Progress 10/01/08 to 09/30/12
Outputs
OUTPUTS: This project involved the teaching and mentoring of graduate students on the relationships between dietary iron intake and the adaptive responses of liver, muscle and tissue culture cells to low iron status. This included conducting and analyzing three types of experiments. First, the impact of dietary iron intake on the abundance of mRNAs encoding components of the systems that cells use to assemble iron-sulfur (Fe-S) cofactors in mitochondrial and other proteins. This system is required for mitochondrial function in terms of converting food energy into cellular energy in the form of ATP. Second, the mechanisms through which the synthesis of the enzyme mitochondrial aconitase (m-acon) is controlled by iron was determine. m-Acon is a key enzyme in the tricarboxylic acid cycle, a central pathway for converting food energy into metabolic energy as ATP. This included analysis of a regulatory hierarchy through which an iron-regulated RNA binding protein binds to regulatory elements in m-acon and other mRNA. Third, the mechanism through which dietary iron absorption is linked with the rate of red blood cell production was examined. Dissemination included presentations of results at scientific meetings and publications. PARTICIPANTS: Macy Shen was a graduate student who worked mostly on the m-acon part of this project. This project allowed her to obtain her PhD in Jan 2013 TARGET AUDIENCES: The primary target audience of this work is other scientists and clinicians interested in the role of iron in human and animal health. The work was reported primarily at scientific meetings PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Several changes in knowledge have occurred. First, we demonstrated that the mRNA level of many proteins involved in Fe-S biogenesis in muscle are not altered by dietary iron level. In contrast, expression of most of these mRNAs was reduced in iron deficient liver. Second, we demonstrated that m-acon protein was controlled by iron through changes in translation of its mRNA and through changes in its protein stability. Third, we demonstrated the existence of a regulatory RNA binding hierarchy for so-called iron responsive elements in mRNAs like m-acon mRNA. In addition, we examined how the RNA binding protein that binds to these elements is regulated. Fourth, we discovered a new pathway through which the rate of red blood cell formation and intestinal iron absorption is linked. This involves discovery of a new regulator of the expression of the cytokine/hormone erythropoietin which is a key regulator of red cell formation. This latter finding may lead to a change in conditions for treatment of disorders such as the anemia of prematurity in humans.
Publications
- Anderson CP, Shen M, Eisenstein RS, Leibold EA.(2012) Mammalian iron metabolism and its control by iron regulatory proteins. Biochim Biophys Acta. 1823:1468-8. PMID: 22610083.
- Anderson, S.A., Nizzi, C.P., Chang, Yuan-I., Deck, K.M., Schmidt, P., Galy, B., Broman, A.T., Kendziorski, C., Hentze, M.W., Fleming, M.D., Zhang, J. and Eisenstein, R.S. (2013) The IRP1-HIF2α Axis: Coordinating Iron and Oxygen Sensing with Erythropoiesis and Iron Absorption. Cell Metabolism 17:282-290.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: We conducted the following activities in the past year in order to further understand the adaptive processes that occur during iron deficiency. First, we have continued our studies on the role of proteins involved in the formation of iron sulfur cluster cofactors in muscle, liver and other cell types. Much of this work in the past year involved optimization of antibodies directed against key proteins involved in cytosolic iron sulfur cluster formation. Second, we have developed procedures to reduce the activity cytosolic iron sulfur cluster biogenesis proteins in order to understand how this affects the regulation of cellular iron metabolism in response to altered iron availability. Third, we have used mice lacking key regulators of iron metabolism, the iron regulatory proteins (IRP), in order to determine the individual and overlapping roles of IRP1 and IRP2. Fourth, we have finalized our work on the hierarchical regulation of liver mRNAs by IRP. The events we reported this work at include our local weekly seminar series in Nutritional Sciences and the BioIron international meeting in Vancouver, Canada. Fifth, we have continued our studies aimed at determining the how IRP1 is regulated by protein degradation. Sixth, we made significant progress determining the role of IRP2 on the response of liver mitochondria to iron deficiency. Services that were performed included that counseling of graduate students, a technician and a senior researcher on the methodologies involved in the studies, in data analysis and in planning of new experiments. PARTICIPANTS: Richard Eisenstein was the prinicipal investigator on this project. He planned experiments, interpreted data and helped trouble shoot problems. Elisa Ho is a graduate student who developed RNA binding and protein degradation assays for IRP1. Nathan Johnson is a graduate student who developed protocols to detect proteins involved in iron sulfur cluster formation and in reducing the expression of these proteins in cells. Sheila Anderson made diets for animals and assisted with the animal diet studies. Christopher Nizzi performed studies of mRNA translation state in mice lacking IRP1 or IRP2. He also examined the impact of iron deficiency on mitochondrial function in liver of these animals. TARGET AUDIENCES: The target audiences include established scientists as well as young scientists in training. The major effort involved laboratory instruction in methodologies for studying how altered iron available affects specific physiological processes such as mitochondrial ability to oxidize substrates for energy generation. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The following changes in knowledge occurred. First, we determined that there are differential effects of the loss of IRP1 versus IRP2 in liver. IRP2 appears to regulate mRNAs encoding proteins of iron metabolism. IRP1 appears to preferentially regulate the mRNA encoding hypoxia inducible factor 2 alpha, a key regulator of erythropoiesis and other cellular responses to low oxygen. Second, we have determined that excessive expression of IRP2 can be toxic to cells. Third, we have demonstrated that IRP1 protein degradation is regulated by the same protein as controls IRP2 protein degradation. Fourth, we have determined that mice lacking IRP1 have higher numbers of red blood cells than wildtype mice suggesting that IRP1 provides a key link between iron status and red cell formation rate. Fifth, we found that IRP2 is required for the optimal adaptive response of liver mitochondria to iron deficiency.
Publications
- No publications reported this period
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: To better understand the adaptive response to iron deficiency, we examined how iron status may hierarchically regulate expression of key proteins involved in controlling the uptake and metabolic fate of iron. Iron regulatory proteins, known as IRP, control the use of six mRNA encoding key proteins involved in iron metabolism or the adaptive response to iron deficiency. IRP bind to iron responsive elements, known as IRE, in these mRNA and in this manner control mRNA translation. Three studies were undertaken. First, we asked if IRP1 differentially binds to the various IRE involved in mRNA translational control. We demonstrated an RNA binding hierarchy involving IRP1 and the IRE from mRNA whose translation is controlled by IRP. The affinity of IRP1 for natural IRE carried over a 9-fold range. Human IRE mutants found in the mRNA encoding the iron storage protein ferritin fell within this hierarchy supporting the concept that IRE binding affinity determines the extent of expression of proteins encoded by IRE containing mRNA. We then introduced a large number of mutations in the IRE in order to better understand how IRP differentially bind natural IRE. Our study identified key areas of the IRE that contribute to the differential recognition by IRP1. Second, we determined the impact of iron deficiency and acute iron overload on translation of IRE containing mRNA in rat liver. This study demonstrated hierarchical control of the translation of IRE containing mRNA in vivo. Additionally, we observed in rapidly growing weanling rats that ferritin protein accumulation was strongly regulated by dietary iron under conditions where ferritin mRNA translation was not altered. This supports that view that increased ferritin protein degradation or secretion contributes to ferritin protein accumulation in liver. Third, we examined the impact of loss of IRP1 or IRP2 on the translation of IRE containing mRNA in liver. Loss of IRP1 did not affect strongly alter IRE mRNA translation. However, loss of IRP2 caused a partial derepression of L-ferritin mRNA. Interesting, H-ferritin mRNA translation was not affected. Fourth, we have continued our studies on the effect of iron deficiency on muscle and liver function. We are currently determining the impact of iron deficiency on mitochondrial division and fusion, both of which are key processes in mitochondrial health. Our outputs are: First, we have had one manuscript published in RNA. Second, we are writing up the translation paper described in part 2 above. We expect this to be submitted in March 2011. PARTICIPANTS: Graduate student Jeremy Goforth worked on the RNA binding. This was his thesis project and he obtained his PhD on this basis. He now works in industry. Graduate student Elisa Ho is working on the role of IRP-dependent and IRP-independent processes in the adaptive response to iron deficiency. Research Specialist Christopher Nizzi worked on this project with the IRP knockout mice. This project will allow him to obtain another publication. Senior Scientist Sheila Anderson coordinated the animal projects with the knockout mice and the iron deficiency study in rats. She will obtain additional publications. PI Richard Eisenstein oversees these projects. TARGET AUDIENCES: The target audience is largely other scientists interested in iron metabolism and metabolic adaptation. Efforts to cause a change in their knowledge base include attendance at the FASEB summer conference on trace elements. Other efforts include publications on this work which were in press this year and will be published in 2011. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The outcome of these studies is that we have demonstrated that proper fine tuned control of the synthesis of proteins involved in iron metabolism is required to maintain iron homeostasis. These studies support the hypothesis that the affinity or tightness with which IRP bind to specific mRNA targets differs and this allows for different degrees of regulation. As such, our findings provide an explanation as to how liver or red cells can keep expression of the iron storage protein ferritin repressed while allowing use of iron for key cellular processes such as formation of the key iron cofactors heme or Fe-S proteins. Only when iron is present at excessive levels is formation of the iron storage protein ferritin accelerated. This type of regulation is key for the proper response of cells and tissues to variations of dietary iron intake or when cells proliferate and must import a lot of iron but divert it to use for cell division.
Publications
- Goforth, J.B., Anderson, S.A., Nizzi, C.P. and Eisenstein, R.S. (2010) Multiple determinants with iron responsive elements dictate iron regulatory protein binding and regulatory hierarchy. RNA, 16(1): 154-169.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: In the past year, we were involved in activities in conducting experiments to understand the regulatory processes involved in the adaptive responses to dietary iron deficiency. First, we examined how iron deficiency affected liver function in terms of the regulation of mitochondrial function. Here we have focused on examining the relationship between iron deficiency and mitochondrial capacity to export citrate. Second, we performed experiments aimed at understanding how iron regulatory proteins (IRP) differentially regulated the translation of six different target mRNA. Here we have determined the RNA binding affinity of IRP1 for various iron responsive elements from these six mRNA. Third, we determined mechanisms through which iron deficiency leads to the generation of RNA binding activity by IRP1. Here we have focused on the studying how the iron sulfur cluster in cytosolic aconitase is removed allowing the generation of IRP1 RNA binding activity. Fourth, to better understand how IRP1 responds to iron deficiency, we made mutant forms IRP1 that we predict will display altered iron regulation due to changes in their rate of protein degradation. Fifth, we determined the rate at which IRP1 dissociates from different iron responsive elements in tightly versus weakly regulated mRNA. PARTICIPANTS: Richard Eisenstein Ph.D. He is the principal investigator who directs the project. Elisha Ho is a graduate student who worked on the RNA binding aspects of this project. Jeremy Goforth was a graduate student who worked on RNA binding aspects of this project. Christopher Nizzi is an associate research specialist who worked on the dietary iron deficiency and liver function aspect of this project. Kathryn Deck is an assistant scientist who worked on the phosphorylation and IRP1 function aspect. TARGET AUDIENCES: Target audiences include other scientists involved in research on trace elements or who are generally interested in nutrition, diet and health. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The following changes in knowledge were obtained in the past year. First, we determined that iron deficiency leads to an increased capacity for mitochondria to export citrate in terms of moles of citrate exported. Second, we determined that IRP1 binds to iron responsive elements in target mRNA in a hierarchical manner that relates to the physiological function of the encoded proteins. Third, using phosphorylation state mimics of IRP1, we determined that the iron sulfur cluster can be removed from cytosolic aconitase in two ways and that phosphorylation accelerates cluster loss. Fourth, we determined that IRP1 dissociates much more quickly from iron responsive elements in weakly regulated mRNA like mitochondrial acontiase compared to the strongly regulated mRNA, L-ferritin.
Publications
- Deck K.M., Vasanthakumar A., Anderson S.A., Goforth J.B., Kennedy M.C., Antholine W.E., Eisenstein R.S. (2009) Evidence that phosphorylation of iron regulatory protein 1 at serine 138 destablizes the [4Fe-4S] cluster in cytosolic aconitase by enhancing 4Fe-3Fe cycling. J. Biol. Chem. 284:12701-9.
- Goforth, J.B., Anderson, S.A., Nizzi, C.P. and Eisenstein, R.S. (2009) Multiple determinants with iron responsive elements dictate iron regulatory protein binding and regulatory hierarchy. RNA. 16:154-169.
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