Homologues of signal peptide peptidase (SPPLs) are putative aspartic proteases that may catalyse regulated intramembrane proteolysis of type II membrane-anchored signalling factors. Here, we show that four human SPPLs are each sorted to a different compartment of the secretory pathway. We demonstrate that SPPL2a and SPPL2b, which are sorted to endosomes and the plasma membrane, respectively, are functional proteases that catalyse intramembrane cleavage of tumour necrosis factor alpha (TNFalpha). The two proteases promoted the release of the TNFalpha intracellular domain, which in turn triggers expression of the pro-inflammatory cytokine interleukin-12 by activated human dendritic cells. Our study reveals a critical function for SPPL2a and SPPL2b in the regulation of innate and adaptive immunity.

Gamma-secretase and signal peptide peptidase (SPP) are unusual GxGD aspartyl proteases, which mediate intramembrane proteolysis. In addition to SPP, a family of SPP-like proteins (SPPLs) of unknown function has been identified. We demonstrate that SPPL2b utilizes multiple intramembrane cleavages to liberate the intracellular domain of tumor necrosis factor alpha (TNFalpha) into the cytosol and the carboxy-terminal counterpart into the extracellular space. These findings suggest common principles for regulated intramembrane proteolysis by GxGD aspartyl proteases.

Presenilin is implicated in the pathogenesis of Alzheimer's disease. It is thought to constitute the catalytic subunit of the ␥-secretase complex that catalyzes intramembrane cleavage of ␤-amyloid precursor protein, the last step in the generation of amyloidogenic A␤ peptides. The latter are major constituents of amyloid plaques in the brain of Alzheimer's disease patients. Inhibitors of ␥-secretase are considered potential therapeutics for the treatment of this disease because they prevent production of A␤ peptides. Recently, we discovered a family of presenilin-type aspartic proteases. The founding member, signal peptide peptidase, catalyzes intramembrane cleavage of distinct signal peptides in the endoplasmic reticulum membrane of animals. In humans, the protease plays a crucial role in the immune system. Moreover, it is exploited by the hepatitis C virus for the processing of the structural components of the virion and hence is an attractive target for anti-infective intervention. Signal peptide peptidase and presenilin share identical active site motifs and both catalyze intramembrane proteolysis. These common features let us speculate that ␥-secretase inhibitors directed against presenilin may also inhibit signal peptide peptidase. Here we demonstrate that some of the most potent known ␥-secretase inhibitors efficiently inhibit signal peptide peptidase. However, we found compounds that showed higher specificity for one or the other protease. Our findings highlight the possibility of developing selective inhibitors aimed at reducing A␤ generation without affecting other intramembranecleaving aspartic proteases. Alzheimer's disease (AD)1 is characterized by the formation of senile plaques in the brain. Major constituents of these plaques are the amyloidogenic 40-and 42-residue-long A␤ peptides A␤40 and A␤42, respectively (1). The amyloid cascade hypothesis casually links the generation of amyloid plaques with the neuropathological changes accompanying the symptoms typical of this disease (2). A␤ peptides are generated from the type I transmembrane protein ␤-APP (␤-amyloid precursor protein) by sequential proteolysis (3). The protein is first cleaved in the exoplasmic domain by the ␤-site APP-cleaving enzyme (BACE) to release the ectodomain (4, 5). The residual membrane-anchored stub of 99 residues (C99) is subsequently cleaved in the center of the transmembrane region by ␥-secretase (6). The resulting cleavage products, an A␤ peptide and the amyloid intracellular domain (AICD), are liberated from the lipid bilayer toward the exoplasm and cytosol, respectively (7-9).To date, the majority of characterized familial AD mutations are clustered along the presenilin-1 (PS1) gene (10, 11). They are thought to accelerate disease onset by increasing the A␤42/ A␤40 ratio (12). It is not well understood how these mutations, which are essentially scattered along the entire PS1 gene, can lead to a specific increase in the production of the 42-residuelong peptide that corresponds to the most amyloidogenic form of A␤ (13). ...

The human genome encodes seven intramembranecleaving GXGD aspartic proteases. These are the two presenilins that activate signaling molecules and are implicated in Alzheimer's disease, signal peptide peptidase (SPP), required for immune surveillance, and four SPP-like candidate proteases (SPPLs), of unknown function. Here we describe a comparative analysis of the topologies of SPP and its human homologues, SPPL2a, -2b, -2c, and -3. We demonstrate that their N-terminal extensions are located in the extracellular space and, except for SPPL3, are modified with N-glycans. Whereas SPPL2a, -2b, and -2c contain a signal sequence, SPP and SPPL3 contain a type I signal anchor sequence for initiation of protein translocation and membrane insertion. The hydrophilic loops joining the transmembrane regions, which contain the catalytic residues, are facing the exoplasm. The C termini of all these proteins are exposed toward the cytosol. Taken together, our study demonstrates that SPP and its homologues are all of the same principal structure with a catalytic domain embedded in the membrane in opposite orientation to that of presenilins. Other than presenilins, SPPL2a, -2b, -2c, and -3 are therefore predicted to cleave type II-oriented substrate peptides like the prototypic protease SPP.

Fas ligand (FasL) is a type II transmembrane protein belonging to the tumor necrosis factor family. Its binding to the cognate Fas receptor triggers the apoptosis that plays a pivotal role in the maintenance of immune system homeostasis. The cell deathinducing property of FasL has been associated with its extracellular domain, which can be cleaved off by metalloprotease activity to produce soluble FasL. The fate of the remaining membrane-anchored N-terminal part of the FasL molecule has not been determined. Here we show that post-translational processing of overexpressed and endogenous FasL in T-cells by the disintegrin and metalloprotease ADAM10 generates a 17-kDa N-terminal fragment, which lacks the receptor-binding extracellular domain. This FasL remnant is membrane anchored and further processed by SPPL2a, a member of the signal peptide peptidaselike family of intramembrane-cleaving proteases. SPPL2a cleavage liberates a smaller and highly unstable fragment mainly containing the intracellular FasL domain (FasL ICD). We show that this fragment translocates to the nucleus and is capable of inhibiting gene transcription. With ADAM10 and SPPL2a we have identified two proteases implicated in FasL processing and release of the FasL ICD, which has been shown to be important for retrograde FasL signaling.