Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 3 de 3
Filter
Add more filters










Database
Type of study
Language
Publication year range
1.
Photochem Photobiol ; 89(1): 163-72, 2013.
Article in English | MEDLINE | ID: mdl-22862424

ABSTRACT

Mitochondrial frataxin is involved in various functions such as iron homeostasis, iron-sulfur cluster biogenesis, the protection from oxidative stress and apoptosis and acts as a tumor suppressor protein. We now show that the expression of frataxin is stimulated in a p53-dependent manner and prove that frataxin is a direct p53 target gene by showing that the p53-responsive element in the promoter of the mouse frataxin gene is bound by p53. The bacterial expression of human frataxin stimulated maturation of human ferrochelatase, which catalyzes the insertion of iron into protoporphyrin at the last step of heme biosynthesis. Overexpression of frataxin in human cancer A431 and HeLa cells lowered 5-aminolevulinic acid(ALA)-induced accumulation of protoporphyrin and induced resistance to ALA-induced photo-damage, whereas p53 silencing with siRNA in non tumor HEK293T cells down-regulated the expression of frataxin and increased the accumulation of protoporphyrin. Thus, the decrease of the expression of frataxin unregulated by p53 in tumor cells enhances ALA-induced photo-damage, by down-regulation of mitochondrial functions.


Subject(s)
Aminolevulinic Acid/pharmacology , Iron-Binding Proteins/genetics , Mitochondria/radiation effects , Protoporphyrins/biosynthesis , Tumor Suppressor Protein p53/genetics , Aminolevulinic Acid/metabolism , Animals , Cell Line, Tumor , Ferrochelatase/genetics , Ferrochelatase/metabolism , Gene Expression Regulation, Neoplastic , Humans , Iron/metabolism , Iron-Binding Proteins/metabolism , Mice , Mitochondria/genetics , Mitochondria/metabolism , Promoter Regions, Genetic , Protein Binding , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Response Elements , Signal Transduction , Tumor Suppressor Protein p53/antagonists & inhibitors , Tumor Suppressor Protein p53/metabolism , Frataxin
2.
Biochem Pharmacol ; 71(1-2): 42-9, 2005 Dec 19.
Article in English | MEDLINE | ID: mdl-16288996

ABSTRACT

Photodynamic therapy (PDT) using delta-aminolevulinic acid (ALA)-induced accumulation of protoporphyrin IX is a useful approach to the early detection and treatment of cancers. To investigate the role of ferrochelatase in the accumulation of protoporphyrin, we first made mouse fibroblast Balb/3T3 cells highly expressing ferrochelatase and examined the ALA-induced photo-damage as well as the accumulation of porphyrin in the cells. When the ferrochelatase-transfected cells were treated with ALA and then exposed to visible light, they became resistant to the light without accumulating porphyrins, with a concomitant increase in the formation of heme. The accumulation of protoporphyrin was also abolished in human erythroleukemia K562 cells stably expressing mouse ferrochelatase. When mouse fibrosarcoma MethA cells, mouse fibroblast L929 cells and Balb/3T3 cells were treated with ALA, the greatest accumulation of protoporphyrin and the greatest level of cell death in response to the light were observed in MethA cells. The expression level of ferrochelatase was the lowest in MethA cells, while that of porphobilinogen deaminase was similar among all three cell lines. Moreover, an iron-chelator, desferrioxamine, which sequesters iron preventing the ferrochelatase reaction, enhanced the photo-damage as well as the accumulation of protoporphyrin in ALA-treated L929 cells. Thus, the light-induced cell death was tightly coupled with the accumulation of protoporphyrin caused by a decrease in ferrochelatase. Finally, we examined the uptake of ALA by MethA, L929 and Balb/3T3 cells. The extent of the uptake by MethA and L929 cells was greater, indicating a greater accumulation of protoporphyrin than in the Balb/3T3 cells. Taken together, not only the low level of ferrochelatase but also the augmented uptake of ALA contributes to the ALA-induced accumulation of protoporphyrin IX and subsequent photo-damage in cancer cells.


Subject(s)
Aminolevulinic Acid/pharmacology , Ferrochelatase/metabolism , Photosensitizing Agents/pharmacology , Protoporphyrins/metabolism , Aminolevulinic Acid/metabolism , Animals , BALB 3T3 Cells , Cell Line, Tumor , Humans , Mice , Photosensitizing Agents/metabolism
3.
Hum Mol Genet ; 14(2): 327-34, 2005 Jan 15.
Article in English | MEDLINE | ID: mdl-15574461

ABSTRACT

Erythropoietic protoporphyria (EPP) is an autosomal inherited disease of heme biosynthesis caused by a partial deficiency of the enzyme ferrochelatase. Patients with EPP show only 20-30% normal activity because of mutations in one of the alleles of the ferrochelatase gene. To clarify the molecular mechanisms of this low level of activity, we co-expressed human ferrochelatase carrying His- and HA-tags in a tandem fashion in Escherichia coli. Purification of the His-tag-containing enzyme revealed that the His-enzyme forms an oligomer in association with the HA-enzyme, and analysis by gel-filtration confirmed that the enzyme is a dimer (approximately 80 kDa). Then we expressed homo- and heterodimers composed of the wild-type and engineered mutants of the enzyme (C395Delta, H157A, H263A, H388A) or mutants from EPP patients (I186T, M267I). The levels of homodimeric enzymes produced were low, and the activities of the purified homodimeric mutants were abolished. On the other hand, the heterodimers with wild-type and mutated subunits exhibited potential, but weak, activities without a marked change of Km values for substrates. These results showed that heterodimers containing normal and mutated subunits retain the enzymic activity, which is inconsistent with the hypothesis that ferrochelatase is only active when the dimer contains two normal subunits. Pretreatment at 42 degrees C led to a rapid inactivation of the heterodimeric mutants, indicating instability. Thus, we provide evidence that the instability of the heterodimer containing normal and mutated ferrochelatase as well as the low production levels due to the structural defect of the mutant protein, not the abolishment of the enzymic activity of the heterodimer, causes the weak activity in EPP patients.


Subject(s)
Ferrochelatase/genetics , Protoporphyria, Erythropoietic/genetics , Dimerization , Ferrochelatase/metabolism , Genetic Vectors , Humans , Mutation , Plasmids , Protoporphyria, Erythropoietic/enzymology
SELECTION OF CITATIONS
SEARCH DETAIL
...