Substrate profiling of mitochondrial caseinolytic protease P via a site-specific photocrosslinking approach

The function of proteases is mainly determined by the substrates they are processing. However, substrate identification remains a challenging task. Here, we used genetic code expansion to incorporate a diazirine-lysine amino acid (DiazK) at selected positions of mitochondrial caseinolytic protease P (ClpP) in mammalian cells and determined its substrate scope by proteomic analyses after UV-light dependent photocrosslink. The results confirm its major involvement in maintaining the overall mitochondrial protein homeostasis as evident from alternative approaches. Additionally, novel putative substrates were identified revealing the highly complementary nature of this approach. Furthermore, this allows for the identification of changing substrate dynamics in an unbiased manner. Induction of oxidative stress with rotenone followed by identification of unique proteins corroborate its role in mitochondrial stress response. We hypothesize, that the oxidative stress is counteracted by processing proteins, which are involved in respiratory chain complex proteins synthesis and maturation as well as catabolic enzymes respectively.