G-quadruplexes sequester free heme in living cells

Abstract

Heme is an essential cofactor for many enzymes, but free heme is toxic and its levels are tightly regulated. G-quadruplexes bind heme avidly in vitro, raising the possibility that they may sequester heme in vivo. If so, then treatment that displaces heme from quadruplexes is predicted to induce expression of genes involved in iron and heme homeostasis. Here we show that PhenDC3, a G-quadruplex ligand structurally unrelated to heme, displaces quadruplex-bound heme in vitro and alters transcription in cultured human cells, up-regulating genes that support heme degradation and iron homeostasis, and most strikingly causing a 30-fold induction of heme oxidase 1, the key enzyme in heme degradation. We propose that G-quadruplexes sequester heme to protect cells from the pathophysiological consequences of free heme. This identifies a new function for G-quadruplexes and a new mechanism for protection of cells from heme.

Human ribosomal G-quadruplexes regulate heme bioavailability

“…Human ribosomes bind hemin in vitro. It has been suggested that G4s might associate with heme in vivo (10,11,35). In vitro, heme binds with high affinity to G4s by end-stacking (10)(11)(12)(36)(37)(38) (Figure 1B).…”

Human Ribosomal G-Quadruplexes Regulate Heme Bioavailability

“…o more abundant and more expansive G-tracts on the LSU than the SSU (8), o greater conservation over phylogeny of LSU G-tracts than SSU G-tracts (7,8), o higher thermodynamic stability of LSU G4s than SSU G4s (7,8), o greater heme binding to G4 oligomers from the LSU than those from the SSU (Figure 3A, Figure S.1B), o greater enrichment of LSU than SSU particles in BioTASQ pulldowns (Figure 2D These responses were interpreted to support a model in which G4s sequester and detoxify heme in cells (29). Here, by exploiting differential interactions of apo-rRNA and heme-rRNA with hemin-agarose, we developed more direct methods to establish in vivo heme-G4 interactions, with a focus on rRNA G4s.…”

“…Human ribosomes bind hemin in vitro. It has been suggested that G4s might associate with heme in vivo (29). In vitro, heme binds with high affinity to G4s by endstacking (30-32) ( Figure 1B).…”

“…The binding is specific for G4s as a mutant oligonucleotide, mutes3, that lacks G- PhenDC3 was used to confirm binding of hemin to ribosomal G4s. PhenDC3, like hemin, end-stacks on G4s (29) and therefore competes with heme for binding to G4s.…”

“…Decades of in vitro biophysical and chemical characterization of G4-heme complexes have found that they interact with high affinity (KD ~ 10 nM) and are potent redox catalysts, facilitating peroxidase and peroxygenase reactions(4)(5)(6). However, it remained unclear if heme-G4 complexes are formed in vivo and if heme-G4 catalyzed reactions were physiologically relevant.Gray and coworkers(29) recently proposed that heme binds to G4s in vivo, based on the transcriptional response of cells to PhenDC3. PhenDC3 up-regulates heme degrading enzymes like heme oxygenase, and other iron and heme homeostatic factors.…”