PROTEINS IN HEME TRANSPORT AND GENE REGULATION

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Heme transport by the plasma glycoprotein hemopexin (HPX) is a specific, membrane receptor-mediated process in which both the transport protein and its receptor recycle after endocytosis, like iron transport by transferrin. Heme is catabolized by heme oxygenase (HO) in the endoplasmic reticulum. HPX is a vital part of the body's acute phase defence against injury and pathogens; acts to slow oxidative damage which contributes to aging; and conserves iron. Heme-HPX exerts effects on liver, neurons, cells of the neural retina and immune system (e.g. T-lymphocytes). The overall goal of this research program is to determine the mechanisms for the pleiotropic cellular responses after the heme-HPX complex binds to its membrane receptor, which include coordinated gene regulation. The two principal areas of this research project are to define the role of proteins in intracellular heme transport from the surface HPX receptor and to elucidate the mechanisms whereby the genes for proteins involved in heme and iron metabolism including HO, HPX, transferrin and the transferrin receptor are coordinately and differentially regulated in response to heme-HPX as well as other genes which encode proteins important for cellular defenses against oxidative stress, such as metallothionein (MT). These activities are biologically important in maintaining cell homeostasis under physiologically relevant changes in intracellular heme and iron which take place during trauma, infection, hemolytic conditions, inflammation and reperfusion injury in surgery. To achieve these goals, a comprehensive approach is being taken in which the mechanisms of gene regulation in response to HPX receptor occupancy alone or to heme uptake and intracellular transport will be defined for HO, MT and HPX itself. HPX receptor occupancy activates protein kinase C and reactive oxygen intermediates are generated, perhaps during heme release and transport, thus providing a means to influence nuclear events by activating transcription factors (e.g. Jun, Fos and NFkappaB). Progress has already been made towards identifying the cis-acting elements and trans-acting proteins that operate in heme-HPX and heme-mediated gene regulation. The specific aims are: l) and 2) to continue to define the cis-acting elements in the promoter of the HO-1 and the MT-1 genes together with the trans-acting factors involved in transcriptional regulation by heme-HPX and heme; 3) to establish the mechanisms whereby the expression of HPX itself is regulated in response to heme-HPX or heme: 4) to characterize the HPX receptor isolated by affinity chromatography which we successfully used to purify the HPX receptor from Haemophilus influenzae, and to clone and sequence this protein; 5) to continue to elucidate the role of cytochrome b5 and other heme-binding proteins in intracellular heme transport using biochemical, immunological and morphological techniques.