Proteomics and phosphoproteomics study of LCMT1 overexpression and oxidative stress: overexpression of LCMT1 arrests H₂O₂-induced lose of cells viability

<b>Objectives:</b> Protein phosphatase 2A (PP2A), a major serine/threonine phosphatase, is also known to be a target of ROS. The methylation of PP2A can be catalyzed by leucine carboxyl methyltransferase-1 (LCMT1), which regulates PP2A activity and substrate specificity. <b>Methods:</b> In the previous study, we have showed that LCMT1-dependent PP2Ac methylation arrests H₂O₂-induced cell oxidative stress damage. To explore the possible protective mechanism, we performed iTRAQ-based comparative quantitative proteomics and phosphoproteomics studies of H₂O₂-treated vector control and LCMT1-overexpressing cells. <b>Results:</b> A total of 4480 non-redundant proteins and 3801 unique phosphopeptides were identified by this means. By comparing the H₂O₂-regulated proteins in LCMT1-overexpressing and vector control cells, we found that these differences were mainly related to protein phosphorylation, gene expression, protein maturation, the cytoskeleton and cell division. Further investigation of LCMT1 overexpression-specific regulated proteins under H₂O₂ treatment supported the idea that LCMT1 overexpression induced ageneral dephosphorylation of proteins and indicated increased expression of non-erythrocytic hemoglobin, inactivation of MAPK3 and regulation of proteins related to Rho signal transduction, which were known to be linked to the regulation of the cytoskeleton. <b>Discussion:</b> These data provide proteomics and phosphoproteomics insights into the association of LCMT1-dependent PP2Ac methylation and oxidative stress and indirectly indicate that the methylation of PP2A plays an important role against oxidative stress.**研究目的**：蛋白磷酸酶2A（Protein phosphatase 2A, PP2A）作为一类主要的丝氨酸/苏氨酸磷酸酶，同时也是活性氧（reactive oxygen species, ROS）的作用靶点。PP2A的甲基化可由亮氨酸羧基甲基转移酶1（leucine carboxyl methyltransferase-1, LCMT1）催化，该过程可调控PP2A的活性与底物特异性。 **研究方法**：既往研究已证实，亮氨酸羧基甲基转移酶1依赖的PP2A催化亚基甲基化可抑制过氧化氢（hydrogen peroxide, H₂O₂）诱导的细胞氧化应激损伤。为探究其潜在的保护机制，本研究对经H₂O₂处理的空载对照细胞与过表达LCMT1的细胞开展了基于同位素标记相对和绝对定量（isobaric tags for relative and absolute quantitation, iTRAQ）的比较定量蛋白质组学与磷酸化蛋白质组学分析。 **研究结果**：本研究共鉴定得到4480个非冗余蛋白与3801条独特磷酸肽。通过对比过表达LCMT1与空载对照细胞中受H₂O₂调控的蛋白，我们发现这些差异蛋白主要参与蛋白磷酸化、基因表达、蛋白质成熟、细胞骨架及细胞分裂等生物学过程。进一步针对H₂O₂处理下LCMT1过表达特异性调控蛋白的分析表明，LCMT1过表达可诱导整体蛋白质去磷酸化，并可观察到非红细胞血红蛋白表达上调、丝裂原活化蛋白激酶3（mitogen-activated protein kinase 3, MAPK3）失活以及与Rho信号转导相关蛋白的调控变化，而后者已知与细胞骨架调控密切相关。 **讨论**：本研究的蛋白质组学与磷酸化蛋白质组学数据揭示了LCMT1依赖的PP2A催化亚基甲基化与氧化应激之间的关联，并间接表明PP2A甲基化在对抗氧化应激过程中发挥重要作用。