A study on the autophagy receptor p62/SQSTM1. Impact of ATF4 and 5´untranslated region of mRNA upon amino acid deprivation
ForfatterFredriksen, Line Mari
Autophagy is crucial for sustainment of cellular homeostasis, but there is limited knowledge of how autophagy is regulated. The present study examined characteristics of the first mammalian autophagy receptor described, the sequestosome-1 or p62/ SQSTM1, regarding transcriptional and translational expression under stress by amino acid starvation in the mammalian Hek293 cells. Reviews report p62 to be restored during prolonged starvation in mouse embryonic fibroblasts and HepG2 cells. Initially p62 is also degraded by autophagy, but after 4-8 hours the level is restored. The transcription factor ATF4 is induced by starvation. Three aims were defined in the present study; first to test whether the p62 promoter is up-regulated by ATF4 compared to the inducing transcription factor NRF2, second to perform bioinformatics studies of the sequences in the p62 5´UTR and examine whether structures as “hairpins” related to the 5´UTR may explain levels of the p62 translation, and third to determine if different lengths of the 5´UTR have impact on the p62 translation under starvation. Measures of expression of p62 promoter in Hek293 cells co-transfected with ATF4 expression plasmid compared to NRF2 as control, were assessed by reporter gene assay. To find whether ATF4 is involved in the upregulation of p62, and if translation of p62 is regulated by sequences in 5´UTR, bioinformatics and luciferase analyses were performed. In the experimental set-up of Hek293 cells transfected with ATF4 and p62 promoter region, showed no enhancing effect on the p62 promoter to be detected. Bioinformatic analysis showed that the 5`UTR was unusually short and occurred in three main variants of 35, 62 and 100 bp. The 100 bp 5`UTR was predicted to form two hairpin structures. The three variants of the 5`UTRs were cloned in front of the luciferase gene, and their effect on luciferase expression was measured under normal condition and upon prolonged starvation. Interestingly, the 5`UTRs seemed to repress protein expression both under normal conditions and during starvation, possible due to formation of hairpin structures. In contrast the p62 5`UTRs co-transfected with an ATF4 expression plasmid did seem to reverse the repression of the p62 5`UTRs. Western blot of all extracts from starved Hek293 cells showed that the level of p62 decreased with 1- 4 hours of starvation, but after 6 hours an increase occurred. However, the LC3B lipidation level was modest. Taken together, the restoration of the p62 expression upon prolonged starvation may be due to ATF4 action on p62 5`UTR in a direct or indirect way.
ForlagUiT Norges arktiske universitet
UiT The Arctic University of Norway
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