A gamma-secretase-like intramembrane cleavage of TNFalpha by the GxGD aspartyl protease SPPL2b.

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.

SPPL2a and SPPL2b promote intramembrane proteolysis of TNFalpha in activated dendritic cells to trigger IL-12 production.

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.

Consensus analysis of signal peptide peptidase and homologous human aspartic proteases reveals opposite topology of catalytic domains compared with presenilins.

The human genome encodes seven intramembrane-cleaving 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.

Targeting presenilin-type aspartic protease signal peptide peptidase with gamma-secretase inhibitors.

Presenilin is implicated in the pathogenesis of Alzheimer's disease. It is thought to constitute the catalytic subunit of the gamma-secretase complex that catalyzes intramembrane cleavage of beta-amyloid precursor protein, the last step in the generation of amyloidogenic Abeta peptides. The latter are major constituents of amyloid plaques in the brain of Alzheimer's disease patients. Inhibitors of gamma-secretase are considered potential therapeutics for the treatment of this disease because they prevent production of Abeta 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 gamma-secretase inhibitors directed against presenilin may also inhibit signal peptide peptidase. Here we demonstrate that some of the most potent known gamma-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 Abeta generation without affecting other intramembrane-cleaving aspartic proteases.

A selective interaction between OS-9 and the carboxyl-terminal tail of meprin beta.

OS-9, a protein previously uncharacterized, was shown to interact specifically with the intracellular region of the membrane proteinase meprin beta found in brush border membranes of kidney and small intestine. We have shown previously that this cytoplasmic region is indispensable for the maturation of meprin beta, which included an endoplasmic reticulum (ER)-to-Golgi translocation. We characterized OS-9 and found that it is associated with ER membranes and that it is exposed to the cytoplasm. Consistent with the kinetics of maturation of meprin beta, OS-9 associates with meprin beta only transiently, coinciding with ER-to-Golgi transport of meprin beta. The OS-9-binding site in the cytoplasmic domain of meprin beta overlaps the region essential for this transport. We characterized alternatively spliced forms of rat and mouse OS-9, and we found that only the non-spliced form of OS-9 binds to meprin beta, implicating the spliced out segment in the binding, and suggesting the possible mechanism of the regulation of OS-9 function. Taken together, our results indicated that OS-9 may be involved in the ER-to-Golgi transport of meprin beta. Ubiquitous expression of OS-9 raises the possibility that it may interact with other membrane proteins that possess the cytoplasmic moiety homologous to that of meprin beta during their ER-to-Golgi transition.

YOS9, the putative yeast homolog of a gene amplified in osteosarcomas, is involved in the endoplasmic reticulum (ER)-Golgi transport of GPI-anchored proteins.

The OS-9 gene maps to a region (q13-15) of chromosome 12 that is highly amplified in human osteosarcomas and encodes a protein of unknown function. Here we have characterized a homolog designated as YOS9 (YDR057w) from Saccharomyces cerevisiae. The yeast protein (Yos9) is a membrane-associated glycoprotein that localizes to the endoplasmic reticulum (ER). YOS9 interacts genetically with genes involved in ER-Golgi transport, particularly SEC34, whose temperature-sensitive mutant is rescued by YOS9 overexpression. Interestingly, Yos9 appears to play a direct role in the transport of glycosylphosphatidylinositol (GPI)-anchored proteins to the Golgi apparatus. Yos9 binds directly to Gas1 and Mkc7 and accelerates Gas1 transport and processing in cells overexpressing YOS9. Correspondingly, Gas1 processing is slowed in cells bearing a deletion in YOS9. No effect upon the transport and processing of non-GPI-anchored proteins (e.g. invertase and carboxypeptidase Y) was detected in cells either lacking or overexpressing Yos9. As Yos9 is not a component of the Emp24 complex, it may act as a novel escort factor for GPI-anchored proteins in ER-Golgi transport in yeast and possibly in mammals.