A cellular assay system revealed the deamidase-mediated regulation of the Arg-mediated N-end rule protein degradation pathway

Regulated protein production and degradation are essential for maintaining proteostasis in eukaryotic cells. The N-end rule, or N-degron pathway, is a protein degradation machinery in which the N-terminal amino acid is the mark of the protein degradation via the proteasome. The N-end rule is a conserved protein disposal machinery in eukaryotic cells. However, the precise cellular mechanisms and their physiological roles are not fully understood. Herein, we report the development of an Arg-mediated N-end rule assay system using artificial substrates expressed in cultured cell lines. We demonstrated that the N-end rule degradation is significantly influenced by the expression levels of N-terminal amino acid-modifying enzymes, including NTAN1, NTAQ1, and ATE1. In the N-terminal Asn protein pathway, an increase in NTAN1 or ATE1 expression promotes its disposal via the N-end rule degradation pathway. Interestingly, overexpression of NTAQ1 decreased the degradation of the protein bearing Gln at the N-terminus. Computational prediction of NTAQ1 and ATE1 complex formation suggests that the outer loop region of NTAQ1 is involved in its interaction with ATE1 and that the NTAQ1 overexpression may negatively affect this interaction. Our findings suggest that the degradation activity of the Arg/N-end rule pathway is positively or negatively regulated by deamidase expression levels and propose a higher degree of control of protein degradation by the Arg/N-end rule within cells.