Development of GABARAP family protein-sensitive LIR-based probes for neuronal autophagy
Development of GABARAP family protein-sensitive LIR-based probes for neuronal autophagy
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DOI：
10.1186/s13041-019-0458-z
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发表时间：
2019-04-08
期刊：
影响因子：
3.6
通讯作者：
Lee, Jin-A
中科院分区：
文献类型：
--
作者：
Jeon, Pureum;Park, Ju-Hui;Lee, Jin-A
自噬允许溶酶体细胞降解胞质成分。特别是，神经元自噬对于细胞稳态和神经元存活至关重要，并且受到有丝分裂后神经元中几种自噬相关（ATG）蛋白的严格调节。在这些 ATG 蛋白中，已知 LC3/GABARAP 蛋白可调节自噬体生物发生/成熟和货物识别。然而，尽管 GABARAP 家族蛋白在有丝分裂后神经元中大量表达，但人们对它们在神经元自噬中的作用知之甚少。我们之前开发了基于 HyD（疏水结构域）-LIR（LC3 相互作用区域）的自噬体标记。在这项研究中，为了监测有丝分裂后神经元自噬体中的 GABARAP 家族蛋白，我们通过在 LIR 探针中添加一个 LIR 基序来提高探针特异性检测内源 GABARAP 家族蛋白的灵敏度。我们测试了 ULK2 和 Stbd1 两种不同 LIR 在细胞定位至自噬体方面的效率。 HyD-2xLIR(ULK2)-GFP 和 HyD-2xLIR(Stbd1)-GFP 以自噬依赖性方式表现出相对于 MEF/HeLa 细胞中 LC3B 阳性自噬体的 GABARAP 阳性自噬体的特异性定位。事实上，HyD-2xLIR(Stbd1)-GFP 可以有效检测培养的皮质神经元中的 GABARAP 阳性自噬体。我们改进的 GABARAP 敏感探针将有助于了解 GABARAP 家族蛋白在神经元自噬中的具体作用。
Autophagy allows for lysosomal cellular degradation of cytosolic components. In particular, neuronal autophagy is essential for cellular homeostasis and neuronal survival and is tightly regulated by several autophagy-related (ATG) proteins in post-mitotic neurons. Among these ATG proteins, the LC3/GABARAP proteins are known to regulate autophagosome biogenesis/maturation and cargo recognition. However, little is known about the role of GABARAP family proteins in neuronal autophagy despite their abundant expression in post-mitotic neurons. We have previously developed HyD (Hydrophobic Domain)-LIR (LC3-interacting region)-based autophagosome markers. In this study, to monitor GABARAP family proteins in autophagosomes of post-mitotic neurons, we improved the sensitivity of the probes for specifically detecting endogenous GABARAP family proteins by adding one more LIR motif to the LIR probes. We have tested the efficiency of two different LIRs, from ULK2 and Stbd1, in regard to their cellular localization to autophagosomes. HyD-2xLIR(ULK2)-GFP and HyD-2xLIR(Stbd1)-GFP demonstrated specific localization to GABARAP-positive autophagosomes relative to LC3B-positive autophagosomes in MEF/HeLa cells in an autophagy-dependent manner. Indeed, HyD-2xLIR(Stbd1)-GFP could efficiently detect GABARAP-positive autophagosomes in cultured cortical neurons. Our improved GABARAP-sensitive probes will contribute toward understanding the specific role of GABARAP family proteins in regard to neuronal autophagy.