Conveying a deadly message

Reactive oxygen species (ROS) are implicated in apoptosis, and the p66 isoform of the adaptor protein Shc regulates ROS metabolism and apoptosis. So could there be a direct connection between p66^Shc and ROS release? Giorgio et al. reveal all in Cell.

p66^Shc is known to be required for mitochondrial depolarization and for cytochrome c release in response to pro-apoptotic stimuli. And, because blocking the permeability transition pore of mitochondria inhibited this pro-apoptotic function, the group's first step was to see whether p66^Shc could directly induce permeability transition. When allowed to enter the mitochondrial intermembrane space, recombinant p66^Shc indeed increased the permeability of isolated mitochondria. p66^Shc is known to be enriched in the inner membrane fraction of mitochondria and, consistent with this, the authors found it within the inner mitochondrial space.

With the localization of p66^Shc pinpointed, Giorgio et al. then showed that p66^Shc was essential for the increase in mitochondrial ROS that occurs after treatment with pro-apoptotic stimuli and that a functional electron transport chain was a requisite. They ruled out the possibility that the increase in ROS was the result of decreased scavenging or occurred indirectly through mitochondrial swelling.

Unlike antimycin A, for example, which generates the ROS superoxide (O₂^−·) and hydrogen peroxide (H₂O₂) by blocking the electron transport chain (ETC), p66^Shc didn't inhibit respiration. It required a functional ETC to stimulate the production of H₂O₂ (O₂^−· was not generated), which implies that p66^Shc reduced oxygen using certain components of the ETC. Could p66^Shc itself mediate electron transfer? The results from the group suggested that it could, and indicated a redox potential for p66^Shc of −35 mV. As this value is closest — among the redox systems present in mitochondria — to that of cytochrome c, the authors investigated whether p66^Shc could exchange electrons with cytochrome c, and found that it could. Specifically, p66^Shc could interact with, and oxidize, cytochrome c in vitro. In doing so, oxygen was reduced and H₂O₂ was formed in vitro and in vivo.

The authors then used the fact that neither the p46 nor p52 isoforms of Shc influence ROS regulation or apoptosis to implicate the N-terminal region (which contains the collagen-homologous-2 (CH2) and phosphotyrosine binding (PTB) domains) of p66^Shc in cytochrome c binding. Further probing uncovered a 44-residue region just N-terminal to the PTB domain which mediated cytochrome c binding, with the identification of E132, E133 and W134 as essential residues for redox activity and cytochrome c binding. Mutations that impair this redox activity not only impaired the ability of p66^Shc to mediate ROS production, but also abrogated its ability to induce mitochondrial permeability transition and subsequent apoptosis. So p66^Shc seems to be “an atypical signal transducer that converts proapoptotic into redox signals”.