The potent BECN2-ATG14 coiled-coil interaction is selectively critical for endolysosomal degradation of GPRASP1/GASP1-associated GPCRs
The potent BECN2-ATG14 coiled-coil interaction is selectively critical for endolysosomal degradation of GPRASP1/GASP1-associated GPCRs
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DOI：
10.1080/15548627.2023.2233872
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发表时间：
2023-07
期刊：
影响因子：
13.3
通讯作者：
Xianxiu Qiu;Na Li;Qifan Yang;Shuai Wu;Xiaohua Li;Xuehua Pan;Soh Yamamoto;Xiaozhe Zhang;Jincheng Zeng;Jiahao Liao;Congcong He;Renxiao Wang;Yanxiang Zhao
中科院分区：
文献类型：
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作者：
Xianxiu Qiu;Na Li;Qifan Yang;Shuai Wu;Xiaohua Li;Xuehua Pan;Soh Yamamoto;Xiaozhe Zhang;Jincheng Zeng;Jiahao Liao;Congcong He;Renxiao Wang;Yanxiang Zhao
BECN 2是BECN 1的哺乳动物特异性同源物。这两种蛋白质都作为III类磷脂酰肌醇3-激酶复合物（PtdIns 3 K）中的支架分子，以促进大自噬/自噬和内溶酶体运输。我们先前的研究已经表明，BECN 1卷曲螺旋结构域形成亚稳态同源二聚体，并且容易自解离以形成BECN 1-ATG 14或BECN 1-UVRAG卷曲螺旋复合物，作为PtdIns 3 K复合物的支架“臂”的一部分。在这里，我们报告的晶体结构的BECN 2卷曲螺旋结构域，它形成了一个亚稳的同源二聚体类似BECN 1，但显示出降低的稳定性和大的偏差，从理想的卷曲螺旋几何形状由于额外的“不完美”的残基。我们还报道了BECN 2-ATG 14卷曲螺旋复合物的晶体结构，它在结构上类似于我们以前报道的BECN 1-UVRAG卷曲螺旋复合物，但功能上不同。有效的BECN 2-ATG 14相互作用对于GPRASP 1相关DRD 2/D2 R的内溶酶体降解选择性至关重要，但对EGFR（依赖于BECN 1-UVRAG相互作用的货物）的影响有限。我们设计了与BECN 2卷曲螺旋结构域选择性相互作用的钉合肽，以增强BECN 2-ATG 14或BECN 1-UVRAG相互作用。一种这样的肽特异性地促进BECN 2依赖性过程，包括DRD 2/D2 R的自噬和内溶酶体降解，但不影响BECN 1依赖性EGFR降解。我们的研究结果表明，尽管与BECN 1具有高序列同一性，但BECN 2卷曲螺旋结构域具有独特的结构特征，并且BECN 2-ATG 14相互作用选择性地促进BECN 2特异性货物（包括GPRASP 1相关的GPCR）的自噬或内溶酶体降解。缩写AMBRA 1 autophagy and beclin 1 regulator 1; ATG14 autophagy related 14; ATG5 autophagy related 5; ATG7 autophagy related 7; BECN 1 beclin 1; BECN 2 beclin 2; CC coiled-coil; CQ chloroquine CNR1/CB1R cannabinoid receptor 1 DAPI 4 β，6-diamidino-2-phenylindole; dCCD delete CCD; DRD 2/D2 R多巴胺受体D2 GPRASP 1/GASP 1 G蛋白偶联受体相关分选蛋白1 GPCR G蛋白偶联受体; ITC等温滴定量热法; IP免疫沉淀; KD敲低; KO敲低; MAP 1 LC 3/LC 3微管相关蛋白1轻链3; NRBF 2核受体结合因子2; OPRD 1/DOR阿片受体δ 1 PIK 3C 3/VPS 34磷脂酰肌醇3-激酶催化亚基3型; PIK 3R 4/VPS 15磷脂酰肌醇-3-激酶调节亚基4; PtdIns 3 K III类磷脂酰肌醇3-激酶; PtdIns 3 P磷脂酰肌醇-3-磷酸; RUBCN rubicon自噬调节剂; SQSTM 1/p62隔离体1; UVRAG UV辐射抗性相关; VPS空泡蛋白分选; WT野生型。
ABSTRACT BECN2 is a mammal-specific homolog of BECN1. Both proteins serve as a scaffolding molecule in the class III phosphatidylinositol 3-kinase complex (PtdIns3K) to promote macroautophagy/autophagy and endolysosomal trafficking. Our previous studies have shown that the BECN1 coiled-coil domain forms a metastable homodimer and readily self-dissociate to form the BECN1-ATG14 or BECN1-UVRAG coiled-coil complex as part of the scaffolding “arm” of the PtdIns3K complex. Here we report the crystal structure of the BECN2 coiled-coil domain, which forms a metastable homodimer similar to BECN1 but shows reduced stability and large deviation from the ideal coiled-coil geometry due to extra “imperfect” residues. We also report the crystal structure of BECN2-ATG14 coiled-coil complex, which is structurally similar to the BECN1-UVRAG coiled-coil complex we reported previously but functionally distinct. The potent BECN2-ATG14 interaction is selectively critical for endolysosomal degradation of the GPRASP1-associated DRD2/D2R but shows limited effect on EGFR, a cargo that depends on the BECN1-UVRAG interaction. We designed stapled peptides that selectively interacted with the BECN2 coiled-coil domain to enhance the BECN2-ATG14 or BECN1-UVRAG interaction. One such peptide specifically promoted BECN2-dependent processes including autophagy and endolysosomal degradation of DRD2/D2R but did not affect BECN1-dependent EGFR degradation. Our findings suggest that, despite high sequence identity to BECN1, BECN2 coiled-coil domain has unique structural features and the BECN2-ATG14 interaction selectively promotes autophagic or endolysosomal degradation of BECN2-specific cargos including GPRASP1-associated GPCRs. Abbreviations AMBRA1 autophagy and beclin 1 regulator 1; ATG14 autophagy related 14; ATG5 autophagy related 5; ATG7 autophagy related 7; BECN1 beclin 1; BECN2 beclin 2; CC coiled-coil; CQ chloroquine CNR1/CB1R cannabinoid receptor 1 DAPI 4ʹ,6-diamidino-2-phenylindole; dCCD delete CCD; DRD2/D2R dopamine receptor D2 GPRASP1/GASP1 G protein-coupled receptor associated sorting protein 1 GPCR G-protein coupled receptor; ITC isothermal titration calorimetry; IP immunoprecipitation; KD knockdown; KO knockout; MAP1LC3/LC3 microtubule associated protein 1 light chain 3; NRBF2 nuclear receptor binding factor 2; OPRD1/DOR opioid receptor delta 1 PIK3C3/VPS34 phosphatidylinositol 3-kinase catalytic subunit type 3; PIK3R4/VPS15 phosphoinositide-3-kinase regulatory subunit 4; PtdIns3K class III phosphatidylinositol 3-kinase; PtdIns3P phosphatidylinositol-3-phosphate; RUBCN rubicon autophagy regulator; SQSTM1/p62 sequestosome 1; UVRAG UV radiation resistance associated; VPS vacuolar protein sorting; WT wild type.