ABSTRACT

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). GLIPR2 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 glipr2 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.

Abbreviations: ATG14: autophagy related 14; Baf A1: bafilomycin A₁; 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

摘要

巨自噬/自噬诱导的关键介质是由 PIK3C3/VPS34、PIK3R4/VPS15、BECN1 和 ATG14 组成的 III 类磷脂酰肌醇 3-激酶复合物 I (PtdIns3K-C1)。尽管已知有几种蛋白质可以增强或降低 PtdIns3K-C1 的活性，但我们对 PtdIns3K-C1 的分子调控的理解仍然不完整。以前，我们在筛选与 BECN1 的氨基酸 267-284 相互作用的蛋白质中鉴定了一种高尔基体相关蛋白 GLIPR2，BECN1 的一个区域在与细胞穿透前导序列融合时足以诱导自噬。在这项研究中，我们使用 CRISPR-Cas9 介导的细胞和小鼠中 GLIPR2 的消耗来研究 GLIPR2 在调节自噬和 PtdIns3K-C1 活性中的作用。GLIPR2敲除导致高尔基体结构不太紧凑，这也在自噬诱导条件下观察到，例如氨基酸饥饿或 Tat-BECN1 肽处理。重要的是，GLIPR2 与纯化的 PtdIns3K-C1 的结合抑制了 PtdIns3K-C1 的体外脂质激酶活性。此外，glipr2敲除小鼠的组织增加了基础自噬通量以及增加了 PtdIns3P 结合蛋白 WIPI2 的募集。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因子。

缩写： ATG14：自噬相关14；Baf A1：巴弗洛霉素 A ₁；BARA：β-α重复，自噬特异性；CQ：氯喹；GFP：绿色荧光蛋白；GLIPR2：GLI发病机制相关2；HBSS：汉克斯平衡盐溶液；KO：淘汰赛；MAP1LC3/LC3：微管相关蛋白1轻链3；PBS：磷酸盐缓冲液；PtdIns3K-C1：磷脂酰肌醇 3-激酶复合物 I；PtdIns3P：3-磷酸磷脂酰肌醇；SEM：均值的标准误；WIPI2：WD 重复结构域，磷酸肌醇相互作用 2