REGULATION OF HEPATIC ER STRESS BY THE E3 UBIQUITIN LIGASE GP78 IN ZEBRAFISH

Abstract

Mammalian gp78 is an E3 ubiquitin ligase that is anchored at the membrane of the endoplasmic reticulum (ER). It regulates protein homeostasis by polyubiquitinating and targeting proteins for proteasomal degradation under both physiologic and stress conditions. To further test its role in vivo, we analyzed the gross embryonic morphology of zebrafish embryos in which gp78 was knocked down using morpholinos and in transgenic fish overexpressing wild-type gp78 or dominant-negative gp78. We show that gp78 is highly conserved among vertebrates. Zebrafish gp78, similar to human gp78, can colocalize with mouse MmUBC7 in HeLa cells. In vitro ubiquitination assays confirmed that zebrafish gp78 is indeed an E3 ubiquitin ligase. Although gp78 was maternally and constitutively expressed during embryonic development, with relatively high expression levels in several tissues, such as liver and brain, the knockdown of endogenous gp78 or overexpression of wild-type or dominant-negative gp78 did not result in developmental defects, suggesting compensation by other E3 ubiquitin ligases during embryonic development.

ER-associated protein degradation (ERAD) activity by the unfolded protein response (UPR) represents one of the mechanisms for restoring ER homeostasis. However, the significance of gp78 in the regulation of hepatic ER stress in vivo remains elusive. Here we report that zebrafish gp78 plays a key role in the regulation of hepatic ER stress under tunicamycin-induced stress, but not under physiologic conditions. Tunicamycin treatment induced ER stress and upregulated the expression of several key components of the gp78-mediated ERAD complex in the liver. Moreover, hepatic-specific overexpression of the dominant-negative form of gp78 (gp78-R2M) rendered livers more sensitive to tunicamycin-induced ER stress, suggesting a role for gp78-mediated ERAD in the regulation of hepatic protein homeostasis. Moreover, the overexpression of gp78-R2M enhanced the expression of sterol response element binding protein (Srebp) target genes in response to ER stress, while this was not observed in fish overexpressing wild-type gp78. Together, these data indicate that gp78 plays a critical role in the regulation of hepatic ER stress and lipid metabolism.