Targeted proteomics addresses selectivity and complexity of protein degradation by autophagy.

Alexandre Leytens, Rocío Benítez-Fernández, Carlos Jiménez-García, Carole Roubaty, Michael Stumpe, Patricia Boya, Jörn Dengjel
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引用次数: 0

Abstract

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 de novo 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.e. 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.Abbreviation: 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.

靶向蛋白质组学解决了自噬降解蛋白质的选择性和复杂性问题。
大自噬/自噬是一种组成性活跃的分解代谢溶酶体降解途径,在人类疾病中经常发现其失调。它通常被认为具有细胞保护作用,在细胞受到应激时通常会上调。自噬的启动在蛋白质水平上受到调控,不需要从头合成蛋白质。自噬一直被认为是非选择性的,但现在很清楚,不同的刺激可通过选择性自噬受体(SAR)导致细胞成分的选择性降解。由于自噬的选择性和多种降解途径的潜在协同作用,对自噬通量(即选择性自噬依赖蛋白降解)的监测应能解决这一复杂问题。在这里,我们介绍了一种靶向蛋白质组学方法,该方法监测 37 种自噬相关蛋白的丰度变化,涵盖了与自噬过程相关的蛋白,如启动复合体和 Atg8 家族蛋白脂化机制,以及大多数已知的 SARs。我们的研究表明,在自噬诱导条件下,参与自噬体生物生成的蛋白质会上调并免于降解,这与 SARs 形成鲜明对比,且与细胞系相关。经典的大量刺激(如营养饥饿)主要诱导依赖泛素的可溶性 SAR 的降解,而不是依赖泛素的膜结合型 SAR 的降解。相反,铁螯合剂去铁酮会导致与有丝分裂和网状吞噬/ER-吞噬相关的泛素依赖型和非依赖型SAR降解。我们的方法是自动化的,支持大规模筛选测定,为(临床前)应用和监测特定自噬通量铺平了道路。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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