David J. Haile

Molecular characterization of a copper transport protein in S. cerevisiae: An unexpected role for copper in iron transport

We report the identification and characterization of CTR1, a gene in the yeast S. cerevisiae that encodes a multispanning plasma membrane protein specifically required for high affinity copper transport into the cell. The predicted protein contains a methionine- and serine-rich domain that includes 11 examples of the sequence Met-X2-Met, a motif noted in proteins involved in bacterial copper metabolism. CTR1 mutants and deletion strains have profound deficiency in ferrous iron uptake, thus revealing a requirement for copper in mediating ferrous transport into the cell. Genetic evidence suggests that the target for this requirement is the FET3 gene (detailed in a companion study), predicted to encode a copper-containing protein that acts as a cytosolic ferro-oxidase. These findings provide an unexpected mechanistic link between the uptake of copper and iron.

HOMEOSTATIC REGULATION OF COPPER UPTAKE IN YEAST VIA DIRECT BINDING OF MAC1 PROTEIN TO UPSTREAM REGULATORY SEQUENCES OF FRE1 AND CTR1

Copper deprivation of Saccharomyces cerevisiae induces transcription of the FRE1 andCTR1 genes. FRE1 encodes a surface reductase capable of reducing and mobilizing copper chelates outside the cell, and CTR1 encodes a protein mediating copper uptake at the plasma membrane. In this paper, the protein encoded by MAC1is identified as the factor mediating this homeostatic control. A novel dominant allele of MAC1, MAC1 up2 , is mutated in a Cys-rich domain that may function in copper sensing (a G to A change of nucleotide 812 resulting in a Cys-271 to Tyr substitution). This mutant is functionally similar to theMAC1 up1 allele in which His-279 in the same domain has been replaced by Gln. Both mutations confer constitutive copper-independent expression of FRE1 and CTR1. A sequence including the palindrome TTTGCTCA … TGAGCAAA, appearing within the 5′-flanking region of the CTR1promoter, is necessary and sufficient for the copper- andMAC1-dependent CTR1 transcriptional regulation. An identical sequence appears as a direct repeat in theFRE1 promoter. The data indicate that the signal resulting from copper deprivation is transduced via the Cys-rich motif of MAC1 encompassing residues 264–279. MAC1 then binds directly and specifically to the CTR1 and FRE1 promoter elements, inducing transcription of those target genes. This model defines the homeostatic mechanism by which yeast regulates the cell acquisition of copper in response to copper scarcity or excess.

Regulation of Genes of Iron Metabolism by the Iron-Response Proteins

Iron is an essential nutrient, yet excess iron can be toxic to cells. The uptake of iron by mammalian cells is post-transcriptionally regulated by the interaction of iron-response proteins (IRP1 and IRP2) with iron-response elements (IREs) found in the mRNAs of genes of iron metabolism, such as ferritin, the transferrin receptor, erythroid aminolevulinic acid synthase, and mitochondrial aconitase. The IRPs are RNA binding proteins that bind to the IRE (found in the mRNAs of the regulated genes) in an iron- dependent manner. Binding of IRPs to the IREs leads to changes in the expression of the regulated genes and subsequent changes in the uptake, utilization, or storage of intracellular iron. Recent work has demonstrated that the binding of the IRPs to the IREs can also be modulated by changes in the redox state or oxidative stress level of the cell. These findings provide an important link between iron metabolism and states of oxidative stress.

An iron-sulfur cluster plays a novel regulatory role in the iron-responsive element binding protein

Post-transcriptional regulation of genes important in iron metabolism, ferritin and the transferrin receptor (TfR), is achieved through regulated binding of a cytosolic protein, the iron-responsive element binding protein (IRE-BP), to RNA stem-loop motifs known as iron-responsive elements (IREs). Binding of the IRE-BP respresses ferritin translation and represses degradation of the TfR mRNA. The IRE-BP senses iron levels and accordingly modifies binding to IREs through a novel sensing mechanism. An iron-sulfur cluster of the IRE-BP reversibly binds iron; when cytosolic iron levels are depleted, the cluster becomes depleted of iron and the IRE-BP acquires the capacity to bind IREs. When cytosolic iron levels are replete, the IRE-BP loses RNA binding capacity, but acquires enzymatic activity as a functional aconitase. RNA binding and aconitase activity are mutually exclusive activities of the IRE-BP, and the state of the iron-sulfur cluster determines how the IRE-BP will function.

Heterogenous distribution of ferroportin-containing neurons in mouse brain.

Iron is crucial for a variety of cellular functions in neuronal cells. Neuronal iron uptake is reflected in a robust and consistent expression of transferrin receptors and divalent metal transporter 1 (DMT 1). Conversely, the mechanisms by which neurons neutralize and possibly excrete iron are less clear. Studies indicate that neurons express ferroportin which could reflect a mechanism for iron export. We mapped the distribution of ferroportin in the adult mouse brain using an antibody prepared from a peptide representing amino acid sequences 223–303 of mouse ferroportin. The antibody specifically detected ferroportin in brain homogenates, whereas homogenates of cultured endothelial cells were devoid of immunoreactivity. In brain sections, ferroportin was confined to neuronal cell bodies and peripheral processes of cerebral cortex, hippocampus, thalamus, brain stem, and cerebellum. In brain stem ferroportin-labeling was particularly high in neurons of cranial nerve nuclei and reticular formation. Ferroportin was hardly detectable in striatum, pallidum, or hypothalamus. Among non-neuronal cells, ferroportin was detected in oligodendrocytes and choroid plexus epithelial cells. A comparison with previous studies on the distribution of transferrin receptors in neurons shows that many neuronal pools coincide with those expressing ferroportin. The data therefore indicate that neuronal iron homeostasis consists of a delicate balance between transferrin receptor-mediated uptake of iron-transferrin and ferroportin-related iron excretion. The findings also suggest a particular high turnover of iron in neuronal regions, such as habenula, hippocampus, reticular formation and cerebellum, as several neurons in these regions exhibit a prominent co-expression of transferrin receptors and ferroportin.

Autologous Stem Cell Transplantation: The Predictive Value of the Morse Fall Scale in Hospitalized Patients

BACKGROUND: Falls are common in hospitalized patients undergoing autologous stem cell transplantation (ASCT). Research demonstrates that preventing patient falls requires knowledge of the risk factors and the circumstances preceding the patients fall. OBJECTIVES: To identify risk factors related to falls in recipients of ASCT and assess the predictive value of the Morse Fall Scale (MFS). METHODS: Of the 288 patients who underwent transplantation during the study period, 14 were fallers. Twenty controls were randomly selected. The study used descriptive case‐control analysis and simple logistic regression to analyze the data. FINDINGS: Eight fallers and four non‐fallers had high MFS scores. The logistic regression model indicated that patients with high MFS scores were 5.3 times more likely to fall and that for each day patients experienced diarrhea, their risk of fall increased 1.2 times.