Role of Prohibitin Nitrosylation in its Neuroprotective Functions

Project Summary/Abstract The mitochondrial protein Prohibitin (PHB) is essential for life. Its importance to cellular activities is attested by the fact that deletion of PHB is embryonic lethal in mice and that to date no mutation has been found in the coding region of PHB in any neurological disease conditions, indicating that PHB integrity is essential and that somatic mutation is detrimental. Our recent work has demonstrated that PHB has remarkable neuroprotective potential against ischemic brain injury with an underlying mitochondrial associated mechanism. Its expression is critical for mitochondrial function in stress situations. However, how this important protein that are stable at both mRNA and protein levels, is functionally regulated in neuroprotection, as well as how it is dysregulated in other neurological conditions, remain surprisingly unknown. In exploring the mechanisms of PHB regulation, we discovered that nitric oxide (NO) is required for PHB expression mediated neuroprotection. Therefore, we investigated the interaction between NO and PHB and found that NO modifies PHB post-translationally, through protein s-nitrosylation, a novel regulatory mechanism similar to protein phosphorylation. In this application, we propose to study the effects of PHB S-nitrosylation and the mechanisms underlying functional regulation of PHB by NO. Our central hypothesis is that nitrosylation is critical for PHB’s neuroprotective function and, consequently, disturbances of PHB nitrosylation are detrimental and contribute to pathology. We will use a novel mutant knock-in mouse, in which the sole cysteine residue of PHB protein is mutated so that PHB cannot be nitrosylated, to analyze the mechanisms of NO regulation and the effects of loss of PHB nitrosylation on PHB function, in the settings of brain ischemic injury in association with mitochondria structural alterations. Three specific aims will systematically test the hypothesis. The results of the proposed studies will reveal a previously unrecognized regulatory mechanism of PHB which we believe is crucial to facilitate the design of potential therapies that could ultimately benefit patients at risk of stroke and other neurological diseases.