A human mitochondrial ferritin encoded by an intronless gene.

Ferritin is a ubiquitous protein that plays a critical role in regulating intracellular iron homoeostasis by storing iron inside its multimeric shell. It also plays an important role in detoxifying potentially harmful free ferrous iron to the less soluble ferric iron by virtue of the ferroxidase activity of the H subunit. Although excess iron is stored primarily in cytoplasm, most of the metabolically active iron in cells is processed in mitochondria. Little is yet known of how these organelles regulate iron homeostasis and toxicity. Here we report an unusual intronless gene on chromosome 5q23.1 that encodes a 242-amino acid precursor of a ferritin H-like protein. This 30-kDa protein is targeted to mitochondria and processed to a 22-kDa subunit that assembles into typical ferritin shells and has ferroxidase activity. Immunohistochemical analysis showed that it accumulates in high amounts in iron-loaded mitochondria of erythroblasts of subjects with impaired heme synthesis. This new ferritin may play an important role in the regulation of mitochondrial iron homeostasis and heme synthesis.

Overexpression of wild type and mutated human ferritin H-chain in HeLa cells: in vivo role of ferritin ferroxidase activity.

Transfectant HeLa cells were generated that expressed human ferritin H-chain wild type and an H-chain mutant with inactivated ferroxidase activity under the control of the tetracycline-responsive promoter (Tet-off). The clones accumulated exogenous ferritins up to levels 14-16-fold over background, half of which were as H-chain homopolymers. This had no evident effect in the mutant ferritin clone, whereas it induced an iron-deficient phenotype in the H-ferritin wild type clone, manifested by approximately 5-fold increase of IRPs activity, approximately 2.5-fold increase of transferrin receptor, approximately 1.8-fold increase in iron-transferrin iron uptake, and approximately 50% reduction of labile iron pool. Overexpression of the H-ferritin, but not of the mutant ferritin, strongly reduced cell growth and increased resistance to H(2)O(2) toxicity, effects that were reverted by prolonged incubation in iron-supplemented medium. The results show that in HeLa cells H-ferritin regulates the metabolic iron pool with a mechanism dependent on the functionality of the ferroxidase centers, and this affects, in opposite directions, cellular growth and resistance to oxidative damage. This, and the finding that also in vivo H-chain homopolymers are much less efficient than the H/L heteropolymers in taking up iron, indicate that functional activity of H-ferritin in HeLa cells is that predicted from the in vitro data.

Immunohistochemistry of HFE in the duodenum of C282Y homozygotes with antisera for recombinant HFE protein.

BACKGROUND AND OBJECTIVE: HFE is a class-I MHC related protein which carries the C282Y mutation in most patients with hereditary hemochromatosis, an iron overload disease. HFE protein is expected to have a relevant role in the regulation of duodenal iron absorption, and HFE protein was immunohistochemically identified in the crypt cells. The aim of the work was to analyze whether the C282Y mutation affects HFE accumulation in the duodenum. DESIGN AND METHODS: We developed antisera for the extracellular portion of recombinant human HFE protein expressed in E. coli. The antisera were specific for HFE protein and the C282Y mutant in immunoblotting, immunoprecipitation and immunocytochemistry experiments of transfected cells, and they did not cross react with HLA antigens in various analyses. The antisera gave positive results in the staining of paraffin-fixed sections of duodenal slices of subjects with hemochromatosis. RESULTS: The antisera stained evident supranuclear granules in all enterocytes of 7 C282Y homozygous subjects, and a dark area in the same region in 3 other C282Y homozygotes. Granular bodies were absent from the duodenal sections of 8 C282Y negative subjects, from 2 C282Y heterozygotes and 3 C282Y homozygotes, with or without hemochromatosis. INTERPRETATION AND CONCLUSIONS: The detection of HFE-protein in granular bodies in the enterocytes of the large majority (77%) of C282Y homozygotes and not in other subjects suggests that the mutation facilitates protein accumulation in the duodenum.

Overexpression of the hereditary hemochromatosis protein, HFE, in HeLa cells induces and iron-deficient phenotype.

A transfectant HeLa cell clone expressing HFE under the control of a tetracycline-repressible promoter was generated. HFE expression was fully repressed by the presence of doxycycline, while it was strongly induced by growth in the absence of doxycycline. HFE accumulation was accompanied by a large (approximately 10-fold) decrease in H- and L-ferritin levels, by a approximately 3-4-fold increase in transferrin receptor, and a approximately 2-fold increase in iron regulatory protein activity. These indices of cellular iron deficiency were reversed by iron supplementation complexes. The overexpressed HFE immunoprecipitated together with transferrin receptor, indicating a physical association which is the likely cause for the observed approximately 30% decrease in 55Fe-transferrin incorporation after 18 h incubation. In the HFE-expressing cells the reduction in transferrin-mediated iron incorporation was partially compensated by a approximately 30% increase in non-transferrin iron incorporation from 55Fe-NTA, evident after prolonged, 18 h, incubations. The findings indicate that HFE binding to transferrin receptor reduces cellular iron availability and regulates the balance between transferrin-mediated and non-transferrin-mediated cellular iron incorporation.

Transient overexpression of human H- and L-ferritin chains in COS cells.

The understanding of the in vitro mechanisms of ferritin iron incorporation has greatly increased in recent years with the studies of recombinant and mutant ferritins. However, little is known about how this protein functions in vivo, mainly because of the lack of cellular models in which ferritin expression can be modulated independently from iron. To this aim, primate fibroblastoid COS-7 cells were transiently transfected with cDNAs for human ferritin H- and L-chains under simian virus 40 promoter and analysed within 66 h. Ferritin accumulation reached levels 300-500-fold higher than background, with about 40% of the cells being transfected. Thus ferritin concentration in individual cells was increased up to 1000-fold over controls with no evident signs of toxicity. The exogenous ferritin subunits were correctly assembled into homopolymers, but did not affect either the size or the subunit composition of the endogenous heteropolymeric fraction of ferritin, which remained essentially unchanged in the transfected and non-transfected cells. After 18 h of incubation with [59Fe]ferric-nitrilotriacetate, cellular iron incorporation was similar in the transfected and non-transfected cells and most of the protein-bound radioactivity was associated with ferritin heteropolymers, while H- and L-homopolymers remained iron-free. Cell co-transfection with cDNAs for H- and L-chains produced ferritin heteropolymers that also did not increase cellular iron incorporation. It is concluded that transient transfection of COS cells induces a high level of expression of ferritin subunits that do not co-assemble with the endogenous ferritins and have no evident activity in iron incorporation/metabolism.

Correlation between conventional disease activity measures in juvenile chronic arthritis.

OBJECTIVE: To estimate in a cross sectional analysis the degree of colinearity among the disease activity measures more commonly used in juvenile chronic arthritis (JCA). METHODS: This study assessed in a single clinical evaluation three subjective variables, three measures of functional capacity, eight articular indices, and two laboratory indicators of systemic inflammation in 55 consecutive children with JCA. The relation between the clinical measures of JCA activity was determined by Pearson correlation coefficients. An r value of 0.7 or greater was considered evidence of colinearity. RESULTS: Among the subjective variables, parent global assessment of overall well being and parent assessment of pain were correlated with each other; the physician assessment of disease activity did not show evidence of colinearity with any other variable. The functional status measures were correlated with each other, but not with the indices of articular inflammation. There was a high degree of colinearity among the articular variables, with the number of active joints and the overall severity score being correlated with each other as well as with all the single articular indices. The laboratory variables were correlated with each other, but not with any of the articular, functional or subjective variables. CONCLUSION: Our results show a high degree of colinearity among the disease activity measures belonging to the same category, whereas this is uncommon for variables that investigate different domains of disease activity. These data underline the need to include the evaluation of each domain in the assessment of JCA activity.

Evidence that residues exposed on the three-fold channels have active roles in the mechanism of ferritin iron incorporation.

Iron is thought to enter the ferritin cavity via the three-fold channel, which is lined in its narrowest part by the residues Asp-131 and Glu-134. We describe here variants of human ferritins with active and inactive ferroxidase centres having Asp-131 and Glu-134 substituted with Ala and Ala or with Ile and Phe respectively. The two types of substitution had similar effects on ferritin functionality: (i) they decreased the amount of iron incorporated from Fe(II) solutions and decreased ferroxidase activity by about 50%; (ii) they inhibited iron incorporation from Fe(III) citrate in the presence of ascorbate; (iii) they resulted in loss of Fe and Tb binding sites; and (iv) they resulted in a marked decrease in the inhibition of iron oxidation by Tb (but not by Zn). In addition, it was found that substitution with Ala of Cys-130 and His-118, both of which face the three-fold channel, decreased the capacity of H-ferritin to bind terbium and to incorporate iron from Fe(III) citrate in the presence of ascorbate. The results indicate that: (i) in three-fold channels are the major sites of iron transfer into the cavity of H- and L-ferritins; (ii) at least two metal binding sites are located on the channels which play an active role in capturing and transferring iron into the cavity; and (iii) the permeability of the channel is apparently not affected by the hydrophilicity of its narrowest part. In addition, it is proposed that iron incorporation from Fe(III) citrate complexes in the presence of ascorbate is a reliable, and possibly more physiological, approach to the study of ferritin functionality.

Evidence that the specificity of iron incorporation into homopolymers of human ferritin L- and H-chains is conferred by the nucleation and ferroxidase centres.

Mammalian ferritins are iron-storage proteins made of 24 subunits of two types: the H- and L-chains. L-chains, in contrast with H-chains, lack detectable ferroxidase activity. When ferritins were subjected to iron loading in vitro with increments near the saturation limit of 4000 Fe atoms per molecule, the homopolymers of human H-chains formed insoluble aggregates, caused by non-specific iron hydrolysis, whereas the homopolymers of L-chains remained soluble and incorporated most of the available iron. To analyse the molecular reasons for the difference, Glu-57 and Glu-60, which are conserved and exposed on the cavity of L-chains, were substituted with His, as in H-chains. The double substitution made the L-homopolymers as sensitive as the H-homopolymers to the iron-induced aggregation, whereas the opposite substitution in the H-chain increased homopolymer resistance to the aggregation only marginally. Millimolar concentrations of citrate and phosphate increased iron incorporation in H-homopolymers by reducing non-specific iron hydrolysis, but inhibited that in L-homopolymers by sequestering available iron. The data indicate that the specific iron incorporation into L-homopolymers is mainly due to the iron-nucleation capacity of Glu-57, Glu-60 and other carboxyl groups exposed on the cavity; in contrast, the specificity of iron incorporation into H-homopolymers is related to its ferroxidase activity, which determines rapid Fe(III) accumulation inside the cavity. The finding that ferroxidase centres are essential for the incorporation of iron in the presence of likely candidates of cellular iron transport, such as phosphate and citrate, confirms their importance in ferritin function in vivo.

Construction of a ferroxidase center in human ferritin L-chain.

Ferritins are 24-mer proteins which store and detoxify intracellular iron. Mammalian ferritins are made of two subunit types, the H- and L-chains, with different functional specificity. The H-chain has a metal-binding site (the ferroxidase center) which confers ferroxidase activity to the protein and accelerates iron incorporation. In the L-chain the center is substituted by a salt bridge. We performed several site-directed mutageneses in the L-chain with the aim to construct the center and confer ferroxidase activity to the protein. Most variants were insoluble and did not refold into homopolymers, probably due to electrostatic repulsion introduced by the substitutions. However, they formed hybrids when they were renatured together with the L- or H-chains. The heteropolymers made of 90% L-chain and 10% of an L-variant with all the ligand residues of the H-chain center had 25-30% of the ferroxidase activity of the H-chain homopolymer. This corresponds to the activity of an H/L heteropolymer with 7% H-chain. It is concluded that: (i) it is possible to construct a ferroxidase center in the L-chain with an activity equivalent to that of the H-chain, (ii) the residues of the center interfere with the folding/assembly of the L-, but not of the H-chain, (iii) heteropolymers can be made even between ferritin subunits with large differences of refolding rates.

The role of the L-chain in ferritin iron incorporation. Studies of homo and heteropolymers.

Mammalian ferritins are 24-meric proteins composed of variable proportions of H and L-subunits. The L-chain, in contrast to the H-chain, lacks detectable ferroxidase activity, and its role in ferritin iron incorporation is unclear. In this study, apoferritins were subjected to iron loading with large iron increments to favour spontaneous iron hydrolysis. The homopolymers of the wild-type H-chain, and of a mutant H-chain with an inactivated ferroxidase centre, formed massive protein aggregates, while the L-chain homopolymers remained mostly soluble. The difference between H and L-ferritins was not related to the rate of iron oxidation or to the presence of preformed iron cores. Heteropolymers were constructed in vitro by co-renaturing different proportions of the H-chain with the L-chain or mutant H-chain with an inactivated ferroxidase centre. After loading with high iron increments, protein aggregation of the heteropolymers was reduced when the L-chain content was above 70 to 80%, either in combination with the wild-type H-chain or with the inactivated mutant H-chain. Under acidic conditions (pH 5.5, 1000 Fe atoms per molecule) the heteropolymers with about 20% H and 80% L-chains incorporated three to fourfold more iron into soluble 24-mers than the homopolymers. The data indicate that ferritins with more than 18 L-chains per molecule have the capacity to lower non-specific iron hydrolysis in bulk solution. This property is possibly due to a specific attraction of the incoming oxidized iron into the cavity and may be related to an effect of the L-chain on the cavity microenvironment. It is concluded that under high iron increments the ferritins with high L:H-chain ratios are the most efficient in incorporating iron, and this goes some way to explain why iron storage tissues contain L-rich isoferritins.

Synthesis and biological activity of digitoxigenin aminoesters.

A series of 3 beta-esters of digitoxigenin (3 beta-hydroxy-14 beta-hydroxy-5 beta-card-20(22)-enolide) with alpha-aminoacids, were synthesized and tested for inotropic activity on the guinea-pig isolated heart and by slow infusion in the cat in comparison with digitoxigenin, Lanatoside C and Strophantin K. Esterification of the 3 beta-hydroxy group of digitoxigenin with various amino acids led to compounds still retaining inotropic activity with low in vivo potency and short duration of action. The compounds are inactive when administered orally.

Synthesis of 6alpha-methyldigitoxigenin 3-acetate.

In order to determine the influence of a 6alpha-methyl group activity, the 6alpha-methyl derivative of digitoxigenin 3-acetate 14 was prepared and pharmacologically tested in comparison with digitoxigenen 3-acetate. The synthesis of 6alpha-methyldigitoxigenin 3-acetate (14) was performed starting from 21-hydroxy-4-pregnene-3,20-dione (1). According to the cardiac activity determined on guinea-pig isolated heart and by slow infusion in the cat, the 6alpha-methyldigitoxigenin 3-acetate (14) is not more active than digitoxigenin 3-acetate.