Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, United States.
C. Kenneth and Dianne Wright Center for Clinical and Translational Research, Virginia Commonwealth University, Richmond, Virginia 23298, United States.
Biochemistry. 2023 Jan 17;62(2):543-553. doi: 10.1021/acs.biochem.2c00583. Epub 2022 Dec 22.
Nonalcoholic fatty liver disease is a major risk factor for hepatocellular carcinoma (HCC). Astrocyte elevated gene-1/Metadherin (AEG-1/MTDH) augments lipid accumulation (steatosis), inflammation, and tumorigenesis, thereby promoting the whole spectrum of this disease process. Targeting AEG-1 is a potential interventional strategy for nonalcoholic steatohepatitis (NASH) and HCC. Thus, proper understanding of the regulation of this molecule is essential. We found that AEG-1 is palmitoylated at residue cysteine 75 (Cys75). Mutation of Cys75 to serine (Ser) completely abolished AEG-1 palmitoylation. We identified ZDHHC6 as a palmitoyltransferase catalyzing the process in HEK293T cells. To obtain insight into how palmitoylation regulates AEG-1 function, we generated knock-in mice by CRISPR/Cas9 in which Cys75 of AEG-1 was mutated to Ser (AEG-1-C75S). No developmental or anatomical abnormality was observed between AEG-1-wild type (AEG-1-WT) and AEG-1-C75S littermates. However, global gene expression analysis by RNA-sequencing unraveled that signaling pathways and upstream regulators, which contribute to cell proliferation, motility, inflammation, angiogenesis, and lipid accumulation, were activated in AEG-1-C75S hepatocytes compared to AEG-1-WT. These findings suggest that AEG-1-C75S functions as dominant positive and that palmitoylation restricts oncogenic and NASH-promoting functions of AEG-1. We thus identify a previously unknown regulatory mechanism of AEG-1, which might help design new therapeutic strategies for NASH and HCC.
非酒精性脂肪性肝病是肝细胞癌（HCC）的一个主要危险因素。星形细胞上调基因-1/Metadherin（AEG-1/MTDH）增加脂质积累（脂肪变性）、炎症和肿瘤发生，从而促进整个疾病过程。针对 AEG-1 是治疗非酒精性脂肪性肝炎（NASH）和 HCC 的一种潜在的干预策略。因此，正确理解该分子的调节机制至关重要。我们发现 AEG-1 在半胱氨酸残基 75（Cys75）处被棕榈酰化。将 Cys75 突变为丝氨酸（Ser）完全消除了 AEG-1 的棕榈酰化。我们鉴定出 ZDHHC6 是一种棕榈酰转移酶，可在 HEK293T 细胞中催化该过程。为了深入了解棕榈酰化如何调节 AEG-1 的功能，我们使用 CRISPR/Cas9 在 AEG-1 中生成了 Cys75 突变为 Ser 的敲入小鼠（AEG-1-C75S）。在 AEG-1-WT 和 AEG-1-C75S 同窝仔鼠之间未观察到发育或解剖异常。然而，通过 RNA 测序进行的全基因组基因表达分析表明，与 AEG-1-WT 相比，信号通路和上游调控因子被激活，这些因子有助于细胞增殖、迁移、炎症、血管生成和脂质积累。这些发现表明，AEG-1-C75S 作为显性阳性发挥作用，棕榈酰化限制了 AEG-1 的致癌和促进 NASH 的功能。因此，我们确定了 AEG-1 的一个以前未知的调节机制，这可能有助于设计针对 NASH 和 HCC 的新治疗策略。
