Astrocyte Elevated Gene-1 Cys75 S-Palmitoylation by ZDHHC6 Regulates Its Biological Activity

Abstract

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 RNAsequencing 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.

The endoplasmic reticulum–localized enzyme zDHHC6 mediates S-acylation of short transmembrane constructs from multiple type I and II membrane proteins

“…Among the few experimentally established substrates of zDHHC6, many are either type I or type II transmembrane proteins: CANX ( 25 , 27 ), TfR ( 25 , 26 ), the interferon-induced transmembrane protein 3 (IFITM3) ( 38 ), the tight junction protein angulin-1 ( 39 ), or the recently identified substrates AEG1 and CLIMP-63/CKAP4 ( 40 , 41 ). All these proteins display one or several cysteines in very close proximity to their TMD.…”

Spatial transcriptomics unravels palmitoylation and zonation-dependent gene regulation by AEG-1 in mouse liver

“…The generation of AEG-1-C75S mouse by CRISPR/Cas9 technology has been described ( 16 ). AEG-1-C75S heterozygote x heterozygote mating was performed to generate AEG-1-WT and AEG-1-C75S littermates.…”

“…AEG-1 functions as a scaffold in multiple steps in the activation process of NF-κB, a master regulator of inflammatory cytokines, and by activating NF-κB AEG-1 promotes inflammation ( 13 , 14 , 15 ). AEG-1 is a 582 amino acid protein that has a single cysteine at position 75 (Cys75) and we recently demonstrated that AEG-1 is palmitoylated at Cys75 ( 16 ). Palmitoylation, a post-translational modification in which fatty acids are covalently attached to cysteine residues, is largely recognized as a positive regulator of protein function ( 17 ).…”

“…Palmitoylation, a post-translational modification in which fatty acids are covalently attached to cysteine residues, is largely recognized as a positive regulator of protein function ( 17 ). We interrogated how palmitoylation regulates AEG-1 function by generating a knock-in mouse via CRISPR/Cas9 where Cys75 was mutated to serine (AEG-1-C75S) ( 16 ). RNA-sequencing analysis of AEG-1-WT and AEG-C75S hepatocytes unraveled that palmitoylation negatively regulates AEG-1 function such that genes regulating steatosis, inflammation, and tumorigenesis are significantly more abundant in AEG-C75S hepatocytes compared to AEG-1-WT ( 16 ).…”

Diverse Roles of Protein Palmitoylation in Cancer Progression, Immunity, Stemness, and Beyond

“…Mechanistically, palmitoylation negatively regulates AEG-1 function by reducing the stability of AEG-1 and by reducing its affinity with its interacting proteins. Further, a different research group identified ZDHHC6 as the main palmitoyl transferase for AEG-1 and found that CRISPR/Cas9 knock-in mice with a palmitoylation-defective form of AEG-1 had increased signaling pathways and regulators that contribute to cell proliferation, motility, angiogenesis, and lipid accumulation [ 147 ]. Together, palmitoylation plays a negative regulatory role on AEG-1 function.…”