title: The Vps27/Hrs/STAM (VHS) Domain of the Signal-transducing Adaptor Molecule (STAM) Directs Associated Molecule with the SH3 Domain of STAM (AMSH) Specificity to Longer Ubiquitin Chains and Dictates the Position of Cleavage.
abstract: The deubiquitinating enzyme associated molecule with the SH3 domain of STAM (AMSH) is crucial for the removal of ubiquitin molecules during receptor-mediated endocytosis and lysosomal receptor sorting. AMSH interacts with signal transducing adapter molecule (STAM) 1 or 2, which enhances the activity of AMSH through an unknown mechanism. This stimulation is dependent on the ubiquitin-interacting motif of STAM. Here we investigate the specific mechanism of AMSH stimulation by STAM proteins and the role of the STAM Vps27/Hrs/STAM domain. We show that, in the presence of STAM, the length of the ubiquitin chains affects the apparent cleavage rate. Through measurement of the chain cleavage kinetics, we found that, although the kcat of Lys(63)-linked ubiquitin chain cleavage was comparable for di- and tri-ubiquitin, the Km value was lower for tri-ubiquitin. This increased affinity for longer chains was dependent on the Vps27/Hrs/STAM domain of STAM and required that the substrate ubiquitin chain contain homogenous Lys(63)-linkages. In addition, STAM directed AMSH cleavage toward the distal isopeptide bond in tri-ubiquitin chains. Finally, we generated a structural model of AMSH-STAM to show how the complex binds Lys(63)-linked ubiquitin chains and cleaves at the distal end. These data show how a deubiquitinating enzyme-interacting protein dictates the efficiency and specificity of substrate cleavage.
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel and.
authors.forename: Nardeen
authors.initials: N
authors.lastname: Baiady
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel and.
authors.forename: Prasanth
authors.initials: P
authors.lastname: Padala
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel and.
authors.forename: Bayan
authors.initials: B
authors.lastname: Mashahreh
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel and.
authors.forename: Einav
authors.initials: E
authors.lastname: Cohen-Kfir
authors.affiliation: the Department of Chemistry and Biochemistry, James Madison University, Harrisonburg, Virginia 22807.
authors.forename: Emily A
authors.initials: EA
authors.lastname: Todd
authors.affiliation: the Department of Chemistry and Biochemistry, James Madison University, Harrisonburg, Virginia 22807.
authors.forename: Kelly E
authors.initials: KE
authors.lastname: Du Pont
authors.affiliation: the Department of Chemistry and Biochemistry, James Madison University, Harrisonburg, Virginia 22807.
authors.forename: Christopher E
authors.initials: CE
authors.lastname: Berndsen
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel and. Electronic address: reuvenw@ekmd.huji.ac.il.
authors.forename: Reuven
authors.initials: R
authors.lastname: Wiener
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs; D056827:Endosomal Sorting Complexes Required for Transport / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D025801:Ubiquitin / Q000378:metabolism*; D043222:Ubiquitin Thiolesterase / Q000235:genetics / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D056827:Endosomal Sorting Complexes Required for Transport; D010750:Phosphoproteins; C109775:STAM protein, human; C491647:STAM2 protein, human; C486696:STAMBP protein, human; D025801:Ubiquitin; D043222:Ubiquitin Thiolesterase
keywords: AMSH; STAM; VHS; deubiquitylation (deubiquitination); endosomal sorting complex required for transport (ESCRT); metalloprotease; polyubiquitin chain; ubiquitin; ubiquitin-interacting motif