Researchers Discover New Route To Hemoglobin Synthesis

A research team led byLeonard I. Zon, a Howard Hughes Medical Institute investigator atChildren's Hospital Boston and Harvard Medical School, published itsfindings in the August 18, 2005, issue of the journal Nature. Zon andhis colleagues in Boston collaborated on the studies with researchersfrom the University of Rochester Medical Center and the University ofUtah School of Medicine.

The researchers began their studieshoping to learn why a zebrafish mutant known as shiraz (sir) failed toproduce hemoglobin. The sir mutant zebrafish, which were first isolatedby Zon and colleagues in the Tübingen Screen Consortium in Germany,intrigued the researchers because they die from anemia caused by lackof hemoglobin.

Over the years, Zon and his colleagues havediscovered many zebrafish mutants that fail to make hemoglobin becauseof defects in iron metabolism. As they have teased out the causes ofthese defects, they have learned that the biochemical pathway involvedin hemoglobin synthesis in zebrafish has been largely conserved overthe 300 million years of evolution between fish and humans. Accordingto Zon, the easily manipulable fish constitutes an excellent modelorganism for studying the regulation of heme formation.

In thecurrent study, the researchers traced the hemoglobin defect to the genefor an enzyme known as glutaredoxin 5 (grx5). But the researchers foundearly on that the enzyme was not directly connected to hemoglobinproduction. “Nobody had worked on this gene in vertebrates before, butwe found in the scientific literature that this gene in yeast wasrequired for the production of iron-sulfur clusters in themitochondria,” said Zon. Iron-sulfur clusters are incorporated intocertain proteins to enable their enzymatic functions. In furtherexperiments, the researchers confirmed that versions of grx5 inzebrafish, yeast, mice and humans are functionally equivalent.

“Itseemed like the whole process was evolutionarily conserved,” said Zon.“But the difference is that yeast do not make hemoglobin. So we neededto figure out a mechanism that would explain why these fish that haveproblems making iron-sulfur clusters could not make hemoglobin.”

Otherresearchers' studies had indicated that the presence of iron-sulfurclusters in the cell is important for controlling an enzyme called ironregulatory protein 1 (IRP1). In turn, IRP1 regulates another enzymecalled ALAS2 that plays a key role in heme synthesis. Indeed,experiments by Zon and his colleagues demonstrated that the loss ofgrx5 in the mutant zebrafish inappropriately activates IRP1, whichblocks the synthesis of ALAS2, and thus heme production. For example,when they restored ALAS2 by injecting into the sir mutants a truncatedform of ALAS2 that lacked the portion of the molecule sensitive toIRP1, they complete restored the mutant zebrafish hemoglobin production.

“Peoplehave always thought that hemoglobin synthesis required only enough ironin the cell for heme production to proceed and then just the additionof the globin protein to form hemoglobin,” said Zon. “Now, we've addeda fourth component, iron-sulfur clusters, which are required for hemeproduction. This is a very interesting and unpredicted finding fromwhat we had known before, and our experiments have really defined a newpathway for hemoglobin production,” he said.

Zon said that thefindings could apply to developing new treatments for a rare form ofanemia, known as sideroblastic anemia, in which elevated IRP1 activitycauses a deficiency of ALAS2. In most cases, an increase in IRP1 islikely caused by a mutation in a transporter for iron-sulfur clustersthat traps them in mitochondria, where they cannot interact with IRP1to control it.

In a search for possible treatments for theanemia, Zon and his colleagues are exploring the genetic machinery ofhemoglobin production in zebrafish for targets of drugs that couldrestore normal levels of iron-sulfur clusters. “The pathway that wehave found is very sensitive, so our findings might be extended toenable treatments for other forms of anemia,” said Zon.