Previously, our laboratory identified and characterized the cis and trans elements that regulate iron-dependent alterations in expression of ferritin and the transferrin receptor. Iron-responsive elements (IREs) are RNA stem-loops found in the 5' end of ferritin mRNA and the 3' end of transferrin receptor mRNA. We have cloned, expressed, and characterized two essential iron-sensing proteins, Iron Regulatory Protein 1 (IRP1) and Iron Regulatory Protein 2 (IRP2). IRPs bind to IREs when iron levels are depleted, resulting in both the inhibition of translation of ferritin mRNA and other transcripts with IREs near the 5' end and the prolongation of the half-life of the transferrin receptor mRNA.

Iron-Sulfur Cluster Assembly
Tong
IRP1 is an iron-sulfur protein related to mitochondrial aconitase, a citric acid cycle enzyme that functions as a cytosolic aconitase in cells that are iron-replete. Regulation of the RNA binding activity of IRP1 involves a transition from a form of IRP1 in which a [4Fe-4S] cluster is bound to a form that loses both iron and aconitase activity. The [4Fe-4S]–containing protein does not bind to IREs, and the status of the cluster appears to determine whether IRP1 will bind to RNA. Recently, we have identified mammalian enzymes of iron-sulfur cluster assembly that are homologous to the NifS and Nif U genes implicated in bacterial iron-sulfur cluster assembly. We have shown that these gene products facilitate assembly of the iron-sulfur cluster of IRP1. We discovered that single genes in the human genome encode both mitochondrial and cytosolic forms of the cysteine desulfurase IscS, IscU, and NFU. NFU is abundant and may represent a scaffold for iron-sulfur cluster assembly.

Iron-Dependent Degradation of IRP2 and Other Proteins
Ghosh
IRP2 also binds to IREs in iron-depleted cells, but unlike IRP1, IRP2 is degraded in cells that are replete with iron. Experimental evidence indicates that IRP2 undergoes iron-catalyzed oxidation. The proteasome then selectively ubiquitinates and degrades the oxidized protein. Indirect evidence suggests that numerous other proteins are degraded by a pathway in which oxidative modification is followed by ubiquitination and proteasomal degradation of the ubiquitinated substrate.

Physiology and Regulation of Iron Metabolism
Smith, Cooperman, Grabill, Jui-Chen, Meyron-Holtz, Land, Missirlis
To approach questions about the physiology of iron metabolism, we generated loss-of-function mutations of IRP1 and IRP2 in mice through homologous recombination in embryonic cell lines. In the absence of provocative stimuli, we observed no abnormalities in iron metabolism associated with loss of IRP1 function. IRP2-/-mice develop a progressive movement disorder characterized by gait abnormalities and tremor. Animals accumulate iron in axons and develop axonal degeneration. Ferritin over-expression occurs in affected neurons, and ferritin accumulates in axons. Affected tissues are those that contain cells that depend mainly on IRP2 for repression of ferritin translation. Animals that lack both IRP1 and IRP2 do not survive past the blastocyst stage.

Structural Characterization of IRPs and IREs
Yikilmaz
We have purified milligram quantities of IRP1 and IRP2 and are working on crystallization of each IRP. In addition, we are trying to co-crystallize each IRP in a complex with IRE. We have characterized and over-expressed an IRP-like protein from Plasmodium falciparum. We have also characterized an IRE in the 3'UTR of the iron transporter DMT1 that binds with high affinity to only one of the two IREs.