TP53INP2 contributes to autophagosome formation by promoting LC3-ATG7 interaction
TP53INP2 contributes to autophagosome formation by promoting LC3-ATG7 interaction
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TP53INP2 通过促进 LC3-ATG7 相互作用促进自噬体形成
DOI：
10.1080/15548627.2019.1580510
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发表时间：
2019-08-03
期刊：
影响因子：
13.3
通讯作者：
Liu, Wei
中科院分区：
文献类型：
--
作者：
You, Zhiyuan;Xu, Yinfeng;Liu, Wei
TP 53 INP 2/DOR（tumor protein p53-inducible nuclear protein 2）通过将核脱乙酰化的MAP 1 LC 3/LC 3携带到细胞质中，参与哺乳动物的自噬。在这里，我们报告说，在细胞质中，TP 53 INP 2通过促进LC 3B-ATG 7相互作用在自噬体生物合成中进一步发挥作用。TP 53 INP 2的N-末端区域（包括LC 3相互作用区域）的细胞质表达有效地触发LC 3B-PE产生和自噬体形成。在细胞质中，TP 53 INP 2共定位于含有ATG 14、ZFYVE 1/DFCP 1或WIPI 2的早期自噬膜结构。虽然TP 53 INP 2的敲低不影响这些自噬结构的形成，但BECN 1或Atg 5的缺失或阻止TP 53 INP 2与LC 3相互作用的突变破坏了TP 53 INP 2的膜结合。TP 53 INP 2与ATG 7直接相互作用，在细胞质中形成LC 3B-TP 53 INP 2-ATG 7复合物。TP 53 INP 2-LC 3或TP 53 INP 2-ATG 7相互作用的丧失显著降低了LC 3B-ATG 7结合。总之，这些结果表明，从细胞核转移后，胞质TP 53 INP 2靶向伴随LC 3的早期自噬膜，在那里它通过介导LC 3-ATG 7相互作用促进自噬体生物发生。缩略语：3-MA，3-甲基腺嘌呤; 3 NES，3个重复的核输出信号; 3 NLS，3个重复的核定位信号; ACTB，肌动蛋白β; ATG，自噬相关; BECN 1，Beclin 1; mCherry，单体红色荧光蛋白mCherry; GFP，绿色荧光蛋白; GST，谷胱甘肽S-转移酶; KO，敲除; LC 3 B/MAP 1 LC 3 B，微管相关蛋白1轻链3 β; LC 3B [G120]，甘氨酸120后缺少氨基酸的LC 3B突变体; LDH，乳酸脱氢酶; LMNB 1，核纤层蛋白B1; LIR，LC 3相互作用区; MTORC 1，雷帕霉素复合物1的机制靶标; PE，磷脂酰乙醇胺; PtdIns 3 K，磷脂酰肌醇3-激酶; PtdIns 3 P，磷脂酰肌醇3-磷酸; rDNA，核糖体DNA; RFP，红色荧光蛋白; RNAi，RNA干扰; SQSTM 1，多价螯合体1; TP 53 INP 2，肿瘤蛋白p53诱导核蛋白2; TP 53 INP 2 [1-28]，含有氨基酸1至28的TP 53 INP 2突变体; TP 53 INP 2 [28-45]，含有氨基酸28至45的TP 53 INP 2突变体; TP 53 INP 2 [LIRΔ]，缺失氨基酸1至144的TP 53 INP 2突变体; TP 53 INP 2 [NLSΔ]，缺失氨基酸145至221的TP 53 INP 2突变体; TP 53 INP 2 W35、I38 A、其中色氨酸35和异亮氨酸38被丙氨酸置换的TP 53 INP 2突变体; TP 53 INP 2 W35、I38 A [NLSΔ]、缺乏第145至221位氨基酸的TP 53 INP 2突变体、以及色氨酸35和异亮氨酸38被替换为丙氨酸; TP 53 INP 2 W35、I38 A [Δ1-28]、[NLSΔ]、缺乏第1至28位氨基酸和第145至221位氨基酸的TP 53 INP 2突变体、以及色氨酸35和异亮氨酸38被替换为丙氨酸; TP 53 INP 2 [Δ1-28]、[NLSΔ]、缺乏氨基酸1至28和氨基酸145至221的TP 53 INP 2突变体; TP 53 INP 2 [Δ112-144]，[NLSΔ]，缺乏氨基酸112至144和氨基酸145至221的TP 53 INP 2突变体; TUBB，I类微管蛋白β; ULK 1，unc-51样自噬活化激酶1; VMP 1，空泡膜蛋白1; WIPI 2，WD重复结构域磷酸肌醇相互作用2; WT，野生型; ZFYVE 1/DFCP 1，含1的锌指FYVE型。
ABSTRACT TP53INP2/DOR (tumor protein p53-inducible nuclear protein 2) contributes to mammalian macroautophagy/autophagy by carrying nuclear deacetylated MAP1LC3/LC3 to the cytoplasm. Here, we report that in the cytoplasm, TP53INP2 further functions in autophagosome biogenesis by promoting LC3B-ATG7 interaction. Cytoplasmic expression of the N-terminal region of TP53INP2, which includes the LC3-interacting region, effectively triggered LC3B–PE production and autophagosome formation. In the cytoplasm, TP53INP2 colocalized to early autophagic membrane structures containing ATG14, ZFYVE1/DFCP1 or WIPI2. While knockdown of TP53INP2 did not affect the formation of these autophagic structures, deletion of BECN1 or Atg5, or mutations preventing TP53INP2 from LC3 interaction, disrupted the membrane binding of TP53INP2. TP53INP2 interacted directly with ATG7 to form a LC3B-TP53INP2-ATG7 complex in the cytoplasm. Loss of TP53INP2-LC3 or TP53INP2-ATG7 interaction significantly reduced LC3B-ATG7 binding. Together, these results suggest that after shifting from the nucleus, cytoplasmic TP53INP2 is targeted to early autophagic membranes accompanied by LC3, where it contributes to autophagosome biogenesis by mediating LC3-ATG7 interaction. Abbreviations: 3-MA, 3-methyladenine; 3NES, 3 repeated nuclear export signal; 3NLS, 3 repeated nuclear localization signal; ACTB, actin beta; ATG, autophagy related; BECN1, Beclin 1; mCherry, monomeric red fluorescent protein mCherry; GFP, green fluorescent protein; GST, glutathione S-transferase; KO, knockout; LC3B/MAP1LC3B, microtubule-associated protein 1 light chain 3 beta; LC3B[G120], LC3B mutant lacking amino acids after glycine 120; LDH, lactate dehydrogenase; LMNB1, lamin B1; LIR, LC3-interacting region; MTORC1, mechanistic target of rapamycin complex 1; PE, phosphatidylethanolamine; PtdIns3K, phosphatidylinositol 3-kinase; PtdIns3P, phosphatidylinositol 3-phosphate; rDNA, ribosomal DNA; RFP, red fluorescent protein; RNAi, RNA interference; SQSTM1, sequestosome 1; TP53INP2, tumor protein p53-inducible nuclear protein 2; TP53INP2[1-28], TP53INP2 mutant containing amino acids 1 to 28; TP53INP2[28-45], TP53INP2 mutant containing amino acids 28 to 45; TP53INP2[LIRΔ], TP53INP2 mutant lacking amino acids 1 to 144; TP53INP2[NLSΔ], TP53INP2 mutant lacking amino acids 145 to 221; TP53INP2W35,I38A, TP53INP2 mutant in which tryptophan 35 and isoleucine 38 are replaced with alanine; TP53INP2W35,I38A[NLSΔ], TP53INP2 mutant lacking amino acids 145 to 221, and tryptophan 35 and isoleucine 38 are replaced with alanine; TP53INP2W35,I38A[Δ1-28],[NLSΔ], TP53INP2 mutant lacking amino acids 1 to 28 and amino acids 145 to 221, and tryptophan 35 and isoleucine 38 are replaced with alanine; TP53INP2[Δ1-28],[NLSΔ], TP53INP2 mutant lacking amino acids 1 to 28 and amino acids 145 to 221; TP53INP2[Δ67-111],[NLSΔ], TP53INP2 mutant lacking amino acids 67 to 111 and amino acids 145 to 221; TP53INP2[Δ112-144],[NLSΔ], TP53INP2 mutant lacking amino acids 112 to 144 and amino acids 145 to 221; TUBB, tubulin beta class I; ULK1, unc-51 like autophagy activating kinase 1; VMP1, vacuole membrane protein 1; WIPI2, WD repeat domain phosphoinositide-interacting 2; WT, wild-type; ZFYVE1/DFCP1, zinc finger FYVE-type containing 1.