Engineering degrons of yeast ornithine decarboxylase as vehicles for efficient targeted protein degradation

• Published in: Biochimica et biophysica acta Vol. 1850; no. 12; pp. 2452 - 2463
• Main Authors: Joshi, Rushikesh G., Kulkarni, Swapnali, Ratna Prabha, C.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.12.2015
• Subjects: biosynthesis; CD spectrum; chimerism; Degradation determinant signal; Degron; engineering; Function of degron; Ornithine decarboxylase; Ornithine Decarboxylase - metabolism; peptides; polyamines; proteasome endopeptidase complex; protein degradation; Proteolysis; recombinant fusion proteins; Saccharomyces cerevisiae - enzymology; Structure of degron; yeasts; Structure of degron; TFE; Degron; AzBE; Degradation determinant signal; Function of degron; Ornithine decarboxylase; yEGFP; CD spectrum; ODC
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2015.09.003

Ornithine decarboxylase (ODC), which catalyzes the first step of polyamine biosynthesis, undergoes rapid targeted degradation (TPD) with the help of its two degron sequences, namely the N-terminal 50 residues (N50) and α/β domain (α/β) housing antizyme binding element (AzBE), in response to increased polyamine levels. Antizyme binds to AzBE of ODC and delivers it to proteasome for degradation. Entire ODC was used as a tag to demonstrate TPD of chimeric proteins. Here we fashioned three peptide sequences from yeast ODC to test their capability to act as degrons, namely N50, α/β and Nα/β (a combination of N50 and α/β), and monitored their degradation potentials in chimeric proteins. We have examined the correlation between degradation potentials and structural integrity of the peptides, to find mechanistic explanations. Nα/β with two signals in tandem is a better degron, under the regulation of antizyme. N50 like N44 reported earlier could drive chimeric proteins to degradation, while α/β could not act as an independent degron. Strong correlation was observed between functional efficacy of the peptides and their structural integrity. N50, which was believed to be unstructured, displayed propensity for helical conformation. Nα/β exhibited optimal structure, while α/β failed to adopt native like conformation. Functional efficacy of the degron Nα/β is a consequence of its structural integrity. Nα/β and N50 could target chimeric proteins to degradation. However, α/β failed in the quest. Nα/β, regulated by antizyme, is better suited than N50 for TPD to understand the function of novel proteins. [Display omitted] •Two degrons in yeast ornithine decarboxylase sequence target it for degradation.•They are N-terminal fifty residues and α/β domain with antizyme binding sequence.•Three peptides N50, α/β and Nα/β were examined for their degradation potentials.•Nα/β regulated by antizyme is a better degron than N50, while α/β failed as a degron.•Functional efficacy of the degrons is a consequence of their structural integrity.