The Co-repressor Hairless Protects RORα Orphan Nuclear Receptor from Proteasome-mediated Degradation

Abstract

ROR␣ is a constitutively active orphan nuclear receptor essential for cerebellar development and is previously shown to regulate genes involved in both myogenesis and adipogenesis. The transcriptional activity of ROR␣ is dependent on the presence of a ubiquitous ligand and can be abolished by interaction with Hairless (Hr), a ligand-oblivious nuclear receptor co-repressor. In this study, we first demonstrate that ROR␣ is a shortlived protein and that treatment with the MG-132 proteasome inhibitor results in the accumulation of ubiquitin-conjugated receptor and inhibition of transcription. These data show that ROR␣ transcriptional activity and degradation are intrinsically linked. In addition, the introduction of inactivation mutations in the ligand-binding pocket and co-regulator-binding surface of ROR␣ significantly increases protein stability, indicating that ligand and/or co-regulator binding perpetuates ROR␣ degradation. Strikingly, expression of the co-repressor Hr results in the stabilization of ROR␣ because of an inhibition of proteasome-mediated degradation of the receptor. Stabilization of ROR␣ by Hr requires intact nuclear receptor recognition LXXLL motifs within Hr. Interestingly, the co-repressor nuclear receptor co-repressor (NCoR) has no effect on ROR␣ protein turnover. This study shows that stabilization of ROR␣ is an essential component of Hr-mediated repression and suggests a molecular mechanism to achieve transcriptional repression by a liganded receptor-co-repressor complex.The ubiquitin-proteasome pathway is the major system employed by eukaryotes for the selective degradation of cellular proteins that play key roles in cellular processes such as cell cycle regulation, differentiation, signal transduction, transcription, and chromosomal stabilization (reviewed in Refs. 1 and 2). Proteolytic degradation by the ubiquitin-proteasome system involves ATP-dependent covalent attachment of a macromolecular chain of ubiquitin (Ub) 1 molecules to the target protein, followed by degradation through the multicatalytic 26 S proteasome. The conjugation of Ub, a highly conserved 8.6-kDa protein, to its target protein is mediated by the serial action of three enzymes: E1, the Ub-activating enzyme, activates Ub in an ATP-dependent manner; E2, the Ub-conjugating enzyme, catalyzes the attachment of Ub to the substrate protein; and E3, the Ub-ligases, serves as a scaffold between E2 and the substrate and provides recognition specificity of the substrate. Ubiquitinylation of a substrate is reversible, and Ub moieties can be cleaved from a target protein by deubiquitinating enzymes. These enzymes assure that the cell is not depleted of a Ub pool. A protein tagged with a polyubiquitin chain is recognized and degraded by the 26 S proteasome complex. This complex is composed of a 19 S regulatory subcomplex, consisting of a "lid" subunit and a "base" subunit, the latter containing the six ATPases required for the degradation executed by the 20 S catalytic subcomplex (2).The Ub-proteasome pathway has recently emerged a...

Rapid degradation of cyclooxygenase-1 and hematopoietic prostaglandin D synthase through ubiquitin–proteasome system in response to intracellular calcium level

“…Accumulated COX-1 protein was detected at 10 min after initiation of treatment with A23187 and MG132, and its level increased in a time-dependent manner (Figure 2A), suggesting that COX-1 is a likely substrate of the ubiquitin–proteasome complex, whereas COX-1 did not accumulate when cells were cultured in medium containing A23187 alone (Figure 2A); these results are consistent with previous results (Moraitis and Giguere, 2003; Mishra et al , 2009). To further investigate COX-1 degradation through the ubiquitin–proteasome system, we examined the time dependence of the effect of MG132 on the ubiquitination of the COX-1 protein in the cells.…”

A unique insertion/duplication in the VDR gene that truncates the VDR causing hereditary 1,25-dihydroxyvitamin D-resistant rickets without alopecia

“…The hairless gene product, HR, like the VDR, is involved in regulating hair growth. HR has recently been shown to function as a corepressor with VDR [24], thyroid hormone receptor (TR) [25], and the retinoic acid receptorrelated orphan receptor α (RORα) [26,27]. It has been suggested that VDR and HR regulate a common pathway(s) involved in the hair cycle and epithelial cell differentiation [24].…”

Disruption of the hedgehog signaling pathway contributes to the hair follicle cycling deficiency in Vdr knockout mice

“…Mice lacking functional hairless (Hr) (Panteleyev et al, 1999; Sundberg et al, 1999) develop a hair follicle phenotype comparable to that seen in the VdrKO mouse. Hairless, which is a known corepressor to some nuclear receptors (Potter et al, 2001, 2002; Moraitis et al, 2002; Moraitis and Giguere, 2003), interacts directly with Vdr (Hsieh et al, 2003; Xie et al, 2006; Wang et al, 2007) and inhibits ligand dependent Vdr mediated transcription. Because of the parallel between VdrKO and hairless mice phenotypes, it has been hypothesized that Hr and Vdr converge to control hair cycling but the role of Hr in modulating ligand independent actions of Vdr as in hair follicle cycling is not known.…”