title: Modulation of the protein kinase Cdelta interaction with the "d" subunit of F1F0-ATP synthase in neonatal cardiac myocytes: development of cell-permeable, mitochondrially targeted inhibitor and facilitator peptides.
abstract: The F(1)F(0)-ATP synthase provides approximately 90% of cardiac ATP, yet little is known regarding its regulation under normal or pathological conditions. Previously, we demonstrated that protein kinase Cdelta (PKCdelta) inhibits F(1)F(0) activity via an interaction with the "d" subunit of F(1)F(0)-ATP synthase (dF(1)F(0)) in neonatal cardiac myocytes (NCMs) (Nguyen, T., Ogbi, M., and Johnson, J. A. (2008) J. Biol. Chem. 283, 29831-29840). We have now identified a dF(1)F(0)-derived peptide (NH(2)-(2)AGRKLALKTIDWVSF(16)-COOH) that inhibits PKCdelta binding to dF(1)F(0) in overlay assays. We have also identified a second dF(1)F(0)-derived peptide (NH(2)-(111)RVREYEKQLEKIKNMI(126)-COOH) that facilitates PKCdelta binding to dF(1)F(0). Incubation of NCMs with versions of these peptides containing HIV-Tat protein transduction and mammalian mitochondrial targeting sequences resulted in their delivery into mitochondria. Preincubation of NCMs, with 10 nm extracellular concentrations of the mitochondrially targeted PKCdelta-dF(1)F(0) interaction inhibitor, decreased 100 nm 4beta-phorbol 12-myristate 13-acetate (4beta-PMA)-induced co-immunoprecipitation of PKCdelta with dF(1)F(0) by 50 +/- 15% and abolished the 30 nm 4beta-PMA-induced inhibition of F(1)F(0)-ATPase activity. A scrambled sequence (inactive) peptide, which contained HIV-Tat and mitochondrial targeting sequences, was without effect. In contrast, the cell-permeable, mitochondrially targeted PKCdelta-dF(1)F(0) facilitator peptide by itself induced the PKCdelta-dF(1)F(0) co-immunoprecipitation and inhibited F(1)F(0)-ATPase activity. In in vitro PKC add-back experiments, the PKCdelta-F(1)F(0) inhibitor blocked PKCdelta-mediated inhibition of F(1)F(0)-ATPase activity, whereas the facilitator induced inhibition. We have developed the first cell-permeable, mitochondrially targeted modulators of the PKCdelta-dF(1)F(0) interaction in NCMs. These novel peptides will improve our understanding of cardiac F(1)F(0) regulation and may have potential as therapeutics to attenuate cardiac injury.
authors.affiliation: Department of Pharmacology and Toxicology, School of Medicine, and Program in Regenerative Medicine, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia 30912-2300, USA.
authors.forename: Tiffany T
authors.initials: TT
authors.lastname: Nguyen
authors.forename: Mourad
authors.initials: M
authors.lastname: Ogbi
authors.forename: Qilin
authors.initials: Q
authors.lastname: Yu
authors.forename: Jordan B
authors.initials: JB
authors.lastname: Fishman
authors.forename: Warren
authors.initials: W
authors.lastname: Thomas
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Harvey
authors.forename: David
authors.initials: D
authors.lastname: Fulton
authors.forename: John A
authors.initials: JA
authors.lastname: Johnson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D002463:Cell Membrane Permeability / Q000187:drug effects*; D015195:Drug Design; D057075:Enzyme Assays; D047468:Immunoprecipitation; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D025261:Mitochondrial Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000201:enzymology*; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects; D051745:Protein Kinase C-delta / Q000737:chemistry / Q000378:metabolism*; D021382:Protein Sorting Signals; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000494:pharmacology
chemical_list: D010455:Peptides; D021382:Protein Sorting Signals; D021122:Protein Subunits; D054322:tat Gene Products, Human Immunodeficiency Virus; D051745:Protein Kinase C-delta; C474451:F1F0-ATP synthase; D025261:Mitochondrial Proton-Translocating ATPases; D013755:Tetradecanoylphorbol Acetate
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