Intramembrane proteases catalyze the signal-generating step of various cell signaling pathways and continue to be implicated in diseases ranging from malaria illness to Parkinsonian neurodegeneration1-3. deleting the amino-terminal EF-hands triggered proteolysis prematurely while residues in cytoplasmic loops linking distal transmembrane segments mediated calcium activation. Rhomboid regulation was not orchestrated by either dimerization or substrate relationships. Instead calcium improved catalytic rate by advertising substrate gating. Substrates with cleavage sites outside the membrane could be cleaved but lost the capacity to be controlled. These observations show substrate gating is not an essential step in catalysis but instead evolved like NNC 55-0396 NNC 55-0396 a mechanism for regulating proteolysis inside the membrane. Moreover these insights provide new methods for studying rhomboid functions by investigating upstream inputs that result in proteolysis. Cell membranes are both NNC 55-0396 controlled borders with the outside world as well as dynamic platforms for organizing cell signaling metabolic pathways and ultrastructure assembly. All of these important events rely on enzymes that reside directly within the cell membrane yet achieving a mechanistic understanding NNC 55-0396 of how these specialized enzymes function within this environment offers proven demanding. Intramembrane proteases catalyze the committed signal-generating step of several important signaling pathways by cleaving transmembrane proteins within the membrane1-3. Their importance is definitely underscored by repeated implication in disease. γ-secretase generates the amyloid-β peptide in Alzheimer’s disease4 5 but more recently has been successfully targeted inside a spectrum of cancers6 because its activating cleavage of the Notch receptor causes signaling2. Site-2 protease family metalloenzymes liberate NNC 55-0396 transcription factors from your membrane to control cholesterol and fatty acid composition of membranes1 and signaling circuits that control virulence in pathogenic bacteria7. Rhomboid serine proteases are a family of expert regulators that initiate epidermal growth HOX1 element (EGF) signaling during development3 8 but more recently have been implicated in cleaving adhesins during malaria invasion9 and regulating mitochondrial quality control to guard against Parkinson’s disease10. Since peptide relationship cleavage is definitely irreversible in the cell exact rules of protease activity is definitely paramount. Yet it’s generally thought that intramembrane proteases are constitutively active enzymes over which the cell cannot exert direct regulation11. Instead two mechanisms NNC 55-0396 control activity. The first is transcriptional as exemplified by rhomboid-1: the constitutively active protease is made only when and where needed3. This mechanism has historically served as a beautiful atlas of EGF transmission initiation during development. The second mechanism is definitely centered on controlling access to substrate by segregating it from protease11. Malaria for example sequesters adhesins in secretory organelles before invasion while their secretion onto the surface leads to the 1st encounter with an active rhomboid protease7. The key property missing from these two mechanisms is the ability to respond rapidly to changing conditions: transcriptional and cell localization changes are ill-adapted to provide immediate reactions that are hallmarks of cell signaling. Moreover it’s essentially unprecedented for proteases to be devoid of direct enzymatic rules in the cell raising the possibility that this apparent discrepancy displays our lack of understanding rather than absence of a regulatory mechanism. Although rhomboid protease GlpG offers served like a tractable model for studying the structure-function of intramembrane proteolysis12 no info is definitely available on its cellular role. This knowledge space prohibits deciphering regulatory mechanisms. Instead as a new approach to this query we searched for rhomboid proteins that contain additional domains with precedent for regulating protein activity and focused on a conserved subset of over two dozen animal rhomboid enzymes with EF-hand domains appended to their cytosolic N-termini (Fig. 1a and Extended Data Fig. 1). EF-hands are helix-loop-helix motifs in which calcium binding in the loop serves either a structural or regulatory part. In the second option calcium binding.