title: A Cell-Based Assay Reveals Nuclear Translocation of Intracellular Domains Released by SPPL Proteases.
abstract: During regulated intramembrane proteolysis (RIP) a membrane-spanning substrate protein is cleaved by an ectodomain sheddase and an intramembrane cleaving protease. A cytoplasmic intracellular domain (ICD) is liberated, which can migrate to the nucleus thereby influencing transcriptional regulation. Signal peptide peptidase-like (SPPL) 2a and 2b have been implicated in RIP of type II transmembrane proteins. Even though SPPL2a might represent a potential pharmacological target for treatment of B-cell-mediated autoimmunity, no specific and potent inhibitors for this enzyme are currently available. We report here on the first quantitative cell-based assay for measurement of SPPL2a/b activity. Demonstrating the failure of standard Gal4/VP16 reporter assays for SPPL2a/b analysis, we have devised a novel system employing β-galactosidase (βGal) complementation. This is based on detecting nuclear translocation of the proteolytically released substrate ICDs, which results in specific restoration of βGal activity. Utilizing this potentially high-throughput compatible new setup, we demonstrate nuclear translocation of the ICDs from integral membrane protein 2B (ITM2B), tumor necrosis factor (TNF) and CD74 and identify secreted frizzled-related protein 2 (SFRP2) as potential transcriptional downstream target of the CD74 ICD. We show that the presented assay is easily adaptable to other intramembrane proteases and therefore represents a valuable tool for the functional analysis and development of new inhibitors of this class of enzymes.
authors.affiliation: Biochemical Institute, Christian Albrechts University of Kiel, Otto-Hahn-Platz 9, D-24118, Kiel, Germany.
authors.forename: Torben
authors.initials: T
authors.lastname: Mentrup
authors.affiliation: Institute of Clinical Molecular Biology, Christian Albrechts University of Kiel, Schittenhelmstr. 12, D-24105, Kiel, Germany.
authors.forename: Robert
authors.initials: R
authors.lastname: Häsler
authors.affiliation: Biomedizinisches Centrum (BMC), Ludwig Maximilians University of Munich, Feodor-Lynen-Strasse 17, D-81377, Munich, Germany.|DZNE - German Center for Neurodegenerative Diseases, Feodor-Lynen-Strasse 17, D-81377, Munich, Germany.
authors.forename: Regina
authors.initials: R
authors.lastname: Fluhrer
authors.affiliation: Biochemical Institute, Christian Albrechts University of Kiel, Otto-Hahn-Platz 9, D-24118, Kiel, Germany.
authors.forename: Paul
authors.initials: P
authors.lastname: Saftig
authors.affiliation: Biochemical Institute, Christian Albrechts University of Kiel, Otto-Hahn-Platz 9, D-24118, Kiel, Germany.
authors.forename: Bernd
authors.initials: B
authors.lastname: Schröder
mesh_terms: D021581:Active Transport, Cell Nucleus; D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000944:Antigens, Differentiation, B-Lymphocyte / Q000378:metabolism; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D057809:HEK293 Cells; D006367:HeLa Cells; D000949:Histocompatibility Antigens Class II / Q000378:metabolism; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000944:Antigens, Differentiation, B-Lymphocyte; D000949:Histocompatibility Antigens Class II; C120083:ITM2B protein, human; D008562:Membrane Glycoproteins; D019913:Nuclear Localization Signals; D014409:Tumor Necrosis Factor-alpha; C045084:invariant chain; D016282:Aspartic Acid Endopeptidases; C524670:SPPL2a protein, human; C526456:SPPL2b protein, human
keywords: intramembrane proteolysis; nuclear translocation; protein fragment complementation; signal peptide peptidase-like protease; β-galactosidase; γ-secretase