Studies of proteins in heme and iron metabolism

Abstract: One aim of the study was to investigate the regulation of the murine erythroid isoform of aminolevulinate synthase (eALAS) protein involved in heme synthesis. Another aim was to characterize the structure of the rat hemochromatosis gene (HFE) gene and its expression in conjunction with other genes involved in the regulation of iron homeostasis during inflammation and iron overload in the rat liver. Most of the iron in the body is consumed via heme synthesis starting with eALAS. We found a new protease activity in bone marrow cells, which proteolytically cleaves an 8-kDa segment of the eALAS protein from its N-terminus. More differentiated erythroid cells isolated from peripheral blood exhibit very little of this protein truncation. We also demonstrated that the newly discovered short form of eALAS is located in the mitochondrial membrane of bone marrow erythroid cells. Most common protease inhibitors did not affect this proteolytic activity. The specific function of eALAS short protein is not clear and needs additional studies. To further investigate eALAS regulation we analyzed the effect of heme on eALAS synthesis in natural erythroid cells. Incubation with heme (5-100 ìM) clearly inhibits eALAS synthesis in bone marrow cells. This inhibitory effect of heme could also be observed in peripheral blood cells at higher concentrations. However, low heme concentrations (1-10 ìM) increased eALAS synthesis. Addition of heme also increased the levels of the cytosolic preform of eALAS in blood cells. This suggests that eALAS synthesis is controlled via a composite of regulatory mechanisms including the iron regulatory proteins (IRPs). Regulation of eALAS in bone marrow cells also seems more tightly controlled in order to prevent oxidative cell damage. While eALAS plays a major role in iron consumption via the heme synthesis pathway there are several other proteins and genes responsible for the regulation of iron homeostasis in the body. HFE has been shown to be involved in iron metabolism but little is known about its regulation. Cloning of the rat HFE gene using library screening and PCR showed that the rat HFE gene contains six exons and five introns. Determination of HFE mRNA levels by Northern blot (tissues) and real-time PCR (isolated liver cells) showed that the HFE gene is expressed in multiple tissues in the rat, including bone marrow, with the highest expression in the liver. In contrast to earlier beliefs our experiments on isolated rat liver cells demonstrated the highest expression of the HFE gene in rat hepatocytes. High levels were also found in Kupffer cells. To better understand the process of hemochromatosis, we investigated the expression of HFE and other genes involved in iron metabolism (TfR1, TfR2, DMT1, IREG1, HAMP), in rat liver during inflammation and iron overload. Expression of HAMP in whole liver was higher in iron-loaded than in normal animals. mRNA expression levels of HAMP in cultured hepatocytes from normal rats were also higher than in freshly isolated hepatocytes. Levels were however clearly lower in cultured hepatocytes from iron-loaded rats, arguing that its expression depends on surrounding cells or factors not present in tissue culture. TfR1 had a different regulation than TfR2 during inflammation and iron overload in hepatocytes. The expression of the other studied genes in animals with both normal and iron diets followed the idea that inflammation and iron loading causes iron to be bound intracellularly.