ABSTRACT

Autophagy summarizes evolutionarily conserved, intracellular degradation processes targeting cytoplasmic material for lysosomal degradation. These encompass constitutive processes as well as stress responses, which are often found dysregulated in diseases. Autophagy pathways help in the clearance of damaged organelles, protein aggregates and macromolecules, mediating their recycling and maintaining cellular homeostasis. Protein-protein interaction networks contribute to autophagosome biogenesis, substrate loading, vesicular trafficking and fusion, protein translocations across membranes and degradation in lysosomes. Hypothesis-free proteomic approaches tremendously helped in the functional characterization of protein-protein interactions to uncover molecular mechanisms regulating autophagy. In this review, we elaborate on the importance of understanding protein-protein-interactions of varying affinities and on the strengths of mass spectrometry-based proteomic approaches to study these, generating new mechanistic insights into autophagy regulation. We discuss in detail affinity purification approaches and recent developments in proximity labeling coupled to mass spectrometry, which uncovered molecular principles of autophagy mechanisms.

Abbreviations: AMPK: AMP-activated protein kinase; AP-MS: affinity purification-mass spectrometry; APEX2: ascorbate peroxidase-2; ATG: autophagy related; BioID: proximity-dependent biotin identification; ER: endoplasmic reticulum; GFP: green fluorescent protein; iTRAQ: isobaric tag for relative and absolute quantification; MS: mass spectrometry; PCA: protein-fragment complementation assay; PL-MS: proximity labeling-mass spectrometry; PtdIns3P: phosphatidylinositol-3-phosphate; PTM: posttranslational modification; PUP-IT: pupylation-based interaction tagging; RFP: red fluorescent protein; SILAC: stable isotope labeling by amino acids in cell culture; TAP: tandem affinity purification; TMT: tandem mass tag.

 抽象的

自噬总结了进化上保守的、针对细胞质物质进行溶酶体降解的细胞内降解过程。这些包括构成过程和应激反应，这些反应通常在疾病中失调。自噬途径有助于清除受损的细胞器、蛋白质聚集体和大分子，介导它们的回收并维持细胞稳态。蛋白质-蛋白质相互作用网络有助于自噬体生物发生、底物负载、囊泡运输和融合、蛋白质跨膜易位和溶酶体降解。无假设的蛋白质组学方法极大地有助于蛋白质-蛋白质相互作用的功能表征，以揭示调节自噬的分子机制。在这篇综述中，我们详细阐述了理解不同亲和力的蛋白质-蛋白质相互作用的重要性，以及基于质谱的蛋白质组学方法研究这些相互作用的优势，从而对自噬调节产生新的机制见解。我们详细讨论了亲和纯化方法以及邻近标记与质谱联用的最新进展，揭示了自噬机制的分子原理。

缩写：AMPK：AMP 激活蛋白激酶； AP-MS：亲和纯化-质谱； APEX2：抗坏血酸过氧化物酶-2； ATG：自噬相关； BioID：邻近依赖性生物素识别； ER：内质网； GFP：绿色荧光蛋白； iTRAQ：用于相对和绝对定量的同量异位标签； MS：质谱； PCA：蛋白质片段互补测定； PL-MS：邻近标记-质谱法； PtdIns3P：3-磷酸磷脂酰肌醇； PTM：翻译后修饰； PUP-IT：基于 pupylation 的交互标记； RFP：红色荧光蛋白； SILAC：细胞培养物中氨基酸的稳定同位素标记； TAP：串联亲和纯化； TMT：串联质量标签。