The cytokine tumor necrosis factor (TNF) is the primary trigger of inflammation. Like many extracellular signaling proteins, TNF is synthesized as a transmembrane protein; the active signal is its ectodomain, which is shed from cells after cleavage by an ADAM family metalloprotease, TACE/ADAM17. We report that iRhom2/RHBDF2, a proteolytically inactive member of the rhomboid family, is required for TNF release in mice. iRhom2 binds TACE and promotes exit from the endoplasmic reticulum. The failure of TACE ER exit in the absence of iRhom2 prevents furin-mediated maturation and its trafficking to the cell surface, the site of TNF cleavage. Given the role of TNF in autoimmune and inflammatory diseases, iRhom2 may represent an attractive therapeutic target.Proteolytic release of the extracellular domain of transmembrane proteins is an important mechanism for generating signals that regulate major aspects of animal development, physiology, immunity and pathology (1, 2). An important example of regulated ectodomain shedding is the cytokine TNF, the primary trigger of inflammatory responses. TNF is associated with many human diseases including rheumatoid arthritis, Crohn's disease, atherosclerosis, psoriasis, sepsis, diabetes, and obesity. Its blockade is licensed as a therapy for a number of conditions, and is being assessed for others (3). Soluble, active TNF is shed from the plasma membrane by the ADAM family metalloprotease TACE (TNFα converting enzyme; also known as ADAM17) (4). Despite the medical importance of TNF and other transmembrane signaling proteins, the regulation of ectodomain shedding remains poorly understood. Both the transmembrane forms of the signaling proteins themselves, and the shedding proteases, are subject to control by posttranslational modification, interaction with specific partners, and regulated intracellular trafficking and compartmentalization (5-9). The relative physiological importance, however, of these different modes of regulation is unclear.We have investigated the regulation of ectodomain shedding by genetic and cellular approaches, both in Drosophila and mammalian cells. This has led to the recent discovery of a new class of polytopic endoplasmic reticulum (ER) proteins, the iRhoms, which are noncatalytic relatives of rhomboid intramembrane proteases (Fig. 1A) (10). Drosophila iRhom regulates epidermal growth factor (EGF) receptor signaling by interacting with EGF family ligands in the ER, shunting them into the ER-associated degradation (ERAD) pathway (11). iRhoms are conserved in all metazoans, and in cell culture assays their mammalian Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts counterparts, iRhom1 and iRhom2, can also promote ERAD of EGF. In mammals, however, their physiological role is unknown. We therefore generated a null mutation in the gene that encodes iRhom2/RHBDF2 in mice (Fig. S1A). iRhom2 −/− mice appeared normal: they were viable and fertile, with no morphological defects. Unlike iRhom1, which is widely expressed, iRhom2 is...

Vaccinia virus, a close relative of the causative agent of smallpox, exploits actin polymerization to enhance its cell-to-cell spread. We show that actin-based motility of vaccinia is initiated only at the plasma membrane and remains associated with it. There must therefore be another form of cytoplasmic viral transport, from the cell centre, where the virus replicates, to the periphery. Video analysis reveals that GFP-labelled intracellular enveloped virus particles (IEVs) move from their perinuclear site of assembly to the plasma membrane on microtubules. We show that the viral membrane protein A36R, which is essential for actin-based motility of vaccinia, is also involved in microtubule-mediated movement of IEVs. We further show that conventional kinesin is recruited to IEVs via the light chain TPR repeats and is required for microtubule-based motility of the virus. Vaccinia thus sequentially exploits the microtubule and actin cytoskeletons to enhance its cell-to-cell spread.

SummaryIntramembrane proteolysis governs many cellular control processes, but little is known about how intramembrane proteases are regulated. iRhoms are a conserved subfamily of proteins related to rhomboid intramembrane serine proteases that lack key catalytic residues. We have used a combination of genetics and cell biology to determine that these “pseudoproteases” inhibit rhomboid-dependent signaling by the epidermal growth factor receptor pathway in Drosophila, thereby regulating sleep. iRhoms prevent the cleavage of potential rhomboid substrates by promoting their destabilization by endoplasmic reticulum (ER)-associated degradation; this mechanism has been conserved in mammalian cells. The exploitation of the intrinsic quality control machinery of the ER represents a new mode of regulation of intercellular signaling. Inactive cognates of enzymes are common, but their functions are mostly unclear; our data indicate that pseudoenzymes can readily evolve into regulatory proteins, suggesting that this may be a significant evolutionary mechanism.

The function of the human Tes protein, which has extensive similarity to zyxin in both sequence and domain organization, is currently unknown. We now show that Tes is a component of focal adhesions that, when expressed, negatively regulates proliferation of T47D breast carcinoma cells. Coimmunoprecipitations demonstrate that in vivo Tes is complexed with actin, Mena, and vasodilator-stimulated phosphoprotein (VASP). Interestingly, the isolated NH2-terminal half of Tes pulls out α-actinin and paxillin from cell extracts in addition to actin. The COOH-terminal half recruits zyxin as well as Mena and VASP from cell extracts. These differences suggest that the ability of Tes to associate with α-actinin, paxillin, and zyxin is dependent on the conformational state of the molecule. Consistent with this hypothesis, we demonstrate that the two halves of Tes interact with each other in vitro and in vivo. Using fibroblasts lacking Mena and VASP, we show that these proteins are not required to recruit Tes to focal adhesions. However, using RNAi ablation, we demonstrate that zyxin is required to recruit Tes, as well as Mena and VASP, but not vinculin or paxillin, to focal adhesions.