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

Abstract

SUMMARY Rhomboid 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 non-substrates by rhomboid proteases. A subclass of drugs known to modulate γ-secretase activity acted on the membrane directly and induced non-substrate 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. Further, some γ-secretase-modulating activity or toxicity could partly result from global membrane effects.

Why cells need intramembrane proteases – a mechanistic perspective

“…not only around the cleavage site as hypothesized initially) might be one of the determining features of IMPR substrates . This is consistent with the observation that manipulating membrane properties pharmacologically, which can possibly impact on the conformational dynamics of single‐TMH proteins, can convert non‐substrates into substrates . The actual character of the conformational features of substrate TMHs that different IMPRs recognize remains unknown, and structural analyses of full transmembrane substrates in the presence and absence of their cognate proteases are required to bridge this knowledge gap.…”

Sprouting angiogenesis is regulated by shedding of the C‐type lectin family 14, member A (CLEC14A) ectodomain, catalyzed by rhomboid‐like 2 protein (RHBDL2)

“…Recent data has suggested that rhomboid proteins lack the need for defined cleavage recognition motifs (35, 36); however, Strisovsky et al (28) demonstrated strong evidence for motif‐directed proteolysis, and we have been able to reproduce this sequence specificity here (Fig. 3).…”

“…2 F ). However, it is possible that the intracellular domains have a conformational or stabilizing role for transmembrane helices, which influences efficient proteolysis of a loose cleavage motif for the rhomboid protease (36).…”

Crystal Structures and Inhibition Kinetics Reveal a Two-Stage Catalytic Mechanism with Drug Design Implications for Rhomboid Proteolysis

“…An important advance is our ability to study rhomboid catalysis with reversible inhibitors, and entirely inside the membrane (both kinetically and crystallographically), which is known to impact the catalytic properties of these membrane-immersed enzymes (Bondar et al, 2009; Moin and Urban, 2012; Urban and Moin, 2014; Vinothkumar, 2011). Ultimately we identified an additional, early step in the kinetic scheme for rhomboid proteolysis, visualized several key features of rhomboid catalysis at atomic resolution, and, unexpectedly, discovered a promising alternative avenue for targeting these enzymes.…”

“…Therefore, to gain a better understanding of the mechanism underlying intramembrane catalysis, we sought to adapt this approach to characterize the inhibition kinetics of substrate-mimicking peptide aldehydes. Next, since mounting evidence indicates that cell membranes affect the properties of rhomboid proteases beyond just serving as their environment (Bondar et al, 2009; Moin and Urban, 2012; Urban and Moin, 2014; Vinothkumar, 2011), we developed conditions to crystallize a catalytically active rhomboid protease in a membrane. Soaking rhomboid crystallized from bicelles with the most potent substrate peptide aldehydes produced high-resolution structures that revealed the characteristics of substrate stabilization during catalysis by a membrane-immersed rhomboid protease.…”