The iron-containing protein, acireductone dioxygenase 1 (ADI1), is a dioxygenase important for polyamine synthesis and proliferation. Using differential proteomics, the studies herein demonstrated that ADI1 was significantly down-regulated by cellular iron depletion. This is important, since ADI1 contains a non-heme, iron-binding site critical for its activity. Examination of multiple human cell-types demonstrated a significant decrease in ADI1 mRNA and protein after incubation with iron chelators. The decrease in ADI1 after iron depletion was reversible upon incubation of cells with the iron salt, ferric ammonium citrate (FAC). A significant decrease in ADI1 mRNA levels was observed after 14 h of iron depletion. In contrast, the chelator-mediated reduction in ADI1 protein occurred earlier after 10 h of iron depletion, suggesting additional post-transcriptional regulation. The proteasome inhibitor, MG-132, prevented the iron chelator-mediated decrease in ADI1 expression, while the lysosomotropic agent, chloroquine, had no effect. These results suggest an iron-dependent, proteasome-mediated, degradation mechanism. Poly r(C)-binding protein (PCBPs) 1 and 2 act as iron delivery chaperones to other iron-containing dioxygenases and were shown herein for the first time to be regulated by iron levels. Silencing of PCBP1, but not PCBP2, led to loss of ADI1 expression. Confocal microscopy co-localization studies and proximity ligation assays both demonstrated decreased interaction of ADI1 with PCBP1 and PCBP2 under conditions of iron depletion using DFO. These data indicate PCBP1 and PCBP2 interact with ADI1, but only PCBP1 plays a role in ADI1 expression. In fact, PCBP2 appeared to play an accessory role, being involved as a potential co-chaperone.

Molecular Pharmacology and Pathology Program Department of Pathology and Bosch Institute University of Sydney Sydney New South Wales 2006 Australia

Molecular Pharmacology and Pathology Program Department of Pathology and Bosch Institute University of Sydney Sydney New South Wales 2006 Australia; BIOCEV Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University Průmyslová 595 252 50 Vestec Czech Republic

Molecular Pharmacology and Pathology Program Department of Pathology and Bosch Institute University of Sydney Sydney New South Wales 2006 Australia; Cancer Drug Resistance Program University of Sydney Sydney New South Wales 2006 Australia

Molecular Pharmacology and Pathology Program Department of Pathology and Bosch Institute University of Sydney Sydney New South Wales 2006 Australia; Department of Pathology and Biological Responses Nagoya University Graduate School of Medicine Nagoya 466 8550 Japan; Centre for Cancer Cell Biology Griffith Institute for Drug Discovery Griffith University Nathan Brisbane Queensland Australia

Molecular Pharmacology and Pathology Program Department of Pathology and Bosch Institute University of Sydney Sydney New South Wales 2006 Australia; Melbourne Dementia Research Centre The Florey Institute of Neuroscience and Mental Health The University of Melbourne Parkville VIC Australia

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