A Subset of Membrane-Altering Agents and γ-Secretase Modulators Provoke Nonsubstrate Cleavage by Rhomboid Proteases

• Rhomboid enzymes catalyze proteolysis of specific protein targets in the membrane
• Perturbing cell membranes pharmacologically leads rhomboid to cleave nonsubstrates
• Several γ-secretase-modulating drugs also provoke rhomboid cleavage of nonsubstrates
• Membranes guide rhomboid specificity but are vulnerable to off-target drug effects

SummaryRhomboid proteases are integral membrane enzymes that regulate cell signaling, adhesion, and organelle homeostasis pathways, making substrate specificity a key feature of their function. Interestingly, we found that perturbing the membrane pharmacologically in living cells had little effect on substrate processing but induced inappropriate cleavage of nonsubstrates by rhomboid proteases. A subclass of drugs known to modulate γ-secretase activity acted on the membrane directly and induced nonsubstrate cleavage by rhomboid proteases but left true substrate cleavage sites unaltered. These observations highlight an active role for the membrane in guiding rhomboid selectivity and caution that membrane-targeted drugs should be evaluated for cross-activity against membrane-resident enzymes that are otherwise unrelated to the intended drug target. Furthermore, some γ-secretase-modulating activity or toxicity could partly result from global membrane effects.

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