Cooperation of N- and C-terminal substrate transmembrane domain segments in intramembrane proteolysis by γ-secretase

Abstract

Intramembrane proteases play a pivotal role in biology and medicine, but how these proteases decode cleavability of a substrate transmembrane (TM) domain remains unclear. Here, we study the role of conformational flexibility of a TM domain, as determined by deuterium/hydrogen exchange, on substrate cleavability by γ-secretase in vitro and in cellulo. By comparing hybrid TMDs based on the natural amyloid precursor protein TM domain and an artificial poly-Leu non-substrate, we find that substrate cleavage requires conformational flexibility within the N-terminal half of the TMD helix (TM-N). Robust cleavability also requires the C-terminal TM sequence (TM-C) containing substrate cleavage sites. Since flexibility of TM-C does not correlate with cleavage efficiency, the role of the TM-C may be defined mainly by its ability to form a cleavage-competent state near the active site, together with parts of presenilin, the enzymatic component of γ-secretase. In sum, cleavability of a γ-secretase substrate appears to depend on cooperating TM domain segments, which deepens our mechanistic understanding of intramembrane proteolysis.

Structure and function of SPP/SPPL proteases: insights from biochemical evidence and predictive modeling

“…The authors conclude that flexibility in N-terminal part as well as in the hinge region promotes efficient cleavage. Flexibility in the C-terminal part of the substrate was suggested to allow for the formation of a cleavage-competent state near the active site [119]. These observations are in our opinion in line with the unwinding model [72,134] and nicely correlate with the data on TNFa and SPPL2a [101].…”

“…For SPPL3, it was suggested that an M or Y in position P1 might be favorable [33]; however, a recent N‐terminomics study on the enzyme did not detect a consensus sequence [118]. Yet, the authors found that when substrate and SPPL3 are located in the same compartment, the exchange of the TM domain can turn a nonsubstrate to a substrate [119]. They conclude that despite the lack of a clear consensus sequence structural properties of the TM domain determine cleavability.…”

“…They conclude that despite the lack of a clear consensus sequence structural properties of the TM domain determine cleavability. For γ‐secretase, it has also recently been shown that an artificial nonsubstrate poly‐leucine TM domain was turned into a substrate by reintroducing a few selected residues from the APP TM domain [119]. The lack of a consensus sequence is a fundamental difference to most soluble proteases and holds true for many other intramembrane proteases, including the members of the presenilin family [120].…”

Cleavage efficiency of the intramembrane protease γ‐secretase is reduced by the palmitoylation of a substrate's transmembrane domain

“…As negative controls we included the TMDs of the nonsubstrate ITGB1 and a synthetic TMD consisting of 21 leucines (poly leucine 21, pL21, same number of amino acids as in the FN14 TMD), which has a strongly reduced conformational flexibility and prevents γsecretase cleavage, as shown for the substrate C99. 13 Full-length proteins in the lysate and the secreted peptides in the conditioned medium were detected on the same membrane by immunoblot using an anti-HA antibody. Immunoblot intensity of the secreted peptides were quantified and normalized to the intensity of the corresponding full-length protein to control for potential differences in transfection efficiency or protein expression.…”

“…11 γ-Secretase does not cleave with a pronounced sequence specificity, but may rather require structural flexibility within a substrate's TMD. [12][13][14] Additionally, membrane proteins require a short extracellular domain (ectodomain) to be recognized and cleaved by γsecretase. [15][16][17] This prevents most known γsecretase substrates from being constitutively cleaved, because they have long ectodomains.…”