Global Profiling of N-terminal Cysteine-Dependent Degradation Mechanisms

Hypoxia, a condition characterized by insufficient oxygen supply, challenges cellular homeostasis and energy production, driving the activation of adaptive responses to maintain survival under these stress-inducing conditions. One key strategy involves enzymatic oxidation of N-terminal cysteine residues coupled with proteolysis through the Cys-Arg/N-degron pathway. Despite the presence of hundreds of proteins with N-terminal cysteine in humans, only two have been identified as substrates of this pathway, and its substrate selectivity remains unclear. Moreover, the biological role of this pathway in the cellular response to hypoxia is not well defined. By employing a systematic proteomics approach, we discovered that nearly half of the cysteine-commencing proteome could be regulated by the Cys-Arg/N-degron pathway. Mutagenesis experiments revealed the specificty of Cys-Arg/N-degron pathway showing a preference for hydrophobic and positively charged residues following cysteine. Furthermore, we uncovered substrates that are regulated by this pathway during hypoxia, including IP6K1. The loss of IP6K1 impaired glucose uptake, glycolytic ATP production, and overall mitochondrial morphology and function. As a result, IP6K1-deficient cells exhibited disrupted metabolic adaptation under hypoxic conditions and decreased survival under stress. These findings underscore the importance of the Cys-Arg/N-degron pathway in regulating metabolic responses and highlight its potential importance in hypoxia-related disorders.