Targeted proteomics addresses selectivity and complexity of protein degradation by autophagy

Macroautophagy/autophagy is a constitutively active catabolic lysosomal degradation pathway, often found dysregulated in human diseases. It is often considered to act in a cytoprotective manner and is commonly upregulated in cells undergoing stress. Its initiation is regulated at the protein level and does not require <i>de novo</i> protein synthesis. Historically, autophagy has been regarded as nonselective; however, it is now clear that different stimuli can lead to the selective degradation of cellular components via selective autophagy receptors (SARs). Due to its selective nature and the existence of multiple degradation pathways potentially acting in concert, monitoring of autophagy flux, <i>i.e</i>. selective autophagy-dependent protein degradation, should address this complexity. Here, we introduce a targeted proteomics approach monitoring abundance changes of 37 autophagy-related proteins covering process-relevant proteins such as the initiation complex and the Atg8-family protein lipidation machinery, as well as most known SARs. We show that proteins involved in autophagosome biogenesis are upregulated and spared from degradation under autophagy-inducing conditions in contrast to SARs, in a cell-line dependent manner. Classical bulk stimuli such as nutrient starvation mainly induce degradation of ubiquitin-dependent soluble SARs and not of ubiquitin-independent, membrane-bound SARs. In contrast, treatment with the iron chelator deferiprone leads to the degradation of ubiquitin-dependent and -independent SARs linked to mitophagy and reticulophagy/ER-phagy. Our approach is automatable and supports large-scale screening assays paving the way to (pre)clinical applications and monitoring of specific autophagy flux. <b>Abbreviation:</b> AMBRA1: autophagy and beclin 1 regulator 1; ATG: autophagy related; BafA1: bafilomycin A₁; BNIP1: BCL2 interacting protein 1; BNIP3: BCL2 interacting protein 3; BNIP3L/NIX: BCL2 interacting protein 3-like; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CCPG1: cell cycle progression 1; CV: coefficients of variations; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; DFP: deferiprone; ER: endoplasmic reticulum; FKBP8: FKBP prolyl isomerase 8; GABARAPL: GABA type A receptor associated protein like; LC: liquid chromatography; LOD: limit of detection; LOQ: limit of quantification; MAP1LC3: microtubule associated protein 1 light chain 3; MS: mass spectrometry; NCOA4: nuclear receptor coactivator 4; NBR1: NBR1 autophagy cargo receptor; NUFIP1: nuclear FMR1 interacting protein 1; OPTN: optineurin; PHB2: prohibitin 2; PNPLA2/ATGL: patatin like phospholipase domain containing 2; POI: protein of interest; PTM: posttranslational modification; PRM: parallel reaction monitoring; RB1CC1/FIP200: RB1 inducible coiled-coil 1; RETREG1/FAM134B: reticulophagy regulator 1; RPS6KB1: ribosomal protein S6 kinase B1; RTN3: reticulon 3; SARs: selective autophagy receptors; SQSTM1/p62: sequestosome 1; STBD1: starch binding domain 1; TAX1BP1: Tax1 binding protein 1; TFEB: transcription factor EB; TNIP1: TNFAIP3 interacting protein 1; TOLLIP: toll interacting protein; ULK1: unc-51 like autophagy activating kinase 1; WBP2: WW domain binding protein 2; WDFY3/Alfy: WD repeat and FYVE domain containing 3; WIPI2: WD repeat domain, phosphoinositide interacting 2.

巨自噬（Macroautophagy）/自噬（autophagy）是一种组成型激活的分解代谢型溶酶体降解通路，在人类疾病中常出现失调现象。其通常被认为发挥细胞保护作用，且在受应激的细胞中普遍会上调。自噬的起始过程在蛋白质层面受到调控，且无需从头合成蛋白质。既往学界曾认为自噬是非选择性的，但如今已有研究证实，不同刺激可通过选择性自噬受体（selective autophagy receptors，SARs）介导细胞组分的选择性降解。鉴于自噬具有选择性特性，且可能存在多条协同作用的降解通路，因此对自噬流（即依赖选择性自噬的蛋白质降解过程）的监测需要兼顾这种复杂性。
本研究介绍一种靶向蛋白质组学方法，可监测37种自噬相关蛋白的丰度变化，涵盖该过程相关的核心蛋白（如起始复合物、Atg8家族蛋白脂化修饰系统）以及绝大多数已报道的选择性自噬受体。研究发现，在自噬诱导条件下，参与自噬体生物发生的蛋白质会上调且免于降解，这一现象与选择性自噬受体的变化相反，且呈现细胞系依赖性。经典的整体自噬诱导刺激（如营养匮乏）主要介导泛素依赖性可溶性选择性自噬受体的降解，而对泛素非依赖性膜结合型选择性自噬受体无明显影响。与之相反，使用铁螯合剂去铁酮（deferiprone）处理细胞，则可介导与线粒体自噬、内质网自噬（reticulophagy/ER-phagy）相关的泛素依赖性及非依赖性选择性自噬受体的降解。该方法具备自动化兼容性，可支持大规模筛选实验，为（预）临床应用以及特异性自噬流的监测奠定了基础。
<b>缩写说明：</b> AMBRA1：自噬与Beclin 1调控因子1（autophagy and beclin 1 regulator 1）；ATG：自噬相关（autophagy related）；BafA1：巴弗洛霉素A₁（bafilomycin A₁）；BNIP1：BCL2相互作用蛋白1（BCL2 interacting protein 1）；BNIP3：BCL2相互作用蛋白3（BCL2 interacting protein 3）；BNIP3L/NIX：BCL2相互作用蛋白3样蛋白（BCL2 interacting protein 3-like）；CALCOCO2/NDP52：钙结合与卷曲螺旋结构域蛋白2（calcium binding and coiled-coil domain 2）；CCPG1：细胞周期进程调控蛋白1（cell cycle progression 1）；CV：变异系数（coefficients of variations）；CCCP：羰基氰化物间氯苯腙（carbonyl cyanide m-chlorophenyl hydrazone）；DFP：去铁酮（deferiprone）；ER：内质网（endoplasmic reticulum）；FKBP8：FKBP脯氨酰异构酶8（FKBP prolyl isomerase 8）；GABARAPL：GABA型受体相关蛋白样蛋白（GABA type A receptor associated protein like）；LC：液相色谱（liquid chromatography）；LOD：检测限（limit of detection）；LOQ：定量限（limit of quantification）；MAP1LC3：微管相关蛋白1轻链3（microtubule associated protein 1 light chain 3）；MS：质谱（mass spectrometry）；NCOA4：核受体辅激活因子4（nuclear receptor coactivator 4）；NBR1：NBR1自噬 cargo受体（NBR1 autophagy cargo receptor）；NUFIP1：核FMR1相互作用蛋白1（nuclear FMR1 interacting protein 1）；OPTN：视神经蛋白（optineurin）；PHB2：抑制素2（prohibitin 2）；PNPLA2/ATGL：patatin样磷脂酶结构域包含2（patatin like phospholipase domain containing 2）；POI：目标蛋白（protein of interest）；PTM：翻译后修饰（posttranslational modification）；PRM：平行反应监测（parallel reaction monitoring）；RB1CC1/FIP200：RB1诱导型卷曲螺旋蛋白1（RB1 inducible coiled-coil 1）；RETREG1/FAM134B：内质网自噬调控因子1（reticulophagy regulator 1）；RPS6KB1：核糖体蛋白S6激酶B1（ribosomal protein S6 kinase B1）；RTN3：网状蛋白3（reticulon 3）；SARs：选择性自噬受体（selective autophagy receptors）；SQSTM1/p62：自噬 cargo受体SQSTM1/p62（sequestosome 1）；STBD1：淀粉结合结构域蛋白1（starch binding domain 1）；TAX1BP1：Tax1结合蛋白1（Tax1 binding protein 1）；TFEB：转录因子EB（transcription factor EB）；TNIP1：TNFAIP3相互作用蛋白1（TNFAIP3 interacting protein 1）；TOLLIP：Toll相互作用蛋白（toll interacting protein）；ULK1：UNC-51样自噬激活激酶1（unc-51 like autophagy activating kinase 1）；WBP2：WW结构域结合蛋白2（WW domain binding protein 2）；WDFY3/Alfy：WD重复与FYVE结构域包含蛋白3（WD repeat and FYVE domain containing 3）；WIPI2：WD重复结构域磷酸肌醇相互作用蛋白2（WD repeat domain, phosphoinositide interacting 2）。