MALDI-TOF Mass Spectrometry for interrogating ubiquitin enzymes.
MALDI-TOF Mass Spectrometry for interrogating ubiquitin enzymes.
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DOI：
10.3389/fmolb.2023.1184934
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发表时间：
2023
影响因子：
5
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泛素与底物的连接（泛素化或泛素化）影响其寿命并调节其在细胞内的功能。几类酶监督泛素与底物的连接：E1活化酶，其使泛素在随后的缀合和连接阶段之前化学敏感，分别由E2缀合酶（E2 s）和E3连接酶（E3 s）介导。人类基因组中编码了大约40个E2和600多个E3，它们的组合和合作行为决定了调节数千种底物所需的严格特异性。泛素的去除是由大约100个去泛素化酶（DUB）的网络协调的。许多细胞过程都受到泛素化的严格控制，这对维持细胞内稳态至关重要。由于泛素化的基本作用，人们有兴趣更好地了解泛素机制的功能和特异性。自2014年以来，已经开发了一系列扩展的基质辅助激光解吸/电离飞行时间（MALDI-TOF）质谱（MS）测定，以系统地鉴定各种泛素酶的体外活性。在这里，我们概括了MALDI-TOF MS如何辅助泛素酶的体外表征和发现新的和意想不到的E2和DUBs功能。鉴于MALDI-TOF MS方法的多功能性，我们预见使用该技术来进一步扩展我们对泛素和泛素样酶的理解。
The attachment of ubiquitin to a substrate (ubiquitination or ubiquitylation) impacts its lifetime and regulates its function within the cell. Several classes of enzymes oversee the attachment of ubiquitin to the substrate: an E1 activating enzyme that makes ubiquitin chemically susceptible prior to the following stages of conjugation and ligation, respectively mediated by E2 conjugating enzymes (E2s) and E3 ligases (E3s). Around 40 E2s and more than 600 E3s are encoded in the human genome, and their combinatorial and cooperative behaviour dictate the tight specificity necessary for the regulation of thousands of substrates. The removal of ubiquitin is orchestrated by a network of about 100 deubiquitylating enzymes (DUBs). Many cellular processes are tightly controlled by ubiquitylation, which is essential in maintaining cellular homeostasis. Because of the fundamental role(s) of ubiquitylation, there is an interest in better understanding the function and specificity of the ubiquitin machinery. Since 2014, an expanding array of Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) Mass Spectrometry (MS) assays have been developed to systematically characterise the activity of a variety of ubiquitin enzymes in vitro. Here we recapitulate how MALDI-TOF MS aided the in vitro characterization of ubiquitin enzymes and the discovery of new and unexpected of E2s and DUBs functions. Given the versatility of the MALDI-TOF MS approach, we foreseen the use of this technology to further expand our understanding of ubiquitin and ubiquitin-like enzymes.