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- iRhom2 and TNF: Partners or enemies?Publication . Badenes, Marina; Adrain, ColiniRhom2 is an essential cofactor for ADAM17, the metalloprotease that sheds both the proinflammatory cytokine tumor necrosis factor-α (TNF-α) and TNF receptors (TNFRs) from the cell surface. In this issue of Science Signaling, Sundaram et al. demonstrate a protective role for iRhom2 in promoting ADAM17-mediated shedding of TNFRs in hepatic stellate cells, which reduces TNFR signaling and liver fibrosis in response to injury.
- iTAP, a novel iRhom interactor, controls TNF secretion by policing the stability of iRhom/TACEPublication . Oikonomidi, Ioanna; Burbridge, Emma; Cavadas, Miguel; Sullivan, Graeme; Collis, Blanka; Naegele, Heike; Clancy, Danielle; Brezinova, Jana; Hu, Tianyi; Bileck, Andrea; Gerner, Christopher; Bolado, Alfonso; Kriegsheim, Alex Von; Martin, Seamus J.; Steinberg, FlorianThe apical inflammatory cytokine TNF regulates numerous important biological processes including inflammation and cell death, and drives inflammatory diseases. TNF secretion requires TACE (also called ADAM17), which cleaves TNF from its transmembrane tether. The trafficking of TACE to the cell surface, and stimulation of its proteolytic activity, depends on membrane proteins, called iRhoms. To delineate how the TNF/TACE/iRhom axis is regulated, we performed an immunoprecipitation/mass spectrometry screen to identify iRhom-binding proteins. This identified a novel protein, that we name iTAP (iRhom Tail-Associated Protein) that binds to iRhoms, enhancing the cell surface stability of iRhoms and TACE, preventing their degradation in lysosomes. Depleting iTAP in primary human macrophages profoundly impaired TNF production and tissues from iTAP KO mice exhibit a pronounced depletion in active TACE levels. Our work identifies iTAP as a physiological regulator of TNF signalling and a novel target for the control of inflammation.
- Phosphorylation of iRhom2 Controls Stimulated Proteolytic Shedding by the Metalloprotease ADAM17/TACEPublication . Cavadas, Miguel; Oikonomidi, Ioanna; Gaspar, Catarina J.; Burbridge, Emma; Badenes, Marina; Félix, Inês; Bolado, Alfonso; Hu, Tianyi; Bileck, Andrea; Gerner, Christopher; Domingos, Pedro M.; von Kriegsheim, Alex; Adrain, ColinCell surface metalloproteases coordinate signaling during development, tissue homeostasis, and disease. TACE (TNF-α-converting enzyme), is responsible for cleavage ("shedding") of membrane-tethered signaling molecules, including the cytokine TNF, and activating ligands of the EGFR. The trafficking of TACE within the secretory pathway requires its binding to iRhom2, which mediates the exit of TACE from the endoplasmic reticulum. An important, but mechanistically unclear, feature of TACE biology is its ability to be stimulated rapidly on the cell surface by numerous inflammatory and growth-promoting agents. Here, we report a role for iRhom2 in TACE stimulation on the cell surface. TACE shedding stimuli trigger MAP kinase-dependent phosphorylation of iRhom2 N-terminal cytoplasmic tail. This recruits 14-3-3 proteins, enforcing the dissociation of TACE from complexes with iRhom2, promoting the cleavage of TACE substrates. Our data reveal that iRhom2 controls multiple aspects of TACE biology, including stimulated shedding on the cell surface.
- Deletion of iRhom2 protects against diet-induced obesity by increasing thermogenesisPublication . Badenes, Marina; Amin, Abdulbasit; González-García, Ismael; Félix, Inês; Burbridge, Emma; Cavadas, Miguel; Ortega, Francisco José; de Carvalho, Érika; Faísca, Pedro; Carobbio, Stefania; Seixas, Elsa; Pedroso, Dora; Neves-Costa, Ana; Moita, Luís F.; Fernández-Real, José Manuel; Vidal-Puig, António; Domingos, Ana; López, Miguel; Adrain, ColinObjective: Obesity is the result of positive energy balance. It can be caused by excessive energy consumption but also by decreased energy dissipation, which occurs under several conditions including when the development or activation of brown adipose tissue (BAT) is impaired. Here we evaluated whether iRhom2, the essential cofactor for the Tumour Necrosis Factor (TNF) sheddase ADAM17/TACE, plays a role in the pathophysiology of metabolic syndrome. Methods: We challenged WT versus iRhom2 KO mice to positive energy balance by chronic exposure to a high fat diet and then compared their metabolic phenotypes. We also carried out ex vivo assays with primary and immortalized mouse brown adipocytes to establish the autonomy of the effect of loss of iRhom2 on thermogenesis and respiration. Results: Deletion of iRhom2 protected mice from weight gain, dyslipidemia, adipose tissue inflammation, and hepatic steatosis and improved insulin sensitivity when challenged by a high fat diet. Crucially, the loss of iRhom2 promotes thermogenesis via BAT activation and beige adipocyte recruitment, enabling iRhom2 KO mice to dissipate excess energy more efficiently than WT animals. This effect on enhanced ther- mogenesis is cell-autonomous in brown adipocytes as iRhom2 KOs exhibit elevated UCP1 levels and increased mitochondrial proton leak.
- Phosphorylation of iRhom2 Controls Stimulated Proteolytic Shedding by the Metalloprotease ADAM17/TACEPublication . Cavadas, Miguel; Oikonomidi, Ioanna; Gaspar, Catarina J.; Burbridge, Emma; Badenes, Marina; Félix, Inês; Bolado, Alfonso; Hu, Tianyi; Bileck, Andrea; Gerner, Christopher; Domingos, Pedro M.; von Kriegsheim, Alex; Adrain, ColinCell surface metalloproteases coordinate signaling during development, tissue homeostasis, and disease. TACE (TNF-α-converting enzyme), is responsible for cleavage ("shedding") of membrane-tethered signaling molecules, including the cytokine TNF, and activating ligands of the EGFR. The trafficking of TACE within the secretory pathway requires its binding to iRhom2, which mediates the exit of TACE from the endoplasmic reticulum. An important, but mechanistically unclear, feature of TACE biology is its ability to be stimulated rapidly on the cell surface by numerous inflammatory and growth-promoting agents. Here, we report a role for iRhom2 in TACE stimulation on the cell surface. TACE shedding stimuli trigger MAP kinase-dependent phosphorylation of iRhom2 N-terminal cytoplasmic tail. This recruits 14-3-3 proteins, enforcing the dissociation of TACE from complexes with iRhom2, promoting the cleavage of TACE substrates. Our data reveal that iRhom2 controls multiple aspects of TACE biology, including stimulated shedding on the cell surface.
- Quantitative proteomics screen identifies a substrate repertoire of rhomboid protease RHBDL2 in human cells and implicates it in epithelial homeostasisPublication . Johnson, Nicholas; Březinová, Jana; Stephens, Elaine; Burbridge, Emma; Freeman, Matthew; Adrain, Colin; Strisovsky, KvidoRhomboids are intramembrane serine proteases conserved in all kingdoms of life. They regulate epidermal growth factor receptor signalling in Drosophila by releasing signalling ligands from their transmembrane tethers. Their functions in mammals are poorly understood, in part because of the lack of endogenous substrates identified thus far. We used a quantitative proteomics approach to investigate the substrate repertoire of rhomboid protease RHBDL2 in human cells. We reveal a range of novel substrates that are specifically cleaved by RHBDL2, including the interleukin-6 receptor (IL6R), cell surface protease inhibitor Spint-1, the collagen receptor tyrosine kinase DDR1, N-Cadherin, CLCP1/DCBLD2, KIRREL, BCAM and others. We further demonstrate that these substrates can be shed by endogenously expressed RHBDL2 and that a subset of them is resistant to shedding by cell surface metalloproteases. The expression profiles and identity of the substrates implicate RHBDL2 in physiological or pathological processes affecting epithelial homeostasis.
- Inactive rhomboid proteins: New mechanisms with implications in health and diseasePublication . Lemberg, Marius K; Adrain, ColinRhomboids, proteases containing an unusual membrane-integral serine protease active site, were first identified in Drosophila, where they fulfill an essential role in epidermal growth factor receptor signaling, by cleaving membrane-tethered growth factor precursors. It has recently become apparent that eukaryotic genomes harbor conserved catalytically inactive rhomboid protease homologs, including derlins and iRhoms. Here we highlight how loss of proteolytic activity was followed in evolution by impressive functional diversification, enabling these pseudoproteases to fulfill crucial roles within the secretory pathway, including protein degradation, trafficking regulation, and inflammatory signaling. We distil the current understanding of the roles of rhomboid pseudoproteases in development and disease. Finally, we address mechanistically how versatile features of proteolytically active rhomboids have been elaborated to serve the sophisticated functions of their pseudoprotease cousins. By comparing functional and structural clues, we highlight common principles shared by the rhomboid superfamily, and make mechanistic predictions.
- Species differential regulation of COX2 can be described by an NFκB-dependent logic AND gatePublication . Nguyen, Lan K; Cavadas, Miguel A S; Kholodenko, Boris N; Frank, Till D; Cheong, AlexCyclooxygenase 2 (COX2), a key regulatory enzyme of the prostaglandin/eicosanoid pathway, is an important target for anti-inflammatory therapy. It is highly induced by pro-inflammatory cytokines in a Nuclear factor kappa B (NFκB)-dependent manner. However, the mechanisms determining the amplitude and dynamics of this important pro-inflammatory event are poorly understood. Furthermore, there is significant difference between human and mouse COX2 expression in response to the inflammatory stimulus tumor necrosis factor alpha (TNFα). Here, we report the presence of a molecular logic AND gate composed of two NFκB response elements (NREs) which controls the expression of human COX2 in a switch-like manner. Combining quantitative kinetic modeling and thermostatistical analysis followed by experimental validation in iterative cycles, we show that the human COX2 expression machinery regulated by NFκB displays features of a logic AND gate. We propose that this provides a digital, noise-filtering mechanism for a tighter control of expression in response to TNFα, such that a threshold level of NFκB activation is required before the promoter becomes active and initiates transcription. This NFκB-regulated AND gate is absent in the mouse COX2 promoter, most likely contributing to its differential graded response in promoter activity and protein expression to TNFα. Our data suggest that the NFκB-regulated AND gate acts as a novel mechanism for controlling the expression of human COX2 to TNFα, and its absence in the mouse COX2 provides the foundation for further studies on understanding species-specific differential gene regulation.
- REST is a hypoxia-responsive transcriptional repressorPublication . Cavadas, Miguel A. S.; Mesnieres, Marion; Crifo, Bianca; Manresa, Mario C.; Selfridge, Andrew C.; Keogh, Ciara E.; Fabian, Zsolt; Scholz, Carsten C.; Nolan, Karen A.; Rocha, Liliane M. A.; Tambuwala, Murtaza M.; Brown, Stuart; Wdowicz, Anita; Corbett, Danielle; Murphy, Keith J.; Godson, Catherine; Cummins, Eoin P.; Taylor, Cormac T.; Cheong, AlexCellular exposure to hypoxia results in altered gene expression in a range of physiologic and pathophysiologic states. Discrete cohorts of genes can be either up- or down-regulated in response to hypoxia. While the Hypoxia-Inducible Factor (HIF) is the primary driver of hypoxia-induced adaptive gene expression, less is known about the signalling mechanisms regulating hypoxia-dependent gene repression. Using RNA-seq, we demonstrate that equivalent numbers of genes are induced and repressed in human embryonic kidney (HEK293) cells. We demonstrate that nuclear localization of the Repressor Element 1-Silencing Transcription factor (REST) is induced in hypoxia and that REST is responsible for regulating approximately 20% of the hypoxia-repressed genes. Using chromatin immunoprecipitation assays we demonstrate that REST-dependent gene repression is at least in part mediated by direct binding to the promoters of target genes. Based on these data, we propose that REST is a key mediator of gene repression in hypoxia.
- Rhomboid intramembrane protease RHBDL4 triggers ER-export and non-canonical secretion of membrane-anchored TGFαPublication . Wunderle, Lina; Knopf, Julia D.; Kühnle, Nathalie; Morlé, Aymeric; Hehn, Beate; Adrain, Colin; Strisovsky, Kvido; Freeman, Matthew; Lemberg, Marius K.Rhomboid intramembrane proteases are the enzymes that release active epidermal growth factor receptor (EGFR) ligands in Drosophila and C. elegans, but little is known about their functions in mammals. Here we show that the mammalian rhomboid protease RHBDL4 (also known as Rhbdd1) promotes trafficking of several membrane proteins, including the EGFR ligand TGFα, from the endoplasmic reticulum (ER) to the Golgi apparatus, thereby triggering their secretion by extracellular microvesicles. Our data also demonstrate that RHBDL4-dependent trafficking control is regulated by G-protein coupled receptors, suggesting a role for this rhomboid protease in pathological conditions, including EGFR signaling. We propose that RHBDL4 reorganizes trafficking events within the early secretory pathway in response to GPCR signaling. Our work identifies RHBDL4 as a rheostat that tunes secretion dynamics and abundance of specific membrane protein cargoes.