis is a regulated form of necrosis that depends on receptor-interacting protein kinase (RIPK)3 and mixed lineage kinase domain-like (MLKL). While danger-associated molecular pattern (DAMP)s are involved in various pathological conditions and released from dead cells, the underlying mechanisms are not fully understood. Here we develop a fluorescence resonance energy transfer (FRET) biosensor, termed SMART (a sensor for MLKL activation by RIPK3 based on FRET). SMART is composed of a fragment of MLKL and monitors necroptosis, but not apoptosis or necrosis. Mechanistically, SMART monitors plasma membrane translocation of oligomerized MLKL, which is induced by RIPK3 or mutational activation. SMART in combination with imaging of the release of nuclear DAMPs and Live-Cell Imaging for Secretion activity (LCI-S) reveals two different modes of the release of High Mobility Group Box 1 from necroptotic cells. Thus, SMART and LCI-S uncover novel regulation of the release of DAMPs during necroptosis.
细胞程序性坏死是一种依赖受体相互作用蛋白激酶（RIPK）3 和混合谱系激酶结构域样蛋白（MLKL）的受调控的细胞坏死形式。虽然危险相关分子模式（DAMP）参与各种病理状况并从死亡细胞中释放，但潜在机制尚未完全阐明。在这里，我们开发了一种荧光共振能量转移（FRET）生物传感器，称为 SMART（一种基于 FRET 的 RIPK3 激活 MLKL 的传感器）。SMART 由 MLKL 的一个片段组成，可监测细胞程序性坏死，但不监测细胞凋亡或细胞坏死。从机制上讲，SMART 监测由 RIPK3 或突变激活诱导的寡聚化 MLKL 的质膜易位。SMART 与核 DAMP 释放的成像和分泌活性的活细胞成像（LCI-S）相结合，揭示了高迁移率族蛋白 B1 从细胞程序性坏死细胞中释放的两种不同模式。因此，SMART 和 LCI-S 揭示了细胞程序性坏死过程中 DAMPs 释放的新调控机制。
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