Alex Lee: “Ribosome-stalling reporters suggest the N-terminus of AGAT acting as a creatine-dependent translation-arrest peptide”

SHORT SUMMARY

In this study, our goal was to investigate how high levels of creatine are able to cause reductions in AGAT expression. Through the use of various reporter constructs, we were able to identify a 23 amino acid sequence within the AGAT protein that plays a key role in allowing it to respond to creatine. When this 23 amino acid sequence is fused to other proteins, high levels of creatine are able to decrease this protein’s expression. With this result, we hypothesize that creatine is able to regulate AGAT expression by interfering with the activity of ribosomes which is the cellular machinery responsible for protein production.

ABSTRACT

Arginine:glycine amidinotransferase (AGAT) is the rate-limiting enzyme in the twostep biosynthetic pathway of creatine (CT). Increasing intracellular levels of CT results in decreased AGAT mRNA, protein expression, and enzyme activity. In previously described experiments, we have used genome editing to produce a HAP1 cell line that expresses a C-terminal tagged AGAT-NanoLuc Luciferase (AGAT-NLuc) reporter to monitor the decrease in AGAT expression with increasing intracellular CT. Co-treatment with CT and a transcriptional inhibitor, Actinomycin D, or a translation inhibitor, cycloheximide, suggested the involvement of a post-transcriptional mechanism controlling in AGAT expression. We have identified a 23 amino acid long sequence on the N-terminus of AGAT (‘AGAT peptide’) that enables the CT responsiveness. Fusing this sequence in-frame or as an upstream open reading frame to Firefly Luciferase and expression in cells results in a dose-dependent decrease in luminescence with increasing CT. We hypothesize that the mechanism by which CT controls AGAT expression involves ribosomal stalling. During translation, CT could interact with the nascent AGAT peptide arresting further AGAT translation and reducing AGAT mRNA stability. We have generated a series translation-arrest reporters consisting of a Nterminal Nanoluc Luciferase and C-terminal Firefly luciferase with InterVening Sequences consisting of Internal Ribosome Entry Sites and/or P2A Translation Reinitiation Sequences along with the wild type or mutated AGAT peptide. Our results are highly suggestive of the AGAT nascent N-terminal amino acid sequence acting as a translation arrest peptide.

This poster is not available; Research remains in progress