Involvement of Arabidopsis Multi-Copper Oxidase-Encoding LACCASE12 in Root-to-Shoot Iron Partitioning: A Novel Example of Copper-Iron Crosstalk

Numerous central biological processes depend on the participation of the essential elements iron (Fe) or copper (Cu), including photosynthesis, respiration, cell wall remodeling and oxidative stress protection. Yet, both Fe and Cu metal cations can become toxic when accumulated in excess. Because of the potent ligand-binding and redox chemistries of these metals, there is a need for the tight and combined homeostatic control of their uptake and distribution. Several known examples pinpoint an inter-dependence of Fe and Cu homeostasis in eukaryotes, mostly in green algae, yeast and mammals, but this is less well understood in multicellular plants to date. In Arabidopsis, Cu deficiency causes secondary Fe deficiency, and this is associated with reduced in vitro ferroxidase activity and decreased root-to-shoot Fe translocation. Here we summarize the current knowledge of the cross-talk between Cu and Fe homeostasis and present a partial characterization of LACCASE12 (LAC12) that encodes a member of the multicopper oxidase (MCO) protein family in Arabidopsis. LAC12 transcript levels increase under Fe deficiency. The phenotypic characterization of two mutants carrying T-DNA insertions suggests a role of LAC12 in root-to-shoot Fe partitioning and in maintaining growth on Fe-deficient substrates. A molecular understanding of the complex interactions between Fe and Cu will be important for combating Fe deficiency in crops and for advancing biofortification approaches.

Novel DNA-protection Protein (Dps) Originated From Deinococcus Geothermalis

We characterized two interrelated proteins from the Deinococcus geothermalis strain with regard to the role of DNA-binding and protection, such as a novel Dps, Dgeo_0257, and a Dps orthologous protein, Dgeo_0281. Despite the lack of conserved amino acid sequence for ferroxidase activity, Dgeo_0257 exhibited high DNA-binding affinity and formed a dodecameric conformation. In contrast, Dgeo_0281 protein showed less effective DNA-binding and was mainly observed monomeric or dimeric forms. Electrophoretic mobility shift assay showed that both purified proteins are non-specifically bound to DNA to protect against DNA degradation and the properties of Dps responding to specific metal ions. In the presence of ferrous and ferric ions, Dgeo_0257 or Dgeo_0281 protein does not readily bind to DNA. However, in Zn and Mn ions are present, both proteins had more stable DNA-binding. From the Dps gene disrupted mutant test, each showed a higher sensitivity to oxidative stress than the wild-type strain. In addition, the expression level of each gene was correlated with the presence of each other. In this study, we characterized and confirmed the functional roles of Dgeo_0257 for sensing metal ions and binding to DNAs along with the Dps orthologous Dgeo_0281. Based on various observational evidence, we propose that Dgeo_0257 is a novel Dps protein with no ferroxidase activity.

Association of Iron Status in Follicular Fluid with Pregnancy Outcomes in Infertile Women Undergoing IVF/ICSI

Iron status may influence the outcome of infertile women under the intracytoplasmic sperm injection (ICSI) technique of in vitro fertilization (IVF). The aim of this study is to evaluate iron status and ceruloplasmin ferroxidase activity in the follicular fluid (FF) and their association with IVF outcomes. The study enrolled fertile women with male cause infertility (n=25), infertile women with polycystic ovary syndrome (PCOS; n=21), infertile women with low AMH level (n=26), and women with unexplained infertility (UI; n=27), all undergoing IVF/ICSI. On the day of oocyte suction, the selection of FF samples was accomplished. Iron, ferritin, and transferrin levels, as well as ceruloplasmin (CP) ferroxidase activity, were measured in the FF. In the PCOS group, iron showed significantly higher level (P<0.05) as compared to the control and UI groups. In the PCOS group, ferritin showed significantly higher level (P<0.05) compared with the control group. In the PCOS group, transferrin showed significantly higher level (P<0.05) when compared with the UI group. Also, Cp. ferroxidase activity in the PCOS group showed a lower level, but non-significant difference, compared with the other groups. In conclusion, the increased iron level in the follicular fluid of women with PCOS may lead to decrease pregnancy success after applying IVF protocol.

Bacterial-type plant ferroxidases tune local phosphate sensing in root development

Fluctuating bioavailability of inorganic phosphate (Pi), often caused by complex Pi-metal interactions, guide root tip growth and root system architecture for maximizing the foraged soil volume. Two interacting genes in Arabidopsis thaliana, PDR2 (P5-type ATPase) and LPR1 (multicopper oxidase), are central to external Pi monitoring by root tips, which is modified by iron (Fe) co-occurrence. Upon Pi deficiency, the PDR2-LPR1 module facilitates cell type-specific Fe accumulation and cell wall modifications in root meristems, inhibiting intercellular communication and thus root growth. LPR1 executes local Pi sensing, whereas PDR2 restricts LPR1 function. We show that native LPR1 displays specific ferroxidase activity and requires a conserved acidic triad motif for high-affinity Fe2+ binding and root growth inhibition under limiting Pi. Our data indicate that substrate availability tunes LPR1 function and implicate PDR2 in maintaining Fe homeostasis. LPR1 represents the prototype of an ancient ferroxidase family, which evolved very early upon bacterial colonization of land. During plant terrestrialization, horizontal gene transfer transmitted LPR1-type ferroxidase from soil bacteria to the common ancestor of Zygnematophyceae algae and embryophytes, a hypothesis supported by homology modeling, phylogenomics, and activity assays of bacterial LPR1-type multicopper oxidases.

Expression of soluble, active, fluorescently tagged hephaestin in COS and CHO cell lines

Hephaestin (Hp) is a trans-membrane protein, which plays a critical role in intestinal iron absorption. Hp was originally identified as the gene responsible for the phenotype of sex-linked anaemia in the sla mouse. The mutation in the sla protein causes ac-cumulation of dietary iron in duodenal cells, causing severe microcytic hypochromic anaemia. Although mucosal uptake of dietary iron is normal, export from the duodenum is inhibited. Hp is homologous to ceruloplasmin (Cp), a member of the family of multi copper fer-roxidases (MCFs) and possesses ferroxidase activity that facilitates iron release from the duodenum and load onto the serum iron trans-port protein transferrin. In the present study, attempts were made to produce biologically active recombinant mouse hephaestin as a secretory form tagged with green fluorescent protein (GFP), Hpsec-GFP. Plasmid expressing Hpsec-GFP was constructed and transfected into COS and CHO cells. The GFP aided the monitoring expression in real time to select the best conditions to maximise expression and provided a tag for purifying and analysing Hpsec-GFP. The protein had detectable oxidase activity as shown by in-gel and solution-based assays. The methods described here can provide the basis for further work to probe the interaction of hephaestin with other proteins using complementary fluorescent tags on target proteins that would facilitate the fluorescence resonance energy transfer measurements, for example with transferrin or colocalisation studies, and help to discover more about hephaestin works at the molecular level.

Effects of Dietary Glucose and Fructose on Copper, Iron, and Zinc Metabolism Parameters in Humans

Alterations of transition metal levels have been associated with obesity, hepatic steatosis, and metabolic syndrome in humans. Studies in animals indicate an association between dietary sugars and copper metabolism. Our group has conducted a study in which young adults consumed beverages sweetened with glucose, fructose, high fructose corn syrup (HFCS), or aspartame for two weeks and has reported that consumption of both fructose- and HFCS-sweetened beverages increased cardiovascular disease risk factors. Baseline and intervention serum samples from 107 participants of this study were measured for copper metabolism (copper, ceruloplasmin ferroxidase activity, ceruloplasmin protein), zinc levels, and iron metabolism (iron, ferritin, and transferrin) parameters. Fructose and/or glucose consumption were associated with decreased ceruloplasmin ferroxidase activity and serum copper and zinc concentrations. Ceruloplasmin protein levels did not change in response to intervention. The changes in copper concentrations were correlated with zinc, but not with iron. The decreases in copper, ceruloplasmin ferroxidase activity, ferritin, and transferrin were inversely associated with the increases in metabolic risk factors associated with sugar consumption, specifically, apolipoprotein CIII, triglycerides, or post-meal glucose, insulin, and lactate responses. These findings are the first evidence that consumption of sugar-sweetened beverages can alter clinical parameters of transition metal metabolism in healthy subjects.

The ferroxidase LPR5 functions in the maintenance of phosphate homeostasis and is required for normal growth and development of rice

Members of the Low Phosphate Root (LPR) family have been identified in rice (Oryza sativa) and expression analyses have been conducted. Here, we investigated the functions of one of the five members in rice, LPR5. qRT-PCR and promoter–GUS reporter analyses indicated that under Pi-sufficient conditions OsLPR5 was highly expressed in the roots, and specific expression occurred in the leaf collars and nodes, and its expression was increased under Pi-deficient conditions. In vitro analysis of the purified OsLPR5 protein showed that it exhibited ferroxidase activity. Overexpression of OsLPR5 triggered higher ferroxidase activity, and elevated concentrations of Fe(III) in the xylem sap and of total Fe in the roots and shoots. Transient expression of OsLPR5 in Nicotiana benthamiana provided evidence of its subcellular localization to the cell wall and endoplasmic reticulum. Knockout mutation in OsLPR5 by means of CRISPR-Cas9 resulted in adverse effects on Pi translocation, on the relative expression of Cis-NATOsPHO1;2, and on several morphological traits, including root development and yield potential. Our results indicate that ferroxidase-dependent OsLPR5 has both a broad-spectrum influence on growth and development in rice as well as affecting a subset of physiological and molecular traits that govern Pi homeostasis.

Effect of Narrowband-UVB Phototherapy Treatment on Total Ferroxidase Activity and Ceruloplasmin Oxidase Activity in Sera of Iraqi Vitiligo Patients

Vitiligo is an acquired pigmentary disorder in which white depigmented macules and patches of skin appear on different parts of the body, affecting all ages and both sexes equally. Focal, segmen-tal, generalized and universal are considered the most characteristic patterns of vitiligo. One of the most effective treatment of this disorder is using light therapy, and in particular ultraviolet (UV) light. Among them narrowband ultraviolet B phototherapy (NB-UVB) is the most widely used. Oxidative stress is considered to be one of the possible pathogenic events in melanocyte loss. Imbalance in the oxidant/antioxidant system have been demonstrated in blood of vitiligo patients. To demonstrate the safety of using NB-UVB phototherapy treatment in vitiligo Iraqi patients. Patients with vitiligo group were treated with NB-UVB regimen as three times weekly on non- consecutive days for a total of 20 sessions (about 2 months). Total ferroxidase (activity and specific activity), ceruloplasmin concentration, ceruloplasmin oxidase (activity and specific activity) and serum copper were determined in all participants before and after NBUVB phototherapy. The above biochemical parameters were measured in serum samples of 30 vitiligo patients before and after treatment with the narrow band UVB phototherapy. NB-UVB treatment was found to be associated with a significant reduction in copper ion concentration, but no significant differences in total ferroxidase (activity and specific activity), ceruloplasmin oxidase (activity and specific activity), and ceruloplasmin concentration. The present results provide evidence which support using narrow band UVB phototherapy as a safe method for vitiligo treatment.

Genetic and Clinical Heterogeneity in Thirteen New Cases with Aceruloplasminemia. Atypical Anemia as a Clue for an Early Diagnosis

Aceruloplasminemia is a rare autosomal recessive genetic disease characterized by mild microcytic anemia, diabetes, retinopathy, liver disease, and progressive neurological symptoms due to iron accumulation in pancreas, retina, liver, and brain. The disease is caused by mutations in the Ceruloplasmin (CP) gene that produce a strong reduction or absence of ceruloplasmin ferroxidase activity, leading to an impairment of iron metabolism. Most patients described so far are from Japan. Prompt diagnosis and therapy are crucial to prevent neurological complications since, once established, they are usually irreversible. Here, we describe the largest series of non-Japanese patients with aceruloplasminemia published so far, including 13 individuals from 11 families carrying 13 mutations in the CP gene (7 missense, 3 frameshifts, and 3 splicing mutations), 10 of which are novel. All missense mutations were studied by computational modeling. Clinical manifestations were heterogeneous, but anemia, often but not necessarily microcytic, was frequently the earliest one. This study confirms the clinical and genetic heterogeneity of aceruloplasminemia, a disease expected to be increasingly diagnosed in the Next-Generation Sequencing (NGS) era. Unexplained anemia with low transferrin saturation and high ferritin levels without inflammation should prompt the suspicion of aceruloplasminemia, which can be easily confirmed by low serum ceruloplasmin levels. Collaborative joint efforts are needed to better understand the pathophysiology of this potentially disabling disease.