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.
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.
Nguyen
Tiffany T
TT
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.
Ogbi
Mourad
M
Yu
Qilin
Q
Fishman
Jordan B
JB
Thomas
Warren
W
Harvey
Brian J
BJ
Fulton
David
D
Johnson
John A
JA
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
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
Proc Natl Acad Sci U S A. 2001 Sep 25;98(20):11114-9
11553773
Curr Protein Pept Sci. 2002 Aug;3(4):451-60
12370007
Science. 1999 Sep 3;285(5433):1569-72
10477521
Methods Enzymol. 2002;345:470-89
11665630
Circulation. 1995 Jun 1;91(11):2810-8
7758188
Am J Physiol Heart Circ Physiol. 2004 Aug;287(2):H937-45
15277208
Am J Physiol Heart Circ Physiol. 2007 Oct;293(4):H2219-30
17660387
J Biol Chem. 1989 Dec 5;264(34):20206-15
2555347
Chem Biol. 2001 Dec;8(12):1123-9
11755391
J Bioenerg Biomembr. 2004 Oct;36(5):447-57
15534392
Circulation. 2008 Feb 19;117(7):886-96
18250271
Circ Res. 1996 Dec;79(6):1086-99
8943947
Biochem J. 2006 Jan 1;393(Pt 1):191-9
16336199
J Control Release. 2007 Feb 12;117(2):148-62
17196289
Proc Natl Acad Sci U S A. 1991 May 1;88(9):3997-4000
1850844
Circ Res. 1995 Apr;76(4):654-63
7895339
Biochem J. 1991 Dec 15;280 ( Pt 3):561-73
1837214
Cardiovasc Res. 1999 Jul;43(1):32-43
10536688
Methods Mol Biol. 2003;233:345-50
12840520
Circulation. 2003 Nov 11;108(19):2304-7
14597593
J Biochem. 1998 Jun;123(6):1010-6
9603986
Biochim Biophys Acta. 2000 May 31;1458(2-3):343-55
10838049
Circ Res. 2000 Jun 9;86(11):1173-9
10850970
Am J Physiol Heart Circ Physiol. 2004 Aug;287(2):H946-56
15277209
Physiol Rev. 2008 Apr;88(2):581-609
18391174
J Biol Chem. 1996 Oct 4;271(40):24962-6
8798776
J Biol Chem. 2004 Nov 12;279(46):47985-91
15339931
Am J Physiol Heart Circ Physiol. 2006 Aug;291(2):H854-60
16501016
Mol Cell Biochem. 2005 Oct;278(1-2):223-32
16180108
Circulation. 2001 Jul 3;104(1):85-90
11435343
J Biol Chem. 2004 May 14;279(20):21233-8
15004034
Biochim Biophys Acta. 2004 Nov 4;1659(1):52-62
15511527
Biochem J. 2005 Sep 1;390(Pt 2):377-94
16108756
Proc Natl Acad Sci U S A. 1995 Jan 17;92(2):492-6
7831317
Mol Genet Metab. 2003 Jun;79(2):71-82
12809636
Arch Biochem Biophys. 2003 Dec 15;420(2):246-54
14654063
Biochim Biophys Acta. 2001 Apr 2;1504(2-3):329-39
11245796
J Mol Biol. 2005 Aug 26;351(4):824-38
16045926
Circulation. 2003 Aug 19;108(7):869-75
12860903
Biochim Biophys Acta. 2006 May-Jun;1757(5-6):286-96
16697972
J Mol Cell Cardiol. 1999 Oct;31(10):1845-55
10525422
J Physiol. 2004 Feb 15;555(Pt 1):1-13
14660709
J Biol Chem. 1987 Feb 15;262(5):2291-7
3818597
Biochem J. 1996 Aug 15;318 ( Pt 1):343-9
8761491
Trends Endocrinol Metab. 2009 Jan;20(1):25-33
19056296
Circ Res. 2003 May 2;92(8):873-80
12663490
Proc Natl Acad Sci U S A. 1994 Feb 1;91(3):839-43
8302854
J Biol Chem. 2008 Oct 31;283(44):29831-40
18725417
Circ Res. 2005 Jul 8;97(1):78-85
15961716
Am J Physiol Heart Circ Physiol. 2005 Oct;289(4):H1343-50
15894568
J Biol Chem. 1997 Dec 5;272(49):30945-51
9388241