Vorschau

PDF, Englisch Download (3MB) | Nutzungsbedingungen

Abstract

During the characterization of lipid-enriched microdomains at the Golgi (GICs) (Gkantiragas, I. et al. 2001), a protein with an apparent molecular mass of 17 kDa was identified. Cloning and preliminary biochemical characterization identified a novel protein, GAPR-1, belonging to the superfamily of PR proteins. Based on the primary amino acidic sequence of this protein, some potentially interesting characteristics were identified. It contains a consensus sequence for myristoylation, a putative caveolin-binding domain, a coiled-coil structure, and an isolelectric point (pI) of 9.4, suggesting that GAPR-1 is a highly hydrophilic protein (Eberle, H. B. et al. 2002). In this thesis, this structural information, was used to i) study the interaction of GAPR-1 with membranes, ii) to obtain structural information on the protein, and iii) to identify proteins that interact with GAPR-1.GAPR-1 was shown to be myristoylated and to interact with Caveolin-1. Myristoylation, together with protein-protein or electrostatic interactions at physiological pH could explain its strong membrane association. The crystal structure of GAPR-1 showed strong structural similarities to other plant pathogenesis-related proteins. Substitution of the most conserved amino acids in GAPR-1 (His54, Glu65, Glu86 and His103) in the putative active center changed the protein behavior in solution. Size exclusion chromatography revealed that the major population of GAPR-1 mutant migrated as a dimer, whereas GAPR-1 wild type behaves predominantly as a monomer. The tendency of GAPR-1 to form dimers was confirmed by crosslink experiments and by the yeast two hybrid system. By affinity chromatography, GAPR-1 was shown to interact with three proteins: Nucleolin, Template activating factor α (TAFIα) and HSAPRIL. In the yeast two hybrid system, the interaction of GARP-1 with Nucleolin was confirmed and shown to be dependent on the most conserved amino acid residues in GAPR-1. The interaction between GAPR-1 and Nucleolin may represent a new mechanism of regulation of innate immunity in mammalian cells.