Thomas B. Bartnikas

Mutant SOD1 causes motor neuron disease independent of copper chaperone–mediated copper loading

Copper-mediated oxidative damage is proposed to play a critical role in the pathogenesis of Cu/Zn superoxide dismutase (SOD1)–linked familial amyotrophic lateral sclerosis (FALS). We tested this hypothesis by ablating the gene encoding the copper chaperone for SOD1 (CCS) in a series of FALS-linked SOD1 mutant mice. Metabolic 64Cu labeling in SOD1-mutant mice lacking the CCS showed that the incorporation of copper into mutant SOD1 was significantly diminished in the absence of CCS. Motor neurons in CCS−/− mice showed increased rate of death after facial nerve axotomy, a response documented for SOD1−/− mice. Thus, CCS is necessary for the efficient incorporation of copper into SOD1 in motor neurons. Although the absence of CCS led to a significant reduction in the amount of copper-loaded mutant SOD1, however, it did not modify the onset and progression of motor neuron disease in SOD1-mutant mice. Hence, CCS-dependent copper loading of mutant SOD1 plays no role in the pathogenesis of motor neuron disease in these mouse models.

Perturbation of hepcidin expression by BMP type I receptor deletion induces iron overload in mice.

Bone morphogenetic protein (BMP) signaling induces hepatic expression of the peptide hormone hepcidin. Hepcidin reduces serum iron levels by promoting degradation of the iron exporter ferroportin. A relative deficiency of hepcidin underlies the pathophysiology of many of the genetically distinct iron overload disorders, collectively termed hereditary hemochromatosis. Conversely, chronic inflammatory conditions and neoplastic diseases can induce high hepcidin levels, leading to impaired mobilization of iron stores and the anemia of chronic disease. Two BMP type I receptors, Alk2 (Acvr1) and Alk3 (Bmpr1a), are expressed in murine hepatocytes. We report that liver-specific deletion of either Alk2 or Alk3 causes iron overload in mice. The iron overload phenotype was more marked in Alk3- than in Alk2-deficient mice, and Alk3 deficiency was associated with a nearly complete ablation of basal BMP signaling and hepcidin expression. Both Alk2 and Alk3 were required for induction of hepcidin gene expression by BMP2 in cultured hepatocytes or by iron challenge in vivo. These observations demonstrate that one type I BMP receptor, Alk3, is critically responsible for basal hepcidin expression, whereas 2 type I BMP receptors, Alk2 and Alk3, are required for regulation of hepcidin gene expression in response to iron and BMP signaling.

Transferrin is a major determinant of hepcidin expression in hypotransferrinemic mice

As a central regulator of iron metabolism, hepcidin inhibits dietary iron absorption and macrophage iron recycling. Its expression is regulated by multiple factors including iron availability and erythropoietic activity. To investigate the role of transferrin (Tf) in the regulation of hepcidin expression by these factors in vivo, we employed the hypotransferrinemic (hpx) mouse. These Tf-deficient mice have severe microcytic anemia, tissue iron overload, and hepcidin deficiency. To determine the relationship of Tf levels and erythropoiesis to hepcidin expression, we subjected hpx mutant and control mice to a number of experimental manipulations. Treatment of hpx mice with Tf injections corrected their anemia and restored hepcidin expression. To investigate the effect of erythropoiesis on hepcidin expression, we suppressed erythropoiesis with blood transfusions or myeloablation with chemotherapeutic drugs. Transfusion of hpx animals with wild-type red blood cells led to increased hepcidin expression, while hepcidin expression in myeloablated hpx mice increased only if Tf was administered postablation. These results suggest that hepcidin expression in hpx mice is regulated both by Tf-restricted erythropoiesis and by Tf through a mechanism independent of its role in erythropoiesis.

Mechanisms of the Copper-dependent Turnover of the Copper Chaperone for Superoxide Dismutase

The copper chaperone for superoxide dismutase (CCS) is an intracellular metallochaperone required for incorporation of copper into the essential antioxidant enzyme copper/zinc superoxide dismutase (SOD1). Nutritional studies have revealed that the abundance of CCS is inversely proportional to the dietary and tissue copper content. To determine the mechanisms of copper-dependent regulation of CCS, copper incorporation into SOD1 and SOD1 enzymatic activity as well as CCS abundance and half-life were determined after metabolic labeling of CCS-/- fibroblasts transfected with wild-type or mutant CCS. Wild-type CCS restored SOD1 activity in CCS-/- fibroblasts, and the abundance of this chaperone in these cells was inversely proportional to the copper content of the media, indicating that copper-dependent regulation of CCS is entirely post-translational. Although mutational studies demonstrated no role for CCS Domain I in this copper-dependent regulation, similar analysis of the CXC motif in Domain III revealed a critical role for these cysteine residues in mediating copper-dependent turnover of CCS. Further mutational studies revealed that this CXC-dependent copper-mediated turnover of CCS is independent of the mechanisms of delivery of copper to SOD1 including CCS-SOD1 interaction. Taken together these data demonstrate a mechanism determining the abundance of CCS that is competitive with the process of copper delivery to SOD1, revealing a unique post-translational component of intracellular copper homeostasis.

Mechanisms of biosynthesis of mammalian copper/zinc superoxide dismutase.

Copper/zinc superoxide dismutase (SOD1) is an abundant intracellular enzyme with an essential role in antioxidant defense. The activity of SOD1 is dependent upon the presence of a bound copper ion incorporated by the copper chaperone for superoxide dismutase, CCS. To elucidate the cell biological mechanisms of this process, SOD1 synthesis and turnover were examined following 64Cu metabolic labeling of fibroblasts derived from CCS+/+ and CCS–/– embryos. The data indicate that copper is rapidly incorporated into both newly synthesized SOD1 and preformed SOD1 apoprotein, that each process is dependent upon CCS and that once incorporated, copper is unavailable for cellular exchange. The abundance of apoSOD1 is inversely proportional to the intracellular copper content and immunoblot and gel filtration analysis indicate that this apoprotein exists as a homodimer that is distinguishable from SOD1. Despite these distinct differences, the abundance and half-life of SOD1 is equivalent in CCS+/+ and CCS–/– fibroblasts, indicating that neither CCS nor copper incorporation has any essential role in the stability or turnover of SOD1 in vivo. Taken together, these data provide a cell biological model of SOD1 biosynthesis that is consistent with the concept of limited intracellular copper availability and indicate that the metallochaperone CCS is a critical determinant of SOD1 activity in mammalian cells. These kinetic and biochemical findings also provide an important framework for understanding the role of mutant SOD1 in the pathogenesis of familial amyotrophic lateral sclerosis.

Human transferrin confers serum resistance against bacillus anthracis

The innate immune system in humans consists of both cellular and humoral components that collaborate to eradicate invading bacteria from the body. Here, we discover that the Gram-positive bacterium Bacillus anthracis, the causative agent of anthrax, does not grow in human serum. Fractionation of serum by gel filtration chromatography led to the identification of human transferrin as the inhibiting factor. Purified transferrin blocks growth of both the fully virulent encapsulated B. anthracis Ames and the non-encapsulated Sterne strain. Growth inhibition was also observed in serum of wild-type mice but not of hypotransferrinemic mice that only have ∼1% circulating transferrin levels. We were able to definitely assign the bacteriostatic activity of transferrin to its iron-binding function: neither iron-saturated transferrin nor a recombinant transferrin mutant unable to bind iron could inhibit growth of B. anthracis. Additional iron could restore bacterial growth in human serum. The observation that other important Gram-positive pathogens are not inhibited by transferrin suggests they have evolved effective mechanisms to circumvent serum iron deprivation. These findings provide a better understanding of human host defense mechanisms against anthrax and provide a mechanistic basis for the antimicrobial activity of human transferrin.

Known and potential roles of transferrin in iron biology

Transferrin is an abundant serum metal-binding protein best known for its role in iron delivery. The human disease congenital atransferrinemia and animal models of this disease highlight the essential role of transferrin in erythropoiesis and iron metabolism. Patients and mice deficient in transferrin exhibit anemia and a paradoxical iron overload attributed to deficiency in hepcidin, a peptide hormone synthesized largely by the liver that inhibits dietary iron absorption and macrophage iron efflux. Studies of inherited human disease and model organisms indicate that transferrin is an essential regulator of hepcidin expression. In this paper, we review current literature on transferrin deficiency and present our recent findings, including potential overlaps between transferrin, iron and manganese in the regulation of hepcidin expression.

How to make a metalloprotein

Copper is an essential transition metal that plays a fundamental role in the biochemistry of all aerobic organisms. Recent work elucidating the structural mechanisms of copper delivery to superoxide dismutase provides insight into the cell biology of copper metabolism and serves as an example of how to understand the principles governing the incorporation of metals into proteins.

Investigating the Role of Transferrin in the Distribution of Iron, Manganese, Copper and Zinc

The essential role of transferrin in mammalian iron metabolism is firmly established. Integral to our understanding of transferrin, studies in hypotransferrinemic mice, a model of inherited transferrin deficiency, have demonstrated that transferrin is essential for iron delivery for erythropoiesis and in the regulation of expression of hepcidin, a hormone that inhibits macrophage and enterocyte iron efflux. Here we investigate a potential role for transferrin in the distribution of three other physiologic metals, manganese, copper, and zinc. We first assessed metal content in transferrin-rich fractions of wild-type mouse sera and demonstrate that although both iron and manganese cofractionated predominantly with transferrin, the absolute levels of manganese are several orders of magnitude lower than those of iron. We next measured metal content in multiple tissues in wild-type and hypotransferrinemic mice of various ages. Tissue metal imbalances were severe for iron and minimal to moderate for some metals in some tissues in hypotransferrinemic mice. Metal levels measured in a transferrin-replete yet hepcidin-deficient and iron-loaded mouse strain suggested that the observed imbalances in tissue copper, zinc, and manganese levels were not all specific to hypotransferrinemic mice or caused directly by transferrin deficiency. Overall, our results suggest that transferrin does not have a primary role in the distribution of manganese, copper, or zinc to tissues and that the abnormalities observed in tissue manganese levels are not attributable to a direct role for transferrin in manganese metabolism but rather are attributable to an indirect effect of transferrin deficiency on hepcidin expression and/or iron metabolism.

A competitive enzyme-linked immunosorbent assay specific for murine hepcidin-1: correlation with hepatic mRNA expression in established and novel models of dysregulated iron homeostasis

Mice have been essential for distinguishing the role of hepcidin in iron homeostasis. Currently, investigators monitor levels of murine hepatic hepcidin-1 mRNA as a surrogate marker for the bioactive hepcidin protein itself. Here, we describe and validate a competitive, enzyme-linked immunosorbent assay that quantifies hepcidin-1 in mouse serum and urine. The assay exhibits a biologically relevant lower limit of detection, high precision, and excellent linearity and recovery. We also demonstrate correlation between serum and urine hepcidin-1 values and validate the competitive enzyme-linked immunosorbent assay by analyzing plasma hepcidin response of mice to physiological challenges, including iron deficiency, iron overload, acute blood loss, and inflammation. Furthermore, we analyze multiple murine genetic models of iron dysregulation, including β-thalassemia intermedia (Hbbth3/+), hereditary hemochromatosis (Hfe−/−, Hjv−/−, and Tfr2Y245X/Y245X), hypotransferrinemia (Trfhpx/hpx), heterozygous transferrin receptor 1 deficiency (Tfrc+/−) and iron refractory iron deficiency anemia (Tmprss6−/− and Tmprss6hem8/hem8). Novel compound iron metabolism mutants were also phenotypically characterized here for the first time. We demonstrate that serum hepcidin concentrations correlate with liver hepcidin mRNA expression, transferrin saturation and non-heme liver iron. In some circumstances, serum hepcidin-1 more accurately predicts iron parameters than hepcidin mRNA, and distinguishes smaller, statistically significant differences between experimental groups.