Intramembrane Proteolytic Cleavage by Human Signal Peptide Peptidase Like 3 and Malaria Signal Peptide Peptidase

The FASEB Journal • Research Communication

Intramembrane proteolytic cleavage by human signal peptide peptidase like 3 and malaria signal peptide peptidase

Andrew C. Nyborg, Thomas B. Ladd, Karen Jansen, Thomas Kukar, and Todd E. Golde1 Department of Neuroscience, Mayo Clinic Jacksonville, Mayo Clinic College of Medicine, Jacksonville, Florida, USA

ABSTRACT Signal peptide peptidase (SPP) is an cleaves type II membrane proteins (5, 6, 8–10). In intramembrane cleaving protease (I-CLiP) identified by addition, SPP and presenilin have inverted active site its cleavage of several type II membrane signal pep- topologies (11–13) and presenilins seem to require tides. To date, only human SPP has been directly shown three additional proteins to function as ␥-secretase to have proteolytic activity. Here we demonstrate that (14–19) whereas SPP appears to function as a ho- the most closely related human homologue of SPP, modimer (11, 12, 20). ␥-Secretase activity cannot be signal peptide peptidase like 3 (SPPL3), cleaves a SPP reconstituted in nonmammalian cells without coexpres- substrate, but a more distantly related homologue, sion of presenilin 1 or 2 with Aph-1, Pen-2, and signal peptide peptidase like 2b (SPPL2b), does not. Nicastrin (16, 19). SPP activity can be reconstituted in These data provide strong evidence that the SPP and yeast by expression of SPP alone (11). ␥-Secretase SPPL3 have conserved active sites and suggest that the inhibitors targeting presenilins are currently under active sites SPPL2b is distinct. We have also synthesized investigation for both Alzheimer therapeutics (21) and a cDNA designed to express the single SPP gene possibly as anticancer agents (22, 23). SPP inhibitors present in Plasmodium falciparum and cloned this into a have been suggested as potential therapeutic agents for mammalian expression vector. When the malaria SPP hepatitis C infection (24). protein is expressed in mammalian cells it cleaves a There are four human homologs of SPP, which have SPP substrate. Notably, several human SPP inhibitors been referred to as presenilin homologs (PSH) (25), block the proteolytic activity of malarial SPP (mSPP). intramembrane proteases (IMPAS) (26), and signal Studies from several model organisms that express peptide peptidase like (SPPL) (11). Besides SPP, which multiple SPP homologs demonstrate that the silencing was identified based on a search for the proteolytic of a single SPP homologue is lethal. Based on these activity that cleaved the signal peptide of MHC class I data, we hypothesize that mSPP is a potential a novel molecules and several viral preproproteins, little is therapeutic target for malaria.—Nyborg, A. C., Ladd, known about the function of the various SPPLs. Several T. B., Jansen, K., Kukar, T., Golde, T. E. Intramem- recent papers suggest that SPPLs play critical roles in brane proteolytic cleavage by human signal peptide development. In Caenorhabditis elegans, deficiency of peptidase like 3 and malaria signal peptide peptidase. ce-imp-2 (a SPP like gene) causes a severe developmen- FASEB J. 20, 1671–1679 (2006) tal phenotype (27). Drosophila deficient in one of two SPP genes, CG11840, had defective trachea and died as Key Words: ␥-secretase ⅐ aspartyl protease ⅐ hepatitis C virus larvae (28). In Danio rerio (Zebrafish), when either the ⅐ MHC class I ⅐ Plasmodium falciparum SPP or SPPL3 homologs were knocked down, a similar cell death phenotype was achieved (29). However, knockdown of the SPPL2b homologue resulted in a Presenilin 1 and 2 and signal peptide peptidase (SPP) distinct developmental phenotype with an enlarged are the prototypic members of a large family of putative caudal vein (29). aspartyl proteases that cleave a variety of transmem- Based on these genetic studies, it is clear that SPP brane substrates. Intramembrane cleaving proteases and its homologs have important physiological roles (I-CLiP) play a variety of important roles in cell signal- essential for normal development. However, the molec- ing (1) and regulation (2), cell surveillance (3), and ular determinants of these functional effects have not intracellular communication (4). Each contains a conserved 1) active site motif of YD and GXGD in adjacent 1 Correspondence: Department of Neuroscience, Mayo transmembrane domains (5, 6) and 2) a PAL motif near Clinic Jacksonville, Mayo Clinic College of Medicine, 4500 the COOH terminus (7) that have been shown to be San Pablo Rd., Jacksonville, Florida 32224, USA. E-mail: critical for activity. Presenilins and SPP differ in that [email protected] presenilins cleave type I membrane proteins and SPP doi: 10.1096/fj.06–5762com

0892-6638/06/0020-1671 © FASEB 1671 been elucidated. Indeed, only SPP has been shown to Malaria SPP (mSPP) was identiﬁed by searching the Plas- cleave a transmembrane substrate directly, and it is modium falciparum genome with the entire human SPP pro- unlikely that the SPP cleavage of the endogenous tein sequence. The malaria amino acid sequence (NP_702432) was then human codon optimized and synthe- substrates identiﬁed to date (HCV core protein, human sized by GenScript. The resulting gene was cloned into leukocyte antigen-E eptitopes) mediates the pheno- pCDNA6, which puts a V5 his epitope tag at the COOH

types associated with SPP deficiency (11, 12, 24, 30). terminus (mSPPCTV5). Plasmodium falciparum (Malaria) effects ϳ300 million SPP and mSPP phylogenetic tree analyses and identities people a year, with Ͼ1 million deaths attributable to were generated using the AlignX@ feature of Vector NTI@. plasmodium infection (31). Treatment of malaria is now complicated by the development of widespread DNA transfection of 293 cells resistance of many Plasmodium species to common affordable drugs such as choloroquine, mefloquine, The luciferase reporter assays were performed as described and sulfadoxanine (32–34). Thus, there is a need to previously (12). In short, HEK 293T cells were plated at 70% confluency and transiently transfected using 100 ␮l serum identify novel targets, a process that can be aided by free Opti MEM® (Life Technologies, Inc., Grand Island, NY, utilizing information generated by the sequencing of USA), 8 ␮l of fugene, 0.02 ␮g of pRL-Null Renilla expression the Plasmodium falciparum genome (3D7) (35). A search plasmid (Promega, Madison, WI, USA), 0.25 ␮g of pGL3 5ϫ ␮ of the Plasmodium falciparum genome for SPP homologs ATF6 reporter plasmid, 0.25 g of pAG3 SPPsub plasmid, and identified only one SPP gene that encodes a putative the indicated amount of a SPP construct expression plasmid ϳ or control plasmid to total 2 ␮g of DNA in each well of 12-well protein (NP_702432) that is 50% homologous to ␮ human SPP. As SPP homologs in C. elegans, Drosophila, plate. Each well of cells were lysed using 100 l of passive lysis buffer (Promega) firefly and Renilla luciferase activities were and Zebrafish appear essential for development, we measured using the Dual-Luciferase® kit (Promega) and a hypothesized that given the complex life cycle of ma- Veritas microplate Luminometer (Turner Biosystems, Sunny- laria, a single malarial SPP may be a novel therapeutic vale, CA, USA) with Veritas 2.0.40 software package. Trans- target. Therefore, we synthesized a full-length cDNA fections were performed in triplicate. Results were normal- encoding this gene and have expressed the malarial ized to the Renilla luciferase activity control. In some SPP (mSPP) in mammalian cells. experiments, where substrate was analyzed by Western blot, transfections were modified such that 2 ␮gofSPP and 2 ␮g In this study we used a cell-based reporter assay for sub of SPPL3 FLAG plasmids were used. intramembrane cleavage of type II proteins to assess NT proteolytic activity of two human homologs of SPP, Antibodies and Western blot SPPL3 and SPPL2b and mSPP. Using this assay we found that SPPL3 and mSPP cleave the type II mem- Anti-V5 (Invitrogen, San Diego, CA, USA) and anti-FLAG brane reporter construct but that SPPL2b, a more (Sigma) antibodies were used at 1:1000. Anti-␤-actin antibody distant homologue, did not. We also show that inhibi- (Ab) (Sigma) was used as a loading control at 1:1000. The tors of human SPP and SPPL3 also inhibited the mSPP. anti-ATF6 Ab (Imgenex, San Diego, CA, USA) was raised to the first 273 amino acids and recognizes the NH2-terminal, pre-transmembrane portion of our substrate. This Ab was used at 1:150. Antipeptide antisera were raised in rabbits to MATERIALS AND METHODS the carboxyl (anti-SPPL3ct, residues 364–384) terminal domains of human SPPL3 (Covance, Princeton, NJ, USA). DNA constructs Peptides corresponding to these domains of SPP were synthesized and coupled to keyhole limpet hemocyanin prior to immunization. SPP and the SPP substrate (SPP ) constructs were described Sub Cells were lysed in 1% triton X 100 (TX-100) with 1ϫ previously (12, 20). Full-length SPPL3 was cloned by amplify- complete protease inhibitor (Roche, Nutley, NJ, USA) unless ing a 1188 bp sequence from a human brain cDNA library. otherwise stated. Cell lysates were then spun at 14,000 rpm for The wild-type SPPL3 cDNA contains the entire coding se- 2 min to remove nuclei. Bio-Rad XT loading buffer with quence of SPPL3, and 17 bases of 5Ј untranslated sequence reducing solution was added to each sample. SDS-PAGE was and 10 bases of 3Ј untranslated sequence, and was cloned into performed using Bio-Rad Criterion gel system. 10% Bis-Tris HindIII (5Ј) and XhoI(3Ј) sites of the pFLAG-cytomeglovi- XT gels were used unless otherwise stated with Bio-Rad MES rus-2 (Sigma, St. Louis, MO, USA) to generate an amino- buffer. Gels were transferred to Millipore lowfluor PVDF for terminally FLAG epitope-tagged SPPL3 (SPPL3- FLAG). In NT 90 min and 160 V. Membranes were blocked in a casein SPPL3- FLAG, full-length SPP was cloned into the HindIII NT solution and primary antibodies were used at the reported (5Ј) and XhoI(3Ј) sites of pFLAG-cytomeglovirus-2 such that a concentration in the blocking solution overnight at 4°C. vector-derived three amino acid spacer (LLA) separates the Odyssey secondary antibodies containing either the 680 or FLAG tag from the amino terminus of SPPL3. Full-length 800 fluorophore were incubated with the membrane for 1 h SPPL2b was cloned by amplifying a 1779 bp sequence from a at room temperature at 1:20,000. Fluorescently labeled pro- human brain cDNA library. The wild-type SPPL2b cDNA tein detection was performed using the Odyssey Scanner. contains the entire coding sequence of SPPL2b and six bases of 5Ј untranslated sequence and six bases of 3Ј untranslated sequence, and was cloned into HindIII (5Ј) and XbaI(3Ј) sites Gradient fractionation of the pAG3 expression vector (36). Full-length SPP was also cloned into the pcDNA6-V5-his vector (Invitrogen, San Di- Sucrose gradients were run (37) and glycerol velocity gradi- ego, CA, USA) to generate a carboxyl terminally V5 ϩ 6 X his ents were run as described previously (38). Briefly, HEK cells

epitope tagged SPP (SPPL2bCTV5). All constructs were veri- stably expressing the SPPCTV5 or SPPL2bCTV5 constructs (5 ﬁed by sequencing. conﬂuent 150 mM plates) were lysed in either CHAPSO or

1672 Vol. 20 August 2006 The FASEB Journal NYBORG ET AL. TX-100. Lysates were spun at 3220 g to remove nuclei, insoluble material, and cellular debris and 1 ml of superna- tant was loaded onto the top of an 11 ml 10–40% linear glycerol gradient with either 0.5% CHAPSO 150 mM NaCl and 25 mM HEPES or 0.1% TX-100 in TBS. The samples were then spun in a SW 41 rotor for 15 h at 110,000 g at 4°C. One ml fractions were collected from the top and analyzed by SDS-PAGE for SPP or SPPL2b proteins. Control gradients were prepared identically but included a combination of commercially available, characterized molecular weight stan- dards (SERVA).

Immunoprecipitation

HEK 293T cells were transiently transfected using a calcium ␮ phosphate transfection method (39). 67 g of SPPL3NTFLAG plasmid was added to each 15 cm plate. Cells were lysed in 1% TX-100 (1ϫTBS) and immunoprecipitation (IP) was performed as described (40).

Data analysis

Data were analyzed using Sigma Stat. For comparison of multiple experimental values relative to controls an ANOVA was performed using a Dunnet’s post hoc t test. Variance is reported as the standard error of the mean.

RESULTS

Sequence analysis and expression of human SPP family members and mSPP

Phylogenetic tree analyses have been used to show that the SPP family members can be placed into two families (13). SPPL3, also known as PSH1 (25) and IMP2 (26), Figure 1. mSPP is most closely related to SPP. a) A phylogenetic tree was generated using the Align X feature in Vector is the nearest evolutionary homologue to SPP (13). @ SPPL2b, also known as PSH4 (25) and IMP4 (26), is a NTI . Percent identity scores were reported relative to mSPP. mSPP is most closely related to SPP and SPPL3 whereas more distant relative of SPP and SPPL3 (13). In fact, SPPL2a, b, and c are more closely related to each other. b) HEK SPPL2a, b, and c are more closely related to each other 293T cells were transiently transfected with SPPNTFLAG, than SPP and SPPL3. We have generated a phyloge- SPPL3NTFLAG, SPPL2bCTV5, or mSPPCTV5. Cells were lysed netic tree to determine the relative identity between in 1% triton X 100; lysates were run on SDS-PAGE and human SPP family members and mSPP. The single SPP Western blots were probed with the antibodies listed. gene present in Plasmodium falciparum (NP_702432) is most closely related to SPP and SPPL3 (30% identity) cipitated SPPL3 FLAG with an anti-SPPL3 Ab raised and is more distantly related to SPPL2a, b, and c (Fig. NT against the COOH terminus of SPPL3. The immuno- 1a). To further characterize these SPP homologs, we precipitated material was then run on SDS-PAGE and expressed SPP, SPPL3, SPPL2b, and mSPP in mammalian cells. Western blotted with anti-FLAG Ab (FLAG epitope is at the NH2 terminus of SPPL3). Two anti-FLAG positive The SPP glycoprotein has been shown to run on ϳ ϳ SDS-PAGE as both a monomer (45 kDa) and a dimer bands were observed at 60 and 30 kDa, indicating (90 kDa) (20), as seen in Fig. 1b. Overexpression of that both contain the SPPL3 holo-protein (data not shown). SPPL2bCTV5 is observed on a Western blot as a SPPL3NTFLAG and analysis by SDS-PAGE and Western blot lead to a predominant band detected at ϳ60 kDa group of diffuse bands from 65 to 95 kDa (Fig. 1b; see (Fig. 1b). In experiments where the amount of Fig. 3b), which is consistent with the immature and mature forms of SPPL2b observed previously (29). One SPPL3NTFLAG was highly overexpressed, a signiﬁcant increase in both the ϳ60 kDa and a smaller ϳ30 kDa predominant and speciﬁc band was detected for ϳ band was observed (13, 29). We suspected that, like mSPPCTV5 by Western blot analysis at 35 kDa (Fig. ϳ SPP, SPPL3NTFLAG might exist as both a monomer 1b). A minor 70 kDa was also detected, although the and homodimer. SPPL3 is predicted to be ϳ38 kDa, ratios of these bands tend to vary from experiment to which is slightly larger than observed by SDS-PAGE and experiment (Fig. 1b; see Fig. 4b). These are consistent Western blot. To show that both the ϳ30 and the ϳ60 with monomer and dimer formation; however, in the kDa bands contain full-length SPPL3, we immunopre- case of mSPP we predominantly detect the smaller ϳ35

INTRAMEMBRANE PROTEOLYSIS BY SPPL3 AND mSPP 1673 kDa band, indicating that the dimer may either be less prevalent or less stable in SDS than human SPP.

Functional SPP activity analyses of SPPL3, SPPL2b, and mSPP

The SPP reporter assay utilizes a SPP cleavable type II membrane domain fused to the COOH terminus of ATF6 transcription factor (SPPsub) (12). Upon cleavage, ATF6 is released from the membrane, translocates to the nucleus, and activates a luciferase reporter construct (12, 41). To determine whether the SPPsub ϫ could be cleaved by SPPL3NTFLAG and activate the 5 ATF6 luciferase reporter construct, we examined whether increased expression of SPPL3NTFLAG would increase the luciferase activity. For these studies, we transiently transfected 293T cells with increasing amounts of SPPL3NTFLAG, as indicated in Fig. 2a, and performed the luciferase assay as described in Materials and Methods. As increasing amounts of SPPL3NTFLAG expression vector were transfected, a significant increase in luciferase activity was observed (Fig. 2a). Addition of a SPP inhibitor (ZLL)2-ketone (ZLL) or LY411,575, a ␥-secretase inhibitor that has been shown to inhibit SPP, inhibited the increase in luciferase activity due to SPPL3NTFLAG overexpression. When comparable amounts of SPPNTFLAG or SPPL3NTFLAG were detected by anti-FLAG Western blot, a similar increase in luciferase activity was observed (Fig. 2a). The increase in activity observed, as an increasing amount of SPPL3NTFLAG expression plasmid was transfected, corresponded to an increase in the amount of SPPL3NTFLAG detected by Western blot (Fig. 2b). The reporter construct for the SPP cell-based assay is a5ϫ repeat of the ATF6 binding region coupled to the luciferase promoter. In the event of a significant en- Figure 2. SPPL3 cleaves a SPP substrate. a) Luciferase activity dogenous unfolded protein response, as a result of increased with increasing SPPL3NTFLAG protein expression. overexpressing a foreign protein there is the potential SPPL3NTFLAG expression plasmid was cotransfected into for endogenous ATF6 NH2-terminal signal peptide HEK 293T cells at the concentrations reported and as de- binding to the 5ϫ ATF6 reporter construct contribut- scribed in Materials and Methods. Normalized reporter activ- ing to an artificially high luciferase signal. Assays with- ity expressed as % of the activity present in cells expressing out SPP were performed to confirm that the increase endogenous levels of SPP (% endogenous activity) was plot- sub ted. SPP inhibitor, ZLL (0.2 ␮M), and ␥-secretase inhibitor, in activity seen as a result of overexpression of SPP LY411,575 (50 nM) both inhibited SPPL3NTFLAG activity constructs was due to SPPSub cleavage and not the result significantly. SPP activity levels increased proportionally with of an endogenous ATF6 unfolded protein response. As increasing levels of SPPNTFLAG expression plasmid. Western seen in Fig. 3a, the significant increase in activity sample were prepared from passive lysis buffer lysates used for observed was due to the inclusion of the SPP . When luciferase measurements. Two distinct SPPL3NTFLAG band Sub ϳ ϳ were observed at 60 kDa and 30 kDa. The SPPNTFLAG was an empty vector was substituted for the SPPsub,no ϳ significant luciferase activity was observed as a result of observed as the dimer (labeled) at 95 kDa and monomer (labeled) at ϳ45 kDa as detected with anti-FLAG. *P Ͻ 0.05. SPPNTFLAG, SPPL3NTFLAG, or SPPL2bCTV5 overex- b) Luciferase activity increases with increasing SPPNTFLAG pression, indicating that these constructs did not elicit protein expression. Normalized reporter activity expressed as a significant endogenous ATF6 unfolded protein re- a % of the activity present in cells expressing endogenous sponse. levels of SPP (percent over endogenous activity) were plotted vs. SPPL3 FLAG protein levels expressed as a fold detected Unlike SPPL3 and SPP, overexpression of SPPL2bCTV5 NT over background (Fold SPPL3 detected over background). did not activate the SPPsub to generate significant activity above endogenous activity (Fig. 3a). In our hands anti-V5 and anti-FLAG recognize the SPP epitope tagged con- The increase in luciferase activity as a result of structs with relatively similar affinities, suggesting that the SPPL3NTFLAG overexpression was further examined by SPPL2b levels may have been slightly higher than SPP and Western blot, the SPPSub with anti-V5. Previously we SPPL3 (Fig. 3b). demonstrated that the anti-V5 signal increased as a

1674 Vol. 20 August 2006 The FASEB Journal NYBORG ET AL. Figure 3. SPPL2b does not cleave the SPP substrate and unfolded protein response does not interfere with the assay. a) Overexpression

of either SPPNTFLAG or SPPL3NTFLAG sig- niﬁcantly increased luciferase activity (% endogenous activity) observed in the reporter assay. Conversely, overexpression of either

SPPL2bCTV5 or SPPL2b (not shown) did not signiﬁcantly increase SPP luciferase activity.

Overexpression of SPPNTFLAG, SPPL3NTFLAG, or SPPL2bCTV5 did not contribute to luciferase activity observed by eliciting an endogenous unfolded protein response. This was deter- mined by overexpressing the SPP construct in the presence of 5ϫ ATF6 reporter but an Ͻ absence of SPPsub.*P 0.05. b) Samples that did not contain SPPsub (lysed in passive lysis buffer) were run on SDS-PAGE and probed by

Western blot with anti-FLAG (SPPNTFLAG and SPPL3NTFLAG) and anti-V5 (SPPL2bCTV5). The amount of SPP and SPPL3 levels were relatively comparable. In our hands anti-V5 and anti-FLAG recognize the SPP epitope tagged constructs with relatively similar afﬁnities, suggesting that the SPPL2b levels may have been slightly higher than SPP and SPPL3. c, d) Previ- ously we showed that unmasking of the V5 epitope was a surrogate marker for SPP cleav-

age (12). The amount anti-V5 positive of SPPsub detectable increased with increasing SPPNT- FLAG expression (12). Though SPPsub levels remained relatively constant (as detected by

anti-ATF6) SPPsub detected by anti-V5 (anti-V5 relative light units, RLU; % increase) increased

signiﬁcantly when SPPL3NTFLAG was overex- Ͻ pressed. *P 0.05. e) SPPsub cleavage was observed directly in cells not transfected with either SPP or SPPL3 by probing the SPPsub with anti-V5. An increase in anti-V5 detected SPPSub was observed when either SPPNTFLAG or SPPL3NTFLAG were transiently overexpressed. The increase in the soluble anti-V5 detectable SPPsub decreased as a result of inhibition by ZLL (an SPP inhibitor).

result of SPPSub cleavage, making the V5 epitope a good and overexpressed SPP with an epitope tag ran compa- indicator for both cleavage and solubilization of SPPSub rably on glycerol velocity gradients (20). We found that (12). An increase in the anti-V5 detectable SPPSub was the SPP dimer distributed primarily in the 100–160 kDa observed as a result of SPPNTFLAG or SPPL3NTFLAG range, with a small portion of the SPP dimer sediment- overexpression (Fig. 3c, d). As shown before, the anti- ing at higher molecular weight. ATF6 detectable substrate remained constant but the To explore whether SPP or SPPL2b exists as high anti-V5 detectable SPPsub increased when conditions molecular weight complexes, we analyzed the distribu- favor substrate cleavage (Fig. 3c). The cleavage de- tion of SPP and SPPL2b in glycerol velocity gradients in tected by anti-V5 Western blot was inhibited by SPP the presence of TX-100 or CHAPSO detergent and inhibitor ZLL (Fig. 3e). A doublet of the SPPsub (pre- compared both to exogenous molecular weight marker sumably phosphorylated and unphosphorylated ATF6 proteins and the distribution of endogenous PS1. A NH terminus; ref 42) was resolved and detected on the similar glycerol velocity sedimentation of SPP was ob- Western blot when a 10% Bis-Tris gel was used (Fig. 3c) served when cells overexpressing SPPCTV5 were lysed in as apposed to the single band observed for the substrate either CHAPSO or TX-100 (Fig. 4a). These results resolved on a 4–15% Tris-HCl gel (Fig. 3e). contrast with the distribution of PS1 under varying detergent lysis conditions. Under conditions that main- Glycerol velocity gradient fractionation and sucrose tain ␥-secretase activity (CHAPSO lysis), the NTF and gradient fractionation of SPPL2b and SPP CTF of PS1 sediment to high molecular weight fraction (Ͼ200 kDa, Fig. 4a). Using detergents that disrupt and Glycerol velocity gradient fractionation can be used to inactivate the ␥-secretase complex (TX-100), PS1 is provide estimates of the molecular weight of proteins found in lower molecular weight fractions. Like PS1, and protein complexes. This technique provided initial SPPL2b appears to exist as a high molecular weight evidence that PS1 existed in a high molecular weight complex that is detergent sensitive. In the presence of complex (38). Previously we showed that both weight TX-100, SPPL2bCTV5 is observed from 25 to 160 kDa

INTRAMEMBRANE PROTEOLYSIS BY SPPL3 AND mSPP 1675 performed due to our inability to generate a SPPL3 overexpressing stable cell line. A variety of different SPPL3 plasmids encoding SPPL3 with various epitope tags at either the NH2 terminus or the COOH terminus have been generated. In all cases these plasmids appear to express SPPL3 transiently in a variety of different cell lines (CHO, HEK, and H4). Though SPPL3 can be overexpressed transiently, stable cell lines are devoid of detectable SPPL3. Fractionation studies were at- tempted by transiently overexpressing SPPL3 and SPP. However, transient overexpression of SPP gave fractionation results that were completely inconsistent with the stable cell lines and endogenous results previously generated and essentially uninterruptible (not shown).

mSPP cleaves the SPPsub and is inhibited by SPP inhibitors

Only one SPP homologue is encoded by the Plasmodium Falciparum genome. We synthesized a cDNA encoding the entire predicted open reading frame (NP_702432) that was codon optimized for expression in mammalian Figure 4. Sucrose gradient fractionation and glycerol velocity gradient sedimentation of SPP and SPPL2b. a) 10–40% cells. The cDNA was then cloned into a mammalian linear, glycerol velocity gradients of HEK cells stably overex- expression vector and either transiently or stably pressing SPPCTV5 or SPPL2bCTV5 lysed in CHAPSO or transfected into mammalian cells. Overexpression of TX-100 were analyzed by SDS-PAGE on 4–15% Tris-HCl gels mSPPCTV5 caused a significant increase in the SPPsub and probed with anti-V5. SPPCTV5 dimer sediments in frac- luciferase activity (Fig. 5a). The increase in activity ϳ tions consistent with 160 kDa complex and a monomer that observed due to mSPP V5 overexpression was inhib- sediments in even smaller fractions (not shown). SPP V5 CT CT itable with either LY411,575 or ZLL, both of which overexpressing cells lysed in either CHAPSO or TX-100 show have been shown to effectively inhibit SPP (12) and a similar sedimentation, but SPPL2bCTV5 sedimentation dif- fers significantly when the cells are lysed in CHAPSO instead SPPL3 activity (Figs. 2, 3). Overexpression of of TX-100. Similarly, significant differences in the PS1 frag- mSPPCTV5 did not generate an endogenous un- ment sedimentation are observed in the two different lysis folded protein response that falsely signals the 5ϫ conditions. Gradient molecular weight kDa at bottom refers ATF6 reporter as observed by expression of the to control proteins loaded on a separate but identically reporter construct and mSPPCTV5 but no SPPsub in poured gradients. Lysate and control gradients have been Fig. 5a. In addition, a significant increase in the V5 performed in triplicate. Comparable results were observed for endogenous SPP from HEK cell shown previously (20). b) epitope of the SPPsub was observed as a result of mSPP V5 overexpression (Fig. 5b, c). Two specific HEK cells stably overexpressing SPPCTV5 or SPPL2bCTV5 CT were lysed in 2% CHAPSO and sucrose gradient fractionated. bands were observed for mSPPCTV5 by Western blot Fractions were collected from the top making 1–4 buoyant analysis at ϳ35 kDa and ϳ70 kDa (Fig. 5b). fractions and 9–12 dense fractions. All fractions were analyzed by SDS-PAGE on 4–15% Tris-HCl gels and probed with anti-V5. Much like PS1, SPPCTV5 and SPPL2bCTV5 fraction- ate to buoyant fractions 4, 5, and 6. DISCUSSION

Using a cell-based reporter assay for SPP, we provide with a small amount of SPPL2b in the higher molecular strong evidence that the nearest evolutionary homo- weight range. In contrast, SPPL2bCTV5 fractionated in logue of SPP, SPPL3, has proteolytic activity. Reporter the presence of CHAPSO migrates from 160 to 450 activity and substrate cleavage (detected by anti-V5 kDa. These results are reminiscent of PS1 and suggest analysis) both increased with increasing SPPL3NTFLAG that SPPL2b may exist as a higher molecular weight expression. Moreover, inhibitors of SPP activity inhib- complex. ited SPPL3 activity. It has been demonstrated that ␥-secretase activity and Though all members of the SPP family have been PS1 are enriched by sucrose gradient fractionation to speculated to be I-CLiPs, overexpression of SPPL2b did buoyant cholesterol rich membranes, also known as not increase SPPsub luciferase activity. Opposite active lipid rafts (37, 43). These buoyant membrane are also site topology between presenilin and SPP has been enriched in GS27, Flotilin, and other raft markers (37, thought to be the key factor responsible for their 43). We found that the majority of SPPCTV5 and differences in cleaving type II membrane proteins and SPPL2bCTV5 are enriched in the these same buoyant type I membrane proteins, respectively (5, 6, 11). All membrane fractions (Fig. 4b). SPP family members appear to have similar topological Comparable experiments with SPPL3 could not be orientation (11–13). Hence, SPPL2b contains the ap-

1676 Vol. 20 August 2006 The FASEB Journal NYBORG ET AL. parent active site orientation necessary for SPPsub cleavage to occur, but no cleavage was detected. These data appear to be consistent with genetic data from Ze- brafish, demonstrating that when the SPPL2b gene was knocked down a unique phenotype was observed relative to the SPP and SPPL3 knockdown phenotype. The absence of detectable substrate cleavage by SPPL2b may be attributable to its inability to cleave the substrate or a lack of colocalization of the SPPsub and SPPL2b. SPP and SPPL3 were both shown to localize to the ER (29), similar to ATF6 (44). However, SPPL2b was found predominantly in vesicular structures, which were positive for LAMP-2, a marker for late endo- somes/lysosomes (29), implying that even if SPPL2b could theoretically cleave the SPPsub, substrate and active protease may not localize to the same cellular compartment. Much like PS, SPPL2b may exist as a high molecular weight complex that is sensitive to various detergents. As seen in the glycerol velocity gradient fractionation, SPPL2b fractionated to a much higher molecular weight when the lysate and sedimentation were performed in the presence of CHAPSO. It is possible that, like PS1, SPPL2b has additional cofactors. In contrast to this, SPP did not appear to be sensitive to either detergent used in these experiments and runs on a glycerol velocity gradient from 100 to 160 kDa. These data do not exclude the possibility that SPP exists as a higher molecular weight complex and has cofactors, but indicate that if cofactors of SPP exist they have different solubilization properties from PS and SPPL2b. Having established a method for assessing whether SPP candidate enzymes exhibit SPP activity in vivo,we cloned the only SPP gene identifiable in the malaria genome and tested for activity. mSPP contains the hallmark features of a SPP protein in that it has multiple transmembrane domains, active site YD/ GXGD aspartates in adjacent transmembrane domains, and a PAL motif near the COOH terminus. Each of these features has been shown to be critical for both presenilin and SPP function (45). The mSPP gene is most closely related to SPP and SPPL3. We found that luciferase activity increased due to cleavage of the SPPsub by mSPPCTV5 overexpression. The increase in activity was inhibitable by both a SPP inhibitor, ZLL, and potent ␥-secretase inhibitor, LY411,575, demonstrating the potential to target mSPP as an antimalaria therapy. As the Malaria parasite becomes increasingly resis- Figure 5. mSPP cleaves the SPPsub. a) Overexpression of tant to traditional drugs, the need for novel targets is mSPPCTV5 provided a significant increase in luciferase increasing. The potential to target novel malarial aspar- SPP activity relative to endogenous activity (expressed sub tyl proteases has been established in the plasmepsin and % control). The activity was inhibitable by two SPP inhibitors (ZLL and LY411,575). *P Ͻ 0.05. b, c) Unmask- protein family, and inhibitors in the low nanomolar ing of the V5 epitope was shown to be a surrogate marker range have shown potential in cell culture and animal for SPPsub cleavage (12). The amount anti-V5 positive of models (46–48). In addition, recent studies have SPPsub detectable increased as a result of mSPPCTV5 over- shown that clinically utilized HIV protease inhibitors expression (12) (anti-V5 relative light units, RLU; % ϳ can inhibit the in vitro growth of Plasmodium falciparum increase). The SPPsub substrate was detectable at 53 kDa. ϳ ϳ at or below concentrations found in human plasma The mSPPCTV5 was detected at 35 kDa and 70 kDa. *P Ͻ 0.05. after oral drug administration (49). We hypothesize that mSPP may be a novel drug target because elimina-

INTRAMEMBRANE PROTEOLYSIS BY SPPL3 AND mSPP 1677 tion or inhibition of SPP activity causes embryonic 5. Martoglio, B., and Golde, T. E. (2003) Intramembrane-cleaving death in C. elgans, Drosophila, and Zebrafish even aspartic proteases and disease: presenilins, signal peptide peptidase and their homologs. Hum. Mol. Genet. 12 Spec. No. 2, though each contains at least two SPP homologs (27– R201–R206 29). Thus, it is possible that mSPP is a critical gene for 6. Xia, W., and Wolfe, M. S. (2003) Intramembrane proteolysis by malaria development. If this is the case, then the presenilin and presenilin-like proteases. J. Cell Sci. 116, 2839– 2844 pharmacologically inhibition of mSPP may be lethal to 7. Wang, J., Brunkan, A. L., Hecimovic, S., Walker, E., and Goate, the parasite. As shown here, mSPP is targetable by A. (2004) Conserved “PAL” sequence in presenilins is essential drug-like compounds. Moreover, numerous inhibitors for gamma-secretase activity, but not required for formation or of ␥-secretase and SPP have been reported in the stabilization of gamma-secretase complexes. Neurobiol. Dis. 15, 654–666 literature, and the pharmaceutical industry has gener- 8. Golde, T. E., and Eckman, C. B. (2003) Physiologic and ated thousands of compounds that target ␥-secretase. pathologic events mediated by intramembranous and jux- Some of these compounds have moved into clinical tamembranous proteolysis. Sci. STKE 2003, RE4 trials, and those that target ␥-secretase do so with 9. Lemberg, M. K., and Martoglio, B. (2004) On the mechanism of SPP-catalysed intramembrane proteolysis; conformational con- incredible potency (subnanomolar IC50s) and excellent trol of peptide bond hydrolysis in the plane of the membrane. tissue distribution, including the central nervous system FEBS Lett. 564, 213–218 (21, 50, 51). 10. Martoglio, B. (2003) Intramembrane proteolysis and post-targeting functions of signal peptides. Biochem. Soc Trans. 31, Due to potential toxicity associated with complete 1243–1247 ␥-secretase inhibition, it would be ideal to develop 11. Weihofen, A., Binns, K., Lemberg, M. K., Ashman, K., and compounds that selectively target mSPP. SPP and pre- Martoglio, B. (2002) Identification of signal peptide peptidase, senilin activities are differentially inhibitable as demon- a presenilin-type aspartic protease. Science 296, 2215–2218 ␥ 12. Nyborg, A. C., Jansen, K., Ladd, T. B., Fauq, A., and Golde, T. E. strated by two structurally similar -secretase inhibitors, (2004) A signal peptide peptidase (SPP) reporter activity assay DAPT and LY411,575, having very different effects on based on the cleavage of type II membrane protein substrates SPP inhibition. LY411,575 is an effective inhibitor of provides further evidence for an inverted orientation of the SPP SPP but DAPT is not (12, 52). As the overall homology active site relative to presenilin. J. Biol. Chem. 279, 43148–43156 13. Friedmann, E., Lemberg, M. K., Weihofen, A., Dev, K. K., of mSPP with human SPP or SPPL3 is low, it should be Dengler, U., Rovelli, G., and Martoglio, B. (2004) Consensus possible to identify compounds that selectively inhibit analysis of signal peptide peptidase and homologous human mSPP and not human SPP. Finally, a similar analysis of aspartic proteases reveals opposite topology of catalytic domains compared with presenilins. J. Biol. Chem. 279, 50790– the genomes of other human parasites that cause 50798 significant morbidity and mortality reveals that each 14. Yu, G., Nishimura, M., Arawaka, S., Levitan, D., Zhang, L., contains a single SPP homologue (Leishmaniasis Tandon, A., Song, Y. Q., Rogaeva, E., Chen, F., Kawarai, T., et al. XP_847754, Trypanosomiasis EAN96276, and Giardia (2000) Nicastrin modulates presenilin-mediated notch/glp-1 signal transduction and betaAPP processing [see comments]. EAA41466). Like malaria, these genes all have greater Nature 407, 48–54 sequence homology to SPP than other SPPs. Thus, if it 15. Francis, R., McGrath, G., Zhang, J., Ruddy, D. A., Sym, M., can be shown that mSPP is a good drug target, similar Apfeld, J., Nicoll, M., Maxwell, M., Hai, B., Ellis, M. C., et al. (2002) aph-1 and pen-2 are required for Notch pathway signal- techniques and reagents could be developed to target ing, gamma-secretase cleavage of betaAPP, and presenilin pro- multiple human parasitic pathogens. The SPP assay tein accumulation. Dev. Cell. 3, 85–97 used to generate the data in this manuscript, or a close 16. Edbauer, D., Winkler, E., Regula, J. T., Pesold, B., Steiner, H., variant, should be useful in developing testing potential and Haass, C. (2003) Reconstitution of gamma-secretase activity. Nat. Cell Biol. 5, 486–488 inhibitors. 17. Takasugi, N., Tomita, T., Hayashi, I., Tsuruoka, M., Niimura, M., Takahashi, Y., Thinakaran, G., and Iwatsubo, T. (2003) The role This work was supported by the Mayo Foundation and an of presenilin cofactors in the gamma-secretase complex. Nature NIH/NINDS grant NS39072 to T.E.G., NS44734 to A.C.N. 422, 438–441 pCGNATF6 1–373 plasmid, and the pGL3 5ϫ ATF6 luciferase 18. Kimberly, W. T., LaVoie, M. J., Ostaszewski, B. L., Ye, W., Wolfe, reporter plasmids were generously provided by Ron Prywes. M. S., and Selkoe, D. J. (2003) Gamma-secretase is a membrane The authors have declared that no competing interests protein complex comprised of presenilin, nicastrin, Aph-1, and Pen-2. Proc. Natl. Acad. Sci. USA 100, 6382–6387 exist. 19. Marlow, L., Canet, R. M., Haugabook, S. J., Hardy, J. A., Lahiri, D. K., and Sambamurti, K. (2003) APH1, PEN2, and Nicastrin increase Abeta levels and gamma-secretase activity. Biochem. Biophys. Res. Commun. 305, 502–509 20. 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C., Accepted for publication March 31, 2006.

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