Rhomboid intramembrane protease RHBDL4 triggers ER-export and non-canonical secretion of membrane-anchored TGFα

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.Baden-Württemberg Stiftung; Network of Aging Research (NAR, University of Heidelberg); Deutsche Forschungsgemeinschaft grant: (SFB 1036, TP 12); EMBO Installation Grant number: (2329); Ministry of Education, Youth and Sports of the Czech Republic projects: (LK11206, LO1302); Medical Research Council programme number: (U105178780); Boehringer-Ingelheim

The stromal cell-surface protease fibroblast activation protein-α localizes to lipid rafts and is recruited to invadopodia

AbstractFibroblast activation protein alpha (FAPα) is a cell surface protease expressed by cancer-associated fibroblasts in the microenvironment of most solid tumors. As there is increasing evidence for proteases having non-catalytic functions, we determined the FAPα interactome in cancer-associated fibroblasts using the quantitative immunoprecipitation combined with knockdown (QUICK) method. Complex formation with adenosin deaminase, erlin-2, stomatin, prohibitin, Thy-1 membrane glycoprotein, and caveolin-1 was further validated by immunoblotting. Co-immunoprecipitation (co-IP) of the known stoichiometric FAPα binding partner dipeptidyl-peptidase IV (DPPIV) corroborated the proteomic strategy. Reverse co-IPs validated the FAPα interaction with caveolin-1, erlin-2, and stomatin while co-IP upon RNA-interference mediated knock-down of DPPIV excluded adenosin deaminase as a direct FAPα interaction partner. Many newly identified FAPα interaction partners localize to lipid rafts, including caveolin-1, a widely-used marker for lipid raft localization. We hypothesized that this indicates a recruitment of FAPα to lipid raft structures. In density gradient centrifugation, FAPα co-fractionates with caveolin-1. Immunofluorescence optical sectioning microscopy of FAPα and lipid raft markers further corroborates recruitment of FAPα to lipid rafts and invadopodia. FAPα is therefore an integral component of stromal lipid rafts in solid tumors. In essence, we provide one of the first interactome analyses of a cell surface protease and translate these results into novel biological aspects of a marker protein for cancer-associated fibroblasts

Rhomboid protease RHBDL4 promotes retrotranslocation of aggregation-prone proteins for degradation

Protein degradation is fundamentally important to ensure cell homeostasis. In the endoplasmic reticulum (ER), the ER-associated degradation (ERAD) pathway targets incorrectly folded and unassembled proteins for turnover by the cytoplasmic proteasome. Previously, we showed that the rhomboid protease RHBDL4, together with p97, mediates membrane protein degradation. However, whether RHBDL4 acts in concert with additional ERAD components is unclear, and its full substrate spectrum remains to be defined. Here, we show that, in addition to membrane proteins, RHBDL4 cleaves aggregation-prone luminal ERAD substrates. Since mutations of the RHBDL4 rhomboid domain led to stabilization of substrates at the cytoplasmic side, we hypothesize that, analogous to the homolog ERAD factor derlin, RHBDL4 is directly involved in substrate retrotranslocation. RHBDL4's interaction with the erlin ERAD complex and reciprocal interaction of rhomboid substrates with erlins suggest that RHBDL4 and erlins form a complex that clips substrates and thereby rescues aggregation-prone peptides in the ER from aggregation

Intramembrane protease RHBDL4 cleaves oligosaccharyltransferase subunits to target them for ER-associated degradation

The Endoplasmic Reticulum (ER)-resident intramembrane rhomboid protease RHBDL4 generates metastable protein fragments and together with the ER-associated degradation (ERAD) machinery provides a clearance mechanism for aberrant and surplus proteins. However, the endogenous substrate spectrum and with that the role of RHBDL4 in physiological ERAD is mainly unknown. Here, we use a substrate trapping approach in combination with quantitative proteomics to identify physiological RHBDL4 substrates. This revealed oligosacharyltransferase (OST) complex subunits such as the catalytic active subunit STT3A as substrates for the RHBDL4-dependent ERAD pathway. RHBDL4-catalyzed cleavage inactivates OST subunits by triggering dislocation into the cytoplasm and subsequent proteasomal degradation. Thereby, RHBDL4 controls the abundance and activity of OST, suggesting a novel link between the ERAD machinery and glycosylation tuning

RHBDL4-triggered downregulation of COPII adaptor protein TMED7 suppresses TLR4-mediated inflammatory signaling

Abstract The toll-like receptor 4 (TLR4) is a central regulator of innate immunity that primarily recognizes bacterial lipopolysaccharide cell wall constituents to trigger cytokine secretion. We identify the intramembrane protease RHBDL4 as a negative regulator of TLR4 signaling. We show that RHBDL4 triggers degradation of TLR4’s trafficking factor TMED7. This counteracts TLR4 transport to the cell surface. Notably, TLR4 activation mediates transcriptional upregulation of RHBDL4 thereby inducing a negative feedback loop to reduce TLR4 trafficking to the plasma membrane. This secretory cargo tuning mechanism prevents the over-activation of TLR4-dependent signaling in an in vitro Mycobacterium tuberculosis macrophage infection model and consequently alleviates septic shock in a mouse model. A hypomorphic RHBDL4 mutation linked to Kawasaki syndrome, an ill-defined inflammatory disorder in children, further supports the pathophysiological relevance of our findings. In this work, we identify an RHBDL4-mediated axis that acts as a rheostat to prevent over-activation of the TLR4 pathway

RHBDL4-triggered downregulation of COPII adaptor protein TMED7 suppresses TLR4-mediated inflammatory signaling

The toll-like receptor 4 (TLR4) is a central regulator of innate immunity that primarily recognizes bacterial lipopolysaccharide cell wall constituents to trigger cytokine secretion. We identify the intramembrane protease RHBDL4 as a negative regulator of TLR4 signaling. We show that RHBDL4 triggers degradation of TLR4's trafficking factor TMED7. This counteracts TLR4 transport to the cell surface. Notably, TLR4 activation mediates transcriptional upregulation of RHBDL4 thereby inducing a negative feedback loop to reduce TLR4 trafficking to the plasma membrane. This secretory cargo tuning mechanism prevents the over-activation of TLR4-dependent signaling in an in vitro Mycobacterium tuberculosis macrophage infection model and consequently alleviates septic shock in a mouse model. A hypomorphic RHBDL4 mutation linked to Kawasaki syndrome, an ill-defined inflammatory disorder in children, further supports the pathophysiological relevance of our findings. In this work, we identify an RHBDL4-mediated axis that acts as a rheostat to prevent over-activation of the TLR4 pathway

Skin Barrier Defects Caused by Keratinocyte-Specific Deletion of ADAM17 or EGFR Are Based on Highly Similar Proteome and Degradome Alterations

Keratinocyte-specific deletion of ADAM17 in mice impairs terminal differentiation of keratinocytes leading to severe epidermal barrier defects. Mice deficient for ADAM17 in keratinocytes phenocopy mice with a keratinocyte-specific deletion of epidermal growth factor receptor (EGFR), which highlights the role of ADAM17 as a “ligand sheddase” of EGFR ligands. In this study, we aim for the first proteomic/degradomic approach to characterize the disruption of the ADAM17–EGFR signaling axis and its consequences for epidermal barrier formation. Proteomic profiling of the epidermal proteome of mice deficient for either ADAM17 or EGFR in keratinocytes at postnatal days 3 and 10 revealed highly similar protein alterations for ADAM17 and EGFR deficiency. These include massive proteome alterations of structural and regulatory components important for barrier formation such as transglutaminases, involucrin, filaggrin, and filaggrin-2. Cleavage site analysis using terminal amine isotopic labeling of substrates revealed increased proteolytic processing of S100 fused-type proteins including filaggrin-2. Alterations in proteolytic processing are supported by altered abundance of numerous proteases upon keratinocyte-specific <i>Adam17</i> or <i>Egfr</i> deletion, among them kallikreins, cathepsins, and their inhibitors. This study highlights the essential role of proteolytic processing for maintenance of a functional epidermal barrier. Furthermore, it suggests that most defects in formation of the postnatal epidermal barrier upon keratinocyte-specific ADAM17 deletion are mediated via EGFR

CEO Risk-Related Incentives and Income Smoothing

We investigate whether risk-related incentives of executive stock option (ESO) compensation plans are associated with income smoothing. Given that risk has both potential benefits and costs, including possible losses and/or large fluctuations that affect reported financial outcomes, flexibilities in financial reporting enable a manager to make apparent risk lower while masking the underlying real risk. As such, income smoothing can be a means by which a manager can reduce the unintended consequences of risk taking without at the same time reducing its intended consequences. Using a sample of approximately 7,000 firm-years, we find that risk-taking incentives and income smoothing are positively related. Our results are robust to alternate specifications of income smoothing and risk-taking, and to various firm-level characteristics, including governance structures, CEO share and option holdings. Additionally, we find that our results are especially pronounced in firms whose risk and risk-taking behavior are high