Mammalian cells express hundreds of metalloproteins. Most contain the abundant metals iron and zinc, while others contain various trace metals such as copper, manganese, molybdenum, and cobalt (Waldron et al., 2009
). These metals are essential nutrients because metal cofactors activate enzymes and proteins that perform critical functions in virtually every major cellular process (Dupont et al., 2010
). Several factors complicate incorporation of the correct metal ion into a metalloprotein. First, the binding sites for different metals within metalloproteins can be structurally very similar, and incorporation of the noncognate metal ion is easily achieved in vitro for many of these proteins. Second, pools of “free” metal ions in cells may be vanishingly small and the metals largely unavailable, as most zinc and copper ions are tightly bound to cytosolic proteins (Outten and O'Halloran, 2001
). Third, redox-active metals, such as iron and copper ions, can catalyze the production of damaging reactive oxygen species, and cells must maintain tight control over these metals in order to use them while simultaneously avoiding their toxic effects. Fortunately, the majority of metalloproteins receive the correct metal ion in vivo, as incorporation of the wrong metal ion typically inactivates the protein.
Although the incorporation of the appropriate metal ion(s) into cellular metalloproteins is a critical, essential process, the mechanism by which most metalloproteins receive their specific cofactor is unknown. Some proteins rely on metallochaperones: proteins that specifically bind metal ions and deliver them to target enzymes and transporters through direct protein-protein interactions (Rosenzweig, 2002
). Metallochaperones delivering nickel and copper have been described in prokaryotes and eukaryotes, but much less is known about the delivery of iron and zinc. Frataxin, the protein lacking in the neurodegenerative disease Friedreich's Ataxia, is a mitochondrial protein that is thought to function as an iron chaperone for the assembly of iron-sulfur clusters (Stemmler et al., 2010
More recently, we identified poly (rC) binding protein 1 (PCBP1) as a cytosolic iron chaperone that delivers iron to ferritin (Shi et al., 2008
). In mammals, ferritin is a heteropolymer consisting of 24 subunits of heavy (H) and light (L) peptides that assemble into a hollow sphere into which iron is deposited (Crichton, 2009
; Hintze and Theil, 2006
). PCBP1 binds Fe(II) with micromolar affinity in a 3 Fe:1 PCBP1 molar ratio. PCBP1 binds ferritin in vivo and can enhance iron incorporation into ferritin in vitro and in vivo. Mammalian cells lacking PCBP1 exhibit defects in the incorporation of iron into ferritin as well as an increase in the labile pool of cytosolic iron and an increase in the iron-mediated degradation of iron-regulatory protein 2.
PCBP1 (also called α-CP1 or hnRNP E1) has previously been found to function as an RNA- and DNA- binding protein (Chaudhury et al., 2010
; Makeyev and Liebhaber, 2002
; Ostareck-Lederer and Ostareck, 2004
). PCBP1 is one member of a family of four homologous proteins containing three heterogeneous nuclear ribonucleoprotein K-homology (KH) domains, an ancient and conserved RNA binding module. PCBP1, an intronless gene, likely arose from the retrotranspositon of a splice variant of PCBP2 mRNA, and became fixed in the genome because it encoded a unique function not shared by the other PCBPs. PCBP1 and 2 bind to cytosolic and viral RNAs, thereby affecting their translation or stability. PCBPs also have a role in transcriptional regulation and participate in several protein-protein interactions.
Numerous cellular proteins require iron for activity. Iron in the form of heme and iron-sulfur clusters are cofactors for proteins involved in a host of metabolic and regulatory functions. Enzymes of the “nonheme” iron families directly coordinate iron ions as cofactors. These families include the diiron monooxygenases, such as the δ-9-fatty acid desaturase and the small subunit of ribonucleotide reductase (Shanklin et al., 2009
). A second family is the Fe(II)- and 2-oxoglutarate (2-OG)-dependent dioxygenases (Kaelin and Ratcliffe, 2008
; Loenarz and Schofield, 2008
; Ozer and Bruick, 2007
). This family is a large, evolutionarily conserved class of enzymes that can oxidatively modify a variety of substrates. In mammals, four members of this class regulate the activity of the transcription factors that control the mammalian response to hypoxia.
Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor that binds DNA at specific sites, termed hypoxia response elements (HREs), and activates the expression of more than 100 genes involved in the adaptation to reduced oxygen levels (Kaelin and Ratcliffe, 2008
; Loenarz and Schofield, 2008
; Ozer and Bruick, 2007
). Under hypoxic conditions, the alpha subunit (HIF1α or HIF2α) accumulates and binds to the beta subunit (HIF1β, also called ARNT) to form the active transcription factor. Under conditions of normoxia or hyperoxia, HIF1α is hydroxylated on proline residues 402 and 564, which allows the protein to be recognized by the von Hippel-Lindau tumor suppressor protein (pVHL), thus targeting HIF1α for ubiquitin-mediated degradation in the proteasome (Ivan et al., 2001
; Jaakkola et al., 2001
). Three HIF prolyl hydroxylases, PHD1, 2, and 3 (also called HPH-3, -2, and -1 or EGLN2, 1, and 3, respectively) mediate the hydroxylation of proline residues on HIF1α (Bruick and McKnight, 2001
; Epstein et al., 2001
), although PHD2 is responsible for nearly all (>95%) of the activity in cultured cells (Berra et al., 2003
). HIF1α is also hydroxylated on Asn803 by an asparaginyl hydroxylase, factor inhibiting HIF (FIH1) (Hewitson et al., 2002
; Lando et al., 2002a
). Hydroxylation of Asn803 inhibits the binding of transcriptional coactivators with HIF1α and represents a second mechanism for inhibition of HIF activity (Lando et al., 2002b
). The activities of the HIF hydroxylases are regulated by the availability of the cosubstrates, 2-OG and oxygen. Because the hydroxylases exhibit changes in oxygen binding and activity over the range of oxygen concentrations present in tissues, these enzymes are hypothesized to function directly as oxygen sensors.
The activities of the HIF hydroxylases may also be regulated by the availability of iron. HIF hydroxylase activity is stimulated by the addition of Fe(II) in vitro, and, in cultured cells, activity is inhibited by iron chelators (Kaelin and Ratcliffe, 2008
). In mice, HIF2α accumulates in duodenal enterocytes in response to iron deprivation, which may reflect a localized decrease in HIF hydroxylase activity (Shah et al., 2009
). Cellular factors that control the incorporation of iron into the HIF hydroxylases are unknown.
Here we have addressed the question of whether PCBP1, or its paralog PCBP2, is involved in the delivery of iron to the Fe(II)-dependent prolyl and asparaginyl hydroxylases regulating HIF. We found that iron-deprived cells lacking PCBP1 or PCBP2 exhibited increased levels of HIF1α, which was due to a decrease in prolyl hydroxylation and VHL-mediated degradation. The loss of prolyl hydroxylase activity was traced to a decrease in iron loading of the enzyme, which could be restored with recombinant PCBP1. PCBP1 physically interacted with PHD2, indicating that PCBP1 likely acts as an iron chaperone for PHD2. Our studies also suggest a direct role for PCBPs in the activation of the asparaginyl hydroxylase FIH1.