N‐ and C‐terminal degradation of ecdysteroid receptor isoforms, when transiently expressed in mammalian CHO cells, is regulated by the proteasome and cysteine and threonine proteases

Abstract

Transcriptional activity of nuclear receptors is the result of transactivation capability and the concentration of the receptor protein. The concentration of ecdysteroid receptor (EcR) isoforms, constitutively expressed in mammalian CHO cells, is dependent on a number of factors. As shown previously, ligand binding stabilizes receptor protein concentration. In this paper, we investigate the degradation of EcR isoforms and provide evidence that N-terminal degradation is modulated by isoform-specific ubiquitination sites present in the A/B domains of EcR-A and -B1. This was demonstrated by the increase in EcR concentration by treatment with carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132), an inhibitor of ubiquitin-mediated proteasomal degradation and by deletion of ubiquitination sites. In addition, EcR is degraded by the peptidyl-dipeptidase cathepsin B (CatB) and the endopeptidase cathepsin S (CatS) at the C-terminus in an isoform-specific manner, despite identical C-termini. Ubiquitin-proteasome-mediated degradation and the proteolytic action are modulated by heterodimerization with Ultraspiracle (USP). The complex regulation of receptor protein concentration offers an additional opportunity to regulate transcriptional activity in an isoform- and target cell-specific way and allows the temporal limitation of hormone action.

A novel mosquito ubiquitin targets viral envelope protein for degradation and reduces virion production during dengue virus infection

“…Infection levels were analyzed using isolated RNA and qRT-PCR analysis. MG132 has successfully been used to inhibit the proteasome in insect cells previously [52] and we found no cellular toxicity in our assays at the levels used. Surprisingly, blocking proteasomal activity did not reduce the antiviral effects of Ub3881 ( Fig.…”

Mating induces switch from hormone-dependent to hormone-independent steroid receptor–mediated growth in Drosophila secondary cells

“…Unlike the endogenously expressed isoforms of EcR, overexpression of EcR-C promoted growth of SCs (Fig 7A, 7C and 7E, S1 Data) and produced high levels of EcR protein in the nuclei and cytosol of SCs when expressed alone (Fig 7F and 7H). A simple explanation of our data, given the known role of EcR NTD sequences in protein stability [48], is that BMP signalling activity regulates levels of different EcR isoforms via a genetic interaction with each of their unique NTDs. Alternatively, BMP signalling may affect transcript levels specifically via the sequences encoding each NTD, which are absent in the EcR-C transcript.…”

“…Previous in vitro studies have revealed that when the Drosophila EcR-A and EcR-B1 isoforms are expressed in CHO cells, the different NTDs of these proteins, which include the activation function 1 (AF1) domain, one of the two transcriptional activation domains in these receptors, partially destabilise the proteins through a ubiquitination-dependent mechanism [48]. Although these experiments were performed in a heterologous system and did not reveal similar regulation for the EcR-B2 isoform, which has a much shorter AF1 domain, we tested whether the NTD plays a role in SC-specific, BMP-dependent control of EcR protein levels.…”

“…Either these different 5′ sequences are all independently targeted by a mechanism that mediates BMP-dependent control of EcR protein levels or the regulation is posttranslational. The latter appears more likely because the NTDs of both EcR-A and EcR-B1 are involved in degradative mechanisms that control EcR protein levels [48]. However, because we have not been able to assess EcR transcript levels in SCs in different genetic backgrounds, we cannot exclude a transcript-specific regulatory mechanism.…”

An E3 Ubiquitin Ligase Scaffolding Protein Is Proviral during Chikungunya Virus Infection in Aedes aegypti

“…To elucidate if there was any role of proteasomal pathway in CHIKV replication, we used a well-known proteasome inhibitor MG132 in pre and posttreatment assays. MG132 has also previously been employed as a proteasome inhibitor in Aag2 cells ( 40 , 41 ). Prior to the assay, cell viability upon MG132 treatment was tested in Aag2 cells, and it was found that the cell viability remained above 80% at the three drug concentrations tested, namely, 0.1 μM, 1 μM, and 10 μM ( Fig.…”