Resonant Mode Analysis of the Nanoscale Surface Plasmon Polariton Waveguide Filter with Rectangle Cavity

The resonant mode characteristics of the nanoscale surface plasmon polaritons (SPP) waveguide filter with rectangle cavity are studied theoretically. By using the finite difference time domain method, both the band-stop- and band-pass-type rectangle SPP filters are analyzed. The results show that the whispering gallery mode (WGM) and the Fabry–Perot (FP) mode can be supported by the rectangle SPP resonator. Furthermore, both traveling-wave mode and standing-wave mode can be realized by the WGM, while only standing-wave mode can be introduced by the FP mode. The traveling-wave mode can only be realized by the square-shaped SPP resonator, and the traveling-wave mode is splitted into two standing-wave modes by transforming the cavity shape from square to rectangle. Also, the effects of the cavity shape, cavity size, and coupling gap size on the transmission spectra of the SPP resonators are analyzed in detail. This simple SPP waveguide filter is very promising for the high-density SPP waveguide integrations.     

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.     

Distinct pharmacological effects of inhibitors of signal peptide peptidase and gamma-secretase

Signal peptide peptidase (SPP) and gamma-secretase are intramembrane aspartyl proteases that bear similar active site motifs but with opposite membrane topologies. Both proteases are inhibited by the same aspartyl protease transition-state analogue inhibitors, further evidence that these two enzymes have the same basic cleavage mechanism. Here we report that helical peptide inhibitors designed to mimic SPP substrates and interact with the SPP initial substrate-binding site (the "docking site") inhibit both SPP and gamma-secretase, but with submicromolar potency for SPP. SPP was labeled by helical peptide and transition-state analogue affinity probes but at distinct sites. Nonsteroidal anti-inflammatory drugs, which shift the site of proteolysis by SPP and gamma-secretase, did not affect the labeling of SPP or gamma-secretase by the helical peptide or transition-state analogue probes. On the other hand, another class of previously reported gamma-secretase modulators, naphthyl ketones, inhibited SPP activity as well as selective proteolysis by gamma-secretase. These naphthyl ketones significantly disrupted labeling of SPP by the helical peptide probe but did not block labeling of SPP by the transition-state analogue probe. With respect to gamma-secretase, the naphthyl ketone modulators allowed labeling by the transition-state analogue probe but not the helical peptide probe. Thus, the naphthyl ketones appear to alter the docking sites of both SPP and gamma-secretase. These results indicate that pharmacological effects of the four different classes of inhibitors (transition-state analogues, helical peptides, nonsteroidal anti-inflammatory drugs, and naphthyl ketones) are distinct from each other, and they reveal similarities and differences with how they affect SPP and gamma-secretase.     

Structural properties of the chloroplast stromal processing peptidase required for its function in transit peptide removal

The stromal processing peptidase (SPP) catalyzes removal of transit peptides from a diversity of precursor proteins imported into chloroplasts. SPP contains an HXXEH zinc-binding motif characteristic of members of the metallopeptidase family M16. We previously found that the three steps of precursor processing by SPP (i.e. transit peptide binding, removal, and conversion to a degradable subfragment) are mediated by features that reside in the C-terminal 10-15 residues of the transit peptide. In this study, we performed a mutational analysis of SPP to identify structural elements that determine its function. SPP loses the ability to proteolytically remove the transit peptide when residues of the HXXEH motif, found in an N-terminal region, are mutated. Deletion of 240 amino acids from its C terminus also abolishes activity. Interestingly, however, SPP can still carry out the initial binding step, recognizing the C-terminal residues of the transit peptide. Hence, transit peptide binding and removal are two separable steps of the overall processing reaction. Transit peptide conversion to a subfragment also depends on the HXXEH motif. The precursor of SPP, containing an unusually long transit peptide itself, is not proteolytically active. Thus, the SPP precursor is synthesized as a latent form of the metallopeptidase.     

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.     

The genome of B. subtilis phage SPP1:

Summary DNA molecules of B. subtilis phage SPP1 exhibit terminal redundancy and are partially circularly permuted. This was established by the hybridization of selected EcoRI restriction fragments to single strands of SPP1 DNA and by an analysis of the distribution of denaturation loops in partially denatured SPP1 DNA molecules. Deletions in SPP1 DNA are not compensated by an increase in terminally repetitious DNA. This finding, which is unique to SPP1, is discussed in terms of a modification of the Streisinger/Botstein model of phage maturation.     

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.     

Transducing activity of bacteriophage SPP1.

The generalized transduction system mediated by SPP1 lysates was analyzed. After removal of contaminating PBS particles, SPP1 lysates retain the same levels of transducing activity. The SPP1 particles of normal size, shape and buoyant density carrying bacterial DNA, are vectors in a true transduction process.     

Requirements for signal peptide peptidase-catalyzed intramembrane proteolysis

The presenilin-type aspartic protease signal peptide peptidase (SPP) can cleave signal peptides within their transmembrane region. SPP is essential for generation of signal peptide-derived HLA-E epitopes in humans and is exploited by Hepatitis C virus for processing of the viral polyprotein. Here we analyzed requirements of substrates for intramembrane cleavage by SPP. Comparing signal peptides that are substrates with those that are not revealed that helix-breaking residues within the transmembrane region are required for cleavage, and flanking regions can affect processing. Furthermore, signal peptides have to be liberated from the precursor protein by cleavage with signal peptidase in order to become substrates for SPP. We propose that signal peptides require flexibility in the lipid bilayer to exhibit an accessible peptide bond for intramembrane proteolysis.     

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.     

Allergen particle binding by human primary bronchial epithelial cells is modulated by surfactant protein D

Background

Allergen-containing subpollen particles (SPP) are released from whole plant pollen upon contact with water or even high humidity. Because of their size SPP can preferentially reach the lower airways where they come into contact with surfactant protein (SP)-D. Our previous work demonstrated that SP-D increases the uptake of SPP by alveolar macrophages. In the present study, we investigated the uptake of SPP in human primary epithelial cells and the potential modulation by SP-D. The patho-physiological consequence was evaluated by measurement of pro-inflammatory mediators.

Methods

SPP were isolated from timothy grass and subsequently fluorescently labelled. Human primary bronchial epithelial cells were incubated with SPP or polystyrene particles (PP) in the presence and absence of surfactant protein D. In addition, different sizes and surface charges of the PP were studied. Particle uptake was evaluated by flow cytometry and confocal microscopy. Soluble mediators were measured by enzyme linked immunosorbent assay or bead array.

Results

SPP were taken up by primary epithelial cells in a dose dependent manner. This uptake was coincided with secretion of Interleukin (IL)-8. SP-D increased the fraction of bronchial epithelial cells that bound SPP but not the fraction of cells that internalized SPP. SPP-induced secretion of IL-8 was further increased by SP-D. PP were bound and internalized by epithelial cells but this was not modulated by SP-D.

Conclusions

Epithelial cells bind and internalize SPP and PP which leads to increased IL-8 secretion. SP-D promotes attachment of SPP to epithelial cells and may thus be involved in the inflammatory response to inhaled allergen.     

The genome of B. subtilis phage SSP1: the topology of DNA molecules.

DNA molecules of B. subtilis phage SPP1 exhibit terminal redundancy and are partially circularly permuted. This was established by the hybridization of selected EcoRI restriction fragments to single strands of SPP1 DNA and by an analysis of the distribution of denaturation loops in partially denatured SPP1 DNA molecules. Deletions in SSP1 DNA are not compensated by an increase in terminally repetitious DNA. This finding, which is unique to SPP1, is discussed in terms of a modification of the Streisinger/Botstein model of phage maturation.     

Structure of Bacillus subtilis bacteriophage SPP1 DNA in relation to its transfection activity.

The availability of a detailed restriction map of SPP1 DNA allowed defined manipulations of such molecules. These were performed to investigate structural requirements for SPP1 transfection. (i) The transfection activity of SPP1 DNA was destroyed by degradation with restriction enzymes. Biological activity could be regenerated when transfection was performed with a combination of two different restriction endonuclease digests, provided that such digests generated widely overlapping DNA fragments. (ii) Unique DNA molecules were constructed from the natural population of circularly permuted SPP1 DNA molecules by using genetic engineering techniques. Such molecules had the same specific transfection activity as did the circularly permuted SPP1 DNA. These results are discussed in the context of current models of DNA processing in transfection.     

Analysis of the Scattering of Surface Plasmon Polariton Waves from a Perfectly Conducting Circular Cylinder

Surface plasmon polariton (SPP) waves are the most extensively studied waves among various types of surface waves because they are easy to excite and have been used in many optical applications particularly for plasmonic communication, sensing, and harvesting solar energy. In all these applications, especially on-chip plasmonic communication, scattering can be an important issue to deal with. Therefore, this paper aimed to theoretically inspect the scattering pattern of SPP waves from a perfect electric conductor (PEC) cylindrical scatterer. The cylindrical wave approach is used to solve the scattering problem by a cylindrical object placed below a metallic layer. The scattering is investigated thoroughly by changing the size of the scatterer and its distance from the interface along which the SPP wave is excited. As the size of the scatterer increases, the scattering increases significantly. On the other hand, when the distance of the scatterer from the interface is increased, the scattered field becomes small. Two-dimensional field maps are produced for the incident angle at which SPP is excited. Numerical results are also presented for other incident angles to make a comparison. Furthermore, the forward and backward far-fields are significantly enhanced if the SPP wave is scattered in comparison with when the SPP wave is not present.

     

Characterization of bacteriophage SPP1 transducing particles

Bacillus subtilis lysates produced by virulent bacteriophage SPP1 retained their transducing ability upon purification from contaminating PBSX particles. The buoyant density in CsC1 of the transducing activity was indistinguishable from that of the SPP1 plaque-forming units and the sedimentation behaviour in sucrose gradients of purified transducing particles was the same as that of SPP1 phage particles. Further, high concentrations of anti-SPP1 serum inactivated transducing particles and SPPl plaque-forming units at the same rate. The transduction process was resistant to DNAase treatment, but was enhanced by temperatures that did not allow transformation. It was concluded that particles of the size, shape, density and serum-sensitivity characteristic of SPP1, but carrying bacterial DNA, are vectors in a true transduction process. Cell survival upon SPP1 infection is discussed.     

Modulation of cell interactions with extracellular matrix by lysophosphatidic acid and sphingosine 1-phosphate

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (SPP) are lipid mediators released upon platelet activation. The concentration of LPA in serum is estimated at 1-10 microM whereas the concentration in plasma is considerably less. The SPP concentration in serum is 0.5 microM, approximately two-fold higher than the plasma concentration. The lipids are present during tissue injury and promote cellular processes involved in wound repair. LPA and SPP have multiple effects on cells, many of which are pertinent to wound healing and require that the cells interact in some fashion with components of the extracellular matrix. This review focuses on modulation of cell adhesion, cell migration, collagen gel contraction, and fibronectin matrix assembly by LPA and SPP.     

Signal peptide peptidase cleavage of GB virus B core protein is required for productive infection in vivo

Chronic infection by hepatitis C virus (HCV) is a leading cause of liver disease for which better therapies are urgently needed. Because a clearer understanding of the viral life cycle may suggest novel anti-viral approaches, we studied the role of host signal peptide peptidase (SPP) in viral infection. This intramembrane protease cleaves within a C-terminal signal sequence in the viral core protein, but the molecular determinants of cleavage and whether it is required for infection in vivo are unknown. To answer these questions, we studied SPP processing in GB virus B (GBV-B) infection. GBV-B is the closest phylogenetic relative of HCV and offers an accurate surrogate model for HCV infection. We demonstrate that SPP also processes GBV-B core protein and that a serine residue in the hydrophobic region of the signal sequence (present also in HCV) is critical for efficient SPP cleavage. The small size of the serine side chain combined with its ability to form intra- and interhelical hydrogen bonds likely contributes to recognition of the signal sequence as a substrate for SPP. By introducing mutations with differing effects on SPP processing into an infectious GBV-B molecular clone, we demonstrate that SPP processing of the core protein is required for productive infection in primates. These results broaden our understanding of the mechanism and requirements for SPP cleavage and reveal a functional role in vivo for intramembrane proteolysis in host-pathogen interactions. Moreover, they identify SPP as a potential therapeutic target for reducing the impact of HCV infection.     

Involvement of focal adhesion kinase in inhibition of motility of human breast cancer cells by sphingosine 1-phosphate

Sphingosine 1-phosphate (SPP), a bioactive sphingolipid metabolite, inhibits chemoinvasiveness of the aggressive, estrogen-independent MDA-MB-231 human breast cancer cell line. As in many other cell types, SPP stimulated proliferation of MDA-MB-231 cells, albeit to a lesser extent. Treatment of MDA-MB-231 cells with SPP had no significant effect on their adhesiveness to Matrigel, and only high concentrations of SPP partially inhibited matrix metalloproteinase-2 activation induced by Con A. However, SPP at a concentration that strongly inhibited invasiveness also markedly reduced chemotactic motility. To investigate the molecular mechanisms by which SPP interferes with cell motility, we examined tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin, which are important for organization of focal adhesions and cell motility. SPP rapidly increased tyrosine phosphorylation of FAK and paxillin and of the paxillin-associated protein Crk. Overexpression of FAK and kinase-defective FAK in MDA-MB-231 cells resulted in a slight increase in motility without affecting the inhibitory effect of SPP, whereas expression of FAK with a mutation of the major autophosphorylation site (F397) abolished the inhibitory effect of SPP on cell motility. In contrast, the phosphoinositide 3'-kinase inhibitor, wortmannin, inhibited chemotactic motility in both vector and FAK-F397-transfected cells. Our results suggest that autophosphorylation of FAK on Y397 may play an important role in SPP signaling leading to decreased cell motility.     

Intracellular localization of sucrose-phosphate phosphatase in storage cells

The intracellular location of sucrose-phosphate phosphatase (SPP; EC 3.1, 3.24) was investigated by comparing the ratios of SPP to vacuolar and cytosolic markers in protoplasts and vacuoles from storage tissues of red beet ( Beta vulgaris ) and turnip ( Brassica rapa L. var. rapa ) hypocotyl, and carrot ( Daucus carota ) root. In all three instances, SPP activity was found not to be associated with vacuolar markers ß-acetyl-glucosaminidase and α-mannosidase, but proportionally associated with the cytosolic marker alcohol dehydrogenase. It was determined that in storage cells of red beet and turnip hypocotyl, as well as in carrot root, SPP is located in the cytoplasm. Discrepancies with earlier reports indicating SPP of vacuolar origin are discussed.