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• W2912646755 abstract "Article7 February 2019free access Source DataTransparent process The intramembrane protease SPPL2c promotes male germ cell development by cleaving phospholamban Johannes Niemeyer Biochemical Institute, Christian-Albrechts-University of Kiel, Kiel, Germany Search for more papers by this author Torben Mentrup Biochemical Institute, Christian-Albrechts-University of Kiel, Kiel, Germany Institute of Physiological Chemistry, Technische Universität Dresden, Dresden, Germany Search for more papers by this author Ronny Heidasch Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Allianz, Heidelberg, Germany Search for more papers by this author Stephan A Müller orcid.org/0000-0003-3414-307X DZNE – German Center for Neurodegenerative Diseases, Munich, Germany Neuroproteomics, School of Medicine, Klinikum rechts der Isar and Institute for Advanced Study, Technical University of Munich, Munich, Germany Search for more papers by this author Uddipta Biswas Institute of Physiological Chemistry, Technische Universität Dresden, Dresden, Germany Search for more papers by this author Rieke Meyer Biochemical Institute, Christian-Albrechts-University of Kiel, Kiel, Germany Search for more papers by this author Alkmini A Papadopoulou Institute for Metabolic Biochemistry, Biomedical Center (BMC) München, Ludwig Maximilians University of Munich, Munich, Germany Search for more papers by this author Verena Dederer Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Allianz, Heidelberg, Germany Search for more papers by this author Martina Haug-Kröper Institute for Metabolic Biochemistry, Biomedical Center (BMC) München, Ludwig Maximilians University of Munich, Munich, Germany Search for more papers by this author Vivian Adamski Biochemical Institute, Christian-Albrechts-University of Kiel, Kiel, Germany Search for more papers by this author Renate Lüllmann-Rauch Institute of Anatomy, Christian-Albrechts-University of Kiel, Kiel, Germany Search for more papers by this author Martin Bergmann Institute of Veterinary Anatomy, Justus Liebig University of Gießen, Gießen, Germany Search for more papers by this author Artur Mayerhofer Cell Biology, Anatomy III, Biomedical Center (BMC) München, Ludwig Maximilians University of Munich, Munich, Germany Search for more papers by this author Paul Saftig orcid.org/0000-0003-2637-7052 Biochemical Institute, Christian-Albrechts-University of Kiel, Kiel, Germany Search for more papers by this author Gunther Wennemuth orcid.org/0000-0003-3313-2475 Institute of Anatomy, University Hospital, Duisburg-Essen University, Essen, Germany Search for more papers by this author Rolf Jessberger Institute of Physiological Chemistry, Technische Universität Dresden, Dresden, Germany Search for more papers by this author Regina Fluhrer orcid.org/0000-0002-9778-4643 DZNE – German Center for Neurodegenerative Diseases, Munich, Germany Institute for Metabolic Biochemistry, Biomedical Center (BMC) München, Ludwig Maximilians University of Munich, Munich, Germany Search for more papers by this author Stefan F Lichtenthaler orcid.org/0000-0003-2211-2575 DZNE – German Center for Neurodegenerative Diseases, Munich, Germany Neuroproteomics, School of Medicine, Klinikum rechts der Isar and Institute for Advanced Study, Technical University of Munich, Munich, Germany Munich Cluster for Systems Neurology (SyNergy), Munich, Germany Search for more papers by this author Marius K Lemberg orcid.org/0000-0002-0996-1268 Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Allianz, Heidelberg, Germany Search for more papers by this author Bernd Schröder Corresponding Author [email protected] orcid.org/0000-0003-0774-6270 Biochemical Institute, Christian-Albrechts-University of Kiel, Kiel, Germany Institute of Physiological Chemistry, Technische Universität Dresden, Dresden, Germany Search for more papers by this author Johannes Niemeyer Biochemical Institute, Christian-Albrechts-University of Kiel, Kiel, Germany Search for more papers by this author Torben Mentrup Biochemical Institute, Christian-Albrechts-University of Kiel, Kiel, Germany Institute of Physiological Chemistry, Technische Universität Dresden, Dresden, Germany Search for more papers by this author Ronny Heidasch Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Allianz, Heidelberg, Germany Search for more papers by this author Stephan A Müller orcid.org/0000-0003-3414-307X DZNE – German Center for Neurodegenerative Diseases, Munich, Germany Neuroproteomics, School of Medicine, Klinikum rechts der Isar and Institute for Advanced Study, Technical University of Munich, Munich, Germany Search for more papers by this author Uddipta Biswas Institute of Physiological Chemistry, Technische Universität Dresden, Dresden, Germany Search for more papers by this author Rieke Meyer Biochemical Institute, Christian-Albrechts-University of Kiel, Kiel, Germany Search for more papers by this author Alkmini A Papadopoulou Institute for Metabolic Biochemistry, Biomedical Center (BMC) München, Ludwig Maximilians University of Munich, Munich, Germany Search for more papers by this author Verena Dederer Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Allianz, Heidelberg, Germany Search for more papers by this author Martina Haug-Kröper Institute for Metabolic Biochemistry, Biomedical Center (BMC) München, Ludwig Maximilians University of Munich, Munich, Germany Search for more papers by this author Vivian Adamski Biochemical Institute, Christian-Albrechts-University of Kiel, Kiel, Germany Search for more papers by this author Renate Lüllmann-Rauch Institute of Anatomy, Christian-Albrechts-University of Kiel, Kiel, Germany Search for more papers by this author Martin Bergmann Institute of Veterinary Anatomy, Justus Liebig University of Gießen, Gießen, Germany Search for more papers by this author Artur Mayerhofer Cell Biology, Anatomy III, Biomedical Center (BMC) München, Ludwig Maximilians University of Munich, Munich, Germany Search for more papers by this author Paul Saftig orcid.org/0000-0003-2637-7052 Biochemical Institute, Christian-Albrechts-University of Kiel, Kiel, Germany Search for more papers by this author Gunther Wennemuth orcid.org/0000-0003-3313-2475 Institute of Anatomy, University Hospital, Duisburg-Essen University, Essen, Germany Search for more papers by this author Rolf Jessberger Institute of Physiological Chemistry, Technische Universität Dresden, Dresden, Germany Search for more papers by this author Regina Fluhrer orcid.org/0000-0002-9778-4643 DZNE – German Center for Neurodegenerative Diseases, Munich, Germany Institute for Metabolic Biochemistry, Biomedical Center (BMC) München, Ludwig Maximilians University of Munich, Munich, Germany Search for more papers by this author Stefan F Lichtenthaler orcid.org/0000-0003-2211-2575 DZNE – German Center for Neurodegenerative Diseases, Munich, Germany Neuroproteomics, School of Medicine, Klinikum rechts der Isar and Institute for Advanced Study, Technical University of Munich, Munich, Germany Munich Cluster for Systems Neurology (SyNergy), Munich, Germany Search for more papers by this author Marius K Lemberg orcid.org/0000-0002-0996-1268 Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Allianz, Heidelberg, Germany Search for more papers by this author Bernd Schröder Corresponding Author [email protected] orcid.org/0000-0003-0774-6270 Biochemical Institute, Christian-Albrechts-University of Kiel, Kiel, Germany Institute of Physiological Chemistry, Technische Universität Dresden, Dresden, Germany Search for more papers by this author Author Information Johannes Niemeyer1,‡, Torben Mentrup1,2,‡, Ronny Heidasch3,‡, Stephan A Müller4,5, Uddipta Biswas2, Rieke Meyer1, Alkmini A Papadopoulou6, Verena Dederer3, Martina Haug-Kröper6, Vivian Adamski1, Renate Lüllmann-Rauch7, Martin Bergmann8, Artur Mayerhofer9, Paul Saftig1, Gunther Wennemuth10, Rolf Jessberger2, Regina Fluhrer4,6, Stefan F Lichtenthaler4,5,11, Marius K Lemberg3 and Bernd Schröder *,1,2 1Biochemical Institute, Christian-Albrechts-University of Kiel, Kiel, Germany 2Institute of Physiological Chemistry, Technische Universität Dresden, Dresden, Germany 3Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Allianz, Heidelberg, Germany 4DZNE – German Center for Neurodegenerative Diseases, Munich, Germany 5Neuroproteomics, School of Medicine, Klinikum rechts der Isar and Institute for Advanced Study, Technical University of Munich, Munich, Germany 6Institute for Metabolic Biochemistry, Biomedical Center (BMC) München, Ludwig Maximilians University of Munich, Munich, Germany 7Institute of Anatomy, Christian-Albrechts-University of Kiel, Kiel, Germany 8Institute of Veterinary Anatomy, Justus Liebig University of Gießen, Gießen, Germany 9Cell Biology, Anatomy III, Biomedical Center (BMC) München, Ludwig Maximilians University of Munich, Munich, Germany 10Institute of Anatomy, University Hospital, Duisburg-Essen University, Essen, Germany 11Munich Cluster for Systems Neurology (SyNergy), Munich, Germany ‡These authors contributed equally to this work *Corresponding author. Tel: +49 351 458 6450; Fax: +49 351 458 6307; E-mail: [email protected] EMBO Rep (2019)20:e46449https://doi.org/10.15252/embr.201846449 See also: AA Papadopoulou et al (March 2019) and HS Young & MJ Lemieux (March 2019) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Signal peptide peptidase (SPP) and the four homologous SPP-like (SPPL) proteases constitute a family of intramembrane aspartyl proteases with selectivity for type II-oriented transmembrane segments. Here, we analyse the physiological function of the orphan protease SPPL2c, previously considered to represent a non-expressed pseudogene. We demonstrate proteolytic activity of SPPL2c towards selected tail-anchored proteins. Despite shared ER localisation, SPPL2c and SPP exhibit distinct, though partially overlapping substrate spectra and inhibitory profiles, and are organised in different high molecular weight complexes. Interestingly, SPPL2c is specifically expressed in murine and human testis where it is primarily localised in spermatids. In mice, SPPL2c deficiency leads to a partial loss of elongated spermatids and reduced motility of mature spermatozoa, but preserved fertility. However, matings of male and female SPPL2c−/− mice exhibit reduced litter sizes. Using proteomics we identify the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2)-regulating protein phospholamban (PLN) as a physiological SPPL2c substrate. Accumulation of PLN correlates with a decrease in intracellular Ca2+ levels in elongated spermatids that likely contribute to the compromised male germ cell differentiation and function of SPPL2c−/− mice. Synopsis The intramembrane protease SPPL2c is critical for the turnover of selected tail-anchored proteins in spermatids and thereby supports differentiation and function of male germ cells. The presenilin-homologue Signal peptide peptidase-like 2c is an ER-resident intramembrane protease endogenously expressed in murine and human spermatids. SPPL2c processes selected tail-anchored proteins with a substrate spectrum distinct from that of Signal peptide peptidase (SPP). SPPL2c-deficient mice show a partial loss of elongated spermatids and reduced motility of mature spermatozoa. Phospholamban represents a physiological SPPL2c substrate in murine testis. Introduction Intramembrane proteases cleave substrate proteins within the hydrophobic environment of the phospholipid bilayer 1. Thereby, cleavage fragments are released into the cytoplasm, which can act as transcriptional regulators after nuclear translocation as exemplified by the paradigmatic SREBP and Notch pathways critically regulating cholesterol homeostasis and cellular differentiation, respectively 2. In addition, intramembrane proteolysis controls the homeostasis of membrane-bound substrate proteins 3. Signal peptide peptidase (SPP) and the SPP-like proteases SPPL2a-c and SPPL3 are aspartyl intramembrane proteases with homology to presenilin 4, 5, the catalytic subunit of the γ-secretase complex. However, SPP/SPPL proteases show specificity for type II-oriented substrate proteins, i.e. single-pass transmembrane proteins with their N-terminus intracellularly as opposed to extracellularly (type I) 6, 7. SPP was initially discovered based on its ability to cleave signal peptides in the endoplasmic reticulum (ER) 4. Since then, multiple functions of this protease have been unravelled 6 including the processing of viral proteins like the hepatitis C virus (HCV) core protein 8 and a role in ER-associated protein degradation (ERAD) 9-11. Recently, selected tail-anchored (TA) proteins like heme oxygenase 1 (HO-1) were identified as proteolytic substrates of SPP 11, 12. With their type II-oriented transmembrane domain and short luminal C-terminus, TA proteins can be cleaved independent of any preceding ectodomain processing, which is necessary for SPP-catalysed turnover of signal peptides 13, or recruitment of the ERAD factor Derlin-1 as has been shown for the type II membrane protein XBP1u 9. However, so far it has not been reported if TA proteins can also be substrates of any of the four SPPL proteases. In contrast to the ER-resident SPP, SPPL2a, SPPL2b and SPPL3 reside in lysosomes/late endosomes, the plasma membrane and the Golgi apparatus, respectively. SPPL3 acts as a major regulator of protein glycosylation and glycosaminoglycan biosynthesis by shedding various glycosyltransferases 14, 15. Its ability to cleave substrates with large ectodomains is a unique property within the SPPL family and distinguishes it from SPPL2a and SPPL2b. In cell-based overexpression set-ups, substrate spectra of SPPL2a and SPPL2b overlap significantly suggesting similar catalytic properties of both proteases. Whereas SPPL2a is of critical importance for the development of B lymphocytes and dendritic cells 16-18, the in vivo function of SPPL2b is currently less clear 19 and the identification of physiological substrates of SPPL2b is still pending. In contrast to the other SPPL2 family members, very little is known so far about SPPL2c. Based on its intronless gene structure, it was hypothesised to represent a non-expressed pseudogene 20, 21. Upon heterologous expression of the SPPL2c open reading frame, the resulting protein was observed in the ER 21. However, endogenous expression of SPPL2c has not been demonstrated so far. SPPL2c exhibits the catalytic YD/FD and GxGD signature motifs, conserved in all intramembrane aspartyl proteases 4, 5. Nevertheless, proteolytic activity of SPPL2c has not been revealed yet. Conspicuously, the proposed ER localisation of SPPL2c suggests that its intracellular distribution overlaps with that of SPP. This leads to the question why two SPP/SPPL proteases in the same cellular compartment have evolved and to what extent their functions overlap. Here, we have systematically analysed expression and function of the orphan intramembrane protease SPPL2c. We demonstrate that SPPL2c is an ER-resident protein, which is specifically expressed in murine and human testis under endogenous conditions. There, it is present in developing germ cells with the highest abundance in spermatids. Consequently, differentiation and function of male germ cells are compromised in SPPL2c-deficient mice. We demonstrate for the first time that SPPL2c exhibits proteolytic activity. Similar to SPP, SPPL2c cleaves selected TA proteins, however with a distinct, only partially overlapping substrate spectrum. Using proteomics, we have identified the SERCA regulating protein phospholamban (PLN) as physiological substrate of SPPL2c, presumably leading to a dysregulation of intracellular Ca2+ handling in SPPL2c−/− male germ cells. Results SPPL2c is a testis-specific ER-resident intramembrane protease As predicted from the amino acid sequence, the murine SPPL2c protein exhibits a nine-transmembrane domain topology (Fig 1A and B) with the active site consensus FD and GFGD motifs present in transmembrane segments 6 and 7 21. The coding sequence of the canonical SPPL2c isoform A protein is present as a contiguous block in the genomic locus (Fig EV1A). In addition, databases list a second SPPL2c transcript, isoform B, with a distinct 3′-end, which codes for a protein with a shorter C-terminus. We screened for endogenous expression of SPPL2c in a variety of murine tissues (Fig 1C) by RT–PCR using primer pairs specific for the two isoforms. We detected mRNA of both SPPL2c isoforms in testis, but not in heart, skeletal muscle, brain, spleen, kidney, liver and lung. Polyclonal antisera against two different epitopes (N-Term, C-Term) of the SPPL2c protein were generated as indicated in Fig 1B and were validated using HEK293 cells overexpressing the two isoforms (Fig EV1B). As expected from the position of the epitopes, the C-terminal antibody specifically recognised the longer isoform A due to the lack of this epitope in the C-terminally truncated isoform B, whereas the N-terminal antiserum was capable of detecting both. With these antibodies, we detected the endogenous SPPL2c protein in testis lysates from wild-type mice by Western blotting (Fig 1D and E) finally excluding SPPL2c being a non-expressed pseudogene. To control for specificity, we analysed samples from SPPL2c−/− mice where the entire coding region had been deleted (Fig EV1A). Thus, we provide evidence for endogenous expression of both SPPL2c isoforms at protein level in murine testis. We also assessed several other tissues from wild-type and SPPL2c−/− mice (Fig EV1C). In support of the transcriptional data (Fig 1C), we did not detect SPPL2c protein in any additional tissue under basal conditions. In agreement with previous reports about human SPPL2c 21, ectopically expressed murine SPPL2c was detected in the ER (Fig 1F), but not in the Golgi. In addition, partial co-localisation with a marker protein of the ER-Golgi intermediate compartment (ERGIC) could be observed. Figure 1. SPPL2c is a testis-specific ER-resident intramembrane protease A. Predicted topology of murine SPPL2c. Positions of the predicted N-glycosylation site and critical aspartate residues of the active centre (asterisks) are indicated. B. Scheme of the two murine SPPL2c isoforms A and B. Epitopes (N-term/C-term) used for generation of antisera are marked. SP, predicted signal peptide. C. RT–PCR analysis of SPPL2c expression in different murine tissues. Total RNA from the indicated tissues was either transcribed into cDNA prior to PCR amplification (+RT) or, as negative control, used directly as template (−RT). As indicated in Fig EV1A, a common forward primer in combination with an isoform-specific reverse primer was employed. A fragment of the actin ORF was amplified as control. D, E. Western Blot analysis of total lysates from testis isolated from wild type or SPPL2c−/− using antibodies against C- (D) or N-terminal (E) epitopes of SPPL2c. Lysates from HeLa cells transiently expressing the SPPL2c isoforms were analysed in parallel. Full Western blots are shown in Fig EV1B. *, non-specific band. F. SPPL2c was visualised by indirect immunofluorescence in HeLa cells transiently expressing C-terminally Myc-tagged murine SPPL2c isoform A. For detection of SPPL2c, either the anti-Myc or the SPPL2c antiserum (C-terminal epitope) was employed as indicated together with anti-KDEL, anti-GM130 or anti-ERGIC53 as indicated in order to label the ER, the cis-Golgi apparatus or the ER-Golgi intermediate compartment (ERGIC), respectively. Scale bars, 10 μm. G. Lysates from HEK293 cells transiently transfected with murine SPPL2c-myc (isoform A) were treated with the glycosidases PNGase F or Endo H as indicated prior to Western blot analysis with anti-SPPL2c (C-term). Deglycosylation of endogenously expressed LIMP2 was analysed as control. H. N-glycosylation of endogenous SPPL2c is similar to the overexpressed protein. Total lysates from testis of wild-type or SPPL2c−/− mice were treated and analysed as in (G). I. Subcellular fractionation of post-nuclear supernatant from murine testis using a self-forming Percoll density gradient. Fractions were collected from bottom to top and subjected to Western blot analysis for SPPL2c. Protein disulphide isomerase (PDI), cathepsin D and CD44 were detected as organelle marker proteins. Source data are available online for this figure. Source Data for Figure 1 [embr201846449-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Generation and validation of SPPL2c−/− mice and SPPL2c antisera Scheme of the murine SPPL2c genomic locus and the employed targeting strategy of the allele designed by Velocigene. Upon deletion of the entire SPPL2c coding region, a β-galactosidase reporter gene (β-Gal) and a floxed neomycin resistance cassette (Neo) were inserted. The latter was excised by breeding with Cre-Deleter mice. Prior to analysis of SPPL2c−/− mice, the Cre transgene was removed again by breeding. Positions of primers used for genotyping or RT–PCR are indicated. For genotyping, two PCRs amplifying specific fragments from the wild type (WT) or the post-Cre Knockout allele were performed as depicted from a representative set of mice. HEK293 cells were transiently transfected with SPPL2c isoform A or B fused to a C-terminal Myc epitope or empty vector. Western blot detection was performed with the newly generated, affinity-purified antisera against an N- or C-terminal epitope of SPPL2c. In parallel, anti-Myc was employed as a control and cofilin was detected to confirm equal protein loading. *non-specific band. SPPL2c protein was not detected in the major murine tissues. Total tissue lysates from wild-type (+/+) or SPPL2c−/− mice as indicated were analysed by Western blotting using the SPPL2c antiserum generated against the C-terminus of the protein. Testis lysates were included as positive control, and Actin or EEF2 was visualised to confirm equal protein loading. *non-specific band. Additional deglycosylation controls for Fig 1E. To control for proper deglycosylation of lysates of SPPL2c-transfected HEK cells, shifts in the bands for Tetraspanin-3 (Tspan3) and Transferrin receptor 1 (TfR1) were detected by Western blotting using specific antibodies. N-glycosylation of SPPL2c isoform B is similar to that of isoform A. Lysates of HEK293 cells transiently expressing murine SPPL2c isoform B were treated with endoglycosidase H (Endo H) or peptidyl N-glycosidase F (PNGase F) prior to Western blot analysis with the SPPL2c antiserum against an N-terminal epitope, which also detects isoform B. As control for successful deglycosylation, we also visualised the lysosomal integral membrane protein LIMP-2. *, non-specific band. Murine SPPL2c is N-glycosylated at N106. HEK293 cells were transiently transfected with wild-type SPPL2c isoform A (Iso A) or a N106A mutant inactivating the putative N-glycosylation consensus site. To reveal N-glycosylation of the expressed proteins, lysates were treated with Endo H or PNGase F prior to Western blotting. SPPL2c was detected with the antiserum against the C-terminal epitope validate in (B) and LIMP-2 served as control for the deglycosylation. *, non-specific band. Additional deglycosylation controls for Fig 1F. To control for proper deglycosylation of murine testis lysates, shifts in the bands for Tspan3, TfR1 and SPPL2b were detected by Western blotting using specific antibodies. *, non-specific band. Source data are available online for this figure. Download figure Download PowerPoint Based on the presence of an N-glycosylation consensus site within the N-terminal luminal domain of the protein (Fig 1B), we confirmed glycosylation of overexpressed SPPL2c by treatment with N-glycosidase F (PNGase F) and endoglycosidase H (Endo H) (Figs 1G and EV1D and E). The observed sensitivity of SPPL2c to EndoH treatment indicates that its glycans are not of the complex type in accordance with the detected ER localisation of the protein (Figs 1G and EV1D and E). Mutagenesis of N106 completely abolished N-glycosylation of the SPPL2c protein and thereby confirmed glycosylation of this specific site (Fig EV1F). Importantly, we could show that also SPPL2c from murine testis was modified with EndoH-sensitive N-glycans providing a first indication that also endogenous SPPL2c resides in the ER (Figs 1H and EV1G). This was further supported by co-sedimentation of SPPL2c with the ER marker protein disulphide isomerase (PDI) in a subcellular fractionation of murine testis (Fig 1I). SPPL2c cleaves distinct tail-anchored proteins with a substrate spectrum partially overlapping with that of SPP We aimed to unravel proteolytic activity of SPPL2c and therefore assessed a potential cleavage of known substrates of SPPL2a/b, which are the most closely related proteases within the SPP/SPPL family. Since we have so far not identified any cell line with endogenous SPPL2c expression, we used co-expression assays to assess a potential processing of tumour necrosis factor (TNF-α) (Fig EV2A) 22, 23, neuregulin 1 type III (Fig EV2B) 24 and Bri2 (Fig EV2C) 25 by SPPL2c. In contrast to SPPL2a and SPPL2b, no cleavage indicated by a major NTF depletion and/or release of any intracellular domain (ICD) of the proteins tested was detected in cells co-expressing catalytically active SPPL2c. Since processing of these proteins takes place in the late secretory pathway or at the plasma membrane, where SPPL2c is not present, we also assessed known substrates of the ER-resident homologue SPP. The HCV core protein was very efficiently processed by endogenous SPP 8, which was blocked by expression of catalytically inactive (D/A) SPP (Fig EV2D) that has been shown previously to act as a dominant negative construct 26. In contrast, expression of catalytically inactive SPPL2c (SPPL2c D/A) did not abolish processing of the HCV core protein (Fig EV2D), indicating that SPPL2c does not functionally interact with the HCV core protein. Similarly, also the SPP substrate XBP1u 9 was not cleaved by SPPL2c (Fig EV2E). Click here to expand this figure. Figure EV2. SPPL2c does not cleave known substrates of SPPL2a, SPPL2b and SPP A–C. The SPPL2a/b substrates TNF-α (A), neuregulin 1 (NRG1) type III (B) and Bri2 (C) are not processed by co-expressed SPPL2c. T-Rex™-293 cells stably expressing catalytically active human SPPL2a, SPPL2b, SPPL2c or SPPL3 under a doxycycline-inducible promoter were transiently transfected with the indicated epitope-tagged substrates. Expression of the proteases upon doxycycline induction was confirmed by Western blotting using the indicated protein-specific antibodies. Full-length (FL) and processed forms (NTF, ICD) of substrates were detected by Western blotting of total membrane preparations. To detect Bri2ΔFC and N-terminal fragments (NTFs), an immunoprecipitation with anti-FLAG was performed prior to Western blot analysis. In addition, cell culture media of TNF-α- and Bri2ΔFC-expressing cells (A, C) were analysed to confirm proteolytic release of the ectodomain (sTNF-α, sBri2), which is a prerequisite for the cleavage by SPPL2a/b. Western blot detection with anti-FLAG (TNF-α, Bri2) or anti-V5 (NRG1 type III; sTNF-α and sBri2) directed against the respective tag-epitopes was conducted. By this means, full-length forms of the substrates as well as the N-terminal fragments (NTF) derived from processing of the ectodomain (sTNF-α, sBri2) or the hairpin structure of NRG1 type III were revealed. Upon expression of SPPL2a or SPPL2b, an intracellular domain (ICD) was released from all three substrates, which was not observed upon SPPL2c expression. D, E. HEK293 cells were transiently transfected with N-terminally FLAG-tagged HCV core-E1/4 protein (D) or XBP1u (E) either alone or in combination with active or inactive (D/A) murine SPP or SPPL2c. To compare the turnover and stability of the respective substrates in the absence or presence of the protease, cycloheximide (CHX) chase experiments were performed. Therefore, transfected cells were treated for 0, 2 or 4 h with 100 μg/ml CHX to block protein synthesis prior to cell harvest. The overexpressed murine SPPL2c was detected with anti-SPPL2c (C-terminal epitope). For detection of overexpressed murine (m) SPP, a polyclonal antiserum cross-reacting with endogenous human SPP was employed. In (D), the different processed forms (C/191, C/179)) of the HCV core-E1/4 protein (CE1/4) were labelled as described previously 13. In these experiments, we predominantly detected a dimeric form of SPP. *, non-specific band. Source data are available online for this figure. Download figure Download PowerPoint In contrast, SPPL2c efficiently processed the TA protein HO-1 that has recently been identified as endogenous SPP substrate 11. Like SPP, SPPL2c co-expression efficiently reduced overall levels of murine HO-1 (Fig 2A). In support, HO-1 was also identified in a proteomic substrate screen in SPPL2c-overexpressing HEK cells (accompanying manuscript by Papadopoulou et al 27). Upon co-expression of human HO-1 with the murine proteases, even a potential cleavage product with a slightly lower molecular weight was detected (Fig EV3A). Without any relevant luminal domain, such a minor shift in size would be consistent with a" @default.
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• W2912646755 title "The intramembrane protease <scp>SPPL</scp> 2c promotes male germ cell development by cleaving phospholamban" @default.
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