Elucidating the Potential Role of Protein Prenylation in Bacterial Infection.

Abstract

Many G-proteins, like Ras, require prenylation for membrane localization and function. Protein farnesyltransferase (FTase) and geranylgeranyltransferase type I (GGTase-I) add 15-carbon farnesyl groups and 20-carbon geranylgeranyl groups to proteins, respectively. Substrate specificity for FTase and GGTase-I is minimally described by the Cza2X paradigm where C is a cysteine residue four amino acids from the C-terminus, followed by two amino acids (z is variable; a2 is aliphatic) and an X group that specifies which prenyltransferase is preferred, though there is significant substrate overlap. Recent experiments have demonstrated that infectious bacterium Legionella pneumophila requires host FTase to induce pulmonary disease. Ankyrin b, an essential Legionella effector, is farnesylated, which is important for endocytic pathway evasion and protein localization. Prenylation of effector proteins may be a general method used by pathogenic bacteria to anchor proteins in hosts. To investigate the prevalence of prenylation of bacterial proteins, we measured the reactivity of mammalian FTase and GGTase-I with peptide sequences derived from proteins expressed in pathogenic bacteria. Kinetic analysis of a bacterial peptide library revealed that 23 of 38 peptides are substrates. Several fusion proteins corresponding to peptides shown to be prenylated in vitro also demonstrated prenyltransferase-dependent membrane localization in human cells, further supporting our hypothesis that bacterial effector prenylation may be a general strategy. We also tested prenyl donor analogs with FTase and GGTase-I to identify compounds with substrate specificity similar to native prenyl donors for use as tools to define the prenylome. Methyl-substituted benzene mimics the terminal isoprene of GGPP, generating an analog with similar activity to the native isoprenoid. Our data also suggest that the first two isoprene units are necessary for retaining wild type activity. These studies will further our understanding of the ways that prenyltransferases regulate cellular processes and how inhibitors may be incorporated into treatment of bacterial infections and other diseases.