InCampylobacter jejunia new type of chaperone receives hemebfrom ferrochelatase
Jordi Zamarreño BeasMarco A.M. VideiraVal KaravaevaFrederico M. LourençoMafalda R. AlmeidaFilipa L. SousaLı́gia M. Saraiva
0
Citation
41
Reference
10
Related Paper
Abstract:
Abstract Intracellular heme formation and trafficking are fundamental processes in living organisms. Three biogenesis pathways are used by bacteria and archaea to produce iron protoporphyrin IX (heme b ) that diverge after the formation of the common intermediate uroporphyrinogen III (uro’gen III). In this work, we identify and provide a detailed characterization of the enzymes involved in the transformation of uro’gen III into heme. We show that in this organism operates the protoporphyrin-dependent pathway (PPD pathway), in which the last reaction is the incorporation of ferrous iron into the porphyrin ring by the ferrochelatase enzyme. In general, following this final reaction, little is known about how the formed heme b reaches the target proteins. In particular, the chaperons that are thought to be required to traffic heme for incorporation into hemeproteins to avoid the cytotoxicity associated to free heme, remain largely unidentified. We identified in C. jejuni a chaperon-like protein, named CgdH2, that binds heme with a dissociation constant of 4.9 ± 1.0 µM, a binding that is impaired upon mutation of residues histidine 45 and 133. We show that C. jejuni CgdH2 establishes protein-protein interactions with ferrochelatase, which should enable for the observed transfer of heme from ferrochelatase to CgdH2. Phylogenetic analysis revealed that C. jejuni CgdH2 is evolutionarily distinct from the currently known chaperones. Therefore, CgdH2 is a novel chaperone and the first protein identified as an acceptor of the intracellularly formed heme, thus enlarging our understanding of bacterial heme homeostasis.Keywords:
Ferrochelatase
Protoporphyrin IX
Chaperone (clinical)
Abstract 5‐Aminolevulinic acid (5‐ALA) is a natural precursor of protoporphyrin IX (PpIX), which can be used as a photosensitizer in photodynamic therapy (PDT). Accumulation of PpIX in benign meningioma cells has been observed previously, its exploitation for PDT, however, was discouraged by inconsistent results. To evaluate PDT for benign meningiomas, we investigated PpIX synthesis in two human meningioma cell lines (HBL‐52 and BEN‐MEN‐1), their respective extracellular loss of PpIX and corresponding ferrochelatase (FECH) activity as well as their susceptibility to PDT. We demonstrated PpIX production after 5‐ALA administration and minor loss to the extracellular space in both cell lines. However, significantly more (up to five times) PpIX was accumulated in BEN‐MEN‐1 as compared with HBL‐52 cells. FECH activity was 2.7‐fold higher in HBL‐52 compared with BEN‐MEN‐1 cells and accordingly higher FECH levels could be shown in HBL‐52 cells by Western blot analysis. BEN‐MEN‐1 cells were much more sensitive to PDT and cells could be almost completely killed by irradiation doses of 2 J cm −2 , whereas HBL‐52 showed only an insufficient response at this irradiation dose. We conclude that differences in intracellular PpIX concentrations between HBL‐52 and BEN‐MEN‐1 benign meningioma cells were mainly due to differences in FECH activity and that these differences correspond to their susceptibility to 5‐ALA‐induced PDT.
Ferrochelatase
Protoporphyrin IX
Cite
Citations (49)
Ferrochelatase
Protoporphyrin IX
Cite
Citations (7)
Protoporphyrin IX
Ferrochelatase
Cite
Citations (4)
Protoporphyrin IX
Ferrochelatase
Cite
Citations (28)
Bovine skin fibroblasts accumulated protoporphyrin IX when incubated in culture with the porphyrin-heme precursor, delta-aminolevulinic acid (ALA). Fibroblasts from cattle homozygous for erythropoietic protoporphyria (EPP) and with the clinical symptoms of the disease accumulated approximately sixfold greater amounts of protoporphyrin IX than cells from normal control animals. Cells from obligatory heterozygous animals, which are clinically normal, accumulated an intermediate level of protoporphyrin IX. When these cells were incubated with ALA and CaMg EDTA, all types of cells accumulated approximately the same amount of protoporphyrin IX (approximately 500 nmol/mg protein), suggesting that ferrochelatase activity was equally low after inhibition by treatment with CaMg EDTA in all cells. Thus the ratio of protoporphyrin IX accumulation from ALA in cultures treated with CaMg EDTA compared with controls treated with ALA alone was greatest in normal cells, least in EPP cells, and intermediate in the heterozygote cells. These findings suggest that the amount of protoporphyrin IX accumulation from ALA reflects the extent of deficiency of ferrochelatase and is proportional to the dosage of abnormal EPP gene in cultured fibroblasts. Similarly, stimulation of porphyrin accumulation by CaMg EDTA reflects diminished ferrochelatase activity in these cells. Thus, the results of this study demonstrate the usefulness of estimating protoporphyrin IX formation from ALA for the detection of an EPP gene defect in cultured bovine skin fibroblasts.
Ferrochelatase
Erythropoietic protoporphyria
Protoporphyrin IX
Cite
Citations (19)
Ferrochelatase
Protoporphyrin IX
Tetrapyrrole
Hemin
Cite
Citations (17)
Protoporphyrin IX
Cite
Citations (0)
Previous research showed that the heme-requiring human pathogen Haemophilus influenzae lacks the first six of the seven enzymes required for heme synthesis, starting with the precursor, 5-amino levulinic acid. In this study, I demonstrated either directly or by reasonable inference that all 57 strains of H. influenzae examined, including 2 unable to grow on protoporphyrin IX, possess ferrochelatase, which catalyzes heme formation by insertion of Fe2+ into the protoporphyrin IX nucleus and which is the last enzyme in the heme synthetic pathway. Further, I showed that this enzyme can also function in the reverse direction, releasing Fe2+ from heme.
Protoporphyrin IX
Ferrochelatase
Levulinic acid
Cite
Citations (42)
Ferrochelatase
Protoporphyrin IX
Porphobilinogen deaminase
Cite
Citations (11)
Ferrochelatase
Protoporphyrin IX
Abcg2
Efflux
Cite
Citations (5)