Sirtuin 2 regulates cellular iron homeostasis via deacetylation of transcription factor NRF2
Xiaoyan Yang, Seong-Hoon Park, Hsiang-Chun Chang, Jason S. Shapiro, Athanassios Vassilopoulos, Konrad T. Sawicki, Chunlei Chen, Meng Shang, Paul W. Burridge, Conrad L. Epting, Lisa D. Wilsbacher, Supak Jenkitkasemwong, Mitchell Knutson, David Gius, Hossein Ardehali
Xiaoyan Yang, Seong-Hoon Park, Hsiang-Chun Chang, Jason S. Shapiro, Athanassios Vassilopoulos, Konrad T. Sawicki, Chunlei Chen, Meng Shang, Paul W. Burridge, Conrad L. Epting, Lisa D. Wilsbacher, Supak Jenkitkasemwong, Mitchell Knutson, David Gius, Hossein Ardehali
View: Text | PDF
Research Article Cell biology Metabolism

Sirtuin 2 regulates cellular iron homeostasis via deacetylation of transcription factor NRF2

Abstract

SIRT2 is a cytoplasmic sirtuin that plays a role in various cellular processes, including tumorigenesis, metabolism, and inflammation. Since these processes require iron, we hypothesized that SIRT2 directly regulates cellular iron homeostasis. Here, we have demonstrated that SIRT2 depletion results in a decrease in cellular iron levels both in vitro and in vivo. Mechanistically, we determined that SIRT2 maintains cellular iron levels by binding to and deacetylating nuclear factor erythroid-derived 2–related factor 2 (NRF2) on lysines 506 and 508, leading to a reduction in total and nuclear NRF2 levels. The reduction in nuclear NRF2 leads to reduced ferroportin 1 (FPN1) expression, which in turn results in decreased cellular iron export. Finally, we observed that Sirt2 deletion reduced cell viability in response to iron deficiency. Moreover, livers from Sirt2–/– mice had decreased iron levels, while this effect was reversed in Sirt2–/– Nrf2–/– double-KO mice. Taken together, our results uncover a link between sirtuin proteins and direct control over cellular iron homeostasis via regulation of NRF2 deacetylation and stability.

Authors

Xiaoyan Yang, Seong-Hoon Park, Hsiang-Chun Chang, Jason S. Shapiro, Athanassios Vassilopoulos, Konrad T. Sawicki, Chunlei Chen, Meng Shang, Paul W. Burridge, Conrad L. Epting, Lisa D. Wilsbacher, Supak Jenkitkasemwong, Mitchell Knutson, David Gius, Hossein Ardehali

×

Figure 5

Lysines 506 and 508 of NRF2 are targeted and deacetylated by SIRT2.

Options: View larger image (or click on image) Download as PowerPoint
Lysines 506 and 508 of NRF2 are targeted and deacetylated by SIRT2. (A) ...
(A) Co-IP experiments in HEK293T cells overexpressing Flag-NRF2 or mutant plasmids and CREBBP together, with or without SIRT2. Lysates were immunoprecipitated with anti-Flag antibody and blotted with anti–acetyl-lysine (α-Ac-K) antibody. The numbers under the gel reflect the degree of protein acetylation in the presence of SIRT2 and CREBBP normalized to CREBBP only. pcDNA3.1 empty vector was used as a control. (B) Luciferase activity in HepG2 cells transfected with a murine Fpn1 promoter–luciferase reporter construct along with NRF2 plasmid or the indicated NRF2 deacetylation mutants with mutations in the indicated NRF2-ECH homology (Neh) domain. SIRT2 was coexpressed as indicated. Fpn1 promoter reporter activity was normalized to Renilla luciferase activity and empty vector (EV). Empty vector was used as a control (n = 6 per group). (C) Protein stability of overexpressed WT NRF2, 6KQ, and 6KR mutants after treatment with CHX in HepG2 cells. Samples were taken 0 and 90 minutes after CHX treatment. Since all of the samples could not be run on the same gel, they were processed on 2 separate gels, with Neh1-6KQ shared between them (n = 3 per group). (D) Luciferase activity in HepG2 cells transfected with a murine Fpn1 promoter–luciferase reporter construct along with NRF2 or NRF2 mutants. Luciferase activity was measured and analyzed as in B. An empty vector was used as a control, and pairwise comparisons were made between the glutamate and arginine mutants for each amino acid site (n = 6 per group). (E) Protein stability of overexpressed 506KQ, 506KR, 508KQ, and 508KR mutants after treatment with 100 μg/ml CHX in HepG2 cells. Samples were taken 0 and 90 minutes after CHX treatment (n = 3 per group). Data are presented as the mean ± SEM. *P < 0.05, by ANOVA with Bonferroni’s correction for multiple comparisons (B and C) or by Student’s t test (D and E).