A C-terminal region of signal peptide peptidase defines a functional domain for intramembrane aspartic protease catalysis

Intramembrane proteolysis is now firmly established as a prominent biological process, and structure elucidation is emerging as the new frontier in the understanding of these novel membrane-embedded enzymes. Reproducing this unusual hydrolysis within otherwise water-excluding transmembrane regions with purified proteins is a challenging prerequisite for such structural studies. Here we show the bacterial expression, purification, and reconstitution of proteolytically active signal peptide peptidase (SPP), a membrane-embedded enzyme in the presenilin family of aspartyl proteases. This finding formally proves that, unlike presenilin, SPP does not require any additional proteins for proteolysis. Surprisingly, the conserved C-terminal half of SPP is sufficient for proteolytic activity; purification and reconstitution of this engineered fragment of several SPP orthologues revealed that this region defines a functional domain for an intramembrane aspartyl protease. The discovery of minimal requirements for intramembrane proteolysis should facilitate mechanistic and structural analysis and help define general biochemical principles of hydrolysis in a hydrophobic environment.     

Signal peptide peptidase and gamma-secretase share equivalent inhibitor binding pharmacology

The enzyme gamma-secretase has long been considered a potential pharmaceutical target for Alzheimer disease. Presenilin (the catalytic subunit of gamma-secretase) and signal peptide peptidase (SPP) are related transmembrane aspartyl proteases that cleave transmembrane substrates. SPP and gamma-secretase are pharmacologically similar in that they are targeted by many of the same small molecules, including transition state analogs, non-transition state inhibitors, and amyloid beta-peptide modulators. One difference between presenilin and SPP is that the proteolytic activity of presenilin functions only within a multisubunit complex, whereas SPP requires no additional protein cofactors for activity. In this study, gamma-secretase inhibitor radioligands were used to evaluate SPP and gamma-secretase inhibitor binding pharmacology. We found that the SPP enzyme exhibited distinct binding sites for transition state analogs, non-transition state inhibitors, and the nonsteroidal anti-inflammatory drug sulindac sulfide, analogous to those reported previously for gamma-secretase. In the course of this study, cultured cells were found to contain an abundance of SPP binding activity, most likely contributed by several of the SPP family proteins. The number of SPP binding sites was in excess of gamma-secretase binding sites, making it essential to use selective radioligands for evaluation of gamma-secretase binding under these conditions. This study provides further support for the idea that SPP is a useful model of inhibitory mechanisms and structure in the SPP/presenilin protein family.     

Signal peptide peptidases: A family of intramembrane-cleaving proteases that cleave type 2 transmembrane proteins

Five genes encode the five human signal peptide peptidases (SPPs), which are intramembrane-cleaving aspartyl proteases (aspartyl I-CLiPs). SPPs have been conserved through evolution with family members found in higher eukaryotes, fungi, protozoa, arachea, and plants. SPPs are related to the presenilin family of aspartyl I-CLiPs but differ in several key aspects. Presenilins (PSENs) and SPPs both cleave the transmembrane region of membrane proteins; however, PSENs cleave type 1 membrane proteins whereas SPPs cleave type 2 membrane proteins. Though the overall homology between SPPs and PSENs is minimal, they are multipass membrane proteins that contain two conserved active site motifs YD and GxGD in adjacent membrane-spanning domains and a conserved PAL motif of unknown function near their COOH-termini. They differ in that the active site YD and GxGD containing transmembrane domains of SPPs are inverted relative to PSENs, thus, orienting the active site in a consistent topology relative to the substrate. At least two of the human SPPs (SPP and SPPL3) appear to function without additional cofactors, but PSENs function as a protease, called γ-secretase, only when complexed with Nicastrin, APH-1 and Pen-2. The biological roles of SPP are largely unknown, and only a few endogenous substrates for SPPs have been identified. Nevertheless there is emerging evidence that SPP family members are highly druggable and may regulate both essential physiologic and pathophysiologic processes. Further study of the SPP family is needed in order to understand their biological roles and their potential as therapeutic targets.     

Signal peptide peptidase forms a homodimer that is labeled by an active site-directed gamma-secretase inhibitor

Presenilin (PS) is the presumptive catalytic component of the intramembrane aspartyl protease gamma-secretase complex. Recently a family of presenilin homologs was identified. One member of this family, signal peptide peptidase (SPP), has been shown to be a protease, which supports the hypothesis that PS and presenilin homologs are related intramembrane-cleaving aspartyl proteases. SPP has been reported as a glycoprotein of approximately 45 kDa. Our initial characterization of SPP isolated from human brain and cell lines demonstrated that SPP is primarily present as an SDS-stable approximately 95-kDa protein on Western blots. Upon heating or treatment of this approximately 95-kDa SPP band with acid, a approximately 45-kDa band could be resolved. Co-purification of two different epitope-tagged forms of SPP from a stably transfected cell line expressing both tagged versions demonstrated that the approximately 95-kDa band is a homodimer of SPP. Pulse-chase metabolic labeling studies demonstrated that the SPP homodimer assembles rapidly and is metabolically stable. In a glycerol velocity gradient, SPP sedimented from approximately 100-200 kDa. Significantly the SPP homodimer was specifically labeled by an active site-directed photoaffinity probe (III-63) for PS, indicating that the active sites of SPP and PS/gamma-secretase are similar and providing strong evidence that the homodimer is functionally active. Collectively these data suggest that SPP exists in vivo as a functional dimer.     

Signal peptide peptidase: biochemical properties and modulation by nonsteroidal antiinflammatory drugs

Signal peptide peptidase (SPP) is an intramembrane aspartyl protease that cleaves remnant signal peptides after their release by signal peptidase. SPP contains active site motifs also found in presenilin, the catalytic component of the gamma-secretase complex of Alzheimer's disease. However, SPP has a membrane topology opposite that of presenilin, cleaves transmembrane substrates of opposite directionality, and does not require complexation with other proteins. Here we show that, upon isolation of membranes and solubilization with detergent, the biochemical characteristics of SPP are remarkably similar to gamma-secretase. The majority of the SPP-catalyzed cleavages occurred at a single site in a synthetic substrate based on the prolactin (Prl) signal sequence. However, as seen with cleavage of substrates by gamma-secretase, additional cuts at other minor sites are also observed. Like gamma-secretase, SPP is inhibited by helical peptidomimetics and apparently contains a substrate-binding site that is distinct from the active site. Surprisingly, certain nonsteroidal antiinflammatory drugs known to shift the site of proteolysis by gamma-secretase also alter the cleavage site of Prl by SPP. Together, these findings suggest that SPP and presenilin share certain biochemical properties, including a conserved drug-binding site for allosteric modulation of substrate proteolysis.     

Differential localization and identification of a critical aspartate suggest non-redundant proteolytic functions of the presenilin homologues SPPL2b and SPPL3

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.     

C-terminal PAL motif of presenilin and presenilin homologues required for normal active site conformation

The Alzheimer's disease-associated beta-amyloid peptide is produced through cleavage of amyloid precursor protein by beta-secretase and gamma-secretase. gamma-Secretase is a complex containing presenilin (PS) as the catalytic component and three essential cofactors: Nicastrin, anterior pharynx defective (APH-1) and presenilin enhancer-2 (PEN-2). PS and signal peptide peptidase (SPP) define a novel family of aspartyl proteases that cleave substrates within the transmembrane domain presumptively using two membrane-embedded aspartic acid residues for catalysis. Apart from the two aspartate-containing active site motifs, the only other region that is conserved between PS and SPP is a PAL sequence at the C-terminus. Although it has been well documented that this motif is essential for gamma-secretase activity, the mechanism underlying such a critical role is not understood. Here we show that mutations in this motif affect the conformation of the active site of gamma-secretase resulting in a complete loss of PS binding to a gamma-secretase transition state analog inhibitor, Merck C. Analogous mutations in SPP significantly inhibit its enzymatic activity. Furthermore, these mutations also abolish SPP binding to Merck C, indicating that SPP and gamma-secretase share a similar active site conformation, which is dependent on the PAL motif. Exploring the amino acid requirements within this motif reveals a very small side chain requirement, which is conserved during evolution. Together, these observations strongly support the hypothesis that the PAL motif contributes to the active site conformation of gamma-secretase and of SPP.     

Inhibitory effect of presenilin inhibitor LY411575 on maturation of hepatitis C virus core protein,production of the viral particle and expression of host proteins involved in pathogenicity

Hepatitis C virus (HCV) core protein is responsible for the formation of infectious viral particles and induction of pathogenicity. The C‐terminal transmembrane region of the immature core protein is cleaved by signal peptide peptidase (SPP) for maturation of the core protein. SPP belongs to the family of presenilin‐like aspartic proteases. Some presenilin inhibitors are expected to suppress HCV infection and production; however, this anti‐HCV effect has not been investigated in detail. In this study, presenilin inhibitors were screened to identify anti‐HCV compounds. Of the 13 presenilin inhibitors tested, LY411575 was the most potent inhibitor of SPP‐dependent cleavage of HCV core protein. Production of intracellular core protein and supernatant infectious viral particles from HCV‐infected cells was significantly impaired by LY411575 in a dose‐dependent manner (half maximum inhibitory concentration = 0.27 μM, cytotoxic concentration of the extracts to cause death to 50% of viable cells > 10 μM). No effect of LY411575 on intracellular HCV RNA in the subgenomic replicon cells was detected. LY411575 synergistically promoted daclatasvir‐dependent inhibition of viral production, but not that of viral replication. Furthermore, LY411575 inhibited HCV‐related production of reactive oxygen species and expression of NADPH oxidases and vascular endothelial growth factor. Taken together, our data suggest that LY411575 suppresses HCV propagation through SPP inhibition and impairs host gene expressions related to HCV pathogenicity.     

Drosophila signal peptide peptidase is an essential protease for larval development

We identified the Drosophila melanogaster Signal peptide peptidase gene (Spp) that encodes a multipass transmembrane aspartyl protease. Drosophila SPP is homologous to the human signal peptide peptidase (SPP) and is distantly related to the presenilins. We show that, like human SPP, Drosophila SPP can proteolyze a model signal peptide and is sensitive to an SPP protease inhibitor and that it localizes to the endoplasmic reticulum. Expression of Drosophila SPP was first apparent at germ band extension, and in late embryos it was robust in the salivary glands, proventriculus, and tracheae. Flies bearing mutations in conserved residues or carrying deficiencies for the Spp gene had defective tracheae and died as larvae.     

Expression and characterization of Drosophila signal peptide peptidase-like (sppL), a gene that encodes an intramembrane protease

Intramembrane proteases of the Signal Peptide Peptidase (SPP) family play important roles in developmental, metabolic and signaling pathways. Although vertebrates have one SPP and four SPP-like (SPPL) genes, we found that insect genomes encode one Spp and one SppL. Characterization of the Drosophila sppL gene revealed that the predicted SppL protein is a highly conserved structural homolog of the vertebrate SPPL3 proteases, with a predicted nine-transmembrane topology, an active site containing aspartyl residues within a transmembrane region, and a carboxy-terminal PAL domain. SppL protein localized to both the Golgi and ER. Whereas spp is an essential gene that is required during early larval stages and whereas spp loss-of-function reduced the unfolded protein response (UPR), sppL loss of function had no apparent phenotype. This was unexpected given that genetic knockdown phenotypes in other organisms suggested significant roles for Spp-related proteases.     

The Human Hepatocyte Nuclear Factor 3/Fork Head Gene FKHL13: Genomic Structure and Pattern of Expression

We describe the isolation and characterization of the cDNA for FKHL13, the human homologue of the mouse hepatocyte nuclear factor 3/fork headhomologue 4 (HFH-4) gene, a member of the HNF-3/fork head(also called winged helix) gene family. Members of this gene family contain a conserved DNA binding region of approx. 110 amino acids and are thought to play an important role in cell-specific differentiation. Previous analysis of the mouse and rat HFH-4 cDNAs revealed a distinct pattern of expression for this gene, suggesting that the gene plays an important role in the differentiation of lung and oviduct/ampulla epithelial cells and testicular spermatids. Analysis of the human FKHL13 gene confirmed this pattern of expression. We also found expression in adult human brain cortex, which we were able to confirm for the mouse. The expression pattern of FKHL13/HFH-4, confined to cilia/flagella-producing cells, leads us to believe that the gene plays an important role in the regulation of axonemal structural proteins. We show that the human gene for FKHL13 lies on chromosome 17 (comparison with the chromosomal location of the mouse gene strongly suggests 17q22–q25) and that the gene, which is approx. 6 kb, contains a single intron disrupting thefork headDNA binding domain. Such a disruption of a functional unit provides strong evidence for the theory of intron insertion during gene evolution. The expression of the gene is probably controlled by the CpG island, which is located in the promoter region of the gene. We also demonstrate that the FKHL13 gene is highly conserved among a wide variety of species, including birds.     

The ancient source of a distinct gene family encoding proteins featuring RING and C(3)H zinc-finger motifs with abundant expression in developing brain and nervous system

Intronless genes can arise by germline retrotransposition of a cDNA originating as mRNA from an intron-containing source gene. Previously, we described several members of a family of intronless mammalian genes encoding a novel class of zinc-finger proteins, including one that shows imprinted expression and one that escapes X-inactivation. We report here the identification and characterization of the Makorin ring finger protein 1 gene (MKRN1), a highly transcribed, intron-containing source for this family of genes. Phylogenetic analyses clearly indicate that the MKRN1 gene is the ancestral founder of this gene family. We have identified MKRN1 orthologs from human, mouse, wallaby, chicken, fruitfly, and nematode, underscoring the age and conservation of this gene. The MKRN gene family encodes putative ribonucleoproteins with a distinctive array of zinc-finger motifs, including two to four C(3)H zinc-fingers, an unusual Cys/His arrangement that may represent a novel zinc-finger structure, and a highly conserved RING zinc-finger. To date, we have identified nine MKRN family loci distributed throughout the human genome. The human and mouse MKRN1 loci map to a conserved syntenic group near the T-cell receptor beta cluster (TCRB) in chromosome 7q34-q35 and chromosome 6A, respectively. MKRN1 is widely transcribed in mammals, with high levels in murine embryonic nervous system and adult testis. The ancient origin of MKRN1, high degree of conservation, and expression pattern suggest important developmental and functional roles for this gene and its expressed family members.     

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.     

Identification and characterization of a novel human sphingosine-1-phosphate phosphohydrolase,hSPP2

Sphingosine 1-phosphate (S1P) is a bioactive lipid molecule that acts as both an extracellular signaling mediator and an intracellular second messenger. S1P is synthesized from sphingosine by sphingosine kinase and is degraded either by S1P lyase or by S1P phosphohydrolase. Recently, mammalian S1P phosphohydrolase (SPP1) was identified and shown to constitute a novel lipid phosphohydrolase family, the SPP family. In this study we have identified a second human S1P phosphohydrolase, SPP2, based on sequence homology to human SPP1. SPP2 exhibited high phosphohydrolase activity against S1P and dihydrosphingosine 1-phosphate. The dihydrosphingosine-1-phosphate phosphohydrolase activity was efficiently inhibited by excess S1P but not by lysophosphatidic acid, phosphatidic acid, or glycerol 3-phosphate, indicating that SPP2 is highly specific to sphingoid base 1-phosphates. Immunofluorescence microscopic analysis demonstrated that SPP2 is localized to the endoplasmic reticulum. Although the enzymatic properties and localization of SPP2 were similar to those of SPP1, the tissue-specific expression pattern of SPP2 was different from that of SPP1. Thus, SPP2 is another member of the SPP family that may play a role in attenuating intracellular S1P signaling.     

Structural, expression, and evolutionary analysis of mouse CIAS1

Mutations in the human CIAS1 (hCIAS1) gene have been identified in a continuum of inflammatory disorders including familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), and neonatal onset multisystem inflammatory disease (NOMID). CIAS1 codes for the protein Cryopyrin, which appears to play a role in innate immune function by regulating the production of proinflammatory cytokines. Human and mouse Cryopyrin are highly conserved and consist of three functional domains including a pyrin domain, an NACHT domain, and a leucine-rich repeat (LRR) domain that are characteristics of the NALP family of proteins. The pyrin and NACHT domains of Cryopyrin and other NALP proteins are highly conserved among primate and nonprimate mammals, suggesting purifying selection throughout mammalian evolution. Cryopyrin expression is also very similar in human and mouse with mouse CIAS1 mRNA expression found primarily in peripheral blood leukocytes consistent with the postulated inflammatory function. We also detected significant expression in mouse eye and skin tissue, which is consistent with symptoms observed in human Cryopyrin-associated diseases.     

Cell-surface expression of a new splice variant of the mouse signal peptide peptidase

The signal peptide peptidase (SPP) is an intramembrane-cleaving aspartyl protease that acts on type II transmembrane proteins. SPP substrates include signal peptides after they have been cleaved from a preprotein, hence the name. The known SPP isoform, which we renamed SPPalpha, contains an endoplasmic reticulum retention signal at the carboxy terminus. We found a new splice variant, SPPbeta, with an additional in-frame exon inserted between exons 11 and 12 of SPPalpha. Insertion of the new exon led to a complete change in the amino-acid sequence of the carboxy tail. A stop codon within this new exon resulted in silencing of exon 12 and eliminated the endoplasmic reticulum retention signal. The new SPP isoform predominantly localised to the cell surface in contrast to the more restricted localisation of SPPalpha in the endoplasmic reticulum. Differential expression in mouse tissues and in subcellular compartments suggests new functions for SPP in addition to cleaving signal peptides.     

人Collectrin同源基因的分离克隆及其在人肾集合管和远曲小管的特异表达

为探讨collectrin在人类疾病中的作用 ,利用人类collectrin同源性引物 ,经末端cDNA快速扩增法分离获得人collectrin基因全长序列并对collectrin进行生物信息学分析及定位表达研究 .结果发现 ,人类collectrin(GenBank登录号为AF2 2 9179)基因全长含 1345bp ,开放阅读框架编码 2 2 2个氨基酸 .在核苷酸和氨基酸水平 ,与小鼠collectrin序列分别有 86 9%和 87 4 %同源性 .生物信息学分析结果提示 ,collectrin为一个 2 5kD的具有一个信号肽和一个跨膜区的跨膜糖蛋白 .人类collectrin与人类血管紧张素转换酶相关的羧基肽酶 (ACE2 )具有 4 7 8%高度同源性 .人多组织Northern杂交结果显示 :collectrin基因为人类肾脏特异性表达基因 .原位杂交及免疫组化证实 ,与小鼠collectrin特异表达于集合管细胞不同 ,人collectrin基因mRNA及其蛋白产物除位于肾脏集合管细胞外 ,远曲肾小管细胞也有表达 .由此推论 ,人类collectrin基因为肾脏特异性表达基因 ,与人类血管紧张素转换酶相关的羧基肽酶具有高度同源性 ,可能为血管紧张素转换酶 (ACE)基因家族的新成员 .