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Abstract The Arabidopsis pi4kβ1,2 mutant is mutated in the phosphatidylinositol 4-kinase (PI4K) β1 and PI4Kβ2 enzymes which are involved in the biosynthesis of phosphatidylinositol 4-phosphate (PI4P), a minor membrane lipid with important signaling roles. pi4kβ1,2 plants display autoimmunity and shorter roots. Though the pi4kβ1,2 mutant has been extensively characterized, the source of its autoimmunity remains largely unknown. In this study, through a genetic suppressor screen, we identified multiple partial loss-of-function alleles of signal peptide peptidase (spp) that can suppress all the defects of pi4kβ1,2. SPP is an intramembrane cleaving aspartic protease. Interestingly, pi4kβ1,2 plants display enhanced ER stress response and mutations in SPP can suppress such phenotype. Furthermore, reduced ER stress responses were observed in the spp single mutants. Overall, our study reveals a previously unknown function of PI4Kβ and SPP in ER stress and plant immunity.

The Arabidopsis pi4kβ1,2 mutant is mutated in the phosphatidylinositol 4-kinase (PI4K) β1 and PI4Kβ2 enzymes which are involved in the biosynthesis of phosphatidylinositol 4-phosphate (PI4P), a minor membrane lipid with important signaling roles. pi4kβ1,2 plants display autoimmunity and shorter roots. Though the pi4kβ1,2 mutant has been extensively characterized, the source of its autoimmunity remains largely unknown. In this study, through a genetic suppressor screen, we identified multiple partial loss-of-function alleles of signal peptide peptidase (spp) that can suppress all the defects of pi4kβ1,2. SPP is an intramembrane cleaving aspartic protease. Interestingly, pi4kβ1,2 plants display enhanced ER stress response and mutations in SPP can suppress such phenotype. Furthermore, reduced ER stress responses were observed in the spp single mutants. Overall, our study reveals a previously unknown function of PI4Kβ and SPP in ER stress and plant immunity. [ABSTRACT FROM AUTHOR]

Signal peptide peptidase (SPP) and the four SPP‐like proteases SPPL2a, SPPL2b, SPPL2c and SPPL3 constitute a family of aspartyl intramembrane proteases with homology to presenilins. The different members reside in distinct cellular localisations within the secretory pathway and the endo‐lysosomal system. Despite individual cleavage characteristics, they all cleave single‐span transmembrane proteins with a type II orientation exhibiting a cytosolic N‐terminus. Though the identification of substrates is not complete, SPP/SPPL‐mediated proteolysis appears to be rather selective. Therefore, according to our current understanding cleavage by SPP/SPPL proteases rather seems to serve a regulatory function than being a bulk proteolytic pathway. In the present review, we will summarise our state of knowledge on SPP/SPPL proteases and in particular highlight recently identified substrates and the functional and/or (patho)‐physiological implications of these cleavage events. Based on this, we aim to provide an overview of the current open questions in the field. These are connected to the regulation of these proteases at the cellular level but also in context of disease and patho‐physiological processes. Furthermore, the interplay with other proteostatic systems capable of degrading membrane proteins is beginning to emerge. [ABSTRACT FROM AUTHOR]

Signal peptide peptidase (SPP) is an aspartic protease with two active sites, YD and GXGD, in the transmembrane domain. SPP cleaves signal peptides, and the released fragments play key roles in the immune system, embryo development and protein turnover in cells. Despite SPP having an important function, a general system to identify the requirements of intramembrane proteolysis by SPP has not been developed because proteolysis occurs in the membrane. In this study, we first established a reporter assay system in yeast to verify the cleavage activity of the Arabidopsis thaliana SPP (AtSPP). Next, we screened candidate substrates of AtSPP from A. thaliana pollen and roots. In the pollen, 13 signal peptides with 'pollen' and 'cell wall' as gene ontology terms were selected. In the roots, mutants overexpressing AtSPP were constructed, and gene expression changes were compared with the wild‐type. Nine signal peptides expressed in the roots were selected. Then we used the candidate substrates in our reporter assay system to determine the requirements for proteolysis by AtSPP. Fifteen of 22 signal peptides were cleaved by AtSPP. The absence of the positively charged amino acids, His and Lys on the C terminus of the signal sequence, was observed in cleaved substrates. Moreover, mutation of a helix breaker‐to‐Leu substitution in the intramembrane region in substrates prevented cleavage by AtSPP. These results indicated that substrates of AtSPP required the helix breaker structure to be cleaved.

Immunoevasins are viral proteins that prevent antigen presentation on major histocompatibility complex (MHC) class I, thus evading host immune recognition. Hepatitis C virus (HCV) evades immune surveillance to induce chronic infection; however, how HCV-infected hepatocytes affect immune cells and evade immune recognition remains unclear. Herein, we demonstrate that HCV core protein functions as an immunoevasin. Its expression interfered with the maturation of MHC class I molecules catalyzed by the signal peptide peptidase (SPP) and induced their degradation via HMG-CoA reductase degradation 1 homolog, thereby impairing antigen presentation to CD8+ T cells. The expression ofMHC class I in the livers of HCV core transgenic mice and chronic hepatitis C patients was impaired but was restored in patients achieving sustained virological response. Finally, we show that the human cytomegalovirus US2 protein, possessing a transmembrane region structurally similar to the HCV core protein, targets SPP to impair MHC class I molecule expression. Thus, SPP represents a potential target for the impairment of MHC class I molecules by DNA and RNA viruses. [ABSTRACT FROM AUTHOR]

Intramembrane proteolysis is more than a mechanism to “clean” the membranes from proteins no longer needed. By non-reversibly modifying transmembrane proteins, intramembrane cleaving proteases hold key roles in multiple signaling pathways and often distinguish physiological from pathological conditions. Signal peptide peptidase (SPP) and signal peptide peptidase-like proteases (SPPLs) recently have been associated with multiple functions in the field of signal transduction. SPP/SPPLs together with presenilins (PSs) are the only two families of intramembrane cleaving aspartyl proteases known in mammals. PS1 or PS2 comprise the catalytic center of the γ-secretase complex, which is well-studied in the context of Alzheimer's disease. The mammalian SPP/SPPL family of intramembrane cleaving proteases consists of five members: SPP and its homologous proteins SPPL2a, SPPL2b, SPPL2c, and SPPL3. Although these proteases were discovered due to their homology to PSs, it became evident in the past two decades that no physiological functions are shared between these two families. Based on studies in cell culture models various substrates of SPP/SPPL proteases have been identified in the past years and recently-developed mouse lines lacking individual members of this protease family, will help to further clarify the physiological functions of these proteases. In this review we concentrate on signaling roles of mammalian intramembrane cleaving aspartyl proteases. In particular, we will highlight the signaling roles of PS via its substrates NOTCH, VEGF, and others, mainly focusing on its involvement in vasculature. Delineating also signaling pathways that are affected and/or controlled by SPP/SPPL proteases. From SPP's participation in tumor progression and survival, to SPPL3's regulation of protein glycosylation and SPPL2c's control over cellular calcium stores, various crossovers between proteolytic activity of intramembrane proteases and cell signaling will be described.

Salinity stress in increasingly becoming a major challenge in current and expanding agricultural ecosystems. Unlike temporal abiotic stresses, plants are usually exposed to salinity stress for an entire lifespan. Therefore, a long term effect (10 weeks) of continuous salinity exposure was investigated for three common fig landraces (Zraki, Mwazi, and Khdari). Both relative water content and chlorophyll content decreased with elevated salinity stress, while stem length barely changed. The most prominent decline was observed in root biomass. The data would align common fig to moderately tolerant threshold slop with a C50 range of 100 to 150 mM NaCl. A high and significant correlation was evident between root biomass and chlorophyll content (85%). Concurrently, differential expression of putative salinity responsive genes in common fig were determined; signal peptide peptidase-like 2B (FcSPPL2B), dehydration responsive element binding protein (FcDREB), calcineurin B-like protein (CBL)-CBL-interacting serine/threonine-protein kinase 11 (FcCIPK11), sorbitol dehydrogenase (FcSORD) and dehydrin (FcDHN). The data were discussed for each gene in respect of its potential role in salinity stress mitigation. The combined physiological and molecular data would conclude Zraki as the most salinity tolerant genotype. The major implication of the data emphasizes the tremendous genotype by environment (salinity stress) interaction in common fig. [ABSTRACT FROM AUTHOR]

ABSTRACT The membrane protease SppA of Bacillus subtilis was first described as a signal peptide peptidase and later shown to confer resistance to lantibiotics. Here, we report that SppA forms octameric complexes with YteJ, a membrane protein of thus-far-unknown function. Interestingly, sppA and yteJ deletion mutants exhibited no protein secretion defects. However, these mutant strains differed significantly in their resistance to antimicrobial peptides. In particular, sppA mutant cells displayed increased sensitivity to the lantibiotics nisin and subtilin and the human lysozyme-derived cationic antimicrobial peptide LP9. Importantly, YteJ was shown to antagonize SppA activity both in vivo and in vitro, and this SppA-inhibitory activity involved the C-terminal domain of YteJ, which was therefore renamed SppI. Most likely, SppI-mediated control is needed to protect B. subtilis against the potentially detrimental protease activity of SppA since a mutant overexpressing sppA by itself displayed defects in cell division. Altogether, we conclude that the SppA-SppI complex of B. subtilis has a major role in protection against antimicrobial peptides. IMPORTANCE Our study presents new insights into the molecular mechanism that regulates the activity of SppA, a widely conserved bacterial membrane protease. We show that the membrane proteins SppA and SppI form a complex in the Gram-positive model bacterium B. subtilis and that SppI inhibits SppA protease activity in vitro and in vivo. Furthermore, we demonstrate that the C-terminal domain of SppI is involved in SppA inhibition. Since SppA, through its protease activity, contributes directly to resistance to lantibiotic peptides and cationic antibacterial peptides, we propose that the conserved SppA-SppI complex could play a major role in the evasion of bactericidal peptides, including those produced as part of human innate immune defenses.

Signal peptide peptidase (SPP) is an aspartic protease with two active sites, YD and GXGD, in the transmembrane domain. SPP cleaves signal peptides, and the released fragments play key roles in the immune system, embryo development and protein turnover in cells. Despite SPP having an important function, a general system to identify the requirements of intramembrane proteolysis by SPP has not been developed because proteolysis occurs in the membrane. In this study, we first established a reporter assay system in yeast to verify the cleavage activity of the Arabidopsis thaliana SPP (AtSPP). Next, we screened candidate substrates of AtSPP from A. thaliana pollen and roots. In the pollen, 13 signal peptides with 'pollen' and 'cell wall' as gene ontology terms were selected. In the roots, mutants overexpressing AtSPP were constructed, and gene expression changes were compared with the wild‐type. Nine signal peptides expressed in the roots were selected. Then we used the candidate substrates in our reporter assay system to determine the requirements for proteolysis by AtSPP. Fifteen of 22 signal peptides were cleaved by AtSPP. The absence of the positively charged amino acids, His and Lys on the C terminus of the signal sequence, was observed in cleaved substrates. Moreover, mutation of a helix breaker‐to‐Leu substitution in the intramembrane region in substrates prevented cleavage by AtSPP. These results indicated that substrates of AtSPP required the helix breaker structure to be cleaved. [ABSTRACT FROM AUTHOR]

Abstract Background Signal peptide peptidases play an important role in the removal of remnant signal peptides in the cell membrane, a critical step for extracellular protein production. Although these proteins are likely a central component for extracellular protein production, there has been a lack of research on whether protein secretion could be enhanced via overexpression of signal peptide peptidases. Results In this study, both nattokinase and α-amylase were employed as prototypical secreted target proteins to evaluate the function of putative signal peptide peptidases (SppA and TepA) in Bacillus licheniformis. We observed dramatic decreases in the concentrations of both target proteins (45 and 49%, respectively) in a sppA deficient strain, while the extracellular protein yields of nattokinase and α-amylase were increased by 30 and 67% respectively in a strain overexpressing SppA. In addition, biomass, specific enzyme activities and the relative gene transcriptional levels were also enhanced due to the overexpression of sppA, while altering the expression levels of tepA had no effect on the concentrations of the secreted target proteins. Conclusions Our results confirm that SppA, but not TepA, plays an important functional role for protein secretion in B. licheniformis. Our results indicate that the sppA overexpression strain, B. licheniformis BL10GS, could be used as a promising host strain for the industrial production of heterologous secreted proteins.

Over the last two decades, a group of unusual proteases, so-called intramembrane proteases, have become increasingly recognized for their unique ability to cleave peptide bonds within cellular membranes. They are found in all kingdoms of life and fulfil versatile functions ranging from protein maturation, to activation of signalling molecules, to protein degradation. In this Cell Science at a Glance article and the accompanying poster, we focus on intramembrane proteases in mammalian cells. By comparing intramembrane proteases in different cellular organelles, we set out to review their functions within the context of the roles of individual cellular compartments. Additionally, we exemplify their mode of action in relation to known substrates by distinguishing cleavage events that promote degradation of substrate from those that release active domains from the membrane bilayer. [ABSTRACT FROM AUTHOR]

Signal peptide peptidase (SPP) and the four homologous SPP-like proteases SPPL2a, SPPL2b, SPPL2c and SPPL3 are GxGD-type intramembrane-cleaving proteases (I-CLIPs). In addition to divergent subcellular localisations, distinct differences in the mechanistic properties and substrate requirements of individual family members have been unravelled. SPP/SPPL proteases employ a catalytic mechanism related to that of the γ-secretase complex. Nevertheless, differential targeting of SPP/SPPL proteases and γ-secretase by inhibitors has been demonstrated. Furthermore, also within the SPP/SPPL family significant differences in the sensitivity to currently available inhibitory compounds have been reported. Though far from complete, our knowledge on pathophysiological functions of SPP/SPPL proteases, in particular based on studies in mice, has been significantly increased over the last years. Based on this, inhibition of distinct SPP/SPPL proteases has been proposed as a novel therapeutic concept e.g. for the treatment of autoimmunity and viral or protozoal infections, as we will discuss in this review. This article is part of a Special Issue entitled: Proteolysis as a Regulatory Event in Pathophysiology edited by Stefan Rose-John. [ABSTRACT FROM AUTHOR]

Signal peptide peptidase (SPP) and the four related SPP-like (SPPL) proteases are homologues of the presenilins, which comprise the catalytic centre of the γ-secretase complex. SPP/SPPL proteases are GxGD-type aspartyl intramembrane proteases selective for substrates with a type II membrane topology. Subcellular localisations of SPP/SPPL proteases range from the early secretory pathway to the plasma membrane and the endocytic system. Similarly diverse are their functional roles at the cellular level covering the turnover of signal peptides and membrane proteins, a contribution to the ERAD pathway as well as the regulation of cellular protein glycosylation and certain signaling pathways. Much less well understood are the physiological functions of SPP/SPPL proteases in complex organisms. Whereas a major role of SPPL2a for homeostasis of B cells and dendritic cells has been documented in mice, in vivo functions of SPP and the other SPPLs remain largely elusive to date. SPP/SPPL proteases contribute to regulated intramembrane proteolysis (RIP), a sequential processing of single-spanning transmembrane proteins by an ectodomain sheddase and an intramembrane-cleaving protease (I-CLIP). However, recent studies reported the cleavage of tail-anchored and multi-pass membrane proteins by SPP as well as the capability of SPPL3 to accept substrates without a preceding ectodomain shedding. This revealed that the mechanistic properties within this family are more diverse than initially thought. With this review, we aim to provide an update on recent achievements in defining the function and (patho-) physiological relevance of SPP/SPPL proteases and to highlight open questions in the field. [ABSTRACT FROM AUTHOR]

Proteases are considered attractive drug targets. Various drugs targeting classical, soluble proteases have been approved for treatment of human disease. Intramembrane proteases ( IMPs) are a more recently discovered group of proteolytic enzymes. They are embedded in lipid bilayers and their active sites are located in the plane of a membrane. All four mechanistic families of IMPs have been linked to disease, but currently, no drugs against IMPs have entered the market. In this review, I will outline the function of IMPs with a focus on the ones involved in human disease, which includes Alzheimer's disease, cancer, and infectious diseases by microorganisms. Inhibitors of IMPs are known for all mechanistic classes, but are not yet very potent or selective - aside from those targeting γ-secretase. I will here describe the different features of IMP inhibitors and discuss a list of issues that need attention in the near future in order to improve the drug development for IMPs. [ABSTRACT FROM AUTHOR]

ABSTRACT 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. [ABSTRACT FROM AUTHOR]

The M genome segment of Bunyamwera virus (BUNV)--the prototype of both the Bunyaviridae family and the Orthobunyavirus genus--encodes the glycoprotein precursor (GPC) that is proteolytically cleaved to yield two viral structural glycoproteins, Gn and Gc, and a nonstructural protein, NSm. The cleavage mechanism of orthobunyavirus GPCs and the host proteases involved have not been clarified. In this study, we investigated the processing of BUNV GPC and found that both NSm and Gc proteins were cleaved at their own internal signal peptides (SPs), in which NSm domain I functions as SPNSm and NSmdomain V as SPGc.Moreover, the domain I was further processed by a host intramembrane-cleaving protease, signal peptide peptidase, and is required for cell fusion activities. Meanwhile, the NSm domain V (SPGc) remains integral to NSm, rendering the NSm topology as a two-membrane-spanning integral membrane protein. We defined the cleavage sites and boundaries between the processed proteins as follows: Gn, from residue 17-312 or nearby residues; NSm, 332-477; and Gc, 478-1433. Our data clarified the mechanism of the precursor cleavage process, which is important for our understanding of viral glycoprotein biogenesis in the genus Orthobunyavirus and thus presents a useful target for intervention strategies. [ABSTRACT FROM AUTHOR]

Previously we described the identification of a Plasmodium falciparum signal peptide peptidase (PfSPP) functioning at the blood stage of malaria infection. Our studies also demonstrated that mammalian SPP inhibitors prevent malaria parasite growth at the late-ring/early trophozoite stage of intra-erythrocytic development. Consistent with its role in development, we tested the hypothesis that PfSPP functions at the endoplasmic reticulum of P. falciparum where it cleaves membrane-bound signal peptides generated following the enzyme activity of signal peptidase. The localization of PfSPP to the endoplasmic reticulum was confirmed by immunofluorescence microscopy and immunogold electron microscopy. Biochemical analysis indicated the existence of monomer and dimer forms of PfSPP in the parasite lysate. A comprehensive bioinformatics screen identified several candidate PfSPP substrates in the parasite genome. Using an established transfection based in vivo luminescence assay, malaria heat shock protein 101 (HSP101) was identified as a substrate of PfSPP, and partial inhibition of PfSPP correlated with the emergence of gametocytes. This finding unveils the first known substrate of PfSPP, and provides new perspectives for the function of intra-membrane proteolysis at the erythrocyte stage of malaria parasite life cycle. [ABSTRACT FROM AUTHOR]

Abstract: Presenilin is the catalytic component of the γ-secretase complex, a membrane-embedded aspartyl protease that plays a central role in biology and in the pathogenesis of Alzheimer’s disease. Upon assembly with its three protein cofactors (nicastrin, Aph-1 and Pen-2), presenilin undergoes autoproteolysis into two subunits, each of which contributes one of the catalytic aspartates to the active site. A family of presenilin homologs, including signal peptide peptidase, possess proteolytic activity without the need for other protein factors, and these simpler intramembane aspartyl proteases have given insight into the action of presenilin within the γ-secretase complex. Cellular and molecular studies support a nine-transmembrane topology for presenilins and their homologs, and small-molecule inhibitors and cysteine scanning with crosslinking have suggested certain presenilin residues and regions that contribute to substrate recognition and handling. Identification of partial complexes has also offered clues to protein–protein interactions within the γ-secretase complex. Biophysical methods have allowed 3D views of the γ-secretase complex and presenilins. Most recently, the crystal structure of a microbial presenilin homolog has confirmed a nine-transmembrane topology and intramembranous location and proximity of the two conserved and essential aspartates. The crystal structure also provides a platform for the formulation of specific hypotheses regarding substrate interaction and catalysis as well as the pathogenic mechanism of Alzheimer-causing presenilin mutations. This article is part of a Special Issue entitled: Intramembrane Proteases. [Copyright &y& Elsevier]

Abstract: Intramembrane proteolysis is widely conserved throughout different forms of life, with three major types of proteases being known for their ability to cleave peptide bonds directly within the transmembrane domains of their substrates. Although intramembrane proteases have been extensively studied in humans and model organisms, they have only more recently been investigated in protozoan parasites, where they turn out to play important and sometimes unexpected roles. Signal peptide peptidases are involved in endoplasmic reticulum (ER) quality control and signal peptide degradation from exported proteins. Recent studies suggest that repurposing inhibitors developed for blocking presenilins may be useful for inhibiting the growth of Plasmodium, and possibly other protozoan parasites, by blocking signal peptide peptidases. Rhomboid proteases, originally described in the fly, are also widespread in parasites, and are especially expanded in apicomplexans. Their study in parasites has revealed novel roles that expand our understanding of how these proteases function. Within this diverse group of parasites, rhomboid proteases contribute to processing of adhesins involved in attachment, invasion, intracellular replication, phagocytosis, and immune evasion, placing them at the vertex of host–parasite interactions. This article is part of a Special Issue entitled: Intramembrane Proteases. [Copyright &y& Elsevier]

Abstract: Signal peptide peptidase (SPP) and the homologous SPP-like (SPPL) proteases SPPL2a, SPPL2b, SPPL2c and SPPL3 belong to the family of GxGD intramembrane proteases. SPP/SPPLs selectively cleave transmembrane domains in type II orientation and do not require additional co-factors for proteolytic activity. Orthologues of SPP and SPPLs have been identified in other vertebrates, plants, and eukaryotes. In line with their diverse subcellular localisations ranging from the ER (SPP, SPPL2c), the Golgi (SPPL3), the plasma membrane (SPPL2b) to lysosomes/late endosomes (SPPL2a), the different members of the SPP/SPPL family seem to exhibit distinct functions. Here, we review the substrates of these proteases identified to date as well as the current state of knowledge about the physiological implications of these proteolytic events as deduced from in vivo studies. Furthermore, the present knowledge on the structure of intramembrane proteases of the SPP/SPPL family, their cleavage mechanism and their substrate requirements are summarised. This article is part of a Special Issue entitled: Intramembrane Proteases. [Copyright &y& Elsevier]

Intramembrane proteases cleave membrane proteins in their transmembrane helices to regulate a wide range of biological processes. They catalyse hydrolytic reactions within the hydrophobic environment of lipid membranes where water is normally excluded. How? Do the different classes of intramembrane proteases share any mechanistic principles? In this review these questions will be discussed in view of the crystal structures of prokaryotic members of the three known catalytic types of intramembrane proteases published over the past 7 years. Rhomboids, the intramembrane serine proteases that are the best understood family, will be the initial area of focus, and the principles that have arisen from a number of structural and biochemical studies will be considered. The site-2 metalloprotease and GXGD-type aspartyl protease structures will then be discussed, with parallels drawn and differences highlighted between these enzymes and the rhomboids. Despite the significant advances achieved so far, to obtain a detailed understanding of the mechanism of any intramembrane protease, high-resolution structural information on the substrate-enzyme complex is required. This remains a major challenge for the field. [ABSTRACT FROM AUTHOR]

The establishment of parasite infection within the human erythrocyte is an essential stage in the development of malaria disease. As such, significant interest has focused on the mechanics that underpin invasion and on characterization of parasite molecules involved. Previous evidence has implicated a presenilin-like signal peptide peptidase ( SPP) from the most virulent human malaria parasite, Plasmodium falciparum, in the process of invasion where it has been proposed to function in the cleavage of the erythrocyte cytoskeletal protein Band 3. The role of a traditionally endoplasmic reticulum ( ER) protease in the process of red blood cell invasion is unexpected. Here, using a combination of molecular, cellular and chemical approaches we provide evidence that PfSPP is, instead, a bona fide ER-resident peptidase that remains intracellular throughout the invasion process. Furthermore, SPP-specific drug inhibition has no effect on erythrocyte invasion whilst having low micromolar potency against intra-erythrocytic development. Contrary to previous reports, these results show that PfSPP plays no role in erythrocyte invasion. Nonetheless, PfSPP clearly represents a potential chemotherapeutic target to block parasite growth, supporting ongoing efforts to develop antimalarial-targeting protein maturation and trafficking during intra-erythrocytic development. [ABSTRACT FROM AUTHOR]

Abstract: Signal peptide peptidase A (SppA) is a membrane-bound self-compartmentalized serine protease that functions to cleave the remnant signal peptides left behind after protein secretion and cleavage by signal peptidases. SppA is found in plants, archaea and bacteria. Here, we report the first crystal structure of a Gram-positive bacterial SppA. The 2.4-Å-resolution structure of Bacillus subtilis SppA (SppABS) catalytic domain reveals eight SppABS molecules in the asymmetric unit, forming a dome-shaped octameric complex. The octameric state of SppABS is supported by analytical size-exclusion chromatography and multi-angle light scattering analysis. Our sequence analysis, mutagenesis and activity assays are consistent with Ser147 serving as the nucleophile and Lys199 serving as the general base; however, they are located in different region of the protein, more than 29 Å apart. Only upon assembling the octamer do the serine and lysine come within close proximity, with neighboring protomers each providing one-half of the catalytic dyad, thus producing eight separate active sites within the complex, twice the number seen within Escherichia coli SppA (SppAEC). The SppABS S1 substrate specificity pocket is deep, narrow and hydrophobic, but with a polar bottom. The S3 pocket, which is constructed from two neighboring proteins, is shallower, wider and more polar than the S1 pocket. A comparison of these pockets to those seen in SppAEC reveals a significant difference in the size and shape of the S1 pocket, which we show is reflected in the repertoire of peptides the enzymes are capable of cleaving. [Copyright &y& Elsevier]

Background: Signal peptide peptidase (SPP), a member of the presenilin-like intra-membrane cleaving aspartyl protease family, migrates on Blue Native (BN) gels as 100 kDa, 200 kDa and 450 kDa species. SPP has recently been implicated in other non-proteolytic functions such as retro-translocation of MHC Class I molecules and binding of misfolded proteins in the endoplasmic reticulum (ER). These high molecular weight SPP complexes might contain additional proteins that regulate the proteolytic activity of SPP or support its non-catalytic functions. Results: In this study, an unbiased iTRAQ-labeling mass spectrometry approach was used to identify SPP-interacting proteins. We found that vigilin, a ubiquitous multi-KH domain containing cytoplasmic protein involved in RNA binding and protein translation control, selectively enriched with SPP. Vigilin interacted with SPP and both proteins co-localized in restricted intracellular domains near the ER, biochemically co-fractionated and were part of the same 450 kDa complex on BN gels. However, vigilin does not alter the protease activity of SPP, suggesting that the SPP-vigilin interaction might be involved in the non-proteolytic functions of SPP. Conclusions: We have identified and validated vigilin as a novel interacting partner of SPP that could play an important role in the non-proteolytic functions of SPP. This data adds further weight to the idea that intramembrane-cleaving aspartyl proteases, such as presenilin and SPPs, could have other functions besides the proteolysis of short membrane stubs. [ABSTRACT FROM AUTHOR]

Presenilin is the catalytic component of γ-secretase, a complex aspartyl protease and a founding member of intramembrane-cleaving proteases. γ-Secretase is involved in the pathogenesis of Alzheimer's disease and a top target for therapeutic intervention. However, the protease complex processes a variety of transmembrane substrates, including the Notch receptor, raising concerns about toxicity. Nevertheless, γ-secretase inhibitors and modulators have been identified that allow Notch processing and signaling to continue, and promising compounds are entering clinical trials. Molecular and biochemical studies offer a model for how this protease hydrolyzes transmembrane domains in the confines of the lipid bilayer. Progress has also been made toward structure elucidation of presenilin and the γ-secretase complex by electron microscopy as well as by studying cysteine-mutant presenilins. The signal peptide peptidase (SPP) family of proteases are distantly related to presenilins. However, the SPPs work as single polypeptides without the need for cofactors and otherwise appear to be simple model systems for presenilin in the γ-secretase complex. SPP biology, structure, and inhibition will also be discussed. [ABSTRACT FROM AUTHOR]