Theses and Dissertations - Department of Chemistry & Biochemistry

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Browsing Theses and Dissertations - Department of Chemistry & Biochemistry by Subject "Biophysics"

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    Iron coordination and protein-protein interactions of the protein frataxin
    (University of Alabama Libraries, 2014) Gentry-Dye, Leslie; Busenlehner, Laura S.; University of Alabama Tuscaloosa
    Frataxin is a mitochondrial iron metallochaperone that transports ferrous iron to proteins that require it for function. This dissertation research explores the iron binding properties of human frataxin and how frataxin interacts with the mitochondrial [Fe-S] cluster scaffold Isu2 to assemble [Fe-S] clusters. Friedreich's ataxia (FA) is a neurodegenerative progressive limb and gait ataxia that is caused by an exaggerated GAA triplet codon repeat that results in depleted levels of the iron metallochaperone frataxin. Depleted levels of frataxin have a two-fold consequence. The first is that the mitochondria do not have a way to bind and transport iron to proteins that require iron for function. The second is that the cell interprets this as an iron shortage and imports more iron into the mitochondria. As a result, there is both iron overload (caused by having excess non-bioavailable iron in ferric aggregates in the mitochondria) and iron deficiency (since this iron cannot be mobilized for [Fe-S] cluster assembly). Frataxin coordinates ferrous iron and transports it to Isu2 for the assembly of [Fe-S] clusters. In this dissertation, human frataxin Fe2+ coordination was characterized and applied to further study how frataxin interacts with Isu2 for iron transfer and [Fe-S] cluster assembly. This research supports that mature human frataxin coordinates 3 ferrous iron ions and interacts with Isu2 in the same vicinity of Fe2+ coordination for the stimulation of [Fe-S] cluster assembly and provides insight into the cause of FA.
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    The roles of iron and cadmium in human health
    (University of Alabama Libraries, 2014) Wang, Yu; Busenlehner, Laura S.; University of Alabama Tuscaloosa
    The trace transition metals in humans are divided into two groups, the essential metals and the non-essential/non-native heavy metals. This dissertation research explores the interactions of two transition metals, iron and cadmium, with protein targets to understand their effects on human health. Iron is an important essential metal and is a component of two inorganic cofactors, heme and Fe/S clusters. Disruption of heme and Fe/S cluster cofactor assembly causes downstream protein dysfunction, oxidative stress, and cellular damage. Many diseases, such as the neurodegenerative disease Friedreich's ataxia (FRDA), are caused by the inability to synthesize Fe/S clusters. FRDA is the result of decreased expression of the mitochondrial protein frataxin; however, its exact function is unclear. In this dissertation, a Schizosaccharomyces pombe fission yeast strain was generated in which the yeast frataxin homologue fxn1 was overexpressed to determine what the function(s) of frataxin is through the affected pathways. Based on this study, we demonstrated that S. pombe Fxn1 overexpression elevated the activities of Fe/S enzymes through the up-regulation of Fe/S cluster synthesis, which led to imbalanced iron metabolism, mitochondrial dysfunction and oxidative stress. This research supports that mitochondrial Fxn1 up-regulates the efficiency of Fe/S cluster assembly and provides insight into the cause of FRDA. Besides diseases caused by dysregulation of essential metals, there are diseases related to chronic exposure to heavy metals. The heavy metal cadmium is linked to breast cancers, but with unknown mechanisms. One proposed mechanism is that Cd2+ activates the estrogen receptor &alpha (hERα) transcriptional regulator by binding to the protein and mimicking the conformational effects of the hormone estrogen. We utilized hydrogen/deuterium exchange mass spectrometry to analyze the structural changes of the hERα ligand binding domain upon estradiol or Cd2+ binding. Estradiol binding leads to conformational changes in the dimer interface, the estradiol binding cavity, and the loop between helix H11 and H12. Cadmium demonstrated similar conformational changes at the dimer interface and helix H12. This is the first direct evidence that hERα LBD undergoes structural changes upon Cd2+ binding that are similar to that caused by hormone binding, lending support for this potential mechanism of Cd2+-induced carcinogenesis.