The role of endogenous heme synthesis and degradation domain cysteines in cellular iron-dependent degradation of IRP2.
Iron regulatory protein 2 (IRP2) is a mammalian cytosolic iron-sensing protein that regulates expression of iron metabolism proteins, including ferritin and transferrin receptor 1. IRP2 is ubiquitinated and degraded by the proteasome in iron-replete cells but is relatively stable in iron-depleted cells. Recent work has shown that IRP2 contains a unique 73-amino-acid domain that binds iron in vitro and undergoes iron-dependent oxidation and cleavage (J. Biol. Chem. 278 (2003), 14857). Several cysteines in the 73-amino-acid domain function as an in vitro iron-binding site. To assess the role of these cysteines in cellular iron- dependent degradation of IRP2, we mutagenized these cysteines in various combinations in the context of full-length protein and generated cell lines in which recombinant IRP2 expression was inducible. Iron-dependent degradation of IRP2 mutagenized at any or all of the cysteines of the putative degradation domain in cells was comparable to wild-type (WT). Both WT and cysteine mutant protein were stabilized in 3% oxygen. Treatment with sodium nitroprusside (SNP), an NO+ donor, caused a decrease in cellular IRP2 concentrations, but the SNP effect was abrogated by simultaneous addition of the iron chelator desferal and was not affected by cysteine mutations. Inhibition of endogenous heme synthesis with succinylacetone significantly inhibited iron- dependent degradation of IRP2. Addition of cobalt chloride inhibited degradation of both WT and mutagenized IRP2. Thus, we could not discern a role for the recently defined in vitro cysteine-dependent iron-binding site of IRP2 in cellular physiology. The early molecular events in iron-dependent degradation of IRP2 remain to be elucidated.
Bourdon
Emmanuel
E
Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, Bethesda, MD 20892, USA.
Kang
Dae-Kyung
DK
Ghosh
Manik C
MC
Drake
Steven K
SK
Wey
Jane
J
Levine
Rodney L
RL
Rouault
Tracey A
TA
D003545:Cysteine / Q000235:genetics / Q000502:physiology*; D006418:Heme / Q000096:biosynthesis*; D006537:Heptanoates / Q000494:pharmacology; D006801:Humans; D007501:Iron / Q000378:metabolism / Q000502:physiology*; D035942:Iron Regulatory Protein 2 / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics
D006537:Heptanoates; D011994:Recombinant Proteins; D006418:Heme; C020804:succinylacetone; D007501:Iron; D035942:Iron Regulatory Protein 2; D003545:Cysteine