Doctor of Philosophy(PhD) / Iron (Fe) is essential for cell growth and replication as many Fe-containing proteins catalyse key reactions involved in energy metabolism (cytochromes, mitochondrial aconitase and Fe-S proteins of the electron transport chain), respiration (hemoglobin and myoglobin) and DNA synthesis (ribonucleotide reductase). If not appropriately shielded, Fe could participate in one-electron transfer reactions that lead to the production of extremely toxic free radicals. The Fe storage protein, ferritin, is essential to protect cells against Fe-mediated oxidative stress by accommodating excess Fe into its protein shell (Xu et al., 2005). However, despite intensive research over the last few decades, many questions relating to intracellular Fe metabolism, e.g. Fe release from ferritin remain unanswered. Therefore, it is important to elucidate the molecular mechanisms of Fe trafficking in cells. At the beginning of my candidature, little was understood regarding the effect of anti-cancer agents, anthracyclines on the Fe-regulated genes, including transferrin receptor-1 (TfR1), N-myc downstream-regulated gene-1 (Ndrg1) and ferritin. Furthermore, the mechanisms of ferritin-Fe release and anthracycline-mediated ferritin-Fe accumulation are unclear. The work presented in Chapters 3 and 4 has addressed these issues. Apart from the studies examining the molecular interactions of anthracyclines with Fe, a mouse model with perturbed Fe metabolism was used and the marked alterations of protein expression in the heart of this knockout mouse model was discussed in Chapter 5. Chapter 3 Anthracyclines are effective anti-cancer agents. However, their use is limited by cardiotoxicity, an effect linked to their ability to chelate iron (Fe) and perturb Fe metabolism (Xu et al., 2005). These effects on Fe-trafficking remain poorly understood, but are important to decipher as treatment for anthracycline cardiotoxicity utilises the chelator, dexrazoxane. Incubation of cells with doxorubicin (DOX) up-regulated mRNA levels of the Fe-regulated genes, transferrin receptor-1 (TfR1) and N-myc downstream-regulated gene-1 (Ndrg1). This effect was mediated by Fe-depletion, as it was reversed by adding Fe and was prevented by saturating the anthracycline metal-binding site with Fe. However, DOX did not act like a typical chelator, as it did not induce cellular Fe mobilisation. In the presence of DOX and 59Fe-transferrin, Fe-trafficking studies demonstrated ferritin-59Fe accumulation and decreased cytosolic-59Fe incorporation. This could induce cytosolic Fe-deficiency and increase TfR1 and Ndrg1 mRNA. Up-regulation of TfR1 and Ndrg1 by DOX was independent of anthracycline-mediated radical generation and occurred via HIF-1α-independent mechanisms. Despite increased TfR1 and Ndrg1 mRNA after DOX treatment, this agent decreased TfR1 and Ndrg1 protein expression. Hence, the effects of DOX on Fe metabolism were complex due to its multiple effector mechanisms. Chapter 4 The Fe storage protein, ferritin, can accommodate up to 4500 atoms of Fe in its protein shell (Harrison and Arosio, 1996). However, the underlying mechanism of ferritin-Fe release remains unknown. Previous studies demonstrated that anti-cancer agents, anthracyclines, led to ferritin-59Fe accumulation (Kwok and Richardson, 2003). The increase in ferritin-59Fe was shown to be due to a decrease in the release of Fe from this protein. It could be speculated that DOX may impair the Fe release pathway by preventing the synthesis of essential ferritin partner proteins that induce Fe release. In this study, a native protein purification technique has been utilised to isolate ferritin-associated partners by combining ultra-centrifugation, anion-exchange chromatography, size exclusion chromatography and native gel electrophoresis. In addition to cells in culture (namely, SK-Mel-28 melanoma cells), liver taken from the mouse was used as a physiological in vivo model, as this organ is a major source of ferritin. Four potential partner proteins were identified along with ferritin, e.g. aldehyde dehydrogenase 1 family, member L1 (ALDH1L1). Future studies are required to clarify the relationship of these proteins with cellular Fe metabolism and ferritin-Fe release. Chapter 5 A frequent cause of death in Friedreich’s ataxia patients is cardiomyopathy, but the molecular alterations underlying this condition are unknown. We performed two dimensional electrophoresis to characterise the changes in protein expression of hearts using the muscle creatine kinase frataxin conditional knockout (KO) mouse. Pronounced changes in the protein expression profile were observed in 9-week-old KO mice with severe cardiomyopathy. In contrast, only a few proteins showed altered expression in asymptomatic 4-week-old KO mice. In hearts from frataxin KO mice, components of the iron-dependent complex-I and -II of the mitochondrial electron transport chain and enzymes involved in ATP homeostasis (creatine kinase, adenylate kinase) displayed decreased expression. Interestingly, the KO hearts exhibited increased expression of enzymes involved in the citric acid cycle, catabolism of branched-chain amino acids, ketone body utilisation and pyruvate decarboxylation. This constitutes evidence of metabolic compensation due to decreased expression of electron transport proteins. There was also pronounced up-regulation of proteins involved in stress protection, such as a variety of chaperones, as well as altered expression of proteins involved in cellular structure, motility and general metabolism. This is the first report of the molecular changes at the protein level which could be involved in the cardiomyopathy of the frataxin KO mouse.

The ability to acquire sufficient iron from the environment is essential for growth of most bacteria, including Escherichia coli and Shigella flexneri. In E. coli, the enterobactin-mediated iron acquisition system is the major way for the cells to get iron under iron-limiting conditions. Enterobactin is a siderophore that is synthesized and secreted in response to iron limitation to scavenge external ferric iron with high affinity. In this work, I showed that the alkyl hydroperoxide reductase (Ahp) system participates in cellular iron metabolism in both E. coli and S. flexneri. The Ahp system is composed of two proteins, AhpC and AhpF. AhpC detoxifies peroxides by converting peroxides to alcohol and water, and AhpF recycles AhpC. In this work, the data showed that the ahpC mutant synthesized and secreted much less enterobactin than the wild type E. coli and had a growth defect in low iron medium. AhpC influenced the first step of enterobactin biosynthesis by either facilitating the delivery of its substrate chorismate to the enterobactin biosynthesis pathway, or maintaining an optimal concentration of chorismate inside E. coli cells. In E. coli, the data showed that deletion of both ahpF and the glutathione reductase gor affected iron uptake or utilization, but not enterobactin biosynthesis, indicating the role of AhpF and Gor in cellular iron metabolism is different from that of AhpC. In S. flexneri, the Ahp system was also found to be involved in cellular iron metabolism; however, AhpC was not required for major steps of S. flexneri virulence: invasion, intracellular replication or cell-cell spread. Overall, the Ahp system participated in multiple steps of cellular iron metabolism. / text

The role of food intake and nutrition in athletic performance has gained much recognition in recent years. Therefore, the purpose of the present investigation was to assess whether female runners who consumed a modified vegetarian diet had different iron parameters than female runners who consumed a diet that included red meat. Methods: Two groups of female runners (n=18) volunteered as subjects for this study. One group was composed of women who regularly consumed a modified vegetarian diet (V group) and the other group was composed of women who consumed a diet which included red meat (RM group). The groups were comparable in age, weight, aerobic capacity, number of pregnancies and years since last pregnancy. Dietary analyses and biochemical analyses for the three stages of iron deficiency characterized by iron depletion, iron deficient erythropoiesis and iron deficiency anemia were performed to compare iron status of the two groups of athletes. Results: Serum ferritin values of the V group were significantly lower (p=.001) than those of the RM group. Of all the athletes examined, 56% had serum ferritin values indicative of storage iron depletion. Mean values for serum iron, total iron binding capacity (TIBC) and percent transferrin saturation were in the normal range for both groups, but TIBC values of the V group were significantly higher (p=.02) than the TIBC values of the RM group. Individually, four of the subjects, 22%, (3 V group and 1 RM group), had iron values indicative of iron deficient erythropoiesis. None of the other iron parameters was significantly different between the groups and none of the subjects was clinically anemic. The mean daily iron intake was not significantly different between the groups, but both groups averaged less than the RDA. The total caloric intake of both groups averaged less than 1800 kcals/day with 50% of the athletes consuming less than 1800 kcals/day, and another 28% consumed between 1800-2100 kcals/day. The V group consumed significantly more carbohydrate (p=.02) and less protein (p=.001) than the RM group, but none of the other dietary variables were significantly different between the groups. Conclusion: The two groups of subjects ingested almost the same amount of iron/day, however, the bioavailability of the iron in the diets may have affected iron absorption as the iron stores of the V group were lower than those of the RM group.

Doctor of Philosophy(PhD) / Iron (Fe) is essential for cell growth and replication as many Fe-containing proteins catalyse key reactions involved in energy metabolism (cytochromes, mitochondrial aconitase and Fe-S proteins of the electron transport chain), respiration (hemoglobin and myoglobin) and DNA synthesis (ribonucleotide reductase). If not appropriately shielded, Fe could participate in one-electron transfer reactions that lead to the production of extremely toxic free radicals. The Fe storage protein, ferritin, is essential to protect cells against Fe-mediated oxidative stress by accommodating excess Fe into its protein shell (Xu et al., 2005). However, despite intensive research over the last few decades, many questions relating to intracellular Fe metabolism, e.g. Fe release from ferritin remain unanswered. Therefore, it is important to elucidate the molecular mechanisms of Fe trafficking in cells. At the beginning of my candidature, little was understood regarding the effect of anti-cancer agents, anthracyclines on the Fe-regulated genes, including transferrin receptor-1 (TfR1), N-myc downstream-regulated gene-1 (Ndrg1) and ferritin. Furthermore, the mechanisms of ferritin-Fe release and anthracycline-mediated ferritin-Fe accumulation are unclear. The work presented in Chapters 3 and 4 has addressed these issues. Apart from the studies examining the molecular interactions of anthracyclines with Fe, a mouse model with perturbed Fe metabolism was used and the marked alterations of protein expression in the heart of this knockout mouse model was discussed in Chapter 5. Chapter 3 Anthracyclines are effective anti-cancer agents. However, their use is limited by cardiotoxicity, an effect linked to their ability to chelate iron (Fe) and perturb Fe metabolism (Xu et al., 2005). These effects on Fe-trafficking remain poorly understood, but are important to decipher as treatment for anthracycline cardiotoxicity utilises the chelator, dexrazoxane. Incubation of cells with doxorubicin (DOX) up-regulated mRNA levels of the Fe-regulated genes, transferrin receptor-1 (TfR1) and N-myc downstream-regulated gene-1 (Ndrg1). This effect was mediated by Fe-depletion, as it was reversed by adding Fe and was prevented by saturating the anthracycline metal-binding site with Fe. However, DOX did not act like a typical chelator, as it did not induce cellular Fe mobilisation. In the presence of DOX and 59Fe-transferrin, Fe-trafficking studies demonstrated ferritin-59Fe accumulation and decreased cytosolic-59Fe incorporation. This could induce cytosolic Fe-deficiency and increase TfR1 and Ndrg1 mRNA. Up-regulation of TfR1 and Ndrg1 by DOX was independent of anthracycline-mediated radical generation and occurred via HIF-1α-independent mechanisms. Despite increased TfR1 and Ndrg1 mRNA after DOX treatment, this agent decreased TfR1 and Ndrg1 protein expression. Hence, the effects of DOX on Fe metabolism were complex due to its multiple effector mechanisms. Chapter 4 The Fe storage protein, ferritin, can accommodate up to 4500 atoms of Fe in its protein shell (Harrison and Arosio, 1996). However, the underlying mechanism of ferritin-Fe release remains unknown. Previous studies demonstrated that anti-cancer agents, anthracyclines, led to ferritin-59Fe accumulation (Kwok and Richardson, 2003). The increase in ferritin-59Fe was shown to be due to a decrease in the release of Fe from this protein. It could be speculated that DOX may impair the Fe release pathway by preventing the synthesis of essential ferritin partner proteins that induce Fe release. In this study, a native protein purification technique has been utilised to isolate ferritin-associated partners by combining ultra-centrifugation, anion-exchange chromatography, size exclusion chromatography and native gel electrophoresis. In addition to cells in culture (namely, SK-Mel-28 melanoma cells), liver taken from the mouse was used as a physiological in vivo model, as this organ is a major source of ferritin. Four potential partner proteins were identified along with ferritin, e.g. aldehyde dehydrogenase 1 family, member L1 (ALDH1L1). Future studies are required to clarify the relationship of these proteins with cellular Fe metabolism and ferritin-Fe release. Chapter 5 A frequent cause of death in Friedreich’s ataxia patients is cardiomyopathy, but the molecular alterations underlying this condition are unknown. We performed two dimensional electrophoresis to characterise the changes in protein expression of hearts using the muscle creatine kinase frataxin conditional knockout (KO) mouse. Pronounced changes in the protein expression profile were observed in 9-week-old KO mice with severe cardiomyopathy. In contrast, only a few proteins showed altered expression in asymptomatic 4-week-old KO mice. In hearts from frataxin KO mice, components of the iron-dependent complex-I and -II of the mitochondrial electron transport chain and enzymes involved in ATP homeostasis (creatine kinase, adenylate kinase) displayed decreased expression. Interestingly, the KO hearts exhibited increased expression of enzymes involved in the citric acid cycle, catabolism of branched-chain amino acids, ketone body utilisation and pyruvate decarboxylation. This constitutes evidence of metabolic compensation due to decreased expression of electron transport proteins. There was also pronounced up-regulation of proteins involved in stress protection, such as a variety of chaperones, as well as altered expression of proteins involved in cellular structure, motility and general metabolism. This is the first report of the molecular changes at the protein level which could be involved in the cardiomyopathy of the frataxin KO mouse.

Thesis (PhD)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: The solute carrier family 11 member 1 gene (SLC11A 1) is a divalent metal ion transporter with various pleiotropic effects on macrophage function. This gene that regulates iron, and is also regulated by cellular iron levels, has previously been linked to many infectious and autoimmune diseases. In this analysis, in vitro studies using the luciferase reporter system as well as case-control association studies were applied to investigate the significance of SLC11 A1 allelic variation in patients with diverse disease phenotypes. For in vitro studies, five different SLC11A 1 promoter constructs were generated, followed by transfection into U937 and THP-1 cells. The inserted fragments included two previously described alleles (alleles 2 and 3), two novel alleles identified in this study (alleles 8 and 9) and a C to T point mutation at nucleotide position -237 in the presence of allele 3. The most striking finding was the opposite effect observed for allele 3 in the presence of the -237C~ T polymorphism, similar to that of allele 2. Although the SLC11A 1 gene has previously been implicated in iron transport, we have demonstrated, for the first time, that the various alleles investigated cause differential expression of the gene upon iron loading. Association studies were performed by investigating diseases including oesophageal cancer (DC), inflammatory bowel disease (lBO) and hereditary haemochromatosis (HH) (or primary iron overload). Significant associations (P<O.05) were observed with allele 3 for all three conditions investigated only after stratification according to the presence of the -237C~ T polymorphism. Re-assessment of the promoter alleles according to expression profiles determined by the in vitro studies, showed statistically significant associations for allele 3 with DC and primary iron overload, compared with the respective population-matched control groups. Additionally, several novel variants were identified in exon 2 (112G~A, 148deIGACCAGCCC, 157insGACCAGCCCAG) and intron 1 (IVS1-28C~T), with variant IVS1-28C~T occurring at a significantly increased frequency in patients with DC compared with population-matched controls (P<O.05). Investigation of the SLC11A 1 gene in individuals presenting with iron overload in the absence of homozygosity for the HFE C282Y mutation, provided further support for the importance of sequence variation in the promoter region of the SLC11A 1 gene in modified risk of iron-related disorders. Genes related to iron homeostasis, including HFE, SLC11A3, HAMP and DCYTB, were investigated in individuals with similar criteria and potential disease-causing mutations were identified in 11% White and 45% Black South African patients. The possible significance of the SLC11A3 and DCYTB genes in iron overload in the Black South African population, and the possible involvement of the DCYTB gene in iron overload in general, are demonstrated for the first time. This study contributed to a better understanding of the function of the SLC11A 1 gene in relation to iron metabolism. The involvement of SLC11A 1 in a range of disease phenotypes including cancer and inflammatory conditions that may involve iron dysregulation, can probably be explained by interaction with external factors such as infectious agents that may affect cellular iron status. Our findings provide both in vivo and in vitro evidence that iron dysregulation mediated by allelic effects of SLC11A 1 may underlie disease susceptibility to infectious and autoimmune conditions. / AFRIKAANSE OPSOMMING: Die opgeloste stof draer familie 11 deel 1 geen (SLC11 A 1) is 'n divalente metaal ioon vervoerder met verskeie pleiotropiese effekte op makrofaagfunksie. Die geen, wat yster reguleer en ook deur sellulêre ystervlakke gereguleer word, is voorheen verbind met verskeie infektiewe en outo-immune siektes. In hierdie studie is in vitro analises, deur middel van die lusiferase verklikker sisteem, asook gevalle-kontrole assosiasie studies gebruik om die rol van SLC11A 1 alleel variasie in pasiënte met diverse siektefenotipes te ondersoek. Vyf verskillende SLC11A 1 promotor variante is geskep vir in vitro studies en gevolg deur transfeksie in U937 en THP-1 sellyne. Die ingevoegde fragmente het twee voorheen beskryfde allele (allele 2 en 3), twee nuwe allele wat in hierdie studie geïdentifiseer is (allele 8 en 9) en In C na T puntmutasie by nukleotied posisie -237 in die teenwoordigheid van alleel 3 ingesluit. Die opvallendste bevinding was die teenoorgestelde effek wat waargeneem is wanneer alleel 3 in die teenwoordigheid van die -237C~ T polimorfisme voorkom, soortgelyk aan alleel 2 uitdrukking. Alhoewel die SLC11A1 geen voorheen geïmpliseer is in yster vervoer, is daar vir die eerste keer aangetoon dat na yster lading, die verskillende allele differensiële uitdrukking van die geen veroorsaak. Verskeie siektes, insluitend slukderm kanker (OC), inflammatoriese dermsiekte (lBO) en oorerflike hemochromatose (HH) (of primêre ysteroorlading), is ondersoek deur middel van assosiasie studies. Betekenisvolle verskille (P<O.05) is waargeneem vir alleel 3 tussen die kontrole- en pasiëntgroepe in al drie siektes wat ondersoek is, maar slegs na stratifikasie volgens die teenwoordigheid van die -237C~ T polimorfisme. Na hersiening van die promotor allele volgens ekspressie profiele verkry met in vitro studies is statisties betekenisvolle assosiasie ook verkry vir alleel 3 met OC en primêre ysteroorlading in vergelyking met die onderskeie populasie kontrolegroepe. Verder is verskeie nuwe variante ook geïdentifiseer in ekson 2 (112G~A, 148deIGACCAGCCC, 157insGACCAGCCCAG) en intron 1 (IVS1- 28C~ T) en 'n statisties betekenisvolle verhoogde frekwensie van variant IVS1- 28C~ T is waargeneem in pasiënte met OC in vergelyking met die populasie kontrolegroep (P<O.05). Die belangrikheid van variasie in die promotor area van die SLC11A 1 geen as 'n modifiserende faktor in ysterverwante siektes, is verder ondersteun deur die SLC11A 1 geen in individue met ysteroorlading in die afwesigheid van homosigositeit vir die HFE C282Y mutasie te ondersoek. Ander gene geassosieerd met yster homeostase, insluitend HFE, SLC11A3, HAMP and DCYTB, is ondersoek in individue met soortgelyke seleksie kriteria en potensiële siekte-verwante mutasies is geïdentifiseer in 11% Wit en 45% Swart Suid-Afrikaanse pasiënte. Die moontlike belang van die SLC11A3 en DCYTB gene in ysteroorlading in die Swart Suid-Afrikaanse populasie en die moontlike betrokkenheid van die DCYTB geen in yster oorlading oor die algemeen, is vir die eerste keer aangetoon. Hierdie studie dra by tot 'n beter insig in die funksie van die SLC11A 1 geen ten opsigte van ystermetabolisme. Die betrokkenheid van SLC11A 1 in 'n reeks siekte fenotipes, wat insluit kanker en inflammatoriese toestande wat verband kan hou met 'n yster wanbalans, kan moontlik verklaar word deur interaksie met eksterne faktore soos infektiewe agente wat die sellulêre yster status kan beïnvloed. Ons bevindinge verskaf beide in vivo en in vitro getuienis dat yster wanbalans, wat bemiddel word deur alleliese effekte van SLC11A1, verantwoordelik mag wees vir vatbaarheid vir infektiewe en outoimmune siekte toestande.

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Seven strains of Actinobacillus suis (ATCC 15557, B49, C84, H89-1173, H91-0380, SO4 and VSB 3714) were investigated with respect to iron acquisition from animal transferrins (Tfs) and haemoglobins (Hbs). Growth assays with porcine, bovine and human Tfs and Hbs revealed that all seven strains could use porcine (but not human or bovine) Tf and all three Hbs as iron sources. In solid phase binding assays, membranes derived from all strains exhibited strong binding of porcine Tf and each of the Hbs. Competition binding assays indicated that all three Hbs were bound by the same receptor(s). Affinity procedures allowed the isolation and identification of iron repressible Tf-binding (~100 kDa and ~63 kDa) and Hb-binding (~105 kDa) polypeptides from all strains. Nucleotide sequence analyses revealed that A. suis strains SO4 and C84 possess genes that encode homologues of the Actinobacillus pleuropneumoniae Tf-binding proteins, TbpA and TbpB, and Hb-binding protein, HgbA. In both strains, tbpB was located immediately upstream of tbpA and was shown to be preceded by tonB, exbB and exbD homologues; hgbA was shown to be preceded by a hugZ homologue. Putative promoter and Fur box sequences were located upstream of tonB and hugZ and RT-PCR revealed that the genes in each of these clusters (tonB-exbB-exbD-tbpB-tbpA; hugZ-hgbA) are co-transcribed and iron-repressible. The molecular masses of the predicted mature TbpA, TbpB and HgbA proteins were calculated to be 104.3, 63.4 and 105.0 kDa, respectively, suggesting that the affinity-isolated, ~100 kDa and ~63 kDa Tf-binding polypeptides represent TbpA and TbpB, respectively, and that the ~105 kDa Hb-binding polypeptide represents HgbA. TbpB of A. suis was expressed in Escherichia coli and the recombinant TbpB (rTbpB) was identified by immunoblotting using swine sera raised against recombinant TbpB (A. pleuropneumoniae). It is envisaged that the acquisition of Tf- and Hb-bound iron by A. suis involves mechan