Inhibitory Proteins Block Substrate Access to the Active Site of Bacillus subtilis Intramembrane Metalloprotease SpoIVFB

Intramembrane proteases of diverse signaling pathways use membrane-embedded active sites to cleave membrane-associated substrates. Interactions of intramembrane metalloproteases with modulators are poorly understood. Inhibition of Bacillus subtilis intramembrane metalloprotease SpoIVFB requires BofA and SpoIVFA, which transiently prevent cleavage of Pro-σK during endosporulation. Three conserved BofA residues (N48, N61, T64) in or near predicted transmembrane segment (TMS) 2 were found to be required for SpoIVFB inhibition. Disulfide cross-linking indicated that BofA TMS2 occupies the SpoIVFB active site region. BofA and SpoIVFA neither prevented SpoIVFB from interacting with Pro-σK in co-purification assays nor interfered with cross-linking between the C-terminal regions of Pro-σK and SpoIVFB. However, BofA and SpoIVFA did interfere with cross-linking between the N-terminal Proregion of Pro-σK and the SpoIVFB active site region and interdomain linker. A BofA variant lacking predicted TMS1, in combination with SpoIVFA, was less effective at interfering with some of the cross-links and slightly less effective at inhibiting cleavage of Pro-σK by SpoIVFB. A structural model was built of SpoIVFB in complex with BofA and parts of SpoIVFA and Pro-σK, using partial homology and constraints from cross-linking and co-evolutionary analyses. The model predicts that N48 in BofA TMS2 interacts with T64 (and possibly N61) of BofA to stabilize a membrane-embedded C-terminal region. SpoIVFA is predicted to bridge the BofA C-terminal region and SpoIVFB. Thus, the two inhibitory proteins block access of the Pro-σK N-terminal region to the SpoIVFB active site region. Our findings may inform efforts to develop selective inhibitors of intramembrane metalloproteases.

Escherichia coli S2P family intramembrane protease RseP is engaged in the regulated sequential cleavages of FecR in the ferric citrate signaling

Escherichia coli RseP, a member of the S2P family of intramembrane proteases, is involved in the activation of the σE extracytoplasmic stress response and elimination of remnant signal peptides. However, whether RseP has additional cellular functions is unclear. In this study, we attempted to identify new RseP substrates to explore still unknown physiological roles of this protease. Our mass spectrometry-based quantitative proteomic analysis revealed that the levels of several Fec system proteins encoded by the fecABCDE operon (fec operon) were significantly decreased in an RseP-deficient strain. The Fec system is responsible for the uptake of ferric citrate, and the transcription of the fec operon is controlled by FecI, an alternative sigma factor, and its regulator FecR, a single-pass transmembrane protein. Assays with the fec operon expression reporter demonstrated that the proteolytic activity of RseP is essential for the ferric citrate-dependent upregulation of the fec operon. Analysis using the FecR protein and FecR-derived model proteins showed that FecR undergoes sequential processing at the membrane and that RseP participates in the last step of this sequential processing to generate the N-terminal cytoplasmic fragment of FecR that participates in the transcription of the fec operon with FecI. Ferric citrate signal-dependent generation of this cleavage product is the essential and sufficient role of RseP in the transcriptional activation of the fec operon. Our study unveiled that E. coli RseP performs the intramembrane proteolysis of FecR, a novel physiological role that is essential for regulating iron uptake by the ferric citrate transport system.

A combination of solid-state NMR and MD simulations reveals the binding mode of a rhomboid protease inhibitor

Intramembrane proteolysis plays a fundamental role in many biological and pathological processes. Intramembrane proteases thus represent promising pharmacological targets, but few selective inhibitors have been identified. This is in contrast...

iRhom2: An Emerging Adaptor Regulating Immunity and Disease

The rhomboid family are evolutionary conserved intramembrane proteases. Their inactive members, iRhom in Drosophila melanogaster and iRhom1 and iRhom2 in mammals, lack the catalytic center and are hence labelled “inactive” rhomboid family members. In mammals, both iRhoms are involved in maturation and trafficking of the ubiquitous transmembrane protease a disintegrin and metalloprotease (ADAM) 17, which through cleaving many biologically active molecules has a critical role in tumor necrosis factor alpha (TNFα), epidermal growth factor receptor (EGFR), interleukin-6 (IL-6) and Notch signaling. Accordingly, with iRhom2 having a profound influence on ADAM17 activation and substrate specificity it regulates these signaling pathways. Moreover, iRhom2 has a role in the innate immune response to both RNA and DNA viruses and in regulation of keratin subtype expression in wound healing and cancer. Here we review the role of iRhom2 in immunity and disease, both dependent and independent of its regulation of ADAM17.

The molecular, cellular and pathophysiological roles of iRhom pseudoproteases

iRhom proteins are catalytically inactive relatives of rhomboid intramembrane proteases. There is a rapidly growing body of evidence that these pseudoenzymes have a central function in regulating inflammatory and growth factor signalling and consequent roles in many diseases. iRhom pseudoproteases have evolved new domains from their proteolytic ancestors, which are integral to their modular regulation and functions. Although we cannot yet conclude the full extent of their molecular and cellular mechanisms, there is a clearly emerging theme that they regulate the stability and trafficking of other membrane proteins. In the best understood case, iRhoms act as regulatory cofactors of the ADAM17 protease, controlling its function of shedding cytokines and growth factors. It seems likely that as the involvement of iRhoms in human diseases is increasingly recognized, they will become the focus of pharmaceutical interest, and here we discuss what is known about their molecular mechanisms and relevance in known pathologies.

Atherogenic LOX-1 signaling is controlled by SPPL2-mediated intramembrane proteolysis

The lectin-like oxidized LDL receptor 1 (LOX-1) is a key player in the development of atherosclerosis. LOX-1 promotes endothelial activation and dysfunction by mediating uptake of oxidized LDL and inducing pro-atherogenic signaling. However, little is known about modulators of LOX-1–mediated responses. Here, we show that the function of LOX-1 is controlled proteolytically. Ectodomain shedding by the metalloprotease ADAM10 and lysosomal degradation generate membrane-bound N-terminal fragments (NTFs), which we identified as novel substrates of the intramembrane proteases signal peptide peptidase–like 2a and b (SPPL2a/b). SPPL2a/b control cellular LOX-1 NTF levels which, following self-association via their transmembrane domain, can activate MAP kinases in a ligand-independent manner. This leads to an up-regulation of several pro-atherogenic and pro-fibrotic targets including ICAM-1 and the connective tissue growth factor CTGF. Consequently, SPPL2a/b-deficient mice, which accumulate LOX-1 NTFs, develop larger and more advanced atherosclerotic plaques than controls. This identifies intramembrane proteolysis by SPPL2a/b as a novel atheroprotective mechanism via negative regulation of LOX-1 signaling.