Separation of cellular iron containing compounds by electrophoresis

Vyoral, Daniel; Petrák, Jir̆ı́; Hradilek, A

doi:10.1007/bf02789087

Cited by 15 publications

(16 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The slower migrating iron labeled bands (*) probably represent a complex of Tf and TfR (Fig. 2, lanes 1–5) [20], [21]. Probing for ferritin shows a major band and minor slower migrating forms probably representing ferritin complexes (Fig.…”

Section: Resultsmentioning

confidence: 97%

Prion Protein Modulates Cellular Iron Uptake: A Novel Function with Implications for Prion Disease Pathogenesis

Singh

Mohan²,

Isaac³

et al. 2009

PLoS ONE

Self Cite

View full text Add to dashboard Cite

Converging evidence leaves little doubt that a change in the conformation of prion protein (PrPC) from a mainly α-helical to a β-sheet rich PrP-scrapie (PrPSc) form is the main event responsible for prion disease associated neurotoxicity. However, neither the mechanism of toxicity by PrPSc, nor the normal function of PrPC is entirely clear. Recent reports suggest that imbalance of iron homeostasis is a common feature of prion infected cells and mouse models, implicating redox-iron in prion disease pathogenesis. In this report, we provide evidence that PrPC mediates cellular iron uptake and transport, and mutant PrP forms alter cellular iron levels differentially. Using human neuroblastoma cells as models, we demonstrate that over-expression of PrPC increases intra-cellular iron relative to non-transfected controls as indicated by an increase in total cellular iron, the cellular labile iron pool (LIP), and iron content of ferritin. As a result, the levels of iron uptake proteins transferrin (Tf) and transferrin receptor (TfR) are decreased, and expression of iron storage protein ferritin is increased. The positive effect of PrPC on ferritin iron content is enhanced by stimulating PrPC endocytosis, and reversed by cross-linking PrPC on the plasma membrane. Expression of mutant PrP forms lacking the octapeptide-repeats, the membrane anchor, or carrying the pathogenic mutation PrP102L decreases ferritin iron content significantly relative to PrPC expressing cells, but the effect on cellular LIP and levels of Tf, TfR, and ferritin is complex, varying with the mutation. Neither PrPC nor the mutant PrP forms influence the rate or amount of iron released into the medium, suggesting a functional role for PrPC in cellular iron uptake and transport to ferritin, and dysfunction of PrPC as a significant contributing factor of brain iron imbalance in prion disorders.

show abstract

Section: Resultsmentioning

confidence: 97%

Prion Protein Modulates Cellular Iron Uptake: A Novel Function with Implications for Prion Disease Pathogenesis

Singh

Mohan²,

Isaac³

et al. 2009

PLoS ONE

Self Cite

View full text Add to dashboard Cite

show abstract

“…The difference between LIP in protists and vertebrate cells is its abundance. In various human cell lines the LIP band corresponded to 5-30 % of cellular iron, while a greater part of intracellular iron is stored in ferritin (Vyoral et al, 1998). In protists, which do not possess ferritin, >60 % of iron appeared as chelationsensitive LIP.…”

Section: Discussionmentioning

confidence: 95%

“…'Mobile' iron is also referred to as the labile-iron pool (LIP) as it is easily removed from ligands by iron chelators. It can be visualized and quantified by means of native gradient electrophoresis followed by storage phosphorimaging (Vyoral & Petrák, 1998b;Vyoral et al, 1998). An alternative model for intracellular iron transport was proposed by Richardson et al (1996).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Incorporation of iron into Tritrichomonas foetus cell compartments reveals ferredoxin as a major iron-binding protein in hydrogenosomes

et al. 2003

Self Cite

View full text Add to dashboard Cite

The intracellular transport of iron and its incorporation into organelles are poorly understood processes in eukaryotes and virtually unknown in parasitic protists. The transport of iron is of particular interest in trichomonads, which possess hydrogenosomes instead of mitochondria. The metabolic functions of hydrogenosomes, which contain a specific set of FeS proteins, entirely depend on iron acquisition. In this work the incorporation of iron into the cattle parasite Tritrichomonas foetus was monitored. Iron was efficiently taken up from 59Fe-nitrilotriacetic acid and accumulated in the cytosol (88·9 %) and hydrogenosomes (4·7 % of the total radioactivity). Using atomic absorption spectrophotometry, an unusually high steady-state iron concentration in hydrogenosomes was determined [54·4±1·1 nmol Fe (mg protein)−1]. The concentration of iron in the cytosol was 13·4±0·5 nmol Fe (mg protein)−1. Qualitative analysis of incorporated iron was performed using native gradient PAGE. The majority of the 59Fe in the cytosol appeared as the labile-iron pool, which represents weakly bound iron associated with compounds of molecular mass ranging from 5000 to 30 000 Da. Ferritin was not observed in Tt. foetus, nor in two other anaerobic protists, Entamoeba histolytica and Giardia intestinalis. Analysis of Tt. foetus hydrogenosomes showed at least nine iron-binding compounds, which were absent in metronidazole-resistant mutants. The major iron-binding compound was identified as [2Fe–2S] ferredoxin of the adrenodoxin type.

show abstract

“…55 Fetransferrin was subjected to degradation by preparations of gingipains. Briefly, equal volumes of 55 Fe-transferrin (1 mg/ml), dithiothreitol (40 mM), Tris-HCl buffer (200 mM, pH 7.2), and gingipains (300 U/ml) were incubated at 37°C for 2 h. To preserve the interaction between iron and transferrin or transferrin fragments, samples were analyzed by nondenaturing nonreducing electrophoresis on a 3 to 20% polyacrylamide gradient gel in the presence of Triton X-100, as previously described by Vyoral et al (39). Following electrophoresis, the gel was dried and exposed at Ϫ80°C for 24 h to Kodak Biomax film (Kodak, Rochester, N.Y.) by using an intensifying screen (Transcreen-Low Energy; Kodak).…”

Section: Methodsmentioning

confidence: 99%

Cleavage of Human Transferrin by Porphyromonas gingivalis Gingipains Promotes Growth and Formation of Hydroxyl Radicals

et al. 2004

View full text Add to dashboard Cite

Porphyromonas gingivalis, a gram-negative anaerobic bacterium associated with active lesions of chronic periodontitis, produces several proteinases which are presumably involved in host colonization, perturbation of the immune system, and tissue destruction. The aims of this study were to investigate the degradation of human transferrin by gingipain cysteine proteinases of P. gingivalis and to demonstrate the production of toxic hydroxyl radicals (HO ⅐ ) catalyzed by the iron-containing transferrin fragments generated or by release of iron itself. Analysis by polyacrylamide gel electrophoresis and Western immunoblotting showed that preparations of Arg-and Lys-gingipains of P. gingivalis cleave transferrin (iron-free and iron-saturated forms) into fragments of various sizes. Interestingly, gingival crevicular fluid samples from diseased periodontal sites but not samples from healthy periodontal sites contained fragments of transferrin. By using 55 Fe-transferrin, it was found that degradation by P. gingivalis gingipains resulted in the production of free iron, as well as iron bound to lower-molecular-mass fragments. Subsequent to the degradation of transferrin, bacterial cells assimilated intracellularly the radiolabeled iron. Growth of P. gingivalis ATCC 33277, but not growth of an Arg-gingipainand Lys-gingipain-deficient mutant, was possible in a chemically defined medium containing 30% ironsaturated transferrin as the only source of iron and peptides, suggesting that gingipains play a critical role in the acquisition of essential growth nutrients. Finally, the transferrin degradation products generated by Arg-gingipains A and B were capable of catalyzing the formation of HO ⅐ , as determined by a hypoxanthine/ xanthine oxidase system and spin trapping-electron paramagnetic resonance spectrometry. Our study indicates that P. gingivalis gingipains degrade human transferrin, providing sources of iron and peptides. The iron-containing transferrin fragments or the release of iron itself may contribute to tissue destruction by catalyzing the formation of toxic HO ⅐ .Periodontitis is a chronic inflammatory disorder of the periodontium initiated by an overgrowth of specific bacterial species and characterized by the destruction of tooth-supporting connective tissues, including the alveolar bone. Porphyromonas gingivalis, a gram-negative anaerobic bacterium, has been implicated as a major etiological agent in the onset and progression of chronic periodontitis (33). Arg-and Lys-gingipain cysteine proteinases, which occur in multiple molecular forms due to proteolytic processing of the initially translated polypeptides, are the main endopeptidases produced by P. gingivalis (12,15,16). Two genes code for Arg-gingipains (rgpA and rgpB), and one gene codes for Lys-gingipain (kgp) (12,15,16). An increasing number of reports have stressed the potential roles of Arg-and Lys-gingipains of P. gingivalis in the pathogenesis of this disease, as they are able to degrade a variety of tissue and plasma proteins and have the potential...

show abstract

Separation of cellular iron containing compounds by electrophoresis

Cited by 15 publications

References 21 publications

Prion Protein Modulates Cellular Iron Uptake: A Novel Function with Implications for Prion Disease Pathogenesis

Prion Protein Modulates Cellular Iron Uptake: A Novel Function with Implications for Prion Disease Pathogenesis

Incorporation of iron into Tritrichomonas foetus cell compartments reveals ferredoxin as a major iron-binding protein in hydrogenosomes

Cleavage of Human Transferrin by Porphyromonas gingivalis Gingipains Promotes Growth and Formation of Hydroxyl Radicals

Contact Info

Product

Resources

About