GLIPR2 是自噬的负调控因子，也是 BECN1-ATG14 包含的磷脂酰肌醇 3-激酶复合物的负调控因子。
GLIPR2 is a negative regulator of autophagy and the BECN1-ATG14-containing phosphatidylinositol 3-kinase complex.
机构信息
Center for Autophagy Research, University of Texas Southwestern Medical Center, Dallas, TX, USA.
Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA.
出版信息
Autophagy. 2021 Oct;17(10):2891-2904. doi: 10.1080/15548627.2020.1847798. Epub 2020 Nov 23.
A key mediator of macroautophagy/autophagy induction is the class III phosphatidylinositol 3-kinase complex I (PtdIns3K-C1) consisting of PIK3C3/VPS34, PIK3R4/VPS15, BECN1, and ATG14. Although several proteins are known to enhance or decrease PtdIns3K-C1 activity, our understanding of the molecular regulation of PtdIns3K-C1 is still incomplete. Previously, we identified a Golgi-associated protein, GLIPR2, in a screen for proteins that interact with amino acids 267-284 of BECN1, a region of BECN1 sufficient to induce autophagy when fused to a cell penetrating leader sequence. In this study, we used CRISPR-Cas9-mediated depletion of GLIPR2 in cells and mice to investigate the role of GLIPR2 in the regulation of autophagy and PtdIns3K-C1 activity. Depletion of GLIPR2 in HeLa cells increased autelophagic flux and generation of phosphatidylinositol 3-phosphate (PtdIns3P). knockout resulted in less compact Golgi structures, which was also observed in autophagy-inducing conditions such as amino acid starvation or Tat-BECN1 peptide treatment. Importantly, the binding of GLIPR2 to purified PtdIns3K-C1 inhibited the in vitro lipid kinase activity of PtdIns3K-C1. Moreover, the tissues of knockout mice had increased basal autophagic flux as well as increased recruitment of the PtdIns3P-binding protein, WIPI2. Taken together, our findings demonstrate that GLIPR2 is a negative regulator of PtdIns3K-C1 activity and basal autophagy. ATG14: autophagy related 14; Baf A1: bafilomycin A; BARA: β-α repeated, autophagy-specific; CQ: chloroquine; GFP: green fluorescent protein; GLIPR2: GLI pathogenesis related 2; HBSS: Hanks' balanced salt solution; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PBS: phosphate-buffered saline; PtdIns3K-C1: phosphatidylinositol 3-kinase complex I; PtdIns3P: phosphatidylinositol-3-phosphate; SEM: standard error of the mean; WIPI2: WD repeat domain, phosphoinositide interacting 2.
自噬诱导的关键介质是 III 类磷脂酰肌醇 3-激酶复合物 I（PtdIns3K-C1），由 PIK3C3/VPS34、PIK3R4/VPS15、BECN1 和 ATG14 组成。尽管已经知道有几种蛋白质可以增强或降低 PtdIns3K-C1 的活性，但我们对 PtdIns3K-C1 的分子调节的理解仍然不完整。以前，我们在筛选与 BECN1 氨基酸 267-284 相互作用的蛋白质时，发现了一种与高尔基相关的蛋白 GLIPR2，该区域的 BECN1 与穿透细胞膜的领导序列融合时足以诱导自噬。在这项研究中，我们使用 CRISPR-Cas9 介导的 GLIPR2 细胞和小鼠耗竭来研究 GLIPR2 在自噬和 PtdIns3K-C1 活性调节中的作用。HeLa 细胞中 GLIPR2 的耗竭增加了自噬体的通量和磷脂酰肌醇 3-磷酸（PtdIns3P）的产生。 敲除导致高尔基结构不那么紧凑，在氨基酸饥饿或 Tat-BECN1 肽处理等诱导自噬的条件下也观察到这种情况。重要的是，GLIPR2 与纯化的 PtdIns3K-C1 结合抑制了 PtdIns3K-C1 的体外脂质激酶活性。此外， 敲除小鼠的组织中自噬通量增加，PtdIns3P 结合蛋白 WIPI2 的募集增加。总之，我们的研究结果表明，GLIPR2 是 PtdIns3K-C1 活性和基础自噬的负调节剂。ATG14：自噬相关 14；Baf A1：巴佛洛霉素 A；BARA：β-α 重复，自噬特异性；CQ：氯喹；GFP：绿色荧光蛋白；GLIPR2：GLI 发病相关 2；HBSS：Hanks' 平衡盐溶液；KO：敲除；MAP1LC3/LC3：微管相关蛋白 1 轻链 3；PBS：磷酸盐缓冲盐水；PtdIns3K-C1：磷脂酰肌醇 3-激酶复合物 I；PtdIns3P：磷脂酰肌醇-3-磷酸；SEM：平均值的标准误差；WIPI2：WD 重复域，磷酸肌醇相互作用 2。
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