title: A recombinant transductor-effector system: in vitro study of G inhibitory protein (G-alpha-i1) direct activators.
abstract: Mutations and altered functionality of the inhibitory subfamily of G proteins (Gi) are involved in pathological states. Compounds able to activate Gi in a receptor-independent manner would be useful to treat these pathological conditions. Aimed to study Gi direct activation we have reconstituted a recombinant transductor-effector complex cloning both the mammalian Galpha(i1) subunit and adenylate cyclase (AC). The myristoylation of Galpha, fundamental for interaction with AC, was obtained in the procaryotic expression host Escherichia coli transformed with a single plasmid containing both the coding sequences for human Galpha(i1) and Saccharomyces cerevisiae myristoyl transferase. AC-V isoform was obtained by the expression of its cytosolic domains. A recent synthesized molecule, named BC5, was tested to evaluate its pharmacological profile in a Gi/AC cell-free complex model. In this functional transductor-effector system BC5 was able to activate Gi signalling, moreover providing a new tool to give a better insight into G-protein receptor-independent modulation.
authors.affiliation: Department of Preclinical and Clinical Pharmacology, University of Florence, Viale Pieraccini 6, 50134 Florence, Italy. lorenzo.mannelli@unifi.it
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Di Cesare Mannelli
authors.forename: Alessandra
authors.initials: A
authors.lastname: Pacini
authors.forename: Annarita
authors.initials: A
authors.lastname: Toscano
authors.forename: Carla
authors.initials: C
authors.lastname: Ghelardini
authors.forename: Dina
authors.initials: D
authors.lastname: Manetti
authors.forename: Fulvio
authors.initials: F
authors.lastname: Gualtieri
authors.forename: Tarun B
authors.initials: TB
authors.lastname: Patel
authors.forename: Alessandro
authors.initials: A
authors.lastname: Bartolini
mesh_terms: D000262:Adenylyl Cyclases / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D014161:Transduction, Genetic / Q000379:methods*
chemical_list: D011994:Recombinant Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D000262:Adenylyl Cyclases