Proteolysis inside the Membrane Is a Rate-Governed Reaction Not Driven by Substrate Affinity

Abstract

SUMMARY Enzymatic cleavage of transmembrane anchors to release proteins from the membrane controls diverse signaling pathways and is implicated in over a dozen diseases. How catalysis works within the viscous, water-excluding, two-dimensional membrane is unknown. We developed an inducible reconstitution system to interrogate rhomboid proteolysis quantitatively within the membrane in real time. Remarkably, rhomboid proteases displayed no physiological affinity for substrates (Kd ~190 μM, or 0.1 mol%). Instead, ~10,000-fold differences in proteolytic efficiency with substrate mutants and diverse rhomboid proteases were reflected in kcat values alone. Analysis of gate-open mutant and solvent isotope effects revealed that substrate gating, not hydrolysis, is rate limiting. Ultimately a single proteolytic event within the membrane normally takes minutes. Rhomboid intramembrane proteolysis is thus a slow, kinetically controlled reaction not driven by transmembrane protein-protein affinity. These properties are unlike those of other studied proteases or membrane proteins but strikingly reminiscent of one subset of DNA-repair enzymes, raising important mechanistic and drug-design implications.

Nicastrin functions to sterically hinder γ-secretase–substrate interactions driven by substrate transmembrane domain

“…The apparently strong interaction between γ-secretase and notch TMD is of particular interest, as it is in stark contrast to the binding mechanism of rhomboid proteases, another family of I-CLiPs, which have no physiologically relevant binding affinity between enzyme and substrate within the lipid bilayer (7). To verify that the apparently high-affinity γ-secretase-substrate interaction that we observed in the detergent-solubilized assay also dominates in a more physiological context, we measured cleavage of notch by γ-secretase within the lipid bilayer of a proteoliposome using the elegant techniques recently established by Dickey et al to study rhomboid intramembrane kinetics (7).…”

“…Rhomboid has no such mechanism, as it is responsible for direct shedding of large ectodomain-containing substrates (8,59,60), without the requirement of prior "activation" of substrate by another protease like γ-secretase substrates are. Thus, rhomboid requires a different mechanism to distinguish substrate from nonsubstrate and has apparently evolved to bind its substrates with very low affinity, only hydrolyzing those substrates whose unstable TMD helices have had time to unfold in the active site and subsequently be cleaved (7). Rhomboid cleavage is therefore dependent on differences in rate rather than binding affinity for distinguishing between substrates, in a mechanism similar to DNA glycosylases (61).…”

“…Recent work on rhomboid proteases has demonstrated that this family of I-CLiPs achieves substrate specificity via a mechanism that is dependent on the transmembrane dynamics of the substrate rather than its sequence of amino acids (6,7). Here, rhomboid possesses a very weak binding affinity for substrate and, in a rate-driven reaction, only cleaves those substrates that have unstable TMD helices that have had time to unfold into the catalytic active site, where they are cleaved before they can dissociate from the enzyme-substrate complex.…”

A Cell‐Based Assay Reveals Nuclear Translocation of Intracellular Domains Released by SPPL Proteases

“…Because in vitro reconstitution of recombinantly expressed SPP/SPPL proteases as it is routinely employed for rhomboid I-CLIPs (17) has not been achieved yet, functional analysis of SPPL2 proteases relies on cell-based systems followed by western blot determination of uncleaved or cleaved forms of substrate proteins. Thus, a sensitive, quantitative and potentially high-throughput-compatible assay for monitoring activity of SPPL2a and other SPPL2 proteases is currently not available.…”

Nicastrin guards Alzheimer’s gate

“…Bolduc et al (3) next beautifully adapted a new technology that allows measuring enzyme kinetics directly inside the cell membrane (12). They ultimately found that just the Notch transmembrane segment alone acted as an efficient substrate for γ-secretase (3).…”