ABSTRACT

Macroautophagy/autophagy is suppressed by MTOR (mechanistic target of rapamycin kinase) and is an anticancer target under active investigation. Yet, MTOR-regulated autophagy remains incompletely mapped. We used proteomic profiling to identify proteins in the MTOR-autophagy axis. Wild-type (WT) mouse cell lines and cell lines lacking individual autophagy genes (Atg5 or Ulk1/Ulk2) were treated with an MTOR inhibitor to induce autophagy and cultured in media with either glucose or galactose. Mass spectrometry proteome profiling revealed an elevation of known autophagy proteins and candidates for new autophagy components, including CALCOCO1 (calcium binding and coiled-coil domain protein 1). We show that CALCOCO1 physically interacts with MAP1LC3C, a key protein in the machinery of autophagy. Genetic deletion of CALCOCO1 disrupted autophagy of the endoplasmic reticulum (reticulophagy). Together, these results reveal a role for CALCOCO1 in MTOR-regulated selective autophagy. More generally, the resource generated by this work provides a foundation for establishing links between the MTOR-autophagy axis and proteins not previously linked to this pathway.

Abbreviations: ATG: autophagy-related; CALCOCO1: calcium binding and coiled-coil domain protein 1; CALCOCO2/NDP52: calcium binding and coiled-coil domain protein 2; CLIR: MAP1LC3C-interacting region; CQ: chloroquine; KO: knockout; LIR: MAP1LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; MLN: MLN0128 ATP-competitive MTOR kinase inhibitor; MTOR: mechanistic target of rapamycin kinase; reticulophagy: selective autophagy of the endoplasmic reticulum; TAX1BP1/CALCOCO3: TAX1 binding protein 1; ULK: unc 51-like autophagy activating kinase; WT: wild-type.

摘要

巨自噬/自噬受到 MTOR（雷帕霉素激酶的机械靶标）的抑制，并且是正在积极研究的抗癌靶标。然而，MTOR 调节的自噬仍未完全定位。我们使用蛋白质组学分析来鉴定 MTOR-自噬轴中的蛋白质。野生型（WT）小鼠细胞系和细胞系缺乏个体的自噬基因（Atg5的或ULK1 / ULK2) 用 MTOR 抑制剂处理以诱导自噬，并在含有葡萄糖或半乳糖的培养基中培养。质谱蛋白质组分析揭示了已知自噬蛋白和新自噬成分候选物的升高，包括 CALCOCO1（钙结合和卷曲螺旋结构域蛋白 1）。我们展示了 CALCOCO1 与 MAP1LC3C 物理相互作用，MAP1LC3C 是自噬机制中的关键蛋白质。CALCOCO1 的基因缺失破坏了内质网的自噬（reticulophagy）。总之，这些结果揭示了 CALCOCO1 在 MTOR 调节的选择性自噬中的作用。更一般地说，这项工作产生的资源为在 MTOR-自噬轴和以前未与该途径相关联的蛋白质之间建立联系提供了基础。

缩写： ATG：自噬相关；CALCOCO1：钙结合和卷曲螺旋结构域蛋白 1；CALCOCO2/NDP52：钙结合和卷曲螺旋结构域蛋白 2；CLIR：MAP1LC3C 相互作用区；CQ：氯喹；KO：淘汰赛；LIR：MAP1LC3 相互作用区；MAP1LC3/LC3：微管相关蛋白1轻链3；MEF：小鼠胚胎成纤维细胞；MLN：MLN0128 ATP 竞争性 MTOR 激酶抑制剂；MTOR：雷帕霉素激酶的机制靶点；reticulophagy：内质网的选择性自噬；TAX1BP1/CALCOCO3：TAX1 结合蛋白 1；ULK：unc 51 样自噬激活激酶；WT：野生型。