定量蛋白质组学揭示酵母去泛素化酶Ubp14生物学功能

Quantitative proteomics reveal the potential biological functions of the deubiquitinating enzyme Ubp14 in Saccharomyces cerevisiae

摘要:

泛素化是一种动态可逆的蛋白质翻译后修饰，泛素分子在泛素激活酶、泛素结合酶和泛素连接酶的级联酶促反应催化下共价连接到底物蛋白上。去泛素化酶将泛素分子从底物上移除，动态可逆地调控泛素化修饰，在成熟泛素的生成、泛素链的移除与修剪、游离泛素链的回收等过程中发挥着关键的调控作用。本文的研究对象是酵母中泛素特异性蛋白酶（ubiquitin specific protease，USP）家族成员Ubp14，负责回收细胞内游离的泛素链。本研究定量比较了酵母细胞中Ubp14缺失对全蛋白质组的影响，进而找出其潜在的调控通路和分子功能。首先，通过同源重组技术构建了ubp14Δ菌株，发现其生长速度低于野生型酵母。利用稳定同位素氨基酸代谢标记技术结合深度覆盖的蛋白质组学分析技术，系统比较了ubp14Δ菌株相对于野生型菌株的差异蛋白，共计鉴定3 685个蛋白，通过统计学分析筛选得到109个差异蛋白。基因本体论分析发现，Ubp14缺失引起的差异蛋白主要参与了包括氨基酸代谢、氧化还原和热应激等生物学过程。本研究为深入探究去泛素化酶Ubp14的生物学功能，进而深刻理解游离泛素的稳态平衡与生物学过程调控提供了高可信的蛋白质组学数据信息。

关键词:去泛素化酶;Ubp14;稳定同位素氨基酸代谢标记技术;泛素

Abstract:

Ubiquitination is one of the reversible protein post-translational modifications, in which ubiquitin molecules bind to the target protein in a cascade reaction of ubiquitin activating enzymes, ubiquitin conjugating enzymes, and ubiquitin ligases. The deubiquitinating enzymes (DUBs) remove ubiquitin residues from the substrates, which play key roles in the formation of mature ubiquitin, the removal and trimming of ubiquitin chains, as well as the recycling of free ubiquitin chains. Ubp14, a member of the ubiquitin specific proteases family in Saccharomyces cerevisiae, is mainly responsible for the recycling of intracellular free ubiquitin chains. To investigate its global biological function, a ubp14Δ mutant was constructed by homologous recombination technique. The growth rate of ubp14Δ mutant was lower than that of the wild-type (WT) strain. Using stable isotope labeling by amino acids in cell culture (SILAC) combined with deep coverage proteomics analysis, the differentially expressed proteins of ubp14Δ mutant relative to the wild-type strain were systematically analyzed. A total of 3 685 proteins were identified in this study, and 109 differentially expressed proteins were filtered out by statistical analysis. Gene ontology analysis found that differentially expressed proteins caused by Ubp14 loss were mainly involved in amino acid metabolism, REDOX, heat shock stress and etc, which shed light on the broad biological function of this DUB. This study provides highly reliable proteomic data for further exploring the biological functions of the deubiquitination enzyme Ubp14, and further understanding the relationship between the free ubiquitin homeostasis and biological process regulation.

Key words:deubiquitinating enzyme;Ubp14;stable isotope labeling by amino acids in cell culture (SILAC);ubiquitin