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Bruno Martoglio - One of the best experts on this subject based on the ideXlab platform.

  • Signal Peptide Peptidase dependent cleavage of type ii transmembrane substrates releases intracellular and extracellular Signals
    European Journal of Pharmacology, 2006
    Co-Authors: Sandipan Chatterjee, Bruno Martoglio, Heinrich Rueeger, Maribel Osinde, Daniela Stauffer, Hannah Morgan, Monika Kobialko, Uwe Jochen Dengler, Giorgio Rovelli
    Abstract:

    Abstract The intramembrane-cleaving proteases (I-CLiPs) presenilin-1 and -2 (PS1 and PS2), Signal Peptide Peptidase (SPP) and the Site-2 protease (S2P) catalyze critical steps in cell Signaling and are implicated in diseases such as Alzheimer's disease, hepatitis C virus (HCV) infection and cholesterol homeostasis. Here we describe the development of a cellular assay based on cleavage of the transmembrane sequence of the HCV core protein precursor, releasing intra- and extra-cellular Signals that represent sequential Signal Peptidase and SPP cleavage, respectively. We find that the SPP inhibitor (Z-LL)2-ketone (IC 50  = 1.33 μM) and the γ-secretase potent inhibitors NVP-AHW700-NX (IC 50  = 51 nM) and LY411575 (IC 50  = 61 nM) but not DAPT dose dependently inhibited SPP but not Signal Peptidase cleavage. Our data confirm that type II orientated substrates, like the HCV transmembrane sequence, are sequentially cleaved by Signal Peptidase then SPP. This dual assay provides a powerful tool to pharmacologically analyze sequential cleavage events of Signal Peptidase and SPP and their regulation.

  • Core Protein of Pestiviruses Is Processed at the C Terminus by Signal Peptide Peptidase
    Journal of Virology, 2006
    Co-Authors: Manuela Heimann, Bruno Martoglio, Gleyder Roman Sosa, Heinz-jürgen Thiel, Till Rümenapf
    Abstract:

    The core protein of pestiviruses is released from the polyprotein by viral and cellular proteinases. Here we report on an additional intramembrane proteolytic step that generates the C terminus of the core protein. C-terminal processing of the core protein of classical swine fever virus (CSFV) was blocked by the inhibitor (Z-LL)(2)-ketone, which is specific for Signal Peptide Peptidase (SPP). The same effect was obtained by overexpression of the dominant-negative SPP D(265)A mutant. The presence of (Z-LL)(2)-ketone reduced the viability of CSFV almost 100-fold in a concentration-dependent manner. Reduction of virus viability was also observed in infection experiments using a cell line that inducibly expressed SPP D(265)A. The position of SPP cleavage was determined by C-terminal sequencing of core protein purified from virions. The C terminus of CSFV core protein is alanine(255) and is located in the hydrophobic center of the Signal Peptide. The intramembrane generation of the C terminus of the CSFV core protein is almost identical to the processing scheme of the core protein of hepatitis C viruses.

  • drosophila Signal Peptide Peptidase is an essential protease for larval development
    Genetics, 2005
    Co-Authors: David J. Casso, Bruno Martoglio, Brian Biehs, Soichi Tanda, Thomas B. Kornberg
    Abstract:

    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.

  • consensus analysis of Signal Peptide Peptidase and homologous human aspartic proteases reveals opposite topology of catalytic domains compared with presenilins
    Journal of Biological Chemistry, 2004
    Co-Authors: Elena Friedmann, Marius K. Lemberg, Andreas Weihofen, Giorgio Rovelli, Uwe Jochen Dengler, Kumlesh K Dev, Bruno Martoglio
    Abstract:

    The human genome encodes seven intramembranecleaving GXGD aspartic proteases. These are the two presenilins that activate Signaling molecules and are implicated in Alzheimer’s disease, Signal Peptide Peptidase (SPP), required for immune surveillance, and four SPP-like candidate proteases (SPPLs), of unknown function. Here we describe a comparative analysis of the topologies of SPP and its human homologues, SPPL2a, -2b, -2c, and -3. We demonstrate that their N-terminal extensions are located in the extracellular space and, except for SPPL3, are modified with N-glycans. Whereas SPPL2a, -2b, and -2c contain a Signal sequence, SPP and SPPL3 contain a type I Signal anchor sequence for initiation of protein translocation and membrane insertion. The hydrophilic loops joining the transmembrane regions, which contain the catalytic residues, are facing the exoplasm. The C termini of all these proteins are exposed toward the cytosol. Taken together, our study demonstrates that SPP and its homologues are all of the same principal structure with a catalytic domain embedded in the membrane in opposite orientation to that of presenilins. Other than presenilins, SPPL2a, -2b, -2c, and -3 are therefore predicted to cleave type II-oriented substrate Peptides like the prototypic protease SPP.

  • A misassembled transmembrane domain of a polytopic protein associates with Signal Peptide Peptidase
    Biochemical Journal, 2004
    Co-Authors: Samuel G. Crawshaw, Bruno Martoglio, Suzanna L. Meacock, Stephen High
    Abstract:

    The endoplasmic reticulum (ER) exerts a quality control over newly synthesized proteins and a variety of components have been implicated in the specific recognition of aberrant or misfolded polyPeptides. We have exploited a site-specific cross-linking approach to search for novel ER components that may specifically recognize the misassembled transmembrane domains present in truncated polytopic proteins. We find that a single probe located in the transmembrane domain of a truncated opsin fragment is cross-linked to several ER proteins. These components are distinct from subunits of the Sec61 complex and represent a ‘post-translocon’ environment. In this study, we identify one of these post-translocon cross-linking partners as the Signal Peptide Peptidase (SPP). We find that the interaction of truncated opsin chains with SPP is mediated by its second transmembrane domain, and propose that this interaction may contribute to the recognition of misassembled transmembrane domains during membrane protein quality control at the ER.

Marius K. Lemberg - One of the best experts on this subject based on the ideXlab platform.

  • Signal Peptide Peptidase-Type Proteases: Versatile Regulators with Functions Ranging from Limited Proteolysis to Protein Degradation
    Journal of molecular biology, 2020
    Co-Authors: Sara Suna Yucel, Marius K. Lemberg
    Abstract:

    Intramembrane proteases catalyze the unusual cleavage of Peptide bonds in the plane of biological membranes. They are categorized according to their active site. The GxGD aspartyl proteases comprise presenilin, the Signal Peptide Peptidase (SPP), and SPP-like (SPPL) proteases. Here we focus on the functionally related SPP and SPPL proteases, and review the current understanding of their substrate specificity and summarize known physiological functions in mammalian cells. We discuss how on the one hand regulated intramembrane proteolysis generates Signaling molecules, and on the other hand how processes such as endoplasmic reticulum-associated degradation controls the quantity and activity of central regulators. While the enzymatic core of GxGD intramembrane proteases is conserved, association with regulatory factors and substrate adaptors may have tailored enzymes for various specific functions.

  • the metastable xbp1u transmembrane domain defines determinants for intramembrane proteolysis by Signal Peptide Peptidase
    Cell Reports, 2019
    Co-Authors: Sara Suna Yucel, Walter Stelzer, Dieter Langosch, Alessandra Lorenzoni, Manfred Wozny, Marius K. Lemberg
    Abstract:

    Unspliced XBP1 mRNA encodes XBP1u, the transcriptionally inert variant of the unfolded protein response (UPR) transcription factor XBP1s. XBP1u targets its mRNA-ribosome-nascent-chain-complex to the endoplasmic reticulum (ER) to facilitate UPR activation and prevents overactivation. Yet, its membrane association is controversial. Here, we use cell-free translocation and cellular assays to define a moderately hydrophobic stretch in XBP1u that is sufficient to mediate insertion into the ER membrane. Mutagenesis of this transmembrane (TM) region reveals residues that facilitate XBP1u turnover by an ER-associated degradation route that is dependent on Signal Peptide Peptidase (SPP). Furthermore, the impact of these mutations on TM helix dynamics was assessed by residue-specific amide exchange kinetics, evaluated by a semi-automated algorithm. Based on our results, we suggest that SPP-catalyzed intramembrane proteolysis of TM helices is not only determined by their conformational flexibility, but also by side-chain interactions near the scissile Peptide bond with the enzyme's active site.

  • The Metastable XBP1u Transmembrane Domain Defines Determinants for Intramembrane Proteolysis by Signal Peptide Peptidase
    Elsevier, 2019
    Co-Authors: Sara Suna Yucel, Walter Stelzer, Dieter Langosch, Alessandra Lorenzoni, Manfred Wozny, Marius K. Lemberg
    Abstract:

    Summary: Unspliced XBP1 mRNA encodes XBP1u, the transcriptionally inert variant of the unfolded protein response (UPR) transcription factor XBP1s. XBP1u targets its mRNA-ribosome-nascent-chain-complex to the endoplasmic reticulum (ER) to facilitate UPR activation and prevents overactivation. Yet, its membrane association is controversial. Here, we use cell-free translocation and cellular assays to define a moderately hydrophobic stretch in XBP1u that is sufficient to mediate insertion into the ER membrane. Mutagenesis of this transmembrane (TM) region reveals residues that facilitate XBP1u turnover by an ER-associated degradation route that is dependent on Signal Peptide Peptidase (SPP). Furthermore, the impact of these mutations on TM helix dynamics was assessed by residue-specific amide exchange kinetics, evaluated by a semi-automated algorithm. Based on our results, we suggest that SPP-catalyzed intramembrane proteolysis of TM helices is not only determined by their conformational flexibility, but also by side-chain interactions near the scissile Peptide bond with the enzyme’s active site. : Using XBP1u and determining its functional association with Sec61, Yücel et al. describe a model in which SPP requires both conformational flexibility and site-specific interactions to proteolyze its substrate. Keywords: regulated intramembrane proteolysis, GxGD aspartic intramembrane protease, ERAD, exosite, subsite, transmembrane helix dynamics, XBP1u, HO1, SP

  • the metastable xbp1u transmembrane domain defines determinants for intramembrane proteolysis by Signal Peptide Peptidase
    bioRxiv, 2018
    Co-Authors: Sara Suna Yucel, Walter Stelzer, Dieter Langosch, Alessandra Lorenzoni, Manfred Wozny, Marius K. Lemberg
    Abstract:

    Summary Unspliced XBP1 mRNA encodes XBP1u, the transcriptionally inert variant of the unfolded protein response (UPR) transcription factor XBP1s. XBP1u targets its mRNA-ribosome-nascent-chain-complex to the endoplasmic reticulum (ER) to facilitate UPR activation and prevents overactivation. Yet, its membrane association is controversial. Here, we use cell-free translocation and cellular assays to define a moderately hydrophobic stretch in XBP1u that is sufficient to mediate insertion into the ER membrane. Mutagenesis of this transmembrane (TM) region reveals residues that target XBP1u for an ER-associated degradation route that is initiated by the Signal Peptide Peptidase (SPP). Furthermore, the impact of these mutations on TM helix dynamics was assessed by residue-specific amide exchange kinetics, evaluated by a semi-automated algorithm. Based on our results, we suggest that SPP-catalyzed intramembrane proteolysis of TM helices is not only determined by their conformational flexibility, but also by side chain interactions near the scissile Peptide bond with the enzyme’s active site.

  • metastable xbp1u transmembrane domain mediates insertion into the er membrane and intramembrane proteolysis by the Signal Peptide Peptidase
    bioRxiv, 2018
    Co-Authors: Sara Suna Yucel, Walter Stelzer, Dieter Langosch, Alessandra Lorenzoni, Manfred Wozny, Marius K. Lemberg
    Abstract:

    Unspliced XBP1 mRNA encodes XBP1u, the transcriptionally inert variant of the unfolded protein response (UPR) transcription factor XBP1s. XBP1u targets its mRNA-ribosome-nascent-chain-complex to the endoplasmic reticulum (ER) to facilitate IRE1-mediated activation splicing and acts as a buffer for UPR overactivation. Yet, its membrane association is controversial. Here, we use cell-free translocation and cellular assays to define a moderately hydrophobic stretch in XBP1u that is sufficient to mediate insertion into the ER membrane. Mutagenesis of this transmembrane (TM) region reveals residues that target XBP1u for an ER-associated degradation route that is initiated by the Signal Peptide Peptidase (SPP). Furthermore, the impact of these mutations on TM helix dynamics was assessed by semi-automated residue-specific amide exchange kinetics. Based on our results, we suggest that SPP- catalyzed intramembrane proteolysis of TM helices is not only determined by their conformational flexibility, but also by side chain interactions near the scissile Peptide bond with the enzymes active site.

Todd E. Golde - One of the best experts on this subject based on the ideXlab platform.

  • differential inhibition of Signal Peptide Peptidase family members by established γ secretase inhibitors
    PLOS ONE, 2015
    Co-Authors: Yong Ran, Gabriela Z Ladd, Carolina Ceballosdiaz, Joo In Jung, Doron Greenbaum, Kevin M Felsenstein, Todd E. Golde
    Abstract:

    The Signal Peptide Peptidases (SPPs) are biomedically important proteases implicated as therapeutic targets for hepatitis C (human SPP, (hSPP)), plasmodium (Plasmodium SPP (pSPP)), and B-cell immunomodulation and neoplasia (Signal Peptide Peptidase like 2a, (SPPL2a)). To date, no drug-like, selective inhibitors have been reported. We use a recombinant substrate based on the amino-terminus of BRI2 fused to amyloid β 1-25 (Aβ1-25) (FBA) to develop facile, cost-effective SPP/SPPL protease assays. Co-transfection of expression plasmids expressing the FBA substrate with SPP/SPPLs were conducted to evaluate cleavage, which was monitored by ELISA, Western Blot and immunoprecipitation/MALDI-TOF Mass spectrometry (IP/MS). No cleavage is detected in the absence of SPP/SPPL overexpression. Multiple γ-secretase inhibitors (GSIs) and (Z-LL)2 ketone differentially inhibited SPP/SPPL activity; for example, IC50 of LY-411,575 varied from 51±79 nM (on SPPL2a) to 5499±122 nM (on SPPL2b), while Compound E showed inhibition only on hSPP with IC50 of 1465±93 nM. Data generated were predictive of effects observed for endogenous SPPL2a cleavage of CD74 in a murine B-Cell line. Thus, it is possible to differentially inhibit SPP family members. These SPP/SPPL cleavage assays will expedite the search for selective inhibitors. The data also reinforce similarities between SPP family member cleavage and cleavage catalyzed by γ-secretase.

  • targeting the erad pathway via inhibition of Signal Peptide Peptidase for antiparasitic therapeutic design
    Proceedings of the National Academy of Sciences of the United States of America, 2012
    Co-Authors: Michael B Harbut, Todd E. Golde, Bhumit Patel, Bryan K S Yeung, Case W Mcnamara, Taylor A Bright, Jaime Ballard, Frantisek Supek, Elizabeth A Winzeler
    Abstract:

    Early secretory and endoplasmic reticulum (ER)-localized proteins that are terminally misfolded or misassembled are degraded by a ubiquitin- and proteasome-mediated process known as ER-associated degradation (ERAD). Protozoan pathogens, including the causative agents of malaria, toxoplasmosis, trypanosomiasis, and leishmaniasis, contain a minimal ERAD network relative to higher eukaryotic cells, and, because of this, we observe that the malaria parasite Plasmodium falciparum is highly sensitive to the inhibition of components of this protein quality control system. Inhibitors that specifically target a putative protease component of ERAD, Signal Peptide Peptidase (SPP), have high selectivity and potency for P. falciparum. By using a variety of methodologies, we validate that SPP inhibitors target P. falciparum SPP in parasites, disrupt the protein’s ability to facilitate degradation of unstable proteins, and inhibit its proteolytic activity. These compounds also show low nanomolar activity against liver-stage malaria parasites and are also equipotent against a panel of pathogenic protozoan parasites. Collectively, these data suggest ER quality control as a vulnerability of protozoan parasites, and that SPP inhibition may represent a suitable transmission blocking antimalarial strategy and potential pan-protozoan drug target.

  • a small molecule inhibitor of Signal Peptide Peptidase inhibits plasmodium development in the liver and decreases malaria severity
    PLOS ONE, 2009
    Co-Authors: Iana Parvanova, Todd E. Golde, Sabrina Epiphanio, Abdul H Fauq, Miguel Prudencio, Maria M Mota
    Abstract:

    The liver stage of Plasmodium's life cycle is the first, obligatory step in malaria infection. Decreasing the hepatic burden of Plasmodium infection decreases the severity of disease and constitutes a promising strategy for malaria prophylaxis. The efficacy of the gamma-secretase and Signal Peptide Peptidase inhibitor LY411,575 in targeting Plasmodium liver stages was evaluated both in human hepatoma cell lines and in mouse primary hepatocytes. LY411,575 was found to prevent Plasmodium's normal development in the liver, with an IC50 of approximately 80 nM, without affecting hepatocyte invasion by the parasite. In vivo results with a rodent model of malaria showed that LY411,575 decreases the parasite load in the liver and increases by 55% the resistance of mice to cerebral malaria, one of the most severe malaria-associated syndromes. Our data show that LY411,575 does not exert its effect via the Notch Signaling pathway suggesting that it may interfere with Plasmodium development through an inhibition of the parasite's Signal Peptide Peptidase. We therefore propose that selective Signal Peptide Peptidase inhibitors could be potentially used for preventive treatment of malaria in humans.

  • Signal Peptide Peptidase spp dimer formation as assessed by fluorescence lifetime imaging microscopy flim in intact cells
    Molecular Neurodegeneration, 2006
    Co-Authors: Andrew C. Nyborg, Thomas B. Ladd, Karen Jansen, Lauren Herl, Oksana Berezovska, Anne V Thomas, Bradley T Hyman, Todd E. Golde
    Abstract:

    Background Signal Peptide Peptidase (SPP) is an intramembrane cleaving protease identified by its cleavage of several type II membrane Signal Peptides. Conservation of intramembrane active site residues demonstrates that SPP, SPP family members, and presenilins (PSs) make up a family of intramembrane cleaving proteases. Because SPP appears to function without additional protein cofactors, the study of SPP may provide structural insights into the mechanism of intramembrane proteolysis by this biomedically important family of proteins. Previous studies have shown that SPP isolated from cells appears to be a homodimer, but some evidence exists that in vitro SPP may be active as a monomer. We have conducted additional experiments to determine if SPP exists as a monomer or dimer in vivo.

  • Signal Peptide Peptidase biochemical properties and modulation by nonsteroidal antiinflammatory drugs
    Biochemistry, 2006
    Co-Authors: Toru Sato, Andrew C. Nyborg, Todd E. Golde, Nobuhisa Iwata, Thekla S Diehl, Takaomi Saido, Michael S Wolfe
    Abstract:

    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.

Homayon Ghiasi - One of the best experts on this subject based on the ideXlab platform.

  • blocking hsv 1 glycoprotein k binding to Signal Peptide Peptidase reduces virus infectivity in vitro and in vivo
    PLOS Pathogens, 2021
    Co-Authors: Shaohui Wang, Ujjaldeep Jaggi, Homayon Ghiasi
    Abstract:

    HSV glycoprotein K (gK) is an essential herpes protein that contributes to enhancement of eye disease. We previously reported that gK binds to Signal Peptide Peptidase (SPP) and that depletion of SPP reduces HSV-1 infectivity in vivo. To determine the therapeutic potential of blocking gK binding to SPP on virus infectivity and pathogenicity, we mapped the gK binding site for SPP to a 15mer Peptide within the amino-terminus of gK. This 15mer Peptide reduced infectivity of three different virus strains in vitro as determined by plaque assay, FACS, and RT-PCR. Similarly, the 15mer Peptide reduced ocular virus replication in both BALB/c and C57BL/6 mice and also reduced levels of latency and exhaustion markers in infected mice when compared with control treated mice. Addition of the gK-15mer Peptide also increased the survival of infected mice when compared with control mice. These results suggest that blocking gK binding to SPP using gK Peptide may have therapeutic potential in treating HSV-1-associated infection.

  • absence of Signal Peptide Peptidase an essential herpes simplex virus 1 glycoprotein k binding partner reduces virus infectivity in vivo
    Journal of Virology, 2019
    Co-Authors: Shaohui Wang, Homayon Ghiasi
    Abstract:

    We previously reported that herpes simplex virus (HSV) glycoprotein K (gK) binds to Signal Peptide Peptidase (SPP), also known as minor histocompatibility antigen H13. Binding of gK to SPP is required for HSV-1 infectivity in vitro SPP is a member of the γ-secretase family, and mice lacking SPP are embryonic lethal. To determine how SPP affects HSV-1 infectivity in vivo, the SPP gene was deleted using a tamoxifen-inducible Cre recombinase driven by the ubiquitously expressed ROSA26 promoter. SPP mRNA was reduced by more than 93% in the cornea and trigeminal ganglia (TG) and by 99% in the liver of tamoxifen-injected mice, while SPP protein expression was reduced by 90% compared to the level in control mice. Mice lacking SPP had significantly less HSV-1 replication in the eye as well as reduced gK, UL20, ICP0, and gB transcripts in the cornea and TG compared to levels in control mice. In addition, reduced infiltration of CD45+, CD4+, CD8+, F4/80+, CD11c+, and NK1.1+ T cells was observed in the cornea and TG of SPP-inducible knockout mice compared to that in control mice. Finally, in the absence of SPP, latency was significantly reduced in SPP-inducible knockout mice compared to that in control mice. Thus, in this study we have generated SPP-inducible knockout mice and shown that the absence of SPP affects virus replication in the eye of ocularly infected mice and that this reduction is correlated with the interaction of gK and SPP. These results suggest that blocking this interaction may have therapeutic potential in treating HSV-1-associated eye disease.IMPORTANCE Glycoprotein K (gK) is an essential and highly conserved HSV-1 protein. Previously, we reported that gK binds to SPP, an endoplasmic reticulum (ER) protein, and blocking this binding reduces virus infectivity in vitro and also affects gK and UL20 subcellular localization. To evaluate the function of gK binding to SPP in vivo, we generated SPP-inducible knockout mice and observed the following in the absence of SPP: (i) that significantly less HSV-1 replication was seen in ocularly infected mice than in control mice; (ii) that expression of various HSV-1 genes and cellular infiltrates in the eye and trigeminal ganglia of infected mice was less than that in control mice; and (iii) that latency was significantly reduced in infected mice. Thus, blocking of gK binding to SPP may be a useful tool to control HSV-1-induced eye disease in patients with herpes stromal keratitis (HSK).

  • inhibitors of Signal Peptide Peptidase spp affect hsv 1 infectivity in vitro and in vivo
    Experimental Eye Research, 2014
    Co-Authors: Sariah J Allen, Kevin R Mott, Homayon Ghiasi
    Abstract:

    Recently we have shown that the highly conserved herpes simplex virus glycoprotein K (gK) binds to Signal Peptide Peptidase (SPP), also known as minor histocompatibility antigen H13. In this study we have demonstrated for the first time that inhibitors of SPP, such as L685,458, (Z-LL)2 ketone, aspirin, ibuprofen and DAPT, significantly reduced HSV-1 replication in tissue culture. Inhibition of SPP activity via (Z-LL)2 ketone significantly reduced viral transcripts in the nucleus of infected cells. Finally, when administered during primary infection, (Z-LL)2 ketone inhibitor reduced HSV-1 replication in the eyes of ocularly infected mice. Thus, blocking SPP activity may represent a clinically effective and expedient approach to the reduction of viral replication and the resulting pathology.

  • binding of hsv 1 glycoprotein k gk to Signal Peptide Peptidase spp is required for virus infectivity
    PLOS ONE, 2014
    Co-Authors: Sariah J Allen, Kohji Moriishi, Yoshiharu Matsuura, Kevin R Mott, Konstantin G Kousoulas, Homayon Ghiasi
    Abstract:

    Glycoprotein K (gK) is a virion envelope protein of herpes simplex virus types 1 (HSV-1) and 2 (HSV-2), which plays important roles in virion entry, morphogenesis and egress. Two-hybrid and pull-down assays were utilized to demonstrate that gK and no other HSV-1 genes specifically binds to Signal Peptide Peptidase (SPP), also known as minor histocompatibility antigen H13. SPP dominant negative mutants, shRNA against SPP significantly reduced HSV-1 replication in vitro. SPP also affected lysosomes and ER responses to HSV-1 infection. Thus, in this study we have shown for the first time that gK, despite its role in fusion and egress, is also involved in binding the cytoplasmic protein SPP. These results also suggest that SPP plays an important role in viral replication and possibly virus pathogenesis. This makes SPP unique in that its function appears to be required by the virus as no other protein can compensate its loss in terms of viral replication.

Kohji Moriishi - One of the best experts on this subject based on the ideXlab platform.

  • insights into the mechanism of isoenzyme specific Signal Peptide Peptidase mediated translocation of heme oxygenase
    PLOS ONE, 2017
    Co-Authors: Bianca Schaefer, Kohji Moriishi, Soenke Behrends
    Abstract:

    It has recently been shown that Signal Peptide Peptidase (SPP) can catalyze the intramembrane cleavage of heme oxygenase-1 (HO-1) that leads to translocation of HO-1 into the cytosol and nucleus. While there is consensus that translocated HO-1 promotes tumor progression and drug resistance, the physiological Signals leading to SPP-mediated intramembrane cleavage of HO-1 and the specificity of the process remain unclear. In this study, we used co-immunoprecipitation and confocal laser scanning microscopy to investigate the translocation mechanism of HO-1 and its regulation by SPP. We show that HO-1 and the closely related HO-2 isoenzyme bind to SPP under normoxic conditions. Under hypoxic conditions SPP mediates intramembrane cleavage of HO-1, but not HO-2. In experiments with an inactive HO-1 mutant (H25A) we show that translocation is independent of the catalytic activity of HO-1. Studies with HO-1 / HO-2 chimeras indicate that the membrane anchor, the PEST-domain and the nuclear shuttle sequence of HO-1 are necessary for full cleavage and subsequent translocation under hypoxic conditions. In the presence of co-expressed exogenous SPP, the anchor and the PEST-domain are sufficient for translocation. Taken together, we identified the domains involved in HO-1 translocation and showed that SPP-mediated cleavage is isoform-specific and independent of HO-activity. A closer understanding of the translocation mechanism of HO-1 is of particular importance because nuclear HO-1 seems to lead to tumor progression and drug resistance.

  • the potential of Signal Peptide Peptidase as a therapeutic target for hepatitis c
    Expert Opinion on Therapeutic Targets, 2017
    Co-Authors: Kohji Moriishi
    Abstract:

    AbstractIntroduction: Chronic infection with hepatitis C virus (HCV) causes liver steatosis, cirrhosis, metabolic syndrome with inflammation, and eventually leads to hepatocellular carcinoma. HCV core protein is a well-known capsid protein and pathogenic factor related to lipid accumulation, type 2 diabetes mellitus, and carcinogenesis. Cleavage of the C-terminal transmembrane region by Signal Peptide Peptidase (SPP) is required for maturation of the core protein.Areas covered: Herein, this review details the general aspects of the structure, lifecycle, pathogenesis, and maturation of the HCV core protein, the function of SPP, and clinically available direct-acting antivirals (DAAs). SPP is classified into a group of GXGD-type intramembrane proteases including presenilin-1, which is a component of γ-secretase complex. Several SPP inhibitors were previously identified from γ-secretase inhibitors, but have not yet been improved based on specificity to SPP. Finally, the author discusses the potential of SPP ...

  • abstract 812 processing of core protein by Signal Peptide Peptidase participates in propagation and pathogenesis of hepatitis c virus
    Cancer Research, 2015
    Co-Authors: Toru Okamoto, Kohji Moriishi, Tatsuya Suzuki, Sayaka Aizawa, Takahisa Kouwaki, Takasuke Fukuhara, Kazuhiko Koike, Yoshiharu Matsuura
    Abstract:

    Hepatitis C virus (HCV) is a major causative agent of chronic liver diseases including steatosis, cirrhosis and hepatocellular carcinoma (HCC). The HCV core protein is cleaved off from a precursor polyprotein by a Signal Peptidase and then further processed by Signal Peptide Peptidase (SPP) within the transmembrane region. However, the biological significance of the processing of HCV core protein by SPP remains largely unknown. Transgenic mice expressing HCV core protein exhibit insulin-resistance, steatosis and finally develop HCC, suggesting that core protein plays crucial roles in pathogenesis of HCV. In this study we have established SPP-knockout (KO) mice and cell lines to examine the roles of SPP on the propagation and pathogenesis of HCV. SPP KO mice were embryonic lethal at around E13.5, therefore we prepared SPP-KO mouse embryonic fibroblast cells (MEFs). In SPP-KO cells, HCV core protein was quickly degraded by proteasome and propagation of HCV was severely impaired. Treatment of cells with LY-411575, an inhibitor for SPP, also induced degradation of HCV core protein and suppressed the production of infectious HCV particles. PA28γ or E6AP, which had been reported to degrade core protein, were not involved in SPP-mediated degradation, suggesting the other mechanisms were involved in SPP-induced core protein degradation. Furthermore, oral administration of LY-411575 into HCV core transgenic mice reduced expression of core protein and cured insulin-resistance. The deletion of one allele of SPP gene in core transgenic mice led to impair the expression of core protein and cure insulin-resistance. These results suggest that SPP is a novel drug target for chronic hepatitis C through suppression of viral propagation and liver failures. Citation Format: Toru Okamoto, Sayaka Aizawa, Takahisa Kouwaki, Tatsuya Suzuki, Takasuke Fukuhara, Kohji Moriishi, Kazuhiko Koike, Yoshiharu Matsuura. Processing of core protein by Signal Peptide Peptidase participates in propagation and pathogenesis of hepatitis C virus. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 812. doi:10.1158/1538-7445.AM2015-812

  • binding of hsv 1 glycoprotein k gk to Signal Peptide Peptidase spp is required for virus infectivity
    PLOS ONE, 2014
    Co-Authors: Sariah J Allen, Kohji Moriishi, Yoshiharu Matsuura, Kevin R Mott, Konstantin G Kousoulas, Homayon Ghiasi
    Abstract:

    Glycoprotein K (gK) is a virion envelope protein of herpes simplex virus types 1 (HSV-1) and 2 (HSV-2), which plays important roles in virion entry, morphogenesis and egress. Two-hybrid and pull-down assays were utilized to demonstrate that gK and no other HSV-1 genes specifically binds to Signal Peptide Peptidase (SPP), also known as minor histocompatibility antigen H13. SPP dominant negative mutants, shRNA against SPP significantly reduced HSV-1 replication in vitro. SPP also affected lysosomes and ER responses to HSV-1 infection. Thus, in this study we have shown for the first time that gK, despite its role in fusion and egress, is also involved in binding the cytoplasmic protein SPP. These results also suggest that SPP plays an important role in viral replication and possibly virus pathogenesis. This makes SPP unique in that its function appears to be required by the virus as no other protein can compensate its loss in terms of viral replication.

  • intramembrane processing by Signal Peptide Peptidase regulates the membrane localization of hepatitis c virus core protein and viral propagation
    Journal of Virology, 2008
    Co-Authors: Kiyoko Okamoto, Kohji Moriishi, Yoshio Mori, Yasumasa Komoda, Toru Okamoto, Masayasu Okochi, Masatoshi Takeda, Tetsuro Suzuki, Yoshiharu Matsuura
    Abstract:

    Hepatitis C virus (HCV) core protein has shown to be localized in the detergent-resistant membrane (DRM), which is distinct from the classical raft fraction including caveolin, although the biological significance of the DRM localization of the core protein has not been determined. The HCV core protein is cleaved off from a precursor polyprotein at the lumen side of Ala191 by Signal Peptidase and is then further processed by Signal Peptide Peptidase (SPP) within the transmembrane region. In this study, we examined the role of SPP in the localization of the HCV core protein in the DRM and in viral propagation. The C terminus of the HCV core protein cleaved by SPP in 293T cells was identified as Phe177 by mass spectrometry. Mutations introduced into two residues (Ile176 and Phe177) upstream of the cleavage site of the core protein abrogated processing by SPP and localization in the DRM fraction. Expression of a dominant-negative SPP or treatment with an SPP inhibitor, L685,458, resulted in reductions in the levels of processed core protein localized in the DRM fraction. The production of HCV RNA in cells persistently infected with strain JFH-1 was impaired by treatment with the SPP inhibitor. Furthermore, mutant JFH-1 viruses bearing SPP-resistant mutations in the core protein failed to propagate in a permissive cell line. These results suggest that intramembrane processing of HCV core protein by SPP is required for the localization of the HCV core protein in the DRM and for viral propagation.