Availability and canonical positioning of key amino acids of ornithine-decarboxylase degron is insufficient for alpha-fetoprotein degradation

Abstract

Ornithine decarboxylase (ODC) degrades in proteasome in a ubiquitin-independent manner with the half life of approximately 2 h. Thirty seven C-terminal amino acids of this enzyme constitute a fragment known as the degradation signal (degron), which is responsible for the effectiveness of protein degradation. Among these amino acids, the key positions have recently been mapped (Cys441 and Ala442). Mutations of the key amino acids led to ODC general stabilization, whereas substitution of other amino acids had no significant influence on the ODC degron activity. In addition, deletions or insertions into the region located between the key amino acids and ODC C-end diminished significantly the rate of protein degradation; hence, the distance (remoteness) of these amino acids from ODC C-end is, probably, of crucial importance. Taking into account these data, we have introduced the key amino acids that determine ODC-degron activity into alpha-fetoprotein with the truncated export signal (ΔAFP) so that their positioning was 20 amino-acid away from the C-end (ΔAFPCAG and ΔAFPLCAG). Secretion of ΔAFP and the modified proteins from cells was impossible because of a removal of the N-terminal export signal. Computer analysis of ΔAFP and the derivative ΔAFPCAG and ΔAFPLCAG revealed no significant changes in protein hydrophobicity or in the secondary structure of C-terminal region. The in vitro experiments on HEK293T cells using MG132 proteasome inhibitor and translation inhibitor cycloheximide have demonstrated similar stability of ΔAFP and the derivative ΔAFPCAG and ΔAFPLCAG in cells. Thus, introduction of the key amino acids of ODC degron at the key positions relative to the C-end of ΔAFP did not change the parameters of protein degradation. Perhaps, some other still unknown amino acids are important for ODC-degron functioning. It may well be that ΔAFP conformation prevents interaction of the protein C-end with proteasome.