scholarly journals Overexpression of Wild Type and Mutated Human Ferritin H-chain in HeLa Cells

2000 ◽  
Vol 275 (33) ◽  
pp. 25122-25129 ◽  
Author(s):  
Anna Cozzi ◽  
Barbara Corsi ◽  
Sonia Levi ◽  
Paolo Santambrogio ◽  
Alberto Albertini ◽  
...  
Keyword(s):  
Hela Cells ◽  
Wild Type ◽  
Human Ferritin ◽  
Ferritin H

scholarly journals Biochemical, Biophysical and Functional Characterization of an Insoluble Iron Containing Hepcidin–Ferritin Chimeric Monomer Assembled Together with Human Ferritin H/L Chains at Different Molar Ratios

2021 ◽  
Vol 44 (1) ◽  
pp. 117-127
Author(s):  
Mohamed Boumaiza ◽  
Imene Fhoula ◽  
Fernando Carmona ◽  
Maura Poli ◽  
Michela Asperti ◽  
...  
Keyword(s):  
Iron Homeostasis ◽  
Functional Characterization ◽  
Wild Type ◽  
E Coli ◽  
Molar Ratios ◽  
Human Ferritin ◽  
Stable Product ◽  
Soluble Molecule ◽  
Ferritin H

Hepcidin and ferritin are key proteins of iron homeostasis in mammals. In this study, we characterize a chimera by fusing camel hepcidin to a human ferritin H-chain to verify if it retained the properties of the two proteins. The construct (HepcH) is expressed in E. coli in an insoluble and iron-containing form. To characterize it, the product was incubated with ascorbic acid and TCEP to reduce and solubilize the iron, which was quantified with ferrozine. HepcH bound approximately five times more iron than the wild type human ferritin, due to the presence of the hepcidin moiety. To obtain a soluble and stable product, the chimera was denatured and renatured together with different amounts of L-ferritin of the H-chain in order to produce 24-shell heteropolymers with different subunit proportions. They were analyzed by denaturing and non-denaturing PAGE and by mass spectroscopy. At the 1:5 ratio of HepcH to H- or L-ferritin, a stable and soluble molecule was obtained. Its biological activity was verified by its ability to both bind specifically cell lines that express ferroportin and to promote ferroportin degradation. This chimeric molecule showed the ability to bind both mouse J774 macrophage cells, as well as human HepG2 cells, via the hepcidin–ferroportin axis. We conclude that the chimera retains the properties of both hepcidin and ferritin and might be exploited for drug delivery.

scholarly journals Effects of modifications near the 2-, 3- and 4-fold symmetry axes on human ferritin renaturation

1997 ◽  
Vol 322 (2) ◽  
pp. 461-468 ◽  
Author(s):  
Paolo SANTAMBROGIO ◽  
Patrizia PINTO ◽  
Sonia LEVI ◽  
Anna COZZI ◽  
Ermanna ROVIDA ◽  
...  
Keyword(s):  
Guanidine Hydrochloride ◽  
Building Blocks ◽  
Fold Axis ◽  
Wild Type ◽  
Chemical Modifications ◽  
Specific Chemical ◽  
Large Hysteresis ◽  
N Terminus ◽  
Human Ferritin ◽  
Ferritin H

Ferritin is a protein of 24 subunits which assemble into a shell with 432 point symmetry. It can be denatured reversibly in acidic guanidine hydrochloride, with the formation of poorly populated renaturation intermediates. In order to increase the accumulation of intermediates and to study the mechanism of ferritin renaturation, we analysed variants of the human ferritin H-chain altered at the N-terminus (Δ1–13), near the 4-fold axis (Leu-169→Arg), the 3-fold axis (Asp-131→Ile+Glu-134→Phe) or the 2-fold axis (Ile-85→Cys). We also carried out specific chemical modifications of Cys-130 (near the 3-fold axis) and Cys-85 (near the 2-fold axis). Renaturation of the modified ferritins yielded assembly intermediates that differed in size and physical properties. Alterations of residues around the 2-, 4- and 3-fold axes produced subunit monomers, dimers and higher oligomers respectively. All these intermediates could be induced to assemble into ferritin 24-mers by concentrating them or by co-renaturing them with wild-type H-ferritin. The results support the hypothesis that the symmetric subunit dimers are the building blocks of ferritin assembly, and are consistent with a reassembly pathway involving the coalescence of dimers, probably around the 4-fold axis, followed by stepwise addition of dimers until the 24-mer cage is completed. In addition they show that assembly interactions are responsible for the large hysteresis of folding and unfolding plots. The implications of the studies for in vivoheteropolymer formation in vertebrates, which have two types of ferritin chain (H and L), are discussed.

Transferrin-induced endocytosis in stably transfected HeLa cells expressing wild-type and C282Y-mutant HFE measured by patch-clamp capacitance techniques

2001 ◽  
Vol 120 (5) ◽  
pp. A564-A565
Author(s):  
L SCHWAKE ◽  
A HENKEL ◽  
H RIEDEL ◽  
B HADASCHIK ◽  
T SCHLENKER ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Hela Cells ◽  
Wild Type

Characterization of human ferritin H chain synthetized in Escherichia coli

Gene ◽  
1987 ◽  
Vol 51 (2-3) ◽  
pp. 269-274 ◽  
Author(s):  
Sonia Levi ◽  
Gianni Cesareni ◽  
Paolo Arosio ◽  
Rolando Lorenzetti ◽  
Marco Soria ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Human Ferritin ◽  
Ferritin H

scholarly journals Multiple Elements Controlling Adherence of Enterohemorrhagic Escherichia coli O157:H7 to HeLa Cells

2003 ◽  
Vol 71 (9) ◽  
pp. 4985-4995 ◽  
Author(s):  
Alfredo G. Torres ◽  
James B. Kaper
Keyword(s):  
Escherichia Coli ◽  
Hela Cells ◽  
Culture Conditions ◽  
Enterohemorrhagic Escherichia Coli ◽  
Differentially Expressed ◽  
Wild Type ◽  
Transposon Insertion ◽  
Reduced Adherence ◽  
Insertion Mutants

ABSTRACT Adherence of enterohemorrhagic Escherichia coli (EHEC) to the intestinal epithelium is essential for initiation of infection. Intimin is the only factor demonstrated to play a role in intestinal colonization by EHEC O157:H7. Other attempts to identify additional adhesion factors in vitro have been unsuccessful, suggesting that expression of these factors is under tight regulation. We sought to identify genes involved in the control of adherence of EHEC O157:H7 to cultured epithelial cells. A total of 5,000 independent transposon insertion mutants were screened for their ability to adhere to HeLa cells, and 7 mutants were isolated with a markedly enhanced adherence. The mutants adhered at levels 113 to 170% that of the wild-type strain, and analysis of the protein profiles of these mutants revealed several proteins differentially expressed under in vitro culture conditions. We determined the sequence of the differentially expressed proteins and further investigated the function of OmpA, whose expression was increased in a mutant with an insertionally inactivated tcdA gene. An isogenic ompA mutant showed reduced adherence compared to the parent strain. Disruption of the ompA gene in the tdcA mutant strain abolished the hyperadherent phenotype, and anti-OmpA serum inhibited adhesion of wild-type and tdcA mutant strains to HeLa cells. Enhanced adhesion mediated by OmpA was also observed with Caco-2 cells, and anti-OmpA serum blocked adherence to HeLa cells of other EHEC O157:H7 strains. Our results indicate that multiple elements control adherence and OmpA acts as an adhesin in EHEC O157:H7.

Structure and expression of the chicken ferritin H-subunit gene

1987 ◽  
Vol 7 (5) ◽  
pp. 1751-1758
Author(s):  
P W Stevens ◽  
J B Dodgson ◽  
J D Engel
Keyword(s):  
Chicken Genome ◽  
Single Copy ◽  
Transcription Unit ◽  
Subunit Gene ◽  
Conserved Sequence ◽  
Coding Regions ◽  
Mature Transcript ◽  
Multiple Copies ◽  
Human Ferritin ◽  
Ferritin H

Although the genomes of many species contain multiple copies of ferritin heavy (H)- and light (L)-chain sequences, the chicken genome contains only a single copy of the H-subunit gene. The primary transcription unit of this gene is 4.6 kilobase pairs and contains four exons which are posttranscriptionally spliced to generate a mature transcript of 869 nucleotides. Chicken and human ferritin H-subunit genomic loci are organized with similar exon-intron boundaries. They exhibit approximately 85% nucleotide identity in coding regions, which yield proteins 93% identical in amino acid sequence. We have identified a sequence of 22 highly conserved nucleotides in the 5' untranslated sequences of chicken, human, and tadpole ferritin H-subunit genes and propose that this conserved sequence may regulate iron-modulated translation of ferritin H-subunit mRNAs.

scholarly journals Regulation of transferrin-induced endocytosis by wild-type and C282Y-mutant HFE in transfected HeLa cells

2002 ◽  
Vol 282 (5) ◽  
pp. C973-C979 ◽  
Author(s):  
Lukas Schwake ◽  
Andreas W. Henkel ◽  
Hans D. Riedel ◽  
Thorsten Schlenker ◽  
Matthias Both ◽  
...  
Keyword(s):  
Hela Cells ◽  
Single Cells ◽  
Hereditary Hemochromatosis ◽  
Inhibitory Effect ◽  
Membrane Capacitance ◽  
Wild Type ◽  
Time Resolved ◽  
Cell Membrane Capacitance ◽  
Endocytic Vesicles ◽  
In Cells

The hereditary hemochromatosis protein HFE is known to complex with the transferrin receptor; however, its function regarding endocytosis of transferrin is unclear. We performed patch-clamp capacitance measurements in transfected HeLa cells carrying wild-type or C282Y-mutant HFE cDNA under the control of a tetracycline-sensitive promoter. Whole cell experiments in cells with suppressed expression of wild-type HFE revealed a decrease in membrane capacitance, reflecting predominance of endocytosis in the presence of transferrin. Cells overexpressing C282Y-mutant HFE displayed less intense capacitance decreases, whereas no significant decrease was observed in cells overexpressing wild-type HFE. The formation of single endocytic vesicles in cells with suppressed expression of wild-type HFE was greatly increased in the presence of transferrin as revealed by cell-attached recordings. According to their calculated diameters, many of these vesicles corresponded to clathrin-coated vesicles. These results suggest that wild-type HFE negatively modulates the endocytic uptake of transferrin. This inhibitory effect is attenuated in cells expressing C282Y-mutant HFE. Time-resolved measurements of cell membrane capacitance provide a powerful tool to study transferrin-induced endocytosis in single cells.

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