FUNCTIONAL INSIGHTS INTO HRG-1-MEDIATED HEME TRANSPORT

Heme is an essential cofactor involved in various biological processes including oxygen transport, xenobiotic detoxification, oxidative metabolism, gas sensing, circadian rhythm, signal transduction, microRNA processing and thyroid hormone synthesis. Heme is also an essential nutrient for parasites and is the major dietary iron source for humans. Despite our extensive understanding of the mechanisms of heme synthesis and degradation in eukaryotes, little is known as to how heme is transported and trafficked in eukaryotes. Recently, CeHRG-1 and CeHRG-4 were identified as the first bona fide heme importers/transporters using the heme auxotroph, Caenorhabditis elegans. To gain mechanistic insights into the heme transport function of HRG-1-related proteins, we conducted a structure-function analysis of CeHRG-1 and CeHRG-4 by exploiting yeast mutants that are genetically defective in heme synthesis. Our studies reveal that HRG-1-related proteins transport heme across membranes through the coordinated actions of strategically placed amino acids that are topologically conserved in both, the worm and human proteins. To further dissect the functional elements that dictate their intracellular localization, we generated a series of chimeras by swapping the amino and carboxy terminal segments of CeHRG-1 and CeHRG-4. Our analysis in yeast and mammalian cells demonstrate that the C-terminal domains are essential for membrane localization of the protein, while the N-terminal domains are important for proper function, and plausibly multimerization of HRG-1-related proteins. Currently, there are no pharmacological means to aid in the study of the cellular and physiological roles of eukaryotic heme transporters. We, for the first time, developed and executed a high-throughput screen of 233,360 compounds, to identify potential antagonists of HRG-1-related proteins by utilizing parasite heme transporters as the primary screening bait. Subsequent study in parasites will provide novel drug candidates against helminths that infect humans, livestock, and plants, as well as against genetic disorders of heme and iron metabolism in humans. Taken together, results from our studies will significantly advance novel functional and therapeutic insights into HRG-1 mediated heme transport in health and disease

Amyloid precursor protein (APP) and copper homeostasis in the human neuroblastoma cell line SH-SY5Y

PhD ThesisAlzheimer’s disease (AD) is characterised by cerebellar accumulation and aggregation of amyloid beta (Aβ), a cleavage product of the transmembrane amyloid precursor protein (APP). APP contains a range of functional domains in its large extracellular portion, among which are two copper-binding motifs and one zinc-binding motif. The copper-binding motifs are present in the amino-terminal region of APP and within the Aβ region of the protein and readily reduce Cu(II) to Cu(I), thus APP and its cleavage products are linked to copper metabolism and have been hypothesised to participate in cellular copper homeostasis. In this project human neuroblastoma cell lines SH-SY5Y were utilised to determine the effect of expressing a familial AD mutation on intracellular copper concentrations and possible functional alterations or deficits of enzymes that require copper as a co-factor. The familial AD mutation first found in a Swedish population was previously shown to increase the total amount of released Aβ. Direct phenotypic comparison between SH-SY5Y APPWT cell lines expressing endogenous levels of APP and APPswe Coupling native two dimensional liquid chromatography with metal analysis, SDS-PAGE and Principal Component Analysis identified one major copper and zinc containing pool as copper-zinc superoxide dismutase (SOD1) in soluble whole cell protein extracts. Comparative analysis of metal content between APP cell lines overexpressing APP carrying the Swedish mutation was performed in standard culture and manipulated copper concentrations. WT and APPswe cultures indicated a difference in metallation of SOD1 with copper. APPswe cultures displayed reduced metallation of SOD1, whereas SOD1 metallation with zinc remained unaltered. Functional analysis of copper-binding enzymes, such as SOD1 and cytochrome c oxidase (CCO), using standard biochemical approaches, identified lower activities for both enzymes during standard cell culture in APPswe cells. Upon treatment of cultures with increasing concentrations of exogenous copper APPWT enzyme activities remained unaltered but enzyme activities in APPswe cultures increased in direct correlation with increasing copper concentrations. Combined with phenotypic analysis of growth, survival and intracellular metal content it appears that APPswe cultures are copper deficient, but this can be overcome by copper supplementation. Lower copper accumulation also enables greater survival of APP swe Overall, these data suggest that APP cells in elevated copper. swe overexpression in SH-SY5Y cells results in functional copper deficiency which can be rescued by supplementation of cultures with exogenous copper. APPswe further confers resistance to copper toxicity not only via increased Aβ release, but also via increased copper delivery to enzymatic target proteins improving cellular antioxidant response and energy metabolism. These data are consistent with a function of APP in copper efflux either in a regulatory capacity or directly contributing to copper egress

REGULATORY MECHANISMS OF SLC39A4 (ZIP4) AND SLC39A5 (ZIP5) IN THE ADAPTIVE RESPONSE TO ZINC AVAILABILITY

The aims of this research were to determine how Zip4 and Zip5 are regulated in response to zinc availability and how Zip4 impacts development. Loss of Zip4 resulted in embryonic lethality. Heterozygosity negatively affected eye, heart, and brain development. Excess zinc did not rescue lethality but ameliorated the heterozygous effects. Zip4 and Zip5 had reciprocal regulation in response to zinc availability. Zip4 expression was regulated by stability of the mRNA and protein: both accumulate during zinc deficiency; ZIP4 was rapidly internalized then degraded following zinc repletion. The Zip5 mRNA levels did not change with zinc availability and were polysome-associated. ZIP5 accumulated on the basolateral membranes after zinc repletion. miRNAs predicted to target Zip5 in an accessible region of the conserved 3' UTR were polysome-associated in all tissues that regulate Zip5. Zip4 and Zip5 are both regulated by post-transcriptional mechanisms in response to zinc availability and Zip4 is essential for development

Copper and zinc ligands of pisum sativum and expression of psMT(_A)

The gene, PsMT(_A) is highly expressed in the roots of the garden pea, Pisum sativum. The predicted primary structures of homologues of the PsMT(_A) gene from a range of plant species were compared. Common features are amino and carboxyl terminal domains of approximately 20 residues which are rich in cysteine residues many of which are arranged in the cysteine-Xaa-cysteine (Xaa is not a cysteine) motifs characteristic of metallothionein. The greatest degree of sequence conservation between these predicted gene products occurs within the cysteine rich domains. Two principal (most highly represented in the available sample) categories of sequence were identified on the basis of the arrangement of the cysteine residues within the amino termina domain. A secondary structure motif (β-strand) was predicted (using a computer algorithm) to occur in a conserved position in the central domain linking the two cysteine rich domains of these predicted proteins. This feature is not apparent in the structure of metallothioneins from other species. A recombinant GST-PsMT(_A) fusion protein was isolated from crude lysates of Escherichia coli. containing the plasmid pGEX3X with PsMT(_A) coding sequence. When isolated from Escherichia coli cells grown in zinc supplemented media it was demonstrated that zinc was associated with the PsMT(_A) moiety of the fusion protein. In aqueous solution the PsMT(_A) moiety of the fusion protein formed discreet degradation products. The fusion protein was insoluble at concentrations greater than 20 mg ml(^-1) which rendered it unsuitable for a structural study of the putative metal binding site(s) by (^113)Cd NMR. An antibody to the GST- PsMT(_A) fusion protein was characterised and the epitope was found to lie within the GST moiety. Comparison of the arrangement of cysteines in the amino terminal domains with the different domains of mammalian metallothionein suggested that the two principal categories of predicted plant metallothionein- like gene products may have different affinities for zinc. The predicted products of the metallothionein-like gene highly expressed in the leaves of Arabidopsis thali ana (AtMT-t2) and the PsMT(_A) gene are representative of the two principal categories identified by sequence analysis. The AtMT-t2 coding sequence was amplified from an Arabidopsis thaliana leaf cDNA library and cloned into the pGEX3X plasmid to allow expression of the protein as a fusion to GST in Escherichia coli. Zinc was associated with the AtMT-t2 moiety of the fusion protein. The pH of 50 % displacement for the GST-AtMT-t2 fusion protein with respect to zinc was 0.25 pH units lower than that for the GST-PsMT(_A) fusion protein indicating that putative metallothionein-like protein from Arabidopsis thaliana may have a higher affinity for zinc. It is feasible that this difference allows AtMT-t2 to compete with endogenous ligands of zinc more effectively than PsMT(_A). Expression of the AtMT-t2 gene in a zinc metallothionein deficient strain of Synechococcus (in collaboration with Dr. J.S. Turner) partially restored zinc tolerance to the transformed cells. The similarity of the cysteine rich domains of the predicted metallothionein-like proteins with metallothionein and the demonstration of metal binding in vitro suggested that these genes may be metal regulated. The effect of variations in the exogenous concentration of the trace metals copper zinc and iron on the expression of the PsMT(_A) gene in the roots of Pisum sativum seedlings was investigated by northern analysis. Induction of PsMT(_A) expression was seen with increasing iron concentrations up to 2.0 µM iron chelate. At this concentration of iron chelate, and above, expression of PsMT(_A), decreased. At concentrations of copper above 100 nM induction of PsMT(_A) expression was seen. Below 100 nM copper PsMT(_A) expression increased with decreasing copper concentrations. This response has not been reported for metallothioneins from other species and may be significant for the role of PsMT(_A) in root tissue. In the presence of 2.0 µM iron zinc concentrations above 5.0 µM induced expression of PsMT(_A). The response to changes in exogenous metal concentrations was rapid. Complete repression of PsMT(_A) transcription occurred within 1 h of transfer to media supplemented with 2.0 µM iron. To date no translational products of plant metallothionein-like genes (excluding the E(_c) protein from Tritticum aestivum) have been identified in plant tissue. Two copper and one zinc complex were identified following ion exchange chromatography of soluble extracts from roots of Pisum sativum. The quantity' of the zinc complex eluted from the matrix was reduced by the addition of iron chelate to the growth media. There was no consistent change in the quantity of copper complex recovered in response to iron. Two copper and one zinc component of low molecular weight were identified following gel filtration chromatography of the above complexes. Following polyacrylamide gel electrophoresis of extracts from roots of Pisum sativum, labelled in vivo with (^35)S cysteine, a band was identified with the characteristics predicted for the product of the PsMT(_A) gene. On two dimensional polyacrylamide gels a doublet of spots was identified (migrating to a low pH as predicted for PsMT(_A)) in an extract from roots of seedlings grown in media not supplemented with iron which were not observed on gels of extracts from seedlings grown in media supplemented with iron. The identity of these polypeptides was not established by sequence analysis

Metal Ion Regulation in the Central Nervous System and in Glutamatergic Synapses: Role of the Cellular Prion Protein

Despite many efforts, the molecular mechanisms underlying the pathophysiology of neurodegenerative disorders have not been fully understood. Results published in literature highlight that different neurodegenerative diseases share common features: protein aggregation in neuronal tissue; oxidation of neuronal tissue mediated by redox-active metal ions interaction with a target protein; and functional demise. So, unveiling the physiological function of protein that aggregate in neurodegenerating tissues, as well as their interplay with metal ions, becomes a prominent issue, in order to understand the etiological trigger and to define a possible therapeutic strategy. Metal ions are essential elements for cellular processes, but at the same they are potentially dangerous since they can give rise to Fenton reaction and oxidative/nitrosative stress. So, their homeostasis is strictly regulated in each district of the organism, but in particular in the brain. The brain, having the highest metabolic rate and depending predominantly on oxidative metabolism for its energy, has developed fine mechanisms to compartmentalize, distribute, uptake and excrete the different ionic species. Alterations in one of these mechanisms can lead to great neuronal damages, and maybe neurodegenerative disorders. This work has been focused on the cellular prion protein (PrPC), whose conformational isoform, the scrapie prion protein (PrPSc) is the causative agent of prion disordes and whose function has not been clearly defined, yet. Metal ions are a common denominator to all the cellular pathways in which PrPC seems to be actively involved. In particular, metal ions homeostasis maintainance, neuroprotection in excitotoxic condition and ionotropic receptor modulation have been studied. In the first part of the project, PrPC role in metal ion homeostais maintainance has been investigated. To this aim, copper, manganese, zinc and iron content, as well as metal binding proteins expression have been measured in a PrP knockout murine model, compared to wild-type. The results describe the global rearrangement occurring in the expression of metal binding proteins to maintain trace metals homeostasis, trying to compensate PrPC absence. At the same time, a pronounced decrease in Ceruloplasmin ferroxidase activity has been detected in PrP null mouse serum, pointing out a global impairment in copper metabolism in PrPC absence. In the second part of the project, the importance of the interaction between PrPC and copper ions in excitotoxic conditions and in synapses functionality has been studied. It has been published that PrP null mice show higher levels of neuronal cell death in stressful conditions and when subjected to toxic treatment with glutamate receptor agonists. Moreover, these mice show altered kinetics of N-methyl-D-aspartate (NMDA) receptor current. These alterations appears to be due to an inhibitory regulation that PrPC exerts on NMDA receptors via copper ions, lacking in PrP null hippocampi. First, the enhanced suceptibility to excitotoxicity of PrP knockout mice has been verified and characterised in organotypic hippocampal cultures upon treatment with NMDA. Higher neuronal cell death levels have been detected in all the investigated hippocampal regions. To identify which cellular regulatory mechanism is alterd in PrPC absence, the expression of the proteins mainly involved in excitotoxicity has been compared between PrP knockout and wild-type hippocampi. Among other minor differences, a different modulation of calcium transporters expression has been identified in PrP knockout hippocampi and brains. This global alteration appears to be necessary to maintain calcium homeostasis, since calcium content measurements did not reveal any strong difference between PrP null and wild-type samples. NMDA receptors can be S-nitrosylated on extracellular cysteines and this reaction is always inhibitory. S-nytrosilation requires an electron acceptor to occur, for this reason copper ions are often involved in these kind of reactions. Moreover, copper ions are known to modulate NMDA receptor activity, but the precise mechanism has not been described, yet. Since PrPC is known to support the S-nitrosylation of other membrane proteins, the S-nitrosylation levels of NMDA receptor subunits GluN1 and GluN2A have been measured in PrP knockout hippocampi from adult mice and compared to wild-type ones. Results show that the S-nitrosylated fractions of both GluN1 and GluN2A are reduced in PrP absence. So, this reveals that PrPC modulates NMDA receptor activity providing the copper ions necessary to support their inhibitory S-nitrosylation reaction. Through this mechanism, PrPC contributes to inhibit NMDA receptor currents, as well as to protect neurons in excitotoxic conditions

Bioinorganic Chemistry

This book covers material that could be included in a one-quarter or one-semester course in bioinorganic chemistry for graduate students and advanced undergraduate students in chemistry or biochemistry. We believe that such a course should provide students with the background required to follow the research literature in the field. The topics were chosen to represent those areas of bioinorganic chemistry that are mature enough for textbook presentation. Although each chapter presents material at a more advanced level than that of bioinorganic textbooks published previously, the chapters are not specialized review articles. What we have attempted to do in each chapter is to teach the underlying principles of bioinorganic chemistry as well as outlining the state of knowledge in selected areas. We have chosen not to include abbreviated summaries of the inorganic chemistry, biochemistry, and spectroscopy that students may need as background in order to master the material presented. We instead assume that the instructor using this book will assign reading from relevant sources that is appropriate to the background of the students taking the course. For the convenience of the instructors, students, and other readers of this book, we have included an appendix that lists references to reviews of the research literature that we have found to be particularly useful in our courses on bioinorganic chemistry