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.
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.
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新生儿心肌细胞中蛋白激酶 Cdelta 与 F1F0-ATP 合酶“d”亚基相互作用的调节：细胞渗透性、线粒体靶向抑制剂和促进肽的开发。
DOI：
10.1074/jbc.m109.077578
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发表时间：
2010
期刊：
影响因子：
--
通讯作者：
Johnson,JohnA
中科院分区：
文献类型：
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作者：
Nguyen,TiffanyT;Ogbi,Mourad;Yu,Qilin;Fishman,JordanB;Thomas,Warren;Harvey,BrianJ;Fulton,David;Johnson,JohnA
F1 F0-ATP合酶提供了约90%的心脏ATP，但关于其在正常或病理条件下的调节知之甚少。先前，我们证明了蛋白激酶Cδ（PKCδ）通过与新生心肌细胞（NCMs）中F1 F0-ATP合酶（dF 1F 0）的“d”亚基的相互作用来抑制F1 F0活性（Nguyen，T.，Ogbi，M.，和约翰逊，J.A.（2008）J.Biol.Chem.283，29831-29840）。我们现在已经鉴定了一种dF 1F 0衍生肽（NH 2 - 2AGRKLALKTIDWVSF 16-COOH），其在覆盖测定中抑制PKCδ与dF 1F 0的结合。我们还发现了第二种dF 1F 0衍生肽（NH 2 - 111 RVREYEKQLEKIKNMI 126-COOH），可促进PKCδ与dF 1F 0结合。将NCM与含有HIV-Tat蛋白转导和哺乳动物线粒体靶向序列的这些肽的版本一起孵育，导致其递送到线粒体中。用10 nm细胞外浓度的PKCδ-dF 1F 0相互作用抑制剂预孵育NCMs，可使100 nm 4 β-佛波醇12-肉豆蔻酸酯13-乙酸酯（4β-PMA）诱导的PKCδ与dF 1F 0的免疫共沉淀减少50 ± 15%，并消除30 nm 4 β-PMA诱导的F1 F0-ATP酶活性抑制。含有HIV-Tat和线粒体靶向序列的乱序序列（无活性）肽没有效果。相反，细胞可渗透的、细胞靶向的PKCδ-dF 1F 0促进肽本身诱导PKCδ-dF 1F 0免疫共沉淀并抑制F1 F0-ATP酶活性。在体外PKC反加实验中，PKCδ-F1 F0抑制剂阻断了PKCδ介导的F1 F0-ATP酶活性抑制，而易化剂诱导了抑制。我们已经开发了第一个细胞可渗透的，神经靶向的PKCδ-dF 1F 0相互作用的调节剂在NCMs。这些新的肽将提高我们对心脏F1 F0调节的理解，并可能具有减轻心脏损伤的治疗潜力。
The F1F0-ATP synthase provides ∼90% of cardiac ATP, yet little is known regarding its regulation under normal or pathological conditions. Previously, we demonstrated that protein kinase Cδ (PKCδ) inhibits F1F0activity via an interaction with the “d” subunit of F1F0-ATP synthase (dF1F0) 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 dF1F0-derived peptide (NH2-2AGRKLALKTIDWVSF16-COOH) that inhibits PKCδ binding to dF1F0in overlay assays. We have also identified a second dF1F0-derived peptide (NH2-111RVREYEKQLEKIKNMI126-COOH) that facilitates PKCδ binding to dF1F0. 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 nmextracellular concentrations of the mitochondrially targeted PKCδ-dF1F0interaction inhibitor, decreased 100 nm4β-phorbol 12-myristate 13-acetate (4β-PMA)-induced co-immunoprecipitation of PKCδ with dF1F0by 50 ± 15% and abolished the 30 nm4β-PMA-induced inhibition of F1F0-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 PKCδ-dF1F0facilitator peptide by itself induced the PKCδ-dF1F0co-immunoprecipitation and inhibited F1F0-ATPase activity. Inin vitroPKC add-back experiments, the PKCδ-F1F0inhibitor blocked PKCδ-mediated inhibition of F1F0-ATPase activity, whereas the facilitator induced inhibition. We have developed the first cell-permeable, mitochondrially targeted modulators of the PKCδ-dF1F0interaction in NCMs. These novel peptides will improve our understanding of cardiac F1F0regulation and may have potential as therapeutics to attenuate cardiac injury.