Plant J|拟南芥中由硫柳素寡肽酶介导的蛋白水解分析

蛋白质稳态（proteostation）是细胞正常功能的关键，包括通过控制蛋白质水解产生具有生物功能的肽。蛋白质稳定在维持细胞功能和植物在生理条件下与环境的相互作用中起着重要作用。植物胁迫继续降低农业产量，造成巨大的经济损失；因此，了解植物如何感知胁迫信号，从而引发对生存的反应是至关重要的。

如前所述，在拟南芥中，thimet寡肽酶（TOPs）TOP1（也称为细胞器寡肽酶）和TOP2（也称为胞浆寡肽酶）是植物对病原体反应的重要组成部分，但是，需要对TOPs及其肽底物进行进一步的表征，以了解它们对应激感知和防御信号的贡献。本研究通过液相色谱-串联质谱（LC-MS/MS）的无标记肽组学被用于在拟南芥Col-0野生型和top1top2敲除突变体之间差异定量1111个肽，来自369个蛋白质。这表明350肽在突变体中明显更丰富，代表这些潜在的TOP底物的积累。

用重组TOPs和合成的候选肽进行体外酶活性测定，对10种直接底物进行了验证。这些顶层底物来源于参与光合作用、糖酵解、蛋白质折叠、生物发生和抗氧化防御的蛋白质，除了防御信号外，还涉及TOP参与的其它过程。

序列基序分析显示，切割位点周围的非极性残基优先切割。这些底物的鉴定为顶级信号网络提供了一个框架，通过它可以进一步描述蛋白水解途径和防御信号之间的相互作用。

Protein homeostasis (proteostasis) is crucial for proper cellular function, including the production of peptides with biological functions through controlled proteolysis. Proteostasis has roles in maintenance of cellular functions and plant interactions with the environment under physiological conditions. Plant stress continues to reduce agricultural yields causing substantial economic losses; thus, it is critical to understand how plants perceive stress signals to elicit responses for survival. As previously shown in Arabidopsis thaliana, thimet oligopeptidases (TOPs) TOP1 (also referred to as organellar oligopeptidase) and TOP2 (also referred to as cytosolic oligopeptidase) are essential components in plant response to pathogens, but further characterization of TOPs and their peptide substrates is required to understand their contributions to stress perception and defense signaling. Herein, label‐free peptidomics via liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) was used to differentially quantify 1111 peptides, originating from 369 proteins, between the Arabidopsis Col‐0 wild type and top1top2 knock‐out mutant. This revealed 350 peptides as significantly more abundant in the mutant, representing accumulation of these potential TOP substrates. Ten direct substrates were validated using in vitro enzyme assays with recombinant TOPs and synthetic candidate peptides. These TOP substrates are derived from proteins involved in photosynthesis, glycolysis, protein folding, biogenesis, and antioxidant defense, implicating TOP involvement in processes aside from defense signaling. Sequence motif analysis revealed TOP cleavage preference for nonpolar residues in the positions surrounding the cleavage site. Identification of these substrates provides a framework for TOP signaling networks, through which the interplay between proteolytic pathways and defense signaling can be further characterized.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/tpj.15165?af=R

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