CELLULAR TRAFFICKING

Molecular Regulator Found for Iron Balance, Immune Response

Shared Across Systems, Mon1a Essential for Secreting Variety of Proteins from Cells

The protein produced by a newly discovered gene has emerged in mice as a key regulator of the distinct critical systems for iron balance and immunity. Researchers at HMS, Children’s Hospital Boston, the University of Utah School of Medicine, and other institutions used a variety of genetic approaches to reveal that the gene Mon1a, which also exists in humans, is essential for the secretion of a variety of molecules from cells. The findings, in the August Nature Genetics, indicate that Mon1a is a commonly shared component of the secretory machinery.

Graham Ramsay

Clockwise from left, Fudi Wang, Nancy Andrews, and Kristina Roberts, with collaborators, identified a gene that helps shuttle proteins out of cells.

Genetic defects can increase dietary iron absorption by increasing the transfer of iron across intestinal epithelial cells, leading to iron accumulation. This condition, known as hemochromatosis in humans, is among the most common genetic diseases of white Americans. Patients can absorb up to 30 percent of dietary iron, compared to the 10 percent absorption in healthy individuals. Over time, excess iron builds up, resulting in five to 10 times as much total body iron as found in normal individuals. Without a way to get rid of it, this iron overload can lead to liver failure, diabetes, and heart problems.

But not everyone with a genetic predisposition to this disorder suffers from excess iron. Only about 10 percent of patients susceptible to hemochromatosis show symptoms or distress from the condition, which usually presents itself in patients over 40.

“It can kill by age 20 or be below clinical radar at age 80,” said Nancy Andrews, the George Richards Minot professor of pediatrics at HMS and CHB and senior author on the paper. The variable severity of human iron disorders is not well understood, but apparently is affected by an assortment of genetic and environmental factors, she said. (On Aug. 27, Duke University announced that Andrews would become dean of its medical school later this fall. )

Tracking a Trafficker
Andrews and her research team, including first author Fudi Wang, HMS instructor in pediatrics at CHB, compared the genetic makeup of two inbred mouse strains to find possible genetic flaws contributing to iron buildup. Using quantitative trait locus analysis, the researchers found a certain stretch of DNA on mouse chromosome 9 that correlated with high levels of iron in macrophages from the spleen. Normally, these macrophages phagocytose damaged red blood cells to recycle the iron they contain in hemoglobin. Because these immune scavengers handle very large amounts of iron, they play a major role in determining overall body iron distribution. Comparing spleens from “normal” inbred mouse strains, the geneticists observed a 12-fold difference between the lowest and highest iron levels.
Several years of further genetic probing revealed that the gene of interest within that stretch was Mon1a, encoding a homolog of a molecule in yeast that helps traffic proteins to the vacuole. The researchers suspected a similar role for Mon1a in regulating iron levels in mice, but had no obvious model for how it might behave.

The Andrews group previously showed that the protein ferroportin works as an iron exporter by pumping the mineral out of macrophages. But ferroportin must be on the surface of the cell in order to work; otherwise iron cannot be released, and it builds up. The researchers deduced—and later demonstrated—that Mon1a helps direct the iron exporter from the cytoplasm to its active position at the cell membrane.

Same Piece, Different Puzzles
To investigate the role of Mon1a in iron trafficking, Andrews collaborated with iron metabolism expert Jerry Kaplan, professor of pathology at the University of Utah School of Medicine, who has identified genes for iron metabolism in yeast that have mammalian homologs. The Utah researchers silenced Mon1a activity in macrophages and found that less ferroportin on the cell surface coincided with more iron in the cell.

“The knockdown of Mon1a caused ferroportin to stay in the cell,” said Wang. “With the loss of Mon1a, ferroportin can’t stay on the membrane. Ferroportin loses its function, and the cell retains iron.”

A similar pattern of protein sequestration after Mon1a blockade appeared with the immune system molecule called macrophage migration inhibitory factor, which also built up in macrophages. This parallel suggests that the Mon1a is a shared constituent rather than a dedicated component of iron transport.

Courtesy Fudi Wang

Breakdown. In normal conditions (basal), the iron export protein ferroportin (red) resides in the cytoplasm of spleen macrophages. When cells from a mouse strain with hyperactive Mon1a are treated with iron, ferroportin moves to its active position at the cell membrane, where it shuttles iron out of cells. When cells from a mouse strain with normal Mon1a are treated with iron, most ferroportin remains at its inactive position in the cytoplasm.

This model marks a fundamental change in the understanding of secretion, Kaplan said. His group looked at effects on a variety of secreted molecules to examine Mon1a’s function and found that inflammatory cytokines also accumulated in cells following Mon1a knockdown.

“Without Mon1a, proteins that are usually secreted stay in the cell,” said Andrews, who is also the dean for basic sciences and graduate studies at HMS. “It is generally necessary for secretion.” While the current results were demonstrated in macrophages from the spleen, Andrews said that it is likely that Mon1a has effects in multiple tissues.

—Molly McElroy

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