Signal peptide peptidase (SPP) is an intramembrane cleaving protease (I-CLiP) identified by its cleavage of several type II membrane signal peptides. To date, only human SPP has been directly shown to have proteolytic activity. Here we demonstrate that the most closely related human homologue of SPP, signal peptide peptidase like 3 (SPPL3), cleaves a SPP substrate, but a more distantly related homologue, signal peptide peptidase like 2b (SPPL2b), does not. These data provide strong evidence that the SPP and SPPL3 have conserved active sites and suggest that the active sites SPPL2b is distinct. We have also synthesized a cDNA designed to express the single SPP gene present in Plasmodium falciparum and cloned this into a mammalian expression vector. When the malaria SPP protein is expressed in mammalian cells it cleaves a SPP substrate. Notably, several human SPP inhibitors block the proteolytic activity of malarial SPP (mSPP). Studies from several model organisms that express multiple SPP homologs demonstrate that the silencing of a single SPP homologue is lethal. Based on these data, we hypothesize that mSPP is a potential a novel therapeutic target for malaria.

Presenilins (PS1 and PS2) are supposed to be unusual aspartic proteases and components of the gamma-secretase complex regulating cleavage of type I proteins. Multiple mutations in PS1 are a major cause of familial early-onset Alzheimer's disease (AD). We and others recently identified PS-related families of proteins (IMPAS/PSH/signal peptide peptidases (SPP)). The functions of these proteins are yet to be determined. We found that intramembrane protease-associated or intramembrane protease aspartic protein Impas 1 (IMP1)/SPP induces intramembranous cleavage of PS1 holoprotein in cultured cells coexpressing these proteins. Mutations in evolutionary invariant sites in hIMP1 or specific gamma-secretase inhibitors abolish the hIMP1-mediated endoproteolysis of PS1. In contrast, neither AD-like mutations in hIMP1 nor in PS1 substrate abridge the PS1 cleavage. The data suggest that IMP1 is a bi-aspartic polytopic protease capable of cleaving transmembrane precursor proteins. These data, to our knowledge, are a first observation that a multipass transmembrane protein or the integral protease per se may be a primary substrate for an intramembranous proteolysis.

The US2 and US11 gene products of human cytomegalovirus promote viral evasion by hijacking the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway. US2 and US11 initiate dislocation of newly translocated major histocompatibility complex class I (MHC I) from the ER to the cytosol for proteasome-mediated degradation, thereby decreasing cell surface MHC I. Despite being instrumental in elucidating the mammalian ERAD pathway, the responsible E3 ligase or ligases remain unknown. Using a functional small interfering RNA library screen, we now identify TRC8 (translocation in renal carcinoma, chromosome 8 gene), an ER-resident E3 ligase previously implicated as a hereditary kidney cancer gene, as required for US2-mediated MHC I ubiquitination. Depletion of TRC8 prevents MHC I ubiquitination and dislocation by US2 and restores cell surface MHC I. TRC8 forms an integral part of a novel multiprotein ER complex that contains MHC I, US2, and signal peptide peptidase. Our data show that the TRC8 E3 ligase is required for MHC I dislocation from the ER and identify a new complex associated with mammalian ERAD.

Signal peptide peptidase (SPP) is an unusual aspartyl protease that mediates clearance of signal peptides by proteolysis within the endoplasmic reticulum (ER). Like presenilins, which provide the proteolytically active subunit of the gamma-secretase complex, SPP contains a critical GXGD motif in its C-terminal catalytic center. Although SPP is known to be an aspartyl protease of the GXGD type, several presenilin homologues/SPP-like proteins (PSHs/SPPL) of unknown function have been identified by data base searches. We now investigated the subcellular localization and a putative proteolytic activity of PSHs/SPPLs in cultured cells and in an in vivo model. We demonstrate that SPPL2b is targeted through the secretory pathway to endosomes/lysosomes, whereas SPP and SPPL3 are restricted to the ER. As suggested by the differential subcellular localization of SPPL2b compared with SPP and SPPL3, we found distinct phenotypes upon antisense gripNA-mediated knockdown in zebrafish. spp and sppl3 knockdowns in zebrafish result in cell death within the central nervous system, whereas reduction of sppl2b expression causes erythrocyte accumulation in an enlarged caudal vein. Moreover, expression of D/A mutations of the putative C-terminal active sites of spp, sppl2, and sppl3 produced phenocopies of the respective knockdown phenotypes. Thus, our data suggest that all investigated PSHs/SPPLs are members of the novel family of GXGD aspartyl proteases. Furthermore, SPPL2b is shown to be the first member of the SPP/PSH/SPPL family that is not located within the ER but in endosomal/lysosomal vesicles.

Signal peptide peptidase (SPP) is an intramembrane cleaving protease (I-CLiP) identified by its cleavage of several type II membrane signal peptides. To date, only human SPP has been directly shown to have proteolytic activity. Here we demonstrate that the most closely related human homologue of SPP, signal peptide peptidase like 3 (SPPL3), cleaves a SPP substrate, but a more distantly related homologue, signal peptide peptidase like 2b (SPPL2b), does not. These data provide strong evidence that the SPP and SPPL3 have conserved active sites and suggest that the active sites SPPL2b is distinct. We have also synthesized a cDNA designed to express the single SPP gene present in Plasmodium falciparum and cloned this into a mammalian expression vector. When the malaria SPP protein is expressed in mammalian cells it cleaves a SPP substrate. Notably, several human SPP inhibitors block the proteolytic activity of malarial SPP (mSPP). Studies from several model organisms that express multiple SPP homologs demonstrate that the silencing of a single SPP homologue is lethal. Based on these data, we hypothesize that mSPP is a potential a novel therapeutic target for malaria.

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.

Presenilin, the catalytic component of the gamma-secretase complex, type IV prepilin peptidases, and signal peptide peptidase (SPP) are the founding members of the family of intramembrane-cleaving GXGD aspartyl proteases. SPP-like (SPPL) proteases, such as SPPL2a, SPPL2b, SPPL2c, and SPPL3, also belong to the GXGD family. In contrast to gamma-secretase, for which numerous substrates have been identified, very few in vivo substrates are known for SPP and SPPLs. Here we demonstrate that Bri2 (Itm2b), a type II-oriented transmembrane protein associated with familial British and Danish dementia, undergoes regulated intramembrane proteolysis. In addition to the previously described ectodomain processing by furin and related proteases, we now describe that the Bri2 protein, similar to gamma-secretase substrates, undergoes an additional cleavage by ADAM10 in its ectodomain. This cleavage releases a soluble variant of Bri2, the BRICHOS domain, which is secreted into the extracellular space. Upon this shedding event, a membrane-bound Bri2 N-terminal fragment remains, which undergoes intramembrane proteolysis to produce an intracellular domain as well as a secreted low molecular weight C-terminal peptide. By expressing all known SPP/SPPL family members as well as their loss of function variants, we demonstrate that selectively SPPL2a and SPPL2b mediate the intramembrane cleavage, whereas neither SPP nor SPPL3 is capable of processing the Bri2 N-terminal fragment.

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.

Presenilins (PS1 and PS2) are supposed to be unusual aspartic proteases and components of the gamma-secretase complex regulating cleavage of type I proteins. Multiple mutations in PS1 are a major cause of familial early-onset Alzheimer's disease (AD). We and others recently identified PS-related families of proteins (IMPAS/PSH/signal peptide peptidases (SPP)). The functions of these proteins are yet to be determined. We found that intramembrane protease-associated or intramembrane protease aspartic protein Impas 1 (IMP1)/SPP induces intramembranous cleavage of PS1 holoprotein in cultured cells coexpressing these proteins. Mutations in evolutionary invariant sites in hIMP1 or specific gamma-secretase inhibitors abolish the hIMP1-mediated endoproteolysis of PS1. In contrast, neither AD-like mutations in hIMP1 nor in PS1 substrate abridge the PS1 cleavage. The data suggest that IMP1 is a bi-aspartic polytopic protease capable of cleaving transmembrane precursor proteins. These data, to our knowledge, are a first observation that a multipass transmembrane protein or the integral protease per se may be a primary substrate for an intramembranous proteolysis.