J. Biol. Chem., Vol. 279, Issue 33, 34682-34690, August 13, 2004
Cell-permeable Peptide Antioxidants Targeted to Inner Mitochondrial Membrane inhibit Mitochondrial Swelling, Oxidative Cell Death, and Reperfusion Injury
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 N2A cells (EC50 in nM range). They also decreased mitochondrial ROS production, inhibited MPT and swelling, and prevented cytochrome c release induced by Ca2+ 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
From the discussion:
These SS peptides (SS-02 and SS-31) are the first antioxidants that selectively target and concentrate in the IMM, thereby enabling scavenging of ROS at the site of production. Using these peptide antioxidants, we were able to show that overproduction of ROS underlies the cellular toxicity of tBHP and 3NP. Our studies with isolated mitochondria also demonstrated that ROS mediate cytochrome c release via MPT and rupture of the OMM. By reducing ROS production, these peptide antioxidants were able to prevent mitochondrial depolarization in cells exposed to 3NP. Finally, these peptide antioxidants were able to prevent myocardial stunning associated with reperfusion in the ischemic heart in an ex vivo model. The inability of SS-20, which does not have antioxidant ability, to prevent Ca2+-mediated mitochondrial swelling or reperfusion injury confirms that the protective actions of these peptides are mediated via their antioxidant actions.
...
In summary, we have designed cell-permeable peptide antioxidants that target the site of ROS generation and protect mitochondrial function. Our results demonstrate that ROS play a major role in mediating mitochondrial dysfunction induced by tBHP, Ca2+, and 3NP. These antioxidant peptides may be beneficial in the treatment of aging and diseases associated with oxidative damage such as ischemia-reperfusion injury and neurodegeneration.
The peptides: tetrapeptides with alternating aromatic residues and basic amino acids. SS-02 (Dmt-D-Arg-Phe-Lys-NH2; Dmt = 2',6'-dimethyltyrosine), SS-20 (Phe-D-Arg-Phe-Lys-NH2), SS-31 (D-Arg-Dmt-Lys-Phe-NH2)
Delivery of peptide: Continuous cardiac perfusion with Krebs-Henseleit solution containing various SS peptides.
Key points to take away:
1. the antioxidant localizes in the IMM
2. it is a simple tetrapeptide (much easier to manufacture and create a delivery system for)