Horita Henrick, Law Andy, Middleton Kim
Research and Development Department, Cytoskeleton Inc., Denver, CO 80223, USA.
Proteomes. 2018 May 22;6(2):24. doi: 10.3390/proteomes6020024.
Post-translational modification (PTM) crosstalk is recognized as a major cell-regulatory mechanism, and studies of several proteins have validated the premise that PTMs work in concert. Previous work by our group investigated the potential PTM crosstalk on proteins in the EGFR-Ras-c-Fos axis by utilizing a comprehensive set of PTM reagents termed Signal-Seeker toolkits. In this study, these tools were used to investigate the potential PTM crosstalk that occurs in acetylated mitochondrial proteins in response to a mitochondrial stress-inducing agent hydrogen peroxide (H₂O₂). Mitochondrial protein acetylation has been shown to participate in PTM crosstalk as exemplified by the regulation of the pyruvate dehydrogenase complex via kinase, phosphatase, acetyltransferase, and deacetylase activities. Changes in the acetylated state of mitochondrial proteins were investigated, in response to H₂O₂, using a novel anti acetyl lysine (Ac-K) antibody. Signal-Seeker PTM detection tools were used to validate the acetylation state of ten mitochondrial targets, as well as their endogenous acetylation state in response to H₂O₂. Importantly, the endogenous acetylation, ubiquitination, SUMOylation 2/3, and tyrosine phosphorylation state of four target mitochondrial proteins were also investigated with the toolkit. Each of the four proteins had unique PTM profiles, but diverging acetylation and ubiquitin or SUMO 2/3 signals appeared to be a common theme. This proof-of-concept study identifies the Signal-Seeker toolkits as a useful tool to investigate potential PTM crosstalk.
翻译后修饰（PTM）的相互作用被认为是一种主要的细胞调节机制，对几种蛋白质的研究证实了PTM协同发挥作用的前提。我们团队之前的工作通过使用一套称为信号搜寻工具包的综合PTM试剂，研究了EGFR-Ras-c-Fos轴上蛋白质的潜在PTM相互作用。在本研究中，这些工具被用于研究在乙酰化线粒体蛋白中因线粒体应激诱导剂过氧化氢（H₂O₂）而发生的潜在PTM相互作用。线粒体蛋白乙酰化已被证明参与PTM相互作用，例如通过激酶、磷酸酶、乙酰转移酶和脱乙酰酶活性对丙酮酸脱氢酶复合物的调节。使用一种新型抗乙酰赖氨酸（Ac-K）抗体，研究了线粒体蛋白乙酰化状态对H₂O₂的响应变化。信号搜寻PTM检测工具用于验证十个线粒体靶点的乙酰化状态，以及它们在H₂O₂作用下的内源性乙酰化状态。重要的是，还使用该工具包研究了四种目标线粒体蛋白的内源性乙酰化、泛素化、SUMO化2/3和酪氨酸磷酸化状态。这四种蛋白质中的每一种都有独特的PTM谱，但乙酰化与泛素或SUMO 2/3信号的差异似乎是一个共同的主题。这项概念验证研究确定信号搜寻工具包是研究潜在PTM相互作用的有用工具。
