Wednesday, March 28, 2012

Aconitase or Iron Regulatory Protein 1? Simply put: The Gemini

Cytosolic Aconitase
Aconitase (Figure 1-4) is an important protein in the Citric Acid Cycle, which takes place in the mitochondria of a cell.  Yet, this is not the only location of aconitase.  Aconitase is also found in the cytoplasm and is known as cytosolic aconitase.  In both the mitochondria and cytoplasm aconitase is the protein that converts citrate into isocitrate.  At equilibrium (pH 7.4 and 25 degrees Celcius) the mixture has more citrate than isocitrate.  Yet, isocitrate is used so rapidly in the next step of the Citric Acid Cycle, that the equilibrium is pulled to the right. The iron-sulfur cluster found within the aconitase enzyme binds the substrate as well as helps with the addition or removal of water.  These are key elements in making isocitrate, which is important because it is a part of the pathway that breaks down glucose (sugar) into energy the body can use.  In the mitochondria isocitrate is a part of the Citric Acid Cycle, in the cytoplasm isocitrate is used in other synthetic tasks.

The second role of Cytosolic Aconitase: Iron Regulatory Protein 1
As important as converting citrate into isocitrate is, cytosolic aconitase has another key role in the cytoplasm of the cell.  Cytosolic aconitase is also known as Iron Regulatory Protein 1 (IRP1).  IRP1 is a protein that binds to mRNA, specifically to the Iron Responsive Element (IRE) of the mRNA (Figure 5).  Depending on which end IRP1 binds to, mRNA translation will either be repressed or activated.  If IRP1 binds to the 5’ end of the mRNA, translation will be repressed.  If IRP1 binds to the 3’ end of the mRNA, translation will be activated.  When IRP1 binds to the IRE section of the mRNA, it also helps keep the mRNA from being degradated in the cytoplasm.

The iron-sulfur cluster
Cytosolic aconitase and IRP1 are different only in the fact that cytosolic aconitase contains an iron-sulfur cluster in it.  So, when IRP1 gains the iron-sulfur cluster it becomes aconitase, and when aconitase loses its iron-sulfur cluster it becomes IRP1.  The disassembly of the iron-sulfur cluster happens through oxidants such as H2O2.  In the experiment that Brown and company did, as H2O2 was added to the cell, the iron concentration increased and at the same time aconitase activity decreased.  This lead to the conclusion that oxidation is what lead to the disassembly of the iron-sulfur cluster, and the formation of IRP1 from cytosolic aconitase.  Since the iron-sulfur cluster is solvent accessible it can also be disassembled by O2- and nitrogen species such as NO or ONOO-.  A new iron-sulfur cluster can than enter the IRP1 and causes the molecule to change to aconitase.  This occurs multiple times, as the enzyme switches between its two roles.

Once the iron-sulfur cluster has been removed from cytosolic aconitase and if there is not enough iron in the cell to regenerate the iron-sulfur cluster, the protein stays as IRP1.  In other words, it stays as IRP1 when there are low levels of iron in the cell.  IRP1’s role is to get iron into the cell by binding to different IRE’s on ferritin and transferrin’s mRNAs (Figure 6).  IRP1 inhibits or represses the formation of ferritin, since ferritin stores iron.  In order to do this, it binds to the 5’ end of the ferritin mRNA.  At the same time, IRP1 activates the formation of transferrin receptors by binding to the 3’ end of the transferrin mRNA.  To help in the binding of IRP1 to the IRE site on mRNAs, Ser-138 and Ser-711 are phosphorylated with Protein Kinase C.  This helps IRP1 bind more tightly to the transferrin mRNA, which leads to an increase in the transferrin receptors on the cell surface.  Transferrin receptors allow passage of transferrin, which contains iron, into the cell.  Once there is enough iron in the cell, the iron-sulfur cluster is regenerated and attaches to IRP1, forming cytosolic aconitase again.

In Conclusion
Cytosolic aconitase is an important protein that converts citrate into isocitrate.  Isocitrate is important because it can be used in synthetic tasks.  Yet, when cytosolic aconitase loses its iron-sulfur cluster through oxidation, it becomes Iron Regulatory Protein 1.  IRP1 is involved in the iron homeostasis of a cell.  By attaching to the IRE sites of mRNA, specifically mRNA involved in the translation of ferritin and transferrin receptors, it is able to regulate the iron concentration in the cell. IRP1 can then be easily transformed back into cytosolic aconitase by the addition of the iron-sulfur cluster.  Without cytosolic aconitase the cytosol would not have isocitrate.  Without the iron-sulfur group, that is easily removed and put back in, this protein would not be able to have two very distinct functions.  And without IRP1 the iron levels in the cell would not be regulated as smoothly as IRP1 does by binding to specific mRNA.  The fact that this protein is able to perform these two key tasks in the cytosol is why it is so important.  By being two proteins in one, and the fact the protein is made up of two domains, this is a classic example of a Gemini.