Ubiquitin-proteasome system mediates heme oxygenase-1 degradation through endoplasmic reticulum-associated degradation pathway.
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.
Lin
Pu-Hua
PH
Institute of Pharmacology, School of Medicine, National Yang-Ming University, Taipei, Taiwan.
Chiang
Ming-Tsai
MT
Chau
Lee-Young
LY
D000818:Animals; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism; D051547:Heme Oxygenase-1 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D025801:Ubiquitin / Q000378:metabolism*
D011480:Protease Inhibitors; D034741:RNA, Small Interfering; D025801:Ubiquitin; D051547:Heme Oxygenase-1; D046988:Proteasome Endopeptidase Complex