Use of Peptide Arrays for Identification and Characterization of LIR Motifs
Use of Peptide Arrays for Identification and Characterization of LIR Motifs
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DOI：
10.1007/978-1-4939-8873-0_8
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发表时间：
2019-01-01
期刊：
影响因子：
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通讯作者：
Johansen, Terje
中科院分区：
文献类型：
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作者：
Rasmussen, Mads Skytte;Birgisdottir, Asa Birna;Johansen, Terje
哺乳动物ATG 8蛋白（LC 3A-C/GABARAP，GABARAPL 1和GABARAPL 2）是一种小的泛素样蛋白，主要参与巨自噬。其加工的C-末端与磷脂酰乙醇胺部分后结合，使其能够插入形成自噬体的内膜和外膜的脂质双层中。ATG 8结合多种蛋白质，包括用于选择性自噬的货物受体、核心自噬机制的成员以及参与自噬体的形成、运输和成熟（融合至溶酶体）的其他蛋白质。在大多数情况下，蛋白质与ATG 8的结合是由称为LC 3相互作用区（LIR）的短的保守序列基序介导的。在这里，我们提出了一个协议，用于确定推定的LIR图案在整个蛋白质序列中使用的肽阵列由SPOT合成硝酸纤维素膜。二维肽阵列的使用允许进一步鉴定对LIR结合至关重要的特定残基。
The mammalian ATG8 proteins (LC3A-C/GABARAP, GABARAPL1, and GABARAPL2) are small ubiquitin-like proteins critically involved in macroautophagy. Their processed C-termini are post-translationally conjugated to a phosphatidylethanolamine moiety, enabling their insertion into the lipid bilayers of both the inner and outer membranes of the forming autophagosomes. The ATG8s bind a diverse selection of proteins including cargo receptors for selective autophagy, members of the core autophagy machinery, and other proteins involved in formation, transport, and maturation (fusion to lysosomes) of autophagosomes. Protein binding to the ATG8s is in most cases mediated by short, conserved sequence motifs known as LC3-interacting regions (LIRs). Here, we present a protocol for identifying putative LIR motifs in a whole protein sequence using peptide arrays generated by SPOT synthesis on nitrocellulose membranes. The use of two-dimensional peptide arrays allows for further identification of specific residues critical for LIR binding.