Understanding the Mechanism of Intramembrane Proteases

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(Last Updated On: April 2, 2020)
Dirrerent types of intramembrane proteases
Different types of intramembrane proteases. Image: Langosch et al. 2015

Intramembrane proteolysis is a degradation of proteins within the plane of a lipid bilayer membrane. It is a widespread phenomenon contributing to the functional activation of the substrates and it is also involved in several diseases.


Many families of intramembrane proteases have been discovered and characterized, but their mechanism of discrimination between the substrate and non-substrate is not yet known. How intramembrane proteases (IMPs) achieve site-specific cleavage and what determines the rate of proteolysis still needs to be characterized?

Most of the substrates of the intramembrane proteases possess a single transmembrane domain with a scissile peptide bond and its functional consequences include activation of membrane-tethered transcriptional factors and transcriptional activators along with the secretion of growth factors, bacterial translocation channel maturation, and protein degradation regulation.

Intramembrane proteolysis recognizes and processes its substrate. However, substrate processing requires exposure of the scissile peptide bond which forms a tetrahedral intermediate with catalytic residue leading to the hydrolysis.

Substrate of Intramembrane Proteases
Substrate for the Intramembrane Proteases.
Image: Langosch at al. 2015

As intramembrane proteases are the slow enzymes and, therefore, which of the above step is a rate-limiting step of the proteolysis is not clear. The primary structure of the protease and the conformational dynamics of the substrate and enzyme of the rate-limiting step are also unclear.

To resolve these puzzles and explain how disease-related point mutations affect these properties and the discrimination between the substrate and non-substrate researchers have analyzed two intramembrane proteases γ-secretase and rhomboid proteases.

Different components of the intramembrane proteases

There are different types of intramembrane proteases that differ in their transmembrane topologies and membrane-embedded active site residues. Aspartate intramembrane proteases possess two catalytic Asp residues that comprise presenilins, signal peptide peptidases (SPP), and SPP-like proteases (SPPL).

The aspartate IMP presenilin is a catalytic subunit of the γ-secretase with Nin topology and it is associated with the nicastrin, presenilin enhancer-2 (PEN-2) and anterior pharynx-defective-1 (APH-1) subunits in a 1:1:1:1 stoichiometry to form a γ-secretase complex while SPP and SPPL proteases exhibit an inverted membrane topology (Nout) and they don’t require any cofactor for their activity.

Rhomboid proteases are the serine intramembrane proteases comprising a characteristic Ser-His catalytic dyad. Rhomboid proteases are characterized by their six transmembrane domains with an additional transmembrane domain and in some cases cytosolic domains.

Rhomboid proteases in the eukaryotes and in the bacterial plasma membranes have their active sites in the extracytoplasmic space while mitochondrial rhomboid proteases are also called as presenilin-associated rhomboid-like proteins have an inverted topology with active site exposed toward the mitochondrial matrix. Other intramembrane proteases include site-2 proteases, (zinc metalloproteases) and Ras-converting CAAX endopeptidase-1 (Glutamate intramembrane proteases).

Substrate vs. non-substrate discrimination

Though intramembrane proteases have high substrate specificity, they don’t cleave all of the membrane proteins. As, for example, there are about 1500 potential substrates for the γ-secretase, but in actuality, γ-secretase have only 90 known substrates.

Substrates of γ-secretase have their intracellular domain shedded before intramembrane proteolysis. The shedded intracellular domain is recognized by sheddase enzymes α-and β-secretase. These sheddases may help the initial selection of the substrate for intramembrane proteolysis. The same truth holds with the other intramembrane proteases SPP, SPPL, and S2P, however, substrate recognition about the rhomboid protease is little known.

Intramembrane Protease C99 Catalysis
C99 as an example of Intramembrane Proteases. Image: Langosch et al. 2015

There are some mutations of intramembrane proteases that cause disease. However, it is not known how they affect proteolysis. As, for example, mutation of the transmembrane domain of Protein Kinase PINK1 is linked to Parkinson’s disease affecting the cleavage of Protein Kinase PINK1 by mitochondrial rhomboid protease PARL. While the neuropathy associated mutations in the “myelin protein zero” triggers the degradation by endoplasmic reticulum RHBDL4.

Impact of lipid environment on IMP modulation

The lipid environment of the lipid membrane also regulates the intramembrane proteolysis. For C99, cleavage catalyzed by γ-secretase has been analyzed for the effect of lipid environment where the overall enzyme activity of the γ-secretase was found to be influenced by the presence of cholesterol, sphingolipids, and glycerophospholipids as well as by the length of the acyl chains of the lipids.

In general, lipids in the membrane affect intramembrane proteolysis by binding with substrate and or enzyme and competing with their interaction. Membrane lipids may also alter the substrate diffusion rate influencing the rate of association of the substrate with intramembrane proteases.

Reference: Trends in Biochemical Sciences

Article DOI: 10.1016/j.tibs.2015.04.001

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