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iRhoms are inactive rhomboid-like pseudoproteases that lack essential catalytic residues. Although iRhoms are highly conserved in metazoan species, little is known about their function. In a recent issue of Cell, Zettl et al. (2011) show that iRhoms regulate growth factor signaling through endoplasmic reticulum-associated protein degradation (ERAD).
Highly conserved among all sequenced metazoans, iRhoms are related to rhomboid intramembrane serine proteases but are not active proteases, as they lack the essential catalytic residues required for serine proteases and have no proteolytic activity. Although their high degree of conservation between organisms suggests that these pseudoproteases are under evolutionary selective pressure, their functional significance is largely unknown. A previous study reported that human iRhom1 interacts with the human epidermal growth factor (EGF) when it is overexpressed in Drosophila, suggesting a possible link between an iRhom and EGFR signaling in mammals (Freeman, 2008). In this issue of Developmental Cell, Zettl et al. (2011) show in both Drosophila and mammalian cells that iRhoms regulate growth factor signaling, e.g. EGFR pathways, through the ER quality control machinery that function in endoplasmic reticulum-associated protein degradation (ERAD).
About one third of the eukaryotic genome encodes membrane and secretory proteins, most of which undergo folding and modification in the ER. Protein folding is the most error-prone process in gene expression. When ER homeostasis is disrupted or when cells are stimulated to secrete large amounts of protein, unfolded and misfolded proteins accumulate in the ER and activate the unfolded protein response (UPR). The UPR is a group of adaptive signaling pathways that increase the capacity for ER protein folding, attenuate global mRNA translation, and enhance protein degradation through the transcriptional induction of the ERAD machinery (Ron and Walter, 2007). Eukaryotic cells have evolved a robust ER quality control system that recognizes correctly folded proteins for trafficking to the Golgi compartment. In contrast, unfolded and misfolded proteins are retained in the ER for chaperone-assisted refolding. If misfolding persists, proteins are targeted to the ERAD pathway, which requires retrotranslocation from the ER to the cytoplasm, where they are polyubiquitylated and degraded by the proteasome (Hebert et al., 2010).
In this study, Zettl and colleagues first confirmed previous results by showing that Drosophila, murine, and human iRhoms are catalytically inert against a variety of rhomboid substrates, including Drosophila EGFR ligands and murine EGF. Unlike the active rhomboid proteases that reside in the Golgi and plasma membrane, the iRhoms localize to the ER. To reveal the function of iRhoms, Zetti et al. (2011) then applied Drosophila genetics. In Drosophila embryos, larva, and adults, iRhom RNA is enriched in neuronal cells, suggesting that iRhoms may act in the development and/or function of the nervous system. The authors generated a Drosophila null mutant of iRhom that had no discernible defects during development, although the mutant flies displayed a severe decrease in their daytime activity (as referred to as a “sleep”-like state). This result was confirmed by neuron-specific transgene rescue of iRhom in the mutant flies. Given the established relationship among rhomboid proteases in central nervous system, activation of EGFR signaling, and the sleep-like phenotype in Drosophila (Foltenyi et al., 2007), the authors dissected how iRhoms affect EGFR pathways by analyzing genetic interactions. They found that the rough eye phenotype in Drosophila caused by EGFR hyperactivation through Rhomboid-1 overexpression inversely correlated with the expression level of iRhom. Additionally, a reduction of EGFR signaling reversed the sleep-like phenotype in iRhom mutant flies. No genetic interactions were identified between iRhom and other developmentally significantly pathways, including Wnt, Notch, Hedgehog, and Dpp. These result, together with other synergistic genetic interactions, supported a specific role for Drosophila iRhom in the inhibition of EGFR signaling.
To delineate the mechanism of how Drosophila iRhom inhibits EGFR signaling, the authors first examined whether iRhoms inhibit Rhomboid-1 activity in cell culture. The cleavage and release of Drosophila EGFR ligands by Drosophila Rhomboid-1 in COS7 cells was inhibited by co-expression of Drosophila iRhom. Human and murine iRhoms also inhibited cleavage and release murine EGF. In addition, the release of a metalloprotease substrate was not affected by iRhom, demonstrating specificity of this pseudoprotease in inhibiting Rhomboids. The authors further showed that iRhoms still inhibit the release of EGF in cells that do not harbor rhomboid activity, suggesting that iRhoms act on Rhomboid substrates rather than by inhibiting Rhomboid activity. In support of this, iRhoms act on mutant EGF molecules that are not direct targets of active rhomboid proteases.
The authors then demonstrated that iRhom-induced downregulation of EGF requires proteasome activity. Given that ER-localized EGF needs to be extracted from the ER to the cytoplasm for degradation by the proteasome, and that iRhoms localize and function in the ER, the authors tested whether iRhoms regulate EGF-family ligands through ERAD. They showed that EGF co-immunoprecipitates with murine and human iRhoms. In addition, pulse-chase analysis examining the intracellular kinetics of EGF upon proteasome inhibition showed slowed EGF secretion when iRhom is co-expressed, further supporting direct binding of intracellular EGF to iRhoms. Finally, the authors showed that knockdown of ERAD components Hrd1 and Edem2 abolished the inhibition of EGFR signaling upon overexpression of EGFR inhibitors, demonstrating that ERAD downregulates EGFR signaling under normal conditions.
ERAD is not restricted to misfolded/unfolded proteins under conditions of ER stress, but also operates on short-lived ER proteins and proteins with regulated half-lives, such as HMG CoA reductase, a rate-limiting enzyme in cholesterol metabolism (Brodsky and Wojcikiewicz, 2009). A recent study also suggested that Hedgehog ligand is constitutively degraded by ERAD after self-cleavage in the ER (Chen et al., 2011). The findings of Zettl et al. (2011) now provide additional insight into the function of ERAD in the regulation of intracellular signaling by showing that iRhoms downregulate EGF-family ligands through targeting them to the ERAD machinery. The study also provides evidence that transmembrane pseudoproteases play an important role in ER quality control. There does remain a number of questions. The finding that iRhoms can act on proteins that are not substrates of active rhomboids leads to the question how iRhoms actually recognize their targets. Since ERAD has specifically evolved to target proteins with altered conformations to degradation, is EGF unfolding or misfolding part of the iRhom recognition event? In addition, the precise mechanism by which iRhoms orchestrate ERAD of EGF-family ligands is unknown. To elucidate this process, it would be helpful to define what ERAD components display physical and functional interactions with iRhoms. Do iRhoms simply act as adaptor molecules to assemble substrates with the ERAD machinery or do they actively participate in this process? iRhom-dependent degradation of EGF requires two components of the ERAD machinery, EDEM2 and HRD1. EDEMs (ER-degradation enhancing α-mannosidase-like protein) belong to the α1,2-mannosidase family that are proposed to accelerate ERAD, possibly through cleavage of mannose residues on asparagine(N)-linked glycans (Hebert et al., 2010). Are N-glycans part of the recognition signal for degradation? As HRD1 is an E3 ubiquitin ligase, is ubiquitination required for degradation of EGFR ligands? Finally, since the ERAD machinery is transcriptionally induced by the UPR during periods of ER stress, is iRhom expression similarly regulated by the UPR? Given the conserved function of iRhoms in regulating EGFR signaling in mammals, the dissection of its physiological significance and relevance to human diseases, such as cancer, will be an interest area of future investigation.
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