Glória C. Ferreira

Hypoxia-inducible factor 1 (HIF-1) activates transcription of genes encoding glucose transporters, glycolytic enzymes, vascular endothelial growth factor, and other proteins involved in O2 homeostasis and tumor progression. The expression and transcriptional activity of the HIF-1alpha subunit is regulated by the cellular O2 concentration. We demonstrate that insulin, insulin-like growth factor (IGF)-1, and IGF-2 induce expression of HIF-1alpha, which is required for expression of genes encoding IGF-2, IGF-binding protein (IGFBP)-2 and IGFBP-3. These data provide a novel mechanism by which HIF-1alpha overexpression may occur in tumor cells and contribute to an autocrine growth factor loop.

Hypoxia-inducible factor 1 (HIF-1) transactivates genes the products of which mediate tumor angiogenesis and glycolytic metabolism. Overexpression of the HIF-1 alpha subunit, resulting from intratumoral hypoxia and genetic alterations, has been demonstrated in common human cancers and is correlated with tumor angiogenesis and patient mortality. Here we demonstrate that hypoxia or HIF-1 alpha overexpression stimulates Matrigel invasion by HCT116 human colon carcinoma cells, whereas this process is inhibited by a small interfering RNA directed against HIF-1 alpha. We show that HIF-1 regulates the expression of genes encoding cathepsin D; matrix metalloproteinase 2; urokinase plasminogen activator receptor (uPAR); fibronectin 1; keratins 14, 18, and 19; vimentin; transforming growth factor alpha; and autocrine motility factor, which are proteins that play established roles in the pathophysiology of invasion. Neutralizing antibodies against uPAR block tumor cell invasion induced by hypoxia or HIF-1 alpha overexpression. These results provide a molecular basis for promotion of the invasive cancer phenotype by hypoxia and/or HIF-1 alpha overexpression.

Friedreich ataxia is a severe autosomal-recessive disease characterized by neurodegeneration, cardiomyopathy and diabetes, resulting from reduced synthesis of the mitochondrial protein frataxin. Although frataxin is ubiquitously expressed, frataxin deficiency leads to a selective loss of dorsal root ganglia neurons, cardiomyocytes and pancreatic beta cells. How frataxin normally promotes survival of these particular cells is the subject of intense debate. The predominant view is that frataxin sustains mitochondrial energy production and other cellular functions by providing iron for heme synthesis and iron-sulfur cluster (ISC) assembly and repair. We have proposed that frataxin not only promotes the biogenesis of iron-containing enzymes, but also detoxifies surplus iron thereby affording a critical anti-oxidant mechanism. These two functions have been difficult to tease apart, however, and the physiologic role of iron detoxification by frataxin has not yet been demonstrated in vivo. Here, we describe mutations that specifically impair the ferroxidation or mineralization activity of yeast frataxin, which are necessary for iron detoxification but do not affect the iron chaperone function of the protein. These mutations increase the sensitivity of yeast cells to oxidative stress, shortening chronological life span and precluding survival in the absence of the anti-oxidant enzyme superoxide dismutase. Thus, the role of frataxin is not limited to promoting ISC assembly or heme synthesis. Iron detoxification is another function of frataxin relevant to anti-oxidant defense and cell longevity that could play a critical role in the metabolically demanding environment of non-dividing neuronal, cardiac and pancreatic beta cells.

We have investigated the mechanism of frataxin, a conserved mitochondrial protein involved in iron metabolism and neurodegenerative disease. Previous studies revealed that the yeast frataxin homologue (mYfh1p) is activated by Fe(II) in the presence of O2 and assembles stepwise into a 48-subunit multimer (alpha48) that sequesters >2000 atoms of iron in 2-4-nm cores structurally similar to ferritin iron cores. Here we show that mYfh1p assembly is driven by two sequential iron oxidation reactions: A ferroxidase reaction catalyzed by mYfh1p induces the first assembly step (alpha --> alpha3), followed by a slower autoxidation reaction that promotes the assembly of higher order oligomers yielding alpha48. Depending on the ionic environment, stepwise assembly is associated with accumulation of 50-75 Fe(II)/subunit. Initially, this Fe(II) is loosely bound to mYfh1p and can be readily mobilized by chelators or made available to the mitochondrial enzyme ferrochelatase to synthesize heme. Transfer of mYfh1p-bound Fe(II) to ferrochelatase occurs in the presence of citrate, a physiologic ferrous iron chelator, suggesting that the transfer involves an intermolecular interaction. If mYfh1p-bound Fe(II) is not transferred to a ligand, iron oxidation, and mineralization proceed to completion, Fe(III) becomes progressively less accessible, and a stable iron-protein complex is formed. Iron oxidation-driven stepwise assembly is a novel mechanism by which yeast frataxin can function as an iron chaperone or an iron store.

Protoporhyrinogen oxidase (EC 1.3.3.4), the penultimate enzyme of the heme biosynthetic pathway, catalyzes the removal of six hydrogens from protoporphyrinogen IX to form protoporphyrin IX. The enzyme in eukaryotes is associated with the inner mitochondrial membrane. In the present study we have examined requirements for solubilization of this enzyme and find that it behaves as an intrinsic membrane protein that is solubilized only with detergents such as sodium cholate. The in situ orientation of the enzyme with respect to the inner mitochondrial membrane places the active site on the cytosolic face of this membrane rather than the matrix side where the active site of ferrochelatase, the terminal pathway enzyme, is located. Examination of the kinetics of the two terminal enzymes in mitochondrial membranes demonstrates that substrate channeling occurs between these terminal two-pathway enzymes. However, examination of solubilized and membrane-reconstituted enzymes shows no evidence for a stable complex. Based upon these and previous data a model for the terminal three-pathway enzymes is presented.