Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
Center for Proteome Analysis, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
Biomolecules. 2024 Jul 9;14(7):817. doi: 10.3390/biom14070817.
DZNep (3-deazaneplanocin A) is commonly used to reduce lysine methylation. DZNep inhibits S-adenosyl-l-homocysteine hydrolase (AHCY), preventing the conversion of S-adenosyl-l-homocysteine (SAH) into L-homocysteine. As a result, the SAM-to-SAH ratio decreases, an indicator of the methylation potential within a cell. Many studies have characterized the impact of DZNep on histone lysine methylation or in specific cell or disease contexts, but there has yet to be a study looking at the potential downstream impact of DZNep treatment on proteins other than histones. Recently, protein thermal stability has provided a new dimension for studying the mechanism of action of small-molecule inhibitors. In addition to ligand binding, post-translational modifications and protein-protein interactions impact thermal stability. Here, we sought to characterize the protein thermal stability changes induced by DZNep treatment in HEK293T cells using the Protein Integral Solubility Alteration (PISA) assay. DZNep treatment altered the thermal stability of 135 proteins, with over half previously reported to be methylated at lysine residues. In addition to thermal stability, we identify changes in transcript and protein abundance after DZNep treatment to distinguish between direct and indirect impacts on thermal stability. Nearly one-third of the proteins with altered thermal stability had no changes at the transcript or protein level. Of these thermally altered proteins, CDK6 had a stabilized methylated peptide, while its unmethylated counterpart was unaltered. Multiple methyltransferases were among the proteins with thermal stability alteration, including DNMT1, potentially due to changes in the SAM/SAH levels. This study systematically evaluates DZNep's impact on the transcriptome, the proteome, and the thermal stability of proteins.
DZNep（3-去氮杂胞苷）常用于降低赖氨酸甲基化。DZNep 抑制 S-腺苷-L-同型半胱氨酸水解酶（AHCY），阻止 S-腺苷-L-同型半胱氨酸（SAH）转化为 L-同型半胱氨酸。因此，SAM 与 SAH 的比例降低，这是细胞内甲基化潜力的指标。许多研究已经描述了 DZNep 对组蛋白赖氨酸甲基化的影响，或在特定的细胞或疾病背景下的影响，但尚未有研究关注 DZNep 处理对除组蛋白以外的蛋白质的潜在下游影响。最近，蛋白质热稳定性为研究小分子抑制剂的作用机制提供了一个新的维度。除了配体结合外，翻译后修饰和蛋白质-蛋白质相互作用也会影响热稳定性。在这里，我们使用蛋白质整体可溶性改变（PISA）测定法，试图描述 DZNep 处理在 HEK293T 细胞中诱导的蛋白质热稳定性变化。DZNep 处理改变了 135 种蛋白质的热稳定性，其中超过一半以前报道在赖氨酸残基上被甲基化。除了热稳定性之外，我们还确定了 DZNep 处理后转录物和蛋白质丰度的变化，以区分对热稳定性的直接和间接影响。在热稳定性改变的蛋白质中，近三分之一在转录物或蛋白质水平上没有变化。在这些热稳定性改变的蛋白质中，CDK6 有一个稳定的甲基化肽，而其未甲基化的对应物则没有改变。多个甲基转移酶也在热稳定性改变的蛋白质中，包括 DNMT1，这可能是由于 SAM/SAH 水平的变化。本研究系统地评估了 DZNep 对转录组、蛋白质组和蛋白质热稳定性的影响。
