Regulated intramembrane proteolysis--lessons from amyloid precursor protein processing

Regulated intramembrane proteolysis (RIP) controls the communication between cells and the extracellular environment. RIP is essential in the nervous system, but also in other tissues. In the RIP process, a membrane protein typically undergoes two consecutive cleavages. The first one results in the shedding of its ectodomain. The second one occurs within its transmembrane domain, resulting in secretion of a small peptide and the release of the intracellular domain into the cytosol. The proteolytic cleavage fragments act as versatile signaling molecules or are further degraded. An increasing number of membrane proteins undergo RIP. These include growth factors, cytokines, cell adhesion proteins, receptors, viral proteins and signal peptides. A dysregulation of RIP is found in diseases, such as leukemia and Alzheimer's disease. One of the first RIP substrates discovered was the amyloid precursor protein (APP). RIP processing of APP controls the generation of the amyloid β-peptide, which is believed to cause Alzheimer's disease. Focusing on APP as the best-studied RIP substrate, this review describes the function and mechanism of the APP RIP proteases with the goal to elucidate cellular mechanisms and common principles of the RIP process in general.     

Membrane proteases and tetraspanins

TEMs (tetraspanin-enriched microdomains) are specialized platforms in the plasma membrane that include adhesion receptors and enzymes. Insertion into TEMs dictates the local concentration of these molecules, regulates their internalization rate, their interaction and cross-talk with other receptors at the plasma membrane and provides links with certain signalling pathways. We focus on the associations described for tetraspanins with membrane proteases and their substrates, reviewing the emerging evidence in the literature that suggests that TEMs might be essential platforms for regulating protein shedding, RIP (regulated intramembrane proteolysis) and matrix degradation and assembly.     

Site-2 proteases in prokaryotes: regulated intramembrane proteolysis expands to microbial pathogenesis

Regulated intramembrane proteolysis (RIP) is a widely distributed mechanism of signal transduction in which membrane-bound proteases cleave transmembrane domains of substrate proteins. The site-2 protease (S2P) class of RIP metalloproteases is present in most bacterial genomes but is generally of unknown function except for the well-characterized proteases RseP and SpoIVFB. In this review we will discuss the biochemical functions and physiologic roles of S2P proteases in bacteria and highlight recent data implicating S2P family members in host-pathogen interactions.     

Solution structure and dynamics of the N-terminal cytosolic domain of rhomboid intramembrane protease from Pseudomonas aeruginosa: insights into a functional role in intramembrane proteolysis

Rhomboids are ubiquitous integral membrane proteases that release cellular signals from membrane-bound substrates through a general signal transduction mechanism known as regulated intramembrane proteolysis (RIP). We present the NMR structure of the cytosolic N-terminal domain (NRho) of P. aeruginosa Rhomboid. NRho consists of a novel alpha/beta fold and represents the first detailed structural insight into this class of intramembrane proteases. We find evidence that NRho is capable of strong and specific association with detergent micelles that mimic the membrane/water interface. Relaxation measurements on NRho reveal structural fluctuations on the microseconds-milliseconds timescale in regions including and contiguous to those implicated in membrane interaction. This structural plasticity may facilitate the ability of NRho to recognize and associate with membranes. We suggest that NRho plays a role in scissile peptide bond selectivity by optimally positioning the Rhomboid active site relative to the membrane plane.     

New insights into S2P signaling cascades: Regulation, variation, and conservation

Regulated intramembrane proteolysis (RIP) is a conserved mechanism that regulates signal transduction across the membrane by recruiting membrane‐bound proteases to cleave membrane‐spanning regulatory proteins. As the first identified protease that performs RIP, the metalloprotease site‐2 protease (S2P) has received extensive study during the past decade, and an increasing number of S2P‐like proteases have been identified and studied in different organisms; however, some of their substrates and the related S1Ps remain elusive. Here, we review recent research on S2P cascades, including human S2P, E. coli RseP, B. subtilis SpoIVFB and the newly identified S2P homologs. We also discuss the variation and conservation of characterized S2P cascades. The conserved catalytic motif of S2P and prevalence of amino acids of low helical propensity in the transmembrane segments of the substrates suggest a conserved catalytic conformation and mechanism within the S2P family. The review also sheds light on future research on S2P cascades.     

A fast growing spectrum of biological functions of γ-secretase in development and disease

γ-secretase, which assembles as a tetrameric complex, is an aspartyl protease that proteolytically cleaves substrate proteins within their membrane-spanning domain; a process also known as regulated intramembrane proteolysis (RIP). RIP regulates signaling pathways by abrogating or releasing signaling molecules. Since the discovery, already > 15 years ago, of its catalytic component, presenilin, and even much earlier with the identification of amyloid precursor protein as its first substrate, γ-secretase has been commonly associated with Alzheimer's disease. However, starting with Notch and thereafter a continuously increasing number of novel substrates, γ-secretase is becoming linked to an equally broader range of biological processes. This review presents an updated overview of the current knowledge on the diverse molecular mechanisms and signaling pathways controlled by γ-secretase, with a focus on organ development, homeostasis and dysfunction. This article is part of a Special Issue entitled: Intramembrane Proteases.     

Self-regulated cleavage of the mitochondrial intramembrane-cleaving protease PARL yields Pbeta, a nuclear-targeted peptide

Regulated intramembrane proteolysis (RIP) is an emerging paradigm in signal transduction. RIP is mediated by intramembrane-cleaving proteases (I-CliPs), which liberate biologically active nuclear or secreted domains from their membrane-tethered precursor proteins. The yeast Pcp1p/Rbd1p protein is a Rhomboid-like I-CliP that regulates mitochondrial membrane remodeling and fusion through cleavage of Mgm1p, a regulator of these essential activities. Although this ancient function is conserved in PARL (Presenilins-associated Rhomboid-like protein), the mammalian ortholog of Pcp1p/Rbd1p, the two proteins show a strong divergence at their N termini. However, the N terminus of PARL is significantly conserved among vertebrates, particularly among mammals, suggesting that this domain evolved a distinct but still unknown function. Here, we show that the cytosolic N-terminal domain of PARL is cleaved at positions 52-53 (alpha-site) and 77-78 (beta-site). Whereas alpha-cleavage is constitutive and removes the mitochondrial targeting sequence, beta-cleavage appears to be developmentally controlled and dependent on PARL I-CliP activity supplied in trans. The beta-cleavage of PARL liberates Pbeta, a nuclear targeted peptide whose sequence is conserved only in mammals. Thus, in addition to its evolutionarily conserved function in regulating mitochondrial dynamics, PARL might mediate a mammalian-specific, developmentally regulated mitochondria-to-nuclei signaling through regulated proteolysis of its N terminus and release of the Pbeta peptide.     

Roles of regulated intramembrane proteolysis in virus infection and antiviral immunity

Regulated intramembrane proteolysis (RIP) is a signaling mechanism through which transmembrane precursor proteins are cleaved to liberate their cytoplasmic and/or luminal/extracellular fragments from membranes so that these fragments are able to function at a new location. Recent studies have indicated that this proteolytic reaction plays an important role in host–virus interaction. On one hand, RIP transfers the signal from the endoplasmic reticulum (ER) to nucleus to activate antiviral genes in response to alteration of the ER caused by viral infection. On the other hand, RIP can be hijacked by virus to process transmembrane viral protein precursors and to destroy transmembrane antiviral proteins. Understanding this Yin and Yang side of RIP may lead to new strategies to combat viral infection. This article is part of a Special Issue entitled: Intramembrane Proteases.     

A RIP tide in neuronal signal transduction

The generation of nuclear signaling proteins by regulated intramembrane proteolysis (RIP) is a new paradigm of signal transduction. Mammalian proteins that are processed by RIP include SREBP-1, Notch-1, amyloid precursor protein (APP), and ErbB-4. Intramembranous gamma-secretase cleavage of APP plays a central role in Alzheimer's disease by generating the amyloid beta protein. An intriguing possibility is that the cognate C-terminal fragment generated by gamma-secretase cleavage could also play a role through the regulation of nuclear signaling events. Thus, RIP may contribute to both brain development and degeneration and may provide unexpected diversity to the signaling repertoire of a cell.     

Uncovering a link between a plastid translocon component and rhomboid proteases using yeast mitochondria-based assays

Rhomboid proteases are present in bacteria, insects, yeasts, parasites, mammals and plants. These proteases are part of the regulated intramembrane proteolysis mechanism for controlling processes such as development, stress response, lipid metabolism and mitochondrial membrane remodeling. Specific rhomboid protease substrates linked to these processes have been identified from insects to mammals, but not for plants. Identification of a link is a key step for elucidating the role of each rhomboid protease. Here, using a yeast mitochondria-based approach, we report evidence of a potential link between a plastid translocon component and organellar rhomboid proteases. This identification expands the types of processes involving regulated intramembrane proteolysis potentially to include at least one aspect of plastid protein transport.     

Regulated Intramembrane Proteolysis of the Frontotemporal Lobar Degeneration Risk Factor,TMEM106B,by Signal Peptide Peptidase-like 2a (SPPL2a)

The sequential processing of single pass transmembrane proteins via ectodomain shedding followed by intramembrane proteolysis is involved in a wide variety of signaling processes, as well as maintenance of membrane protein homeostasis. Here we report that the recently identified frontotemporal lobar degeneration risk factor TMEM106B undergoes regulated intramembrane proteolysis. We demonstrate that TMEM106B is readily processed to an N-terminal fragment containing the transmembrane and intracellular domains, and this processing is dependent on the activities of lysosomal proteases. The N-terminal fragment is further processed into a small, rapidly degraded intracellular domain. The GxGD aspartyl proteases SPPL2a and, to a lesser extent, SPPL2b are responsible for this intramembrane cleavage event. Additionally, the TMEM106B paralog TMEM106A is also lysosomally localized; however, it is not a specific substrate of SPPL2a or SPPL2b. Our data add to the growing list of proteins that undergo intramembrane proteolysis and may shed light on the regulation of the frontotemporal lobar degeneration risk factor TMEM106B.     

Sharpening rhomboid specificity by dimerisation and allostery

In this issue of The EMBO Journal, mechanistic analyses of substrate cleavage by rhomboid intramembrane proteases suggest that catalytic efficiency towards natural, transmembrane substrates is allosterically stimulated by initial substrate interaction with an intramembrane exosite, whose formation depends on rhomboid dimerisation. In the realm of intramembrane proteolysis, dimerisation and allosteric cooperativity represent new concepts that, once confirmed more broadly, should radically alter our view of how these proteases work.     

gamma-Secretase cleavage and binding to FE65 regulate the nuclear translocation of the intracellular C-terminal domain (ICD) of the APP family of proteins

Regulated intramembrane proteolysis (RIP) of the amyloid precursor protein (APP) produces amyloid beta-protein (Abeta), the probable causative agent of Alzheimer's disease (AD), and is therefore an important target for therapeutic intervention. However, there is a burgeoning consensus that gamma-secretase, one of the proteases that generates Abeta, is also critical for the signal transduction of APP and a growing list of other receptors. APP is a member of a gene family that includes two amyloid precursor-like proteins, APLP1 and APLP2. Although APP and the APLPs undergo similar proteolytic processing, there is little information about the role of their gamma-secretase-generated intracellular domains (ICDs). Here, we show that APLP1 and 2 undergo presenilin-dependent RIP similar to APP, resulting in the release of a approximately 6 kDa ICD for each protein. Each of the ICDs are degraded by an insulin degrading enzyme-like activity, but they can be stabilized by members of the FE65 family and translocate to the nucleus. Given that modulation of APP processing is a therapeutic target and that the APLPs are processed in a manner similar to APP, any strategy aimed at altering APP proteolysis will have to take into account possible effects on signaling by APLP 1 and 2.