Ubiquitin–proteasome system mediates heme oxygenase-1 degradation through endoplasmic reticulum-associated degradation pathway

Abstract

The present study investigated the cellular mechanism underlying the degradation of heme oxygenase-1 (HO-1), an endoplasmic reticulum (ER)-anchored protein. The turnover of HO-1 induced in vascular smooth muscle cells (VSMCs) was significantly attenuated by proteasome inhibitors, suggesting the involvement of a proteasome-mediated pathway. High molecular weight ubiquitin conjugates were co-immunoprecipitated with HO-1 from VSMCs after proteasome inhibition, and HO-1 ubiquitination was confirmed in HEK293 cells overexpressing His-tagged HO-1 and HA-tagged ubiquitin. Endogenous p97, an ATPase, and Ufd1, both implicated as essential components in the ER-associated degradation pathway (ERAD), were co-eluted with His-tagged HO-1 from metal affinity resin. Knockdown of either p97 or Ufd1 in HEK293 cells using specific siRNA significantly prolonged the half-life of endogenously induced HO-1 and slowed the degradation of ubiquitinated HO-1. HO-1 ubiquitination in HEK293 cells was enhanced by zinc chloride, but suppressed with a zinc chelator (N,N,N',N'-tetrakis(2-pyridylmethyl)ethyl-enediamine), suggesting the involvement of a RING-E3 ligase in this process. Collectively, these data indicate that HO-1 protein turnover is regulated by the ubiquitin-proteasome system through the ERAD pathway.

Ubiquitin-recognition protein Ufd1 couples the endoplasmic reticulum (ER) stress response to cell cycle control

“…However, RNAi-mediated depletion of Ufd1 in mammalian cells has yielded opposite results. While some studies reported impaired ERAD in Ufd1-depleted cells (6,7), others showed accelerated degradation of the classical ERAD substrates, such as cholera toxin and T-cell receptors (8,9). Although these seemingly contradictory observations might stem from the use of distinct cellular systems, they point to greater complexity in the regulation and function of Ufd1 that impinge on the ER stress response.…”

Novel Insights into the Vasoprotective Role of Heme Oxygenase-1

“…The knockdown of both miR-217 and miR-377 increases HO-1 protein expression, while the overexpression of the same miRNAs leads to attenuation of protein expression [40]. Recently, Lin et al show that HO-1 is subjected to posttranslational regulation by the ubiquitin-proteasome system through an endoplasmic reticulum-associated degradation pathway [41]. Proteasome inhibition significantly decreased HO-1 protein degradation.…”

Substance P Induces HO-1 Expression in RAW 264.7 Cells Promoting Switch towards M2-Like Macrophages

“…Therefore, induction of HO-1 has been thought to produce protective effects against a variety of cellular stresses [20]. The expression of HO-1 is regulated mainly at the transcriptional level [21], although a mechanism of HO-1 degradation through the endoplasmic reticulum-associated degradation pathway has been reported [22]. The promoter sequences of HO-1 contain two enhancer regions (E1 and E2) providing binding motifs for a variety of transcription factors, such as activator protein (AP)-1, cAMP-responsive element binding protein (CREB), NF-κB or Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) [23].…”