Conformational surveillance of Orai1 by a rhomboid intramembrane protease prevents inappropriate CRAC channel activation

Calcium influx through plasma membrane calcium release-activated calcium (CRAC) channels, which are formed of hexamers of Orai1, is a potent trigger for many important biological processes, most notably in T cell-mediated immunity. Through a bioinformatics-led cell biological screen, we have identified Orai1 as a substrate for the rhomboid intramembrane protease RHBDL2. We show that RHBDL2 prevents stochastic calcium signaling in unstimulated cells through conformational surveillance and cleavage of inappropriately activated Orai1. A conserved disease-linked proline residue is responsible for RHBDL2’s recognizing the active conformation of Orai1, which is required to sharpen switch-like signaling triggered by store-operated calcium entry. Loss of RHBDL2 control of CRAC channel activity causes severe dysregulation of downstream CRAC channel effectors, including transcription factor activation, inflammatory cytokine expression, and T cell activation. We propose that this surveillance function may represent an ancient activity of rhomboid proteases in degrading unwanted signaling proteins

Sensitive versatile fluorogenic transmembrane peptide substrates for rhomboid intramembrane proteases

Rhomboid proteases are increasingly being explored as potential drug targets, but their potent and specific inhibitors are not available, and strategies for inhibitor development are hampered by the lack of widely usable and easily modifiable in vitro activity assays. Here we address this bottleneck and report on the development of new fluorogenic transmembrane peptide substrates, which are cleaved by several unrelated rhomboid proteases, can be used both in detergent micelles and in liposomes, and contain red-shifted fluorophores that are suitable for high-throughput screening of compound libraries. We show that nearly the entire transmembrane domain of the substrate is important for efficient cleavage, implying that it extensively interacts with the enzyme. Importantly, we demonstrate that in the detergent micelle system, commonly used for the enzymatic analyses of intramembrane proteolysis, the cleavage rate strongly depends on detergent concentration, because the reaction proceeds only in the micelles. Furthermore, we show that the catalytic efficiency and selectivity toward a rhomboid substrate can be dramatically improved by targeted modification of the sequence of its P5 to P1 region. The fluorogenic substrates that we describe and their sequence variants should find wide use in the detection of activity and development of inhibitors of rhomboid proteases. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.15-01948S, GA ČR, Grantová Agentura České Republiky; 2329, EMBO, European Molecular Biology Organization; MOE, Ministry of Education; 232313, Univerzita Karlova v Praze; P208-12-G016, GA ČR, Grantová Agentura České RepublikyEMBO Installation Grant [2329]; Ministry of Education, Youth and Sports of the Czech Republic Projects [LK11206, LO1302]; National Subvention for Development of Research Organizations [RVO: 61388963]; Marie Curie Career Integration Grant [304154

Semaphorin 4B is an ADAM17-cleaved adipokine that inhibits adipocyte differentiation and thermogenesis

Objective: The metalloprotease ADAM17 (also called TACE) plays fundamental roles in homeostasis by shedding key signaling molecules from the cell surface. Although its importance for the immune system and epithelial tissues is well-documented, little is known about the role of ADAM17 in metabolic homeostasis. The purpose of this study was to determine the impact of ADAM17 expression, specifically in adipose tissues, on metabolic homeostasis. Methods: We used histopathology, molecular, proteomic, transcriptomic, in vivo integrative physiological and ex vivo biochemical approaches to determine the impact of adipose tissue-specific deletion of ADAM17 upon adipocyte and whole organism metabolic physiology. Results: ADAM17adipoq-creΔ/Δ mice exhibited a hypermetabolic phenotype characterized by elevated energy consumption and increased levels of adipocyte thermogenic gene expression. On a high fat diet, these mice were more thermogenic, while exhibiting elevated expression levels of genes associated with lipid oxidation and lipolysis. This hypermetabolic phenotype protected mutant mice from obesogenic challenge, limiting weight gain, hepatosteatosis and insulin resistance. Activation of beta-adrenoceptors by the neurotransmitter norepinephrine, a key regulator of adipocyte physiology, triggered the shedding of ADAM17 substrates, and regulated ADAM17 expression at the mRNA and protein levels, hence identifying a functional connection between thermogenic licensing and the regulation of ADAM17. Proteomic studies identified Semaphorin 4B (SEMA4B), as a novel ADAM17-shed adipokine, whose expression is regulated by physiological thermogenic cues, that acts to inhibit adipocyte differentiation and dampen thermogenic responses in adipocytes. Transcriptomic data showed that cleaved SEMA4B acts in an autocrine manner in brown adipocytes to repress the expression of genes involved in adipogenesis, thermogenesis, and lipid uptake, storage and catabolism. Conclusions: Our findings identify a novel ADAM17-dependent axis, regulated by beta-adrenoceptors and mediated by the ADAM17-cleaved form of SEMA4B, that modulates energy balance in adipocytes by inhibiting adipocyte differentiation, thermogenesis and lipid catabolism

iRhom2/ADAM17 deletion protects from obesity associated to increased adipocyte thermogenesis and differentiation and reduced adipocyte Semaphorin 4B shedding

Objectives: The metalloprotease ADAM17 (also called TACE) plays fundamental roles during development, and to promote homeostasis and multiple inflammatory diseases and cancer by shedding key signaling molecules from the cell surface. Its importance in inflammation and growth control is well documented, while little is known about the role of ADAM17 and its regulator iRhom2 in metabolic homeostasis. The purpose of this study was to determine the impact of the sheddase ADAM17/TACE and of its modulator iRhom2 in the pathophysiology of obesity and in adipocyte homeostasis. Materials and Methods: We challenged controls versus iRhom2 global KO or adipocyte specific ADAM17 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 and beige adipocytes to establish the autonomy of the effect of loss of iRhom2 and ADAM17 on differentiation, thermogenesis and respiration. In addition, we used proteomic and transcriptomic analyses to identify and characterize a novel ADAM17 substrate involved. Results: iRhom2 global KO and adipocyte-specific ADAM17 KO mice exhibited a hypermetabolic phenotype characterized by elevated energy consumption and increased levels of adipocyte thermogenic gene expression and browning. This protected mutant mice from obesogenic challenge, limiting weight gain, hepatosteatosis and insulin resistance. Proteomic studies identified Semaphorin 4B (SEMA4B), as a novel ADAM17-shed adipokine, whose expression is regulated by physiological thermogenic cues, that acts to inhibit adipocyte differentiation and dampen thermogenic responses in adipocytes. Conclusion: Our findings identify a novel iRhom2/ADAM17-dependent axis, regulated by beta-adrenoceptors and mediated by the ADAM17-cleaved form of SEMA4B, that modulates energy balance in adipocytes by inhibiting adipocyte differentiation, thermogenesis and lipid catabolism. Keywords: iRhom2, ADAM17/TACE; Semaphorin4B; Thermogenesis; Obesity