Chinmay K. Mukhopadhyay

Ceruloplasmin and cardiovascular disease

Transition metal ion-mediated oxidation is a commonly used model system for studies of the chemical, structural, and functional modifications of low-density lipoprotein (LDL). The physiological relevance of studies using free metal ions is unclear and has led to an exploration of free metal ion-independent mechanisms of oxidation. We and others have investigated the role of human ceruloplasmin (Cp) in oxidative processes because it the principal copper-containing protein in serum. There is an abundance of epidemiological data that suggests that serum Cp may be an important risk factor predicting myocardial infarction and cardiovascular disease. Biochemical studies have shown that Cp is a potent catalyst of LDL oxidation in vitro. The pro-oxidant activity of Cp requires an intact structure, and a single copper atom at the surface of the protein, near His(426), is required for LDL oxidation. Under conditions where inhibitory protein (such as albumin) is present, LDL oxidation by Cp is optimal in the presence of superoxide, which reduces the surface copper atom of Cp. Cultured vascular endothelial and smooth muscle cells also oxidize LDL in the presence of Cp. Superoxide release by these cells is a critical factor regulating the rate of oxidation. Cultured monocytic cells, when activated by zymosan, can oxidize LDL, but these cells are unique in their secretion of Cp. Inhibitor studies using Cp-specific antibodies and antisense oligonucleotides show that Cp is a major contributor to LDL oxidation by these cells. The role of Cp in lipoprotein oxidation and atherosclerotic lesion progression in vivo has not been directly assessed and is an important area for future studies.

Ceruloplasmin Ferroxidase Activity Stimulates Cellular Iron Uptake by a Trivalent Cation-specific Transport Mechanism

The balance required to maintain appropriate cellular and tissue iron levels has led to the evolution of multiple mechanisms to precisely regulate iron uptake from transferrin and low molecular weight iron chelates. A role for ceruloplasmin (Cp) in vertebrate iron metabolism is suggested by its potent ferroxidase activity catalyzing conversion of Fe2+ to Fe3+, by identification of yeast copper oxidases homologous to Cp that facilitate high affinity iron uptake, and by studies of “aceruloplasminemic” patients who have extensive iron deposits in multiple tissues. We have recently shown that Cp increases iron uptake by cultured HepG2 cells. In this report, we investigated the mechanism by which Cp stimulates cellular iron uptake. Cp stimulated the rate of non-transferrin 55Fe uptake by iron-deficient K562 cells by 2–3-fold, using a transferrin receptor-independent pathway. Induction of Cp-stimulated iron uptake by iron deficiency was blocked by actinomycin D and cycloheximide, consistent with a transcriptionally induced or regulated transporter. Cp-stimulated iron uptake was completely blocked by unlabeled Fe3+ and by other trivalent cations including Al3+, Ga3+, and Cr3+, but not by divalent cations. These results indicate that Cp utilizes a trivalent cation-specific transporter. Cp ferroxidase activity was required for iron uptake as shown by the ineffectiveness of two ferroxidase-deficient Cp preparations, copper-deficient Cp and thiomolybdate-treated Cp. We propose a model in which iron reduction and subsequent re-oxidation by Cp are essential for an iron uptake pathway with high ion specificity.

Structure, oxidant activity, and cardiovascular mechanisms of human ceruloplasmin

Ceruloplasmin is the principal carrier of copper in human plasma. It is an abundant protein that participates in the acute phase reaction to stress, but its physiological function(s) is unknown. An antioxidant activity of ceruloplasmin has been described, but recent evidence suggests that the protein may also exhibit potent pro-oxidant activity and cause oxidative modification of low density lipoprotein (LDL). The pro-oxidant activity is highly dependent on the structure of the protein; removal of a single one of the seven integral copper atoms, or a specific proteolytic cleavage event, completely suppresses LDL oxidation. This newly described pro-oxidant activity may help to explain epidemiological studies indicating that ceruloplasmin is an independent risk factor for cardiovascular disease.

Unexpected Link between Iron and Drug Resistance of Candida spp.: Iron Depletion Enhances Membrane Fluidity and Drug Diffusion, Leading to Drug-Susceptible Cells

ABSTRACT Inthis study, we show that iron depletion in Candidaalbicans with bathophenanthrolene disulfonic acid and ferrozine as chelators enhanced its sensitivity to several drugs, including the most common antifungal, fluconazole (FLC). Several other species of Candida also displayed increased sensitivity to FLC because of iron restriction. Iron uptake mutations, namely,Δ ftr1 and Δftr2, as well as the copper transporter mutation Δccc2, which affects high-affinity iron uptake in Candida, produced increased sensitivity to FLC compared to that of the wild type. The effect of iron depletion on drug sensitivity appeared to be independent of the efflux pump proteins Cdr1p and Cdr2p. We found that iron deprivation led to lowering of membrane ergosterol by 15 to 30%. Subsequently, fluorescence polarization measurements also revealed that iron-restricted Candida cells displayed a 29 to 40% increase in membrane fluidity, resulting in enhanced passive diffusion of the drugs. Northern blot assays revealed that the ERG11 gene was considerably down regulated in iron-deprived cells, which might account for the lowered ergosterol content. Our results show a close relationship between cellular iron and drug susceptibilities of C. albicans. Considering that multidrug resistance is a manifestation of multifactorial phenomena, the influence of cellular iron on the drug susceptibilities of Candida suggests iron as yet another novel determinant of multidrug resistance.

Evolutionary Significance of Vitamin C Biosynthesis in Terrestrial Vertebrates

Evolution of vertebrates from aquatic medium to the terrestrial atmosphere containing high concentration of environmental oxygen was accompanied by tissue-specific expression of the gene for L-gulonolactone oxidase (LGO). LGO is the terminal enzyme in the pathway of biosynthesis of ascorbic acid in animals. In this paper we present data to indicate that emergence of LGO is apparently to provide the terrestrial vertebrates with adequate amount of ascorbic acid and thereby protect their tissues against oxygen toxicity. Superoxide dismutase (SOD) was not induced in the early tetrapods. However, SOD activity has increased in the mammals which is accompanied by a decrease in the LGO activity. In fact, there has been an inverse relationship between LGO and SOD in the progress of evolution. SOD activity is markedly high in the guinea pig, flying mammal, monkey and man, the species those lack LGO. The inverse relationship between LGO and SOD is also observed in rats during postnatal development, that is when the new born rats are exposed to high concentration of atmospheric oxygen. Recent results from our laboratory indicate that ascorbic acid is specifically needed for protection of microsomal membranes against cytochrome P450-mediated lipid peroxidation and protein oxidation, where SOD is ineffective. Data presented in this paper also indicate an apparent tissue-specific correlation among LGO activity, P450 level and O2.- production during phylogenetic evolution.

Leishmania donovani depletes labile iron pool to exploit iron uptake capacity of macrophage for its intracellular growth.

Intracellular pathogens employ several strategies for iron acquisition from host macrophages for survival and growth, whereas macrophage resists infection by actively sequestering iron. Here, we show that instead of allowing macrophage to sequester iron, protozoan parasite Leishmania donovani (LD) uses a novel strategy to manipulate iron uptake mechanisms of the host and utilizes the taken up iron for its intracellular growth. To do so, intracellular LD directly scavenges iron from labile iron pool of macrophages. Depleted labile iron pool activates iron sensors iron‐regulatory proteins IRP1 and IRP2. IRPs then bind to iron‐responsive elements present in the 3′ UTR of iron uptake gene transferrin receptor 1 by a post‐transcriptional mRNA stability mechanism. Increased iron‐responsive element–IRP interaction and transferrin receptor 1 expressions in spleen‐derived macrophages from LD‐infected mice confirm that LD employs similar mechanism to acquire iron during infection into mammalian hosts. Increased intracellular LD growth by holo‐transferrin supplementation and inhibited growth by iron chelator treatment confirm the significance of this modulated iron uptake pathway of host in favour of the parasite.

Brain iron homeostasis: from molecular mechanisms to clinical significance and therapeutic opportunities.

Iron has emerged as a significant cause of neurotoxicity in several neurodegenerative conditions, including Alzheimers disease (AD), Parkinsons disease (PD), sporadic Creutzfeldt-Jakob disease (sCJD), and others. In some cases, the underlying cause of iron mis-metabolism is known, while in others, our understanding is, at best, incomplete. Recent evidence implicating key proteins involved in the pathogenesis of AD, PD, and sCJD in cellular iron metabolism suggests that imbalance of brain iron homeostasis associated with these disorders is a direct consequence of disease pathogenesis. A complete understanding of the molecular events leading to this phenotype is lacking partly because of the complex regulation of iron homeostasis within the brain. Since systemic organs and the brain share several iron regulatory mechanisms and iron-modulating proteins, dysfunction of a specific pathway or selective absence of iron-modulating protein(s) in systemic organs has provided important insights into the maintenance of iron homeostasis within the brain. Here, we review recent information on the regulation of iron uptake and utilization in systemic organs and within the complex environment of the brain, with particular emphasis on the underlying mechanisms leading to brain iron mis-metabolism in specific neurodegenerative conditions. Mouse models that have been instrumental in understanding systemic and brain disorders associated with iron mis-metabolism are also described, followed by current therapeutic strategies which are aimed at restoring brain iron homeostasis in different neurodegenerative conditions. We conclude by highlighting important gaps in our understanding of brain iron metabolism and mis-metabolism, particularly in the context of neurodegenerative disorders.

IL-6 and IL-12 specifically regulate the expression of Rab5 and Rab7 via distinct signaling pathways.

Recent studies have shown that phagosome maturation depends on the balance between pro‐inflammatory and anti‐inflammatory cytokines, indicating that cytokine modulates phagosome maturation. However, the mechanism of cytokine‐mediated modulation of intracellular trafficking remains to be elucidated. Here, we have shown that treatment of macrophages with IL‐6 specifically induce the expression of Rab5 through the activation of extracellular signal‐regulated kinase, whereas IL‐12 exclusively upregulate the expression of Rab7 through the activation of p38 MAPK. We have cloned the 5′‐flanking regions of the rab5c or rab7 into the promoterless reporter vector. Our results have shown that cells transfected with rab5c chimera are transactivated by IL‐6, and IL‐12 specifically transactivates cells containing rab7 chimera. Moreover, our results also show that IL‐12 induces lysosomal transport, whereas IL‐6 stimulates the fusion between early compartments in macrophages and accordingly modulates Salmonella trafficking and survival in macrophages. This is the first demonstration showing that cytokine differentially regulates endocytic trafficking by controlling the expression of appropriate Rab GTPase, and provides insight into the mechanism of cytokine‐mediated regulation of intracellular trafficking.

Hepatitis B vaccination with or without hepatitis B immunoglobulin at birth to babies born of HBsAg-positive mothers prevents overt HBV transmission but may not prevent occult HBV infection in babies: a randomized controlled trial

Vertical transmission of Hepatitis B virus HBV can result in a state of chronic HBV infection and its complications. HBV vaccination with or without hepatitis B immunoglobulin (HBIG) prevents transmission of overt infection to the babies. However, whether it also prevents occult HBV infection in babies is not known. Consecutive pregnant women of any gestation found to be HBsAg positive were followed till delivery, and their babies were included in the study. Immediately after delivery, babies were randomized to receive either HBIG or placebo in addition to recombinant HBV vaccine (at 0, 6, 10 and 14 weeks). The primary end‐point of the study, assessed at 18 weeks of age, was remaining free of any HBV infection (either overt or occult) plus the development of adequate immune response to vaccine. The babies were further followed up for a median of 2 years of age to determine their eventual outcome. Risk factors for HBV transmission and for poor immune response in babies were studied. Of the 283 eligible babies, 259 were included in the trial and randomized to receive either HBIG (n = 128) or placebo (n = 131) in addition to recombinant HBV vaccine. Of the 222 of 259 (86%) babies who completed 18 weeks of follow‐up, only 62/222 (28%) reached primary end‐point. Of the remaining, 6/222 (3%) developed overt HBV infection, 142/222 (64%) developed occult HBV infection, and 12/222 (5%) had no HBV infection but had poor immune response. All 6 overt infections occurred in the placebo group (P = 0.030), while occult HBV infections were more common in the HBIG group (76/106 [72%] vs. 66/116 [57%]; P = 0.025). This may be due to the immune pressure of HBIG. There was no significant difference between the two groups in frequency of babies developing poor immune response or those achieving primary end‐point. The final outcome of these babies at 24 months of age was as follows: overt HBV infection 4%, occult HBV infection 42%, no HBV infection but poor immune response 8% and no HBV infection with good immune response 28%. Women who were anti‐HBe positive were a low‐risk group, and their babies were most likely to remain free of HBV infection (occult or overt) and had good immune response to the vaccine. Maternal HBeAg‐positive status and negativity for anti‐HBe predicted not only overt but also any infection (both overt and occult) in babies. In addition, high maternal HBV DNA and treatment with vaccine alone were significant factors for overt HBV infection in babies. The current practice of administration of vaccine with HBIG at birth to babies born of HBsAg‐positive mothers is not effective in preventing occult HBV infection in babies, which may be up to 40%. Because the most important risk factors for mother‐to‐baby transmission of HBV infection are the replicative status and high HBV DNA level in mothers; it will be worthwhile investigating the role of antivirals and HBIG administration during pregnancy to prevent mother‐to‐child transmission of HBV infection.

Dual Role of Insulin in Transcriptional Regulation of the Acute Phase Reactant Ceruloplasmin

Insulin is a potent negative regulator of the response of hepatic cells to pro-inflammatory cytokines, particularly, interleukin (IL)-6. The action of insulin is target-selective because it inhibits transcription of most but not all acute phase genes. We here show that ceruloplasmin (Cp), an acute phase reactant with important functions in iron homeostasis, is subject to a unique dual regulation by insulin. IL-6 increased Cp mRNA expression in HepG2 cells by ∼5-fold. Simultaneous treatment with insulin reduced this stimulation by half. Surprisingly, insulin by itself caused a 2–4-fold induction in Cp mRNA expression. The mechanism of induction by insulin was studied by transfecting into HepG2 cells chimeric constructs of the Cp 5′-flanking region driving luciferase. The activity of a 4800-bp segment of the Cp 5′-flanking region was increased 3-fold by insulin. Deletion and mutation analyses showed the requirement for a single hypoxia-responsive element in a 96-bp segment ∼3600 bp upstream of the initiation site. The domains required for the two activities of insulin were distinct: The distal, hypoxia-responsive element-containing site was sufficient for Cp transactivation by insulin; in contrast, an 848-bp region adjacent to the initiation site was sufficient for IL-6 transactivation of Cp and for the inhibitory activity of insulin. The role of hypoxia-inducible factor-1 in the induction of Cp by insulin was shown by electrophoretic mobility shift assays and by the absence of insulin-stimulated Cp promoter activation in mouse Hepa c4 cells deficient in hypoxia-inducible factor-1 activity. Taken together these results show that insulin functions as a bidirectional, condition-dependent regulator of hepatic cell Cp expression. The unique regulation of Cp may reflect its dual roles in inflammation and iron homeostasis.