Skytte Rasmussen Mads, Mouilleron Stéphane, Kumar Shrestha Birendra, Wirth Martina, Lee Rebecca, Bowitz Larsen Kenneth, Abudu Princely Yakubu, O'Reilly Nicola, Sjøttem Eva, Tooze Sharon A, Lamark Trond, Johansen Terje
a Molecular Cancer Research Group, Department of Medical Biology , University of Tromsø -The Arctic University of Norway , Tromsø , Norway.
b Structural Biology , The Francis Crick Institute , London , UK.
Autophagy. 2017 May 4;13(5):834-853. doi: 10.1080/15548627.2017.1287651. Epub 2017 Feb 15.
The cysteine protease ATG4B cleaves off one or more C-terminal residues of the inactive proform of proteins of the ortholog and paralog LC3 and GABARAP subfamilies of yeast Atg8 to expose a C-terminal glycine that is conjugated to phosphatidylethanolamine during autophagosome formation. We show that ATG4B contains a C-terminal LC3-interacting region (LIR) motif important for efficient binding to and cleavage of LC3 and GABARAP proteins. We solved the crystal structures of the GABARAPL1-ATG4B C-terminal LIR complex. Analyses of the structures and in vitro binding assays, using specific point mutants, clearly showed that the ATG4B LIR binds via electrostatic-, aromatic HP1 and hydrophobic HP2 pocket interactions. Both these interactions and the catalytic site-substrate interaction contribute to binding between LC3s or GABARAPs and ATG4B. We also reveal an unexpected role for ATG4B in stabilizing the unlipidated forms of GABARAP and GABARAPL1. In mouse embryonic fibroblast (MEF) atg4b knockout cells, GABARAP and GABARAPL1 were unstable and degraded by the proteasome. Strikingly, the LIR motif of ATG4B was required for stabilization of the unlipidated forms of GABARAP and GABARAPL1 in cells.
半胱氨酸蛋白酶ATG4B可切割酵母自噬相关蛋白8（Atg8）直系同源物和旁系同源物LC3及GABARAP亚家族蛋白质无活性前体形式的一个或多个C端残基，以暴露出一个C端甘氨酸，该甘氨酸在自噬体形成过程中与磷脂酰乙醇胺结合。我们发现，ATG4B含有一个C端LC3相互作用区域（LIR）基序，该基序对于有效结合并切割LC3和GABARAP蛋白很重要。我们解析了GABARAPL1-ATG4B C端LIR复合物的晶体结构。利用特定点突变体进行的结构分析和体外结合试验清楚地表明，ATG4B的LIR通过静电、芳香族HP1和疏水HP2口袋相互作用进行结合。这些相互作用以及催化位点与底物的相互作用都有助于LC3或GABARAP与ATG4B之间的结合。我们还揭示了ATG4B在稳定未脂化形式的GABARAP和GABARAPL1方面的一个意想不到的作用。在小鼠胚胎成纤维细胞（MEF）atg4b基因敲除细胞中，GABARAP和GABARAPL1不稳定，并被蛋白酶体降解。引人注目的是，ATG4B的LIR基序是细胞中稳定未脂化形式的GABARAP和GABARAPL1所必需的。
