Review
Understanding intramembrane proteolysis: from protein dynamics to reaction kinetics

https://doi.org/10.1016/j.tibs.2015.04.001Get rights and content

Highlights

  • How intramembrane proteases select their substrates is currently unknown.

  • Helix-destabilizing amino acids within transmembrane helices often facilitate cleavage.

  • Transmembrane helix stability does not distinguish substrates from non-substrates.

  • Mutations affecting cleavage influence transmembrane helix bending.

Intramembrane proteolysis – cleavage of proteins within the plane of a membrane – is a widespread phenomenon that can contribute to the functional activation of substrates and is involved in several diseases. Although different families of intramembrane proteases have been discovered and characterized, we currently do not know how these enzymes discriminate between substrates and non-substrates, how site-specific cleavage is achieved, or which factors determine the rate of proteolysis. Focusing on γ-secretase and rhomboid proteases, we argue that answers to these questions may emerge from connecting experimental readouts, such as reaction kinetics and the determination of cleavage sites, to the structures and the conformational dynamics of substrates and enzymes.

Section snippets

Intramembrane proteases are relevant for many biological processes

Intramembrane proteolysis has been first observed in the 1990s. The past two decades have seen a wealth of studies that identified major families of substrates and intramembrane proteases (IMPs, see Glossary) in all kingdoms of life, as well as uncovering the nature and function of some cleavage products (reviewed in 1, 2, 3, 4). Most substrates contain a single transmembrane domain (TMD) harboring the scissile peptide bond. Recently, trimming of lipidated peripheral membrane proteins and

Intramembrane proteases come in different structural families

IMPs are distinguished by their transmembrane topologies and by the nature of their membrane-embedded active site residues (Figure 1). Aspartate IMPs contain two catalytic aspartate residues and comprise presenilins, signal peptide peptidase (SPP), and SPP-like (SPPL) proteases 1, 3. Because the C-terminal aspartate is part of a Gly-X-Gly-Asp active site motif, these proteases are also known as GXGD-type proteases [17]. The aspartate IMP presenilin, the catalytic subunit of γ-secretase, has an N

The mechanism of substrate versus non-substrate discrimination is unclear

Like most soluble proteases, IMPs show high substrate specificity, and most membrane proteins they are exposed to are not cleaved. For example, for γ-secretase currently about 90 substrates have been described, although the physiological significance of their cleavage is not clear in most cases 22, 23, 24 (Figure 2). However, the known substrates represent only a minor fraction of the >1500 potential substrates that correspond to the predicted Nout single-span membrane proteins of the human

The kinetics and products of proteolysis yield insights into the mechanisms of intramembrane proteolysis

There is a striking difference between most IMPs and soluble proteases with regard to the overall kinetics of proteolysis. Many soluble proteases cleave within a fraction of a second as exemplified by membrane type 1 matrix metalloproteinase [29], whereas intramembrane proteolysis by γ-secretase [15] and rhomboid proteases 14, 16 occurs on a timescale of minutes. How can the difference in kinetics displayed by soluble proteases and IMPs be explained?

A mechanistic framework grounded in general

The structure and conformational dynamics of substrates and enzymes

Proteins exhibit an ensemble of conformations that interconvert on timescales ranging from picoseconds to seconds. This is now amply documented for soluble proteins [38] and is an emerging theme for membrane proteins (Box 3) 39, 40, 41. A model involving highly organized transitions between different conformations of substrates and substrate/enzyme complexes at different stages of proteolysis may help to reconcile some apparent conundrums, such as the slow, but site-specific, cleavage by IMPs

The modulatory impact of the lipid environment

Intramembrane proteolysis is also regulated by the lipid environment. This has been analyzed most thoroughly for C99 cleavage by γ-secretase. Both the overall enzyme activity and the ratio of Aβ42 + Aβ43 to total Aβ are influenced to different extents by the presence of cholesterol, sphingolipids, and glycerophospholipids, as well as by the length of the lipid acyl chains 66, 67, 68. Initial studies showed that the lipid environment also potently modulates rhomboid proteases [69].

Concluding remarks

To conclude, it is likely that global conformational changes of substrate and enzyme are required for the recognition of a TMD by an IMP and for subsequent presentation of the TMD scissile bond to the IMP catalytic site. In particular, the efficiency and the specificity by which a TMD helix is subject to intramembrane proteolysis may be governed by the nature of shape fluctuations that include bending motions at an internal hinge region. Future studies will tell whether the intrinsic dynamics

Acknowledgments

We thank Regina Fluhrer, Eliane Küttler, and Alexander Götz for critical reading of the manuscript and their valuable comments. This work was supported by grant LA699/14-1 of the Deutsche Forschungsgemeinschaft, by grant 01GI1004H of the Bundesministerium für Forschung und Technologie (KNDD), the State of Bavaria and the Center of Integrative Protein Science Munich (CIPSM) (D.L.), and by grant 01GI1004A of the KNDD (to H.S.). We also thank the Leibniz Rechenzentrum, Garching, for computing

Glossary

Amyloid precursor protein (APP)
an Nout single-span membrane protein that may have a role in cell–cell adhesion. It serves as substrate for shedding by α- or β-secretase. Shedding by β-secretase produces the C-terminal C99 fragment whose TMD helix is cleaved by γ-secretase. Cleavage yields an intracellular fragment plus a series of Aβ peptides that form cell toxic oligomers and amyloid fibrils that are believed to cause Alzheimer's disease.
Deuterium/hydrogen exchange kinetics
kinetics of the

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