title: Substrate requirements for SPPL2b-dependent regulated intramembrane proteolysis.
abstract: Intramembrane proteolysis is now widely recognized as an important physiological pathway required for reverse signaling and membrane protein degradation. Aspartyl intramembrane cleaving proteases of the GXGD-type play an important regulatory role in health and disease. Besides gamma-secretase/presenilin, signal peptide peptidase (SPP) and SPP-like (SPPL) peptidases also belong to the family of GXGD-type aspartyl proteases. Although recently the first SPPL2a/b substrates have been identified, very little is known about substrate requirements, which allow them to be efficiently processed within the membrane. We demonstrate that similar to gamma-secretase substrates, intramembrane proteolysis of Bri2 (Itm2b) is greatly facilitated by an initial shedding event mediated by ADAM-10. Serial deletions revealed that the length of the ectodomain negatively correlates with efficient intramembrane proteolysis. Bri3 (Itm2c), which is highly homologous to Bri2, fails to be shed. Failure of shedding of Bri3 is accompanied by a lack of intramembrane proteolysis by SPPL2b. Surprisingly, a low molecular weight membrane-retained stub of Bri3 also fails to be processed by SPPL2b, indicating that shedding per se is not sufficient for subsequent intramembrane proteolysis. Extensive domain swapping analysis reveals that primary sequence determinants within the intracellular domain and the transmembrane domain together with short luminal juxtamembrane sequences are required for efficient intramembrane proteolysis.
authors.affiliation: Center for Integrated Protein Science Munich and Adolf-Butenandt-Institute, Department of Biochemistry, Laboratory for Neurodegenerative Disease Research, Ludwig-Maximilians-University, 80336 Munich, Germany.
authors.forename: Lucas
authors.initials: L
authors.lastname: Martin
authors.forename: Regina
authors.initials: R
authors.lastname: Fluhrer
authors.forename: Christian
authors.initials: C
authors.lastname: Haass
mesh_terms: D051722:ADAM Proteins / Q000235:genetics / Q000378:metabolism*; D000072197:ADAM10 Protein; D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases / Q000235:genetics / Q000378:metabolism*; D016282:Aspartic Acid Endopeptidases / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C499959:BRI3 protein, human; C120083:ITM2B protein, human; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D053829:Amyloid Precursor Protein Secretases; D010447:Peptide Hydrolases; D016282:Aspartic Acid Endopeptidases; C526456:SPPL2b protein, human; D051722:ADAM Proteins; D000072197:ADAM10 Protein; C491236:ADAM10 protein, human