The mitochondrial Unfolded Protein Response (UPRMT) is a highly conserved stress pathway that when dysregulated is causally connected to a host of degenerative diseases including Parkinsons, Alzheimers, Freiderichs Ataxia and aging. The recent identification of components in the UPRMT has stimulated interest in this as a “druggable” pathway. Central to the UPRMT is the mitochondrial protease ClpP, required for the turnover of damaged and misfolded proteins in response to cellular stresses. We and others recently identified ClpP as an unexpected target for a novel class of anti-cancer compounds known as imipridones (ONC201) and related analogs (https://www.the-scientist.com/news-opinion/found--a-cancer-drugs-mechanism-of-action- 65918). We showed that these compounds activated ClpP and the UPRMT as determined by the degradation of mitochondrial proteins, impaired mitochondrial respiratory chain activity, and increased integrated stress response (ISR) proteins (CHOP/ATF4). Thus, the main objective of this research is to investigate the basic mechanisms of the UPRMT and elucidate how drug- induced activation of ClpP initiates important stress signals that regulate cell growth and metabolism. We propose three aims to accomplish this: in Aim 1 we will use comprehensive proteomics approaches to identify ClpP substrates and peptides released from the mitochondria. In Aim 2 we will determine how ClpP activation dysregulates mitochondrial metabolism, and affects the consumption of glucose and glutamine by glycolysis and the TCA cycle. In Aim 3 we will determine how ClpP activation alters cytosolic signaling events, namely the activation of the ISR and the resulting reduction in protein synthesis. If successful, our studies will provide significant new insight into the biological functions of the UPRMT and how ClpP regulates this pathway functions to modulate cell stress in normal and disease states.