Background Ubiquitination is a post-translational modification where ubiquitin is covalently attached to lysine residues on substrate proteins to signal their degradation by the 26S proteasome or initiate other non-degradation functions such as cellular trafficking. The diversity of ubiquitin modifications can be attributed to the variable number of ubiquitin molecules attached to a lysine residue (mono- vs. poly-ubiquitin chains), the type of covalent linkages within poly-ubiquitin chains and the number of lysine residues on a substrate that are occupied by ubiquitin at any given time. The integral role ubiquitination plays in cell homeostasis is reflected by the multitude of diseases associated with impaired ubiquitin modification, rendering it the focus of extensive research initiatives and proteomic discovery studies. However, determining the functional role of distinct ubiquitin modifications directly from proteomic data remains challenging and represents a bottleneck in the process of deciphering how ubiquitination at specific substrate sites impacts cell signaling. Methods In this study SILAC coupled with LC-MS/MS is used to identify ubiquitinated proteins in SKOV3 ovarian cancer cells, with the implementation of a computational approach that measures relative ubiquitin occupancy at distinct modification sites upon 26S proteasome inhibition and uses that data to infer functional significance. Results In addition to identifying and quantifying relative ubiquitin occupancy at distinct post-translational modification sites to distinguish degradation from non-degradation signaling, this research led to the discovery of nine ubiquitination sites in the oncoprotein HER2 that have not been previously reported in ovarian cancer. Subsequently the computational approach applied in this study was utilized to infer the functional role of individual HER2 ubiquitin-modified residues. Conclusions In summary, the computational method, previously described for glycosylation analysis, was used in this study for the assessment of ubiquitin stoichiometries and applied directly to proteomic data to distinguish degradation from non-degradation ubiquitin functions.

中文翻译：

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通过响应 26S 蛋白酶体抑制的泛素占据变化对降解泛素信号传导的蛋白质组学分析。

背景泛素化是一种翻译后修饰，其中泛素与底物蛋白上的赖氨酸残基共价连接，以表明它们被 26S 蛋白酶体降解或启动其他非降解功能，例如细胞运输。泛素修饰的多样性可归因于连接到赖氨酸残基的泛素分子数量可变（单泛素链与多泛素链）、多泛素链内共价键的类型和底物上赖氨酸残基的数量在任何给定时间被泛素占据。泛素化在细胞稳态中发挥的不可或缺的作用反映在与泛素修饰受损相关的众多疾病中，使其成为广泛研究计划和蛋白质组学发现研究的重点。然而，直接从蛋白质组学数据确定不同泛素修饰的功能作用仍然具有挑战性，并且代表了破译特定底物位点的泛素化如何影响细胞信号传导的过程中的瓶颈。方法 在本研究中，SILAC 与 LC-MS/MS 结合用于鉴定 SKOV3 卵巢癌细胞中的泛素化蛋白，并采用计算方法测量 26S 蛋白酶体抑制后不同修饰位点的相对泛素占有率，并使用该数据推断功能意义。结果除了识别和量化不同翻译后修饰位点的相对泛素占有率以区分降解和非降解信号外，这项研究导致在癌蛋白 HER2 中发现了 9 个以前在卵巢癌中未曾报道过的泛素化位点。随后，本研究中应用的计算方法被用于推断单个 HER2 泛素修饰残基的功能作用。结论 总之，之前描述的糖基化分析计算方法在本研究中用于评估泛素化学计量，并直接应用于蛋白质组学数据，以区分降解与非降解泛素功能。