Cell-permeable Peptide Antioxidants Targeted to Inner Mitochondrial Membrane inhibit Mitochondrial Swelling, Oxidative Cell Death, and Reperfusion Injury

Abstract

Reactive oxygen species (ROS) play a key role in promoting mitochondrial cytochrome c release and induction of apoptosis. ROS induce dissociation of cytochrome c from cardiolipin on the inner mitochondrial membrane (IMM), and cytochrome c may then be released via mitochondrial permeability transition (MPT)-dependent or MPT-independent mechanisms. We have developed peptide antioxidants that target the IMM, and we used them to investigate the role of ROS and MPT in cell death caused by t-butylhydroperoxide (tBHP) and 3-nitropropionic acid (3NP). The structural motif of these peptides centers on alternating aromatic and basic amino acid residues, with dimethyltyrosine providing scavenging properties. These peptide antioxidants are cell-permeable and concentrate 1000-fold in the IMM. They potently reduced intracellular ROS and cell death caused by tBHP in neuronal N 2 A cells (EC 50 in nM range). They also decreased mitochondrial ROS production, inhibited MPT and swelling, and prevented cytochrome c release induced by Ca 2؉ in isolated mitochondria. In addition, they inhibited 3NP-induced MPT in isolated mitochondria and prevented mitochondrial depolarization in cells treated with 3NP. ROS and MPT have been implicated in myocardial stunning associated with reperfusion in ischemic hearts, and these peptide antioxidants potently improved contractile force in an ex vivo heart model. It is noteworthy that peptide analogs without dimethyltyrosine did not inhibit mitochondrial ROS generation or swelling and failed to prevent myocardial stunning. These results clearly demonstrate that overproduction of ROS underlies the cellular toxicity of tBHP and 3NP, and ROS mediate cytochrome c release via MPT. These IMM-targeted antioxidants may be very beneficial in the treatment of aging and diseases associated with oxidative stress.

Cardioprotection and mitochondrial S-nitrosation: Effects of S-nitroso-2-mercaptopropionyl glycine (SNO-MPG) in cardiac ischemia–reperfusion injury

“…Inhibition of these events is proposed to underlie the cardioprotective effects of mitochondrial K + ATP channel opening [7], uncoupling protein activation [10,11], Na + /H + exchange inhibitors [72], Na + /Ca 2+ exchange inhibitors [73], mitochondrial Ca 2+ uniporter inhibitors [74], and mitochondrially targeted ROS scavengers [75,76]. In support of such a mechanism, SNO-MPG delivered prior to ischemia significantly improved mitochondrial Ca 2+ handling, inhibited ROS generation, and inhibited complex I, at the end of ischemia (Figures 5E & 7).…”

Cell-Permeable, Mitochondrial-Targeted, Peptide Antioxidants

“…16 These SS peptides can scavenge H 2 O 2 and ONOO − and inhibit lipid peroxidation in vitro as demonstrated by the inhibition of linoleic acid oxidation and low-density lipoprotein (LDL) oxidation ( Figure 4 ). Their antioxidant action can be attributed to the tyrosine, or dimethyltyrosine (Dmt), residue.…”

“…SS peptides inhibited mitochondrial swelling (A) and prevented cytochrome c release (B) from isolated mitochondria subjected to calcium overload. 16 Isolated mouse liver mitochondria were exposed to 50 M Ca 2+ and swelling measured by absorbance at 540 nm. The amount of cytochrome c released was expressed as percentage of total cytochrome c in mitochondria.…”

Selective Protection of Normal Cells during Chemotherapy by RY4 Peptides

“…This class of peptide usually localizes to the mitochondrial membrane and demonstrates strong antioxidative ability (11,12). Their antioxidant ability may be attributed to H 2 O 2 and ONOO À scavenging and inhibition of lipid peroxidation.…”