National Engineering Research Center of Edible Fungi, Key Laboratory of Edible Fungi Resources and Utilization (South), Ministry of Agriculture, Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, Shanghai 201403, China; Department of Food Science and Technology, College of Light Industry and Food Engineering, Nanjing Forestry University, Nanjing 210037, China.
National Engineering Research Center of Edible Fungi, Key Laboratory of Edible Fungi Resources and Utilization (South), Ministry of Agriculture, Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, Shanghai 201403, China.
J Proteomics. 2020 Mar 20;215:103668. doi: 10.1016/j.jprot.2020.103668. Epub 2020 Jan 23.
In Volvariella volvacea, an important edible mushroom species, cryogenic autolysis is a typical part of abnormal metabolism; however, the underlying mechanisms remain unclear. Ubiquitylome analysis revealed that chilling stress (CS) affected protein translation and degradation by ubiquitination. Comparative proteomics analysis showed that CS downregulated protein expression in V. volvacea V23 instead of VH3 (improved chilling stress resistance strain). The integrative ubiquitylome, proteomics, and transcriptome analyses indicated that CS reduced protein translation by the ubiquitination of ribosomal proteins. An activity assay of the 20S proteasome showed that CS decreased the degradation efficiency of the ubiquitin-proteasome system. UBEV2, one type of ubiquitin-conjugating enzyme E2 (UBE2) in V. volvacea, was upregulated after cold stress treatment using western blot analysis. GST pull-down experiments of UBEV2 provided evidence that CS affected protein translation by the ubiquitination of ribosomal proteins. Co-IP experiments confirmed that UBEV2 bound to the ubiquitinated SSB2, a ribosome-associated molecular chaperone. An anti-freezing experiment demonstrated that the UBE2 inhibitor could improve the cold stress resistance of V. volvacea. Our observations revealed that CS triggered ubiquitination-mediated autolysis associated with a decrease in protein translation and highlighted the mechanistic role of UBEV2 in facilitating cryogenic autolysis in V. volvacea. SIGNIFICANCE: Volvariella volvacea, the edible straw mushroom, is a highly nutritious food source widely cultivated on a commercial scale in tropical and subtropical regions. The challenges associated with the cryogenic autolysis preservation of V. volvacea have limited its marketability. This issue of cryogenic autolysis is both an interesting scientific problem to solve and a practical economic matter. Integrative ubiquitylome, proteomics, and transcriptome analyses, together with GST pulldown and Co-IP experiments, indicated that chilling stress reduced protein translation by the ubiquitination of ribosomal proteins in V. volvacea. This study significantly contributes to our understanding of ubiquitination-mediated autolysis associated with a decrease in protein translation in V. volvacea. Our data highlight the mechanistic role of UBEV2 in facilitating the cryogenic autolysis of V. volvacea. We provided a new idea for the preservation of V. volvacea by inhibiting UBEV2 to increase its marketability.
在草菇中，低温自溶是一种典型的异常代谢；然而，其潜在的机制尚不清楚。泛素组学分析表明，冷应激（CS）通过泛素化影响蛋白质翻译和降解。比较蛋白质组学分析表明，CS 下调了 V. volvacea V23 中的蛋白质表达，而不是 VH3（改善了冷应激抗性菌株）。整合泛素组学、蛋白质组学和转录组学分析表明，CS 通过核糖体蛋白的泛素化降低了蛋白质翻译。20S 蛋白酶体的活性测定表明，CS 降低了泛素-蛋白酶体系统的降解效率。Western blot 分析表明，冷应激处理后，草菇中的一种泛素结合酶 E2（UBE2）类型 UBEV2 上调。GST 下拉实验为 CS 通过核糖体蛋白的泛素化影响蛋白质翻译提供了证据。Co-IP 实验证实，UBEV2 与核糖体相关分子伴侣 SSB2 的泛素化结合。抗冻实验表明，UBE2 抑制剂可提高草菇的抗冷应激能力。我们的观察结果表明，CS 触发了与蛋白质翻译减少相关的泛素介导的自溶，并强调了 UBEV2 在促进草菇低温自溶中的机制作用。意义：草菇，又称稻草菇，是一种营养丰富的食物，在热带和亚热带地区广泛商业化种植。草菇低温自溶保存所面临的挑战限制了其市场潜力。解决草菇低温自溶问题既是一个有趣的科学问题，也是一个实际的经济问题。整合泛素组学、蛋白质组学和转录组学分析，以及 GST 下拉和 Co-IP 实验表明，冷应激通过草菇核糖体蛋白的泛素化降低了蛋白质翻译。这项研究极大地促进了我们对草菇中与蛋白质翻译减少相关的泛素介导自溶的理解。我们的数据突出了 UBEV2 在促进草菇低温自溶中的机制作用。我们通过抑制 UBEV2 来提高草菇的市场竞争力，为草菇的保存提供了一个新的思路。
