Attenuation of the hypoxia-induced protein kinase Cδ interaction with the ‘d’ subunit of F1Fo-ATP synthase in neonatal cardiac myocytes: implications for energy preservation and survival

2010 ◽  
Vol 429 (2) ◽  
pp. 335-345 ◽  
Author(s):  
Tiffany T. Nguyen ◽  
Mourad Ogbi ◽  
Qilin Yu ◽  
John A. Johnson

The F1Fo-ATP synthase provides most of the heart's energy, yet events that alter its function during injury are poorly understood. Recently, we described a potent inhibitory effect on F1Fo-ATP synthase function mediated by the interaction of PKCδ (protein kinase Cδ) with dF1Fo (‘d’ subunit of the F1Fo-ATPase/ATP synthase). We have now developed novel peptide modulators which facilitate or inhibit the PKCδ–dF1Fo interaction. These peptides include HIV-Tat (transactivator of transcription) protein transduction and mammalian mitochondrial-targeting sequences. Pre-incubation of NCMs (neonatal cardiac myocyte) with 10 nM extracellular concentrations of the mitochondrial-targeted PKCδ–dF1Fo interaction inhibitor decreased Hx (hypoxia)-induced co-IP (co-immunoprecipitation) of PKCδ with dF1Fo by 40±9%, abolished Hx-induced inhibition of F1Fo-ATPase activity, attenuated Hx-induced losses in F1Fo-derived ATP and protected against Hx- and reperfusion-induced cell death. A scrambled-sequence (inactive) peptide, which contained HIV-Tat and mitochondrial-targeting sequences, was without effect. In contrast, the cell-permeant mitochondrial-targeted PKCδ–dF1Fo facilitator peptide, which we have shown previously to induce the PKCδ–dF1Fo co-IP, was found to inhibit F1Fo-ATPase activity to an extent similar to that caused by Hx alone. The PKCδ–dF1Fo facilitator peptide also decreased ATP levels by 72±18% under hypoxic conditions in the presence of glycolytic inhibition. None of the PKCδ–dF1Fo modulatory peptides altered the inner mitochondrial membrane potential. Our studies provide the first evidence that disruption of the PKCδ–dF1Fo interaction using cell-permeant mitochondrial-targeted peptides attenuates cardiac injury resulting from prolonged oxygen deprivation.

2010 ◽  
Vol 285 (29) ◽  
pp. 22164-22173 ◽  
Author(s):  
Tiffany T. Nguyen ◽  
Mourad Ogbi ◽  
Qilin Yu ◽  
Jordan B. Fishman ◽  
Warren Thomas ◽  
...  

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
John A Johnson ◽  
Mourad Ogbi ◽  
Robert W Caldwell

Cardiac ischemia / reperfusion (IR) injury is associated with severe energy deprivation and is the number one cause of death world-wide. Mitochondrial F1Fo ATP synthase produces >90% of cardiac energy in mammals, yet few studies have targeted its role in IR injury. Previously, we identified a hypoxia-induced interaction of delta protein kinase C (dPKC) with the “d” subunit of F1Fo ATP synthase (dF1Fo) in neonatal cardiac myocytes, which inhibits F1Fo function. In the present work we investigated the hypothesis that a novel peptide inhibitor of the dPKC-dF1Fo interaction would preserve ATP and reduce infarct-size in isolated rat hearts subjected to IR injury. This peptide [NH2-YGRKKRRQRRRMLATRALSLIGKRAISTSVC-COOH] contains HIV-Tat protein transduction and mitochondrial targeting domains, the dPKC-dF1Fo inhibitor sequence, and a FLAG epitope. In hearts exposed to global ischemia, or IR, dPKC co-immuno-precipitated with dF1Fo. Pretreatment with the dPKC-dF1Fo inhibitor exacerbated cardiac ATP loss by 1.9-fold (n=5, p<0.03) following 10 min of global ischemia. However; following a pro-longed IR exposure ATP levels were enhanced by 2.1-fold (p<0.02, n=5). These opposing effects of the[[Unable to Display Character: ]]dPKC-dF1Fo inhibitor on ATP levels are likely due to relief of dPKC inhibition of the different modes of the F1Fo complex during ischemia (ATPase) and oxygenated reperfusion (ATP synthase). We next used 2,3,5, tetrazolium chloride staining techniques to determine if the dPKC-dF1Fo inhibitor had infarct-sparing effects following prolonged IR. In hearts exposed to 30 min of global ischemia and 150 min of reperfusion the dPKC-dF1Fo inhibitor reduced infarct size, (expressed as the percentage of total LV area) from 45 + 3 % (n=6) to 22 + 3 % (n=6, p < 0.01). Delivery and stability of the dPKC-dF1Fo inhibitor in hearts was confirmed by FLAG immunoreactivity in western blots conducted on mitochondria isolated the left ventricle. This is the first demonstration that perfusion with the dPKC-dF1Fo inhibitor prior to IR improves ATP recovery and reduces infarction in intact mammalian hearts. Our results support the potential for this peptide as a first-in-class translational agent for combating cardiac IR injury.


2021 ◽  
Vol 1862 (7) ◽  
pp. 148429
Author(s):  
Romero-Aguilar Lucero ◽  
Esparza-Perusquía Mercedes ◽  
Langner Thorsten ◽  
García-Cruz Giovanni ◽  
Feldbrügge Michael ◽  
...  

2003 ◽  
Vol 185 (18) ◽  
pp. 5527-5535 ◽  
Author(s):  
Amaresh Das ◽  
Lars G. Ljungdahl

ABSTRACT The atp operon encoding F1Fo ATP synthase in the fermentative obligate anaerobic bacterium Clostridium pasteurianum was sequenced. It consisted of nine genes arranged in the order atpI(i), atpB(a), atpE(c), atpF(b), atpH(δ), atpA(α), atpG(γ), atpD(β), and atpC(ε), which was identical to that found in many bacteria. Reverse transcription-PCR confirmed the presence of the transcripts of all nine genes. The amount of ATPase activity in the membranes of C. pasteurianum was low compared to what has been found in many other bacteria. The F1Fo complexes solubilized from membranes of C. pasteurianum and Escherichia coli had similar masses, suggesting similar compositions for the F1Fo complexes from the two bacteria. Western blotting experiments with antibodies raised against the purified subunits of F1Fo detected the presence of eight subunits, α, β, γ, δ, ε, a, b, and c, in the F1Fo complex from C. pasteurianum. The F1Fo complex from C. pasteurianum was activated by thiocyanate, cyanate, or sulfhydryl compounds; inhibited by sulfite, bisulfite, or bicarbonate; and had tolerance to inhibition by dicyclohexylcarbodiimide. The target of thiol activation of the F1Fo complex from C. pasteurianum was F1. Thiocyanate and sulfite were noncompetitive with respect to substrate Mg ATP but competitive with respect to each other. The F1 and Fo parts of the F1Fo complexes from C. pasteurianum and E. coli bound to each other, but the hybrid F1Fo complexes were not functionally active.


2017 ◽  
Vol 114 (13) ◽  
pp. 3409-3414 ◽  
Author(s):  
Jiuya He ◽  
Holly C. Ford ◽  
Joe Carroll ◽  
Shujing Ding ◽  
Ian M. Fearnley ◽  
...  

The permeability transition in human mitochondria refers to the opening of a nonspecific channel, known as the permeability transition pore (PTP), in the inner membrane. Opening can be triggered by calcium ions, leading to swelling of the organelle, disruption of the inner membrane, and ATP synthesis, followed by cell death. Recent proposals suggest that the pore is associated with the ATP synthase complex and specifically with the ring of c-subunits that constitute the membrane domain of the enzyme’s rotor. The c-subunit is produced from three nuclear genes, ATP5G1, ATP5G2, and ATP5G3, encoding identical copies of the mature protein with different mitochondrial-targeting sequences that are removed during their import into the organelle. To investigate the involvement of the c-subunit in the PTP, we generated a clonal cell, HAP1-A12, from near-haploid human cells, in which ATP5G1, ATP5G2, and ATP5G3 were disrupted. The HAP1-A12 cells are incapable of producing the c-subunit, but they preserve the characteristic properties of the PTP. Therefore, the c-subunit does not provide the PTP. The mitochondria in HAP1-A12 cells assemble a vestigial ATP synthase, with intact F1-catalytic and peripheral stalk domains and the supernumerary subunits e, f, and g, but lacking membrane subunits ATP6 and ATP8. The same vestigial complex plus associated c-subunits was characterized from human 143B ρ0 cells, which cannot make the subunits ATP6 and ATP8, but retain the PTP. Therefore, none of the membrane subunits of the ATP synthase that are involved directly in transmembrane proton translocation is involved in forming the PTP.


Pharmaceutics ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 763
Author(s):  
Faustino Mollinedo ◽  
Consuelo Gajate

The ether lipid edelfosine induces apoptosis selectively in tumor cells and is the prototypic molecule of a family of synthetic antitumor compounds collectively known as alkylphospholipid analogs. Cumulative evidence shows that edelfosine interacts with cholesterol-rich lipid rafts, endoplasmic reticulum (ER) and mitochondria. Edelfosine induces apoptosis in a number of hematological cancer cells by recruiting death receptors and downstream apoptotic signaling into lipid rafts, whereas it promotes apoptosis in solid tumor cells through an ER stress response. Edelfosine-induced apoptosis, mediated by lipid rafts and/or ER, requires the involvement of a mitochondrial-dependent step to eventually elicit cell death, leading to the loss of mitochondrial membrane potential, cytochrome c release and the triggering of cell death. The overexpression of Bcl-2 or Bcl-xL blocks edelfosine-induced apoptosis. Edelfosine induces the redistribution of lipid rafts from the plasma membrane to the mitochondria. The pro-apoptotic action of edelfosine on cancer cells is associated with the recruitment of F1FO–ATP synthase into cholesterol-rich lipid rafts. Specific inhibition of the FO sector of the F1FO–ATP synthase, which contains the membrane-embedded c-subunit ring that constitutes the mitochondrial permeability transcription pore, hinders edelfosine-induced cell death. Taking together, the evidence shown here suggests that the ether lipid edelfosine could modulate cell death in cancer cells by direct interaction with mitochondria, and the reorganization of raft-located mitochondrial proteins that critically modulate cell death or survival. Here, we summarize and discuss the involvement of mitochondria in the antitumor action of the ether lipid edelfosine, pointing out the mitochondrial targeting of this drug as a major therapeutic approach, which can be extrapolated to other alkylphospholipid analogs. We also discuss the involvement of cholesterol transport and cholesterol-rich lipid rafts in the interactions between the organelles as well as in the role of mitochondria in the regulation of apoptosis in cancer cells and cancer therapy.


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