Progress 10/01/00 to 09/30/04
Outputs
Transferrin transports nonheme iron between tissues. Transferrin is an essential protein since loss of transferrin expression due to mutation can be lethal. We focused on whether or not iron status of cells might regulate the efficiency with which newly synthesized transferrin is released from the cell. While we obtained some initially promising results suggesting that iron status of cells impacted the efficiency of transferrin secretion, we were not able to demonstrate a specific response to iron overload. Consequently, we conclude that high levels of iron were impairing protein secretion in general and not Tf secretion specifically. We also completed some work in a related area of iron metabolism. This concerns the regulation of iron regulatory protein 1 (IRP1) by phosphorylation. Our studies indicate that phosphorylation of IRP1 at serine 711 selectively impairs the ability of the aconitase form of IRP1 to convert citrate to isocitrate. In this way we believe that
phosphorylation promotes the accumulation of cytosolic citrate and its use for gluconeogenesis which is important in the increased need to generate glucose in liver during iron deficiency.
Impacts
Transferrin is a protein critical for the maintenance of normal iron metabolism. We seek to understand the basic processes through which transferrin expression and accumulation in the blood is regulated. We believe this will allow a better understanding of how interorgan iron transport is controlled in health and disease. Iron regulatory protein 1 (IRP1) is critical for normal iron metabolism. Our studies revealed a new role for the aconitase function of IRP1. Our results suggest that phosphorylation of IRP1 can alter the aconitase function in a manner that helps preserve the organic acid citrate. These changes in citrate metabolism may be important in the changes in glucose metabolism in iron deficiency. Citrate can be a source of carbon for glucose formation. Phosphorylation of IRP1 may help promote glucose formation in iron deficiency.
Publications
- Pitula, J.S., Deck, K.M., Clarke, S.L., Anderson, S.A., Vasanthakumar, A. and Eisenstein, R. 2004 Selective Inhibition of the Citrate to Isocitrate Reaction of Cytosolic Aconitase by Phosphomimetic Mutation of Serine 711. Proc. Natl. Acad. Sci. U.S.A.101: 10907-12.
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Progress 01/01/03 to 12/31/03
Outputs
We are interested in how the uptake and metabolic fate of iron is regulated in mammals. Transferrin is a protein required for transport of nonheme iron between tissues. Transferrin is synthesized and secreted into serum mostly by the liver. Synthesis of transferrin is regulated by iron. However, the extent to which iron modulates the efficiency of transferrin secretion and/or intracellular turnover of transferrin has not been determined. In iron overload diseases transferrin levels in blood are depleted and this promotes increases in oxidative damage by iron, since there is not enough transferrin to bind the iron present. We want to determine if this is in part due to reduced efficiency of transferrin synthesis. We have shown that iron overload of a human liver cell line leads to a reduction in the efficiency of transferrin secretion. Furthermore, iron deficiency increases the efficiency of transferrin secretion. However, we have also found that iron overload
decreases synthesis of the control protein albumin. This suggests that some of the effect of iron on transferrin secretion may not be specific but may still explain the response seen in iron overload diseases. We hope to continue this aspect of the work by examining if the synthesis of another control protein, alpha one antitrypsin, is affected by iron. We are also investigating if the basic mechanism of transferrin secretion from cells requires its association with the transferrin receptor. We have obtained cells that do or do not express the transferrin receptor and will determine if Tf secretion is reduced when the transferrin receptor is not expressed.
Impacts
Transferrin is a protein critical for the maintenance of normal iron metabolism. We seek to understand the basic processes through which transferrin expression and accumulation in the blood is regulated. We believe this will allow a better understanding of how interorgan iron transport is controlled in health and disease.
Publications
- Richard Eisenstein and Kerry L. Ross. 2003. Novel Roles for Iron Regulatory Proteins in the Adaptive Response to Iron Deficiency. Journal of Nutrition 133:1510S.
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Progress 01/01/02 to 12/31/02
Outputs
Transferrin transports nonheme iron between tissues. Transferrin is synthesized and secreted into serum primarily by the liver. Iron can regulate transferrin synthesis but relatively little is known of the mechanism of iron action. Furthermore, the extent to which iron modulates the efficiency of transferrin secretion and/or intracellular turnover of transferrin has not been determined. We want to determine the mechanism through which iron modulates the level of Tf present in serum. In iron overload diseases, transferrin levels in blood are depleted and this promotes increased oxidative damage by iron, since there is not enough transferrin to bind the iron present. We have shown that iron overload of a human liver cell line leads to a reduction in the efficiency of transferrin secretion. Furthermore, induction of iron deficiency increases the efficiency of transferrin secretion. We have been trying to examine the secretion of control proteins by these cells to
determine if the effect of iron is specific for transferrin or not. We have been also examining the secretion of other proteins from the liver cells such as alpha one antitrypsin and transthyretin. As these proteins are secreted faster than transferrin so we are continuing to try and find a protein with the same secretion kinetics so that we may compare the effects of iron overload and iron deficiency on transferrin and a control protein over the same time period. We recently obtained a plasmid that will allow us to express transferrin in mammalian cells that do not normally express it. We plan to use this plasmid to see if transferrin secretion is altered in cells that have or do not have the transferrin receptor number one. We have such cells and plan to do this experiment this year.
Impacts
Nearly 1 in 200 Caucasians of Northern European descent have the mutation that causes hereditary hemochromatosis (HH). The extent to which someone with the disease gets HH is quite variable but up to several million people have varying degrees of HH in the United States. Severe HH causes problems with the liver, heart and pancreas. Our studies on the effect of iron overload on serum level of the iron transport protein transferrin should help lead to a better understanding of the negative health effects of HH.
Publications
- No publications reported this period
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Progress 01/01/01 to 12/31/01
Outputs
Transferrin transports nonheme iron between tissues. Transferrin is synthesized and secreted into serum primarily by the liver. Iron can regulate transferrin synthesis but relatively little is known of the mechanism of iron action. Furthermore, the extent to which iron modulates the efficiency of transferrin secretion and/or intracellular turnover of transferrin has not been determined. We want to determine the mechanism through which iron modulates the level of Tf present in serum. In iron overload diseases transferrin levels in blood are depleted and this promotes increases oxidative damage by iron since there is not enough transferrin to bind the iron present. In the past year we have been optimizing conditions for studying transferrin synthesis in a human liver cell line called Hep G2. We are currently continuing our study concerning how iron overload leads to a reduction in the efficiency of transferrin secretion. We plan to determine if iron binding to
transferrin influences its secretion efficiency by making a mutant transferrin that does not bind iron. In addition, we are examining if the transferrin receptor is required for secretion of transferrin. We hypothesize that transferrin binding to its receptor during secretion and is carried out of the cell this way. We have obtained a CHO cell line that does not make transferrin receptor and a derivative of this cell line that can make the receptor. We are working on expressing transferrin in these cells to see if the presence of the receptor affects the efficiency of transferrin secretion. We have obtained a human transferrin cDNA and are in the process of inserting it into a system that will permit us to express it in the CHO cells. The results of these studies may be of use in developing novel means for enhancing Tf expression in humans and production animals
Impacts
Transferrin is the primary protein involved in interorgan distribution of iron. It is critical for transferrin to bind iron present in the serum becuase iron not bound to proteins is toxic. In iron overload such as hemochromatosis iron is present at high levels and can cause dieases like liver cancer. Transferrin levels decline in iron overload states. We are working on understanding how transferrin level in serum is reduced in iron overload and thereby possibly developing means to increase transferrin levels to protect the body from the oxidative damage which occurs when iron levels increase too much.
Publications
- No publications reported this period
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