Background:
The employment of the iron chelator deferasirox (DFX) in transfusion‐dependent patients affected by chronic anaemias occurring in several hematological diseases occasionally results in ...recovery of hematopoiesis with hematopoietic stem cells (HSCs) regaining the ability to differentiate into mature blood cells. Despite a growing body of clinical evidence, the mechanism explaining this interesting peculiarity still remains elusive.
Aims:
Aim of this study was to identify a general molecular mechanism underlying DFX beneficial effect on hematopoiesis both in healthy and in pathological conditions.
Methods:
Human healthy HSCs and two leukemia cell lines, Kasumi‐1 and K562, were treated with DFX 100 μM. N‐Acetyl Cysteine (NAC) was added as antioxidant; fludarabine was administered to inhibit STAT1 activation and interferon signaling. In vitro colony‐forming assays were assessed both in healthy and in leukemia cells to functionally test DFX ability to induce differentiation; the growth of CFU‐GM, BFU‐E and CFU‐GEMM colonies was scored and enumerated. Intracellular and mitochondrial reactive oxygen species (ROS) were assessed by cytofluorimetric analysis after DFX treatment for 24 h. The following differentiation markers were monitored after 24 h and 48 h of DFX treatment by cytofluorimetric analysis: CD14‐APC and CD36‐FITC for Kasumi‐1 cells; CD71‐FITC and CD36‐FITC for K562. Gene expression profile was performed on healthy HSCs using Illumina platform and data were analyzed by Ingenuity Pathway Analysis (IPA); differently expressed genes were validated both in healthy and leukemia cells by Realtime PCR. The expression and phosphorylation state of STAT1 were assessed by western blotting.
Results:
DFX, at clinically relevant concentrations, increased the clonogenic capacity of healthy human CD34+ HSC to form erythroid colonies in a ROS dependent fashion and the whole gene expression analysis of DFX‐treated HSCs unveiled up‐regulation of genes linked to interferon (IFN) signaling. The study, extended on human‐derived leukemia cell lines, revealed a DFX‐mediated overproduction of mitochondria‐generated reactive oxygen species (ROS), accompanied by a ROS‐dependent up‐regulated expression of specific markers of hematopoietic commitment. Similarly to HSCs, DFX treatment of Kasumi‐1 and K562 resulted in a significant up‐regulation of IFN‐related genes expression as well as in a marked hyper‐phosphorylation of the signal transducer and activator of transcription 1 (STAT1), known to be involved, in turn, in the IFN signaling. Most notably, treatment of Kasumi‐1 and K562 cell lines with the antioxidant NAC completely prevented the DFX‐mediated phosphorylation of STAT1 as well as the expression of the IFN‐stimulated genes. Conversely, the inhibition of STAT‐1 alone, by Fludarabine, was not sufficient to affect differentiation processes in both cell lines.
Summary/Conclusion:
The novel findings here reported suggest a hitherto strong impact of ROS signaling and strengthen its role as master regulator of multiple orchestrated events triggered by DFX, like IFN pathway up‐regulation and differentiation induction. Interestingly, DFX property seems to be independent on the physiological or pathological state of the cells. The identification of key factors earlier modulated by DFX and able to activate downstream events involved in the regulation of cell survival, proliferation and differentiation, provides new insights that can be developed to anticipate its administration or to extend the use of the drug also to other categories of patients.
A study is presented on the pH dependence of proton translocation in the oxidative and reductive phases of the catalytic cycle of purified cytochrome c oxidase (COX) from beef heart reconstituted in ...phospholipid vesicles (COV). Protons were shown to be released from COV both in the oxidative and reductive phases. In the oxidation by O2 of the fully reduced oxidase, the H+/COX ratio for proton release from COV (R → O transition) decreased from ≈2.4 at pH 6.5 to ≈1.8 at pH 8.5. In the direct reduction of the fully oxidized enzyme (O → R transition), the H+/COX ratio for proton release from COV increased from ≈0.3 at pH 6.5 to ≈1.6 at pH 8.5. Anaerobic oxidation by ferricyanide of the fully reduced oxidase, reconstituted in COV or in the soluble case, resulted in H+ release which exhibited, in both cases, an H+/COX ratio of 1.7−1.9 in the pH range 6.5−8.5. This H+ release associated with ferricyanide oxidation of the oxidase, in the absence of oxygen, originates evidently from deprotonation of acidic groups in the enzyme cooperatively linked to the redox state of the metal centers (redox Bohr protons). The additional H+ release (O2 versus ferricyanide oxidation) approaching 1 H+/COX at pH ≤ 6.5 is associated with the reduction of O2 by the reduced metal centers. At pH ≥ 8.5, this additional proton release takes place in the reductive phase of the catalytic cycle of the oxidase. The H+/COX ratio for proton release from COV in the overall catalytic cycle, oxidation by O2 of the fully reduced oxidase directly followed by re-reduction (R → O → R transition), exhibited a bell-shaped pH dependence approaching 4 at pH 7.2. A mechanism for the involvement in the proton pump of the oxidase of H+/e- cooperative coupling at the metal centers (redox Bohr effects) and protonmotive steps of reduction of O2 to H2O is presented.
A study is presented of the ←H+/e− stoichiometry for H+ pumping by the cytochrome chain in isolated rat liver mitochondria under level-flow and steady-state conditions. It is shown that the ←H+/e− ...stoichiometry for the cytochrome chain varies under the influence of the flow rate and transmembrane ΔμH+. The rate-dependence is shown to be associated with cytochrome c oxidase, whose ←H+/e− ratio varies from 0 to 1, whilst the ←H+/c− ratio for the span covered by cytochrome c reductase is invariably 2.
Cooperative linkage of solute binding at separate binding sites in allosteric proteins is an important functional attribute of soluble and membrane bound hemoproteins. Analysis of proton/electron ...coupling at the four redox centers, i.e. Cu
A, heme
a, heme
a
3 and Cu
B, in the purified bovine cytochrome
c oxidase in the unliganded, CO-liganded and CN-liganded states is presented. These studies are based on direct measurement of scalar proton translocation associated with oxido-reduction of the metal centers and pH dependence of the midpoint potential of the redox centers.
Heme
a (and Cu
A) exhibits a cooperative proton/electron linkage (Bohr effect). Bohr effect seems also to be associated with the oxygen-reduction chemistry at the heme
a
3–Cu
B binuclear center. Data on electron transfer in cytochrome
c oxidase are also presented, which, together with structural data, provide evidence showing the occurrence of direct electron transfer from Cu
A to the binuclear center in addition to electron transfer via heme
a.
A survey of structural and functional data showing the essential role of cooperative proton/electron linkage at heme
a in the proton pump of cytochrome
c oxidase is presented. On the basis of this and related functional and structural information, variants for cooperative mechanisms in the proton pump of the oxidase are examined.
The H+/e− stoichiometry of protonmotive cytochrome c oxidase, isolated from bovine heart mitochondria and reconstituted in liposomes, has been determined by making use of direct spectrophotometric ...measurements of the initial rates of e− flow and H+ translocation. It is shown that the ←H+/e− ratio for redox-linked proton ejection by the oxidase varies from around 0 to a maximum of 1 as a function of the rate of overall electron flow in the complex.
The four-electron reaction cycle of cytochrome oxidase is comprised of an
eu-oxidase phase in which the enzyme receives the first two electrons and reduces oxygen to bound peroxide and a
peroxidase ...phase in which the peroxy state formed in the
eu-oxidase half of the cycle is reduced by the 3rd and 4th electrons to the ferryl-oxo state and oxidized form, respectively. Here we show that the ferrocyanide-peroxidase activity of cytochrome
c oxidase incorporated in phospholipid vesicles is coupled to proton pumping. The H
+/
e
− ratio for the ferrocyanide-
peroxidase partial reaction is twice higher than for the overall ferrocyanide-
oxidase activity and is close to 2. These results show that proton pumping by COX is confined to the
peroxidase part of the enzyme catalytic cycle (transfer of the 3rd and 4th electron) whereas the
eu-oxidase part (transfer of the first two electrons) may not be proton pumping.
The nuclear gene
OXA1 was first isolated in
Saccharomyces cerevisiae and found to be required at a post-translational step in cytochrome
c oxidase biogenesis, probably at the level of assembly. ...Mutations in
OXA1 lead to a complete respiratory deficiency. The protein Oxa1p is conserved through evolution and a human homolog has been isolated by functional complementation of a yeast
oxa1
−
mutant. In order to further our understanding of the role of Oxa1p, we have constructed two yeast strains in which the
OXA1 open reading frame was almost totally deleted. Cytochrome spectra and enzymatic activity measurements show the absence of heme
aa
3 and of a cytochrome
c oxido-reductase activity and dramatic decrease of the oligomycin sensitive ATPase activity. Analysis of the respiratory complexes in non-denaturing gels reveals that Oxa1p is necessary for the correct assembly of the cytochrome
c oxidase and the ATP synthase complex.
A study is presented on proton transfer associated with the reaction of the fully reduced, purified bovine heart cytochrome c oxidase with molecular oxygen or ferricyanide. The proton consumption ...associated with aerobic oxidation of the four metal centers changed significantly with pH going from ≈3.0 H+/COX at pH 6.2−6.3 to ≈1.2 H+/COX at pH 8.0−8.5. Rereduction of the metal centers was associated with further proton uptake which increased with pH from ≈1.0 H+/COX at pH 6.2−6.3 to ≈2.8 H+/COX at pH 8.0−8.5. Anaerobic oxidation of the four metal centers by ferricyanide resulted in the net release of 1.3−1.6 H+/COX in the pH range 6.2−8.2, which were taken up by the enzyme on rereduction of the metal centers. The proton transfer elicited by ferricyanide represents the net result of deprotonation/protonation reactions linked to anaerobic oxidoreduction of the metal centers. Correction for the ferricyanide-induced pH changes of the proton uptake observed in the oxidation and rereduction phase of the reaction of the reduced oxidase with oxygen gave a measure of the proton consumption in the reduction of O2 to 2H2O. The results show that the expected stoichiometric proton consumption of 4H+ in the reduction of O2 to 2H2O is differently associated, depending on the actual pH, with the oxidation and reduction phase of COX. Two H+/COX are initially taken up in the reduction of O2 to two OH- groups bound to the binuclear Fe a 3−CuB center. At acidic pHs the third and fourth protons are also taken up in the oxidative phase with formation of 2H2O. At alkaline pHs the third and fourth protons are taken up with formation of 2H2O only upon rereduction of COX.
HCV-infection induces a state of oxidative stress more pronounced than in many other inflammatory diseases. Here we propose a temporal sequence of events in the HCV-infected cell whereby the primary ...alteration consists in release of Ca
2+ from the ER followed by uptake into mitochondria. This triggers successive mitochondrial dysfunctions leading to generation of ROS and to a progressive metabolic adaptive response. Pathogenetic implications of the model and new opportunities for therapeutic intervention are discussed.
Michael N. Sack and Paul M. Hwang – National Heart Lung and Blood Institute, National Institute of Health, Bethesda, MD, USA
pH changes associated with the mitochondrial cytochrome oxidase reaction with H2O2 have been studied. In the presence of forricyanide or Tris‐phenanthroline complex of CoIII as electron acceptors, ...reaction or H2O2 with the oxidized cytochrome oxidase is accompanied by a steady proton release with a rate constant of ca. 3 M−1 s−1 at pH 6.8. The acidification is completely inhibited by superoxide dismutase and its pro‐steady‐state kinetics correlates with that of the oxoferryl compound (F) accumulation. Apparently, the proton release is linked to superoxide generation by cytochrome oxidase under these conditions. In the presence of superoxide dismutase and without the electron acceptors, the H2O2‐induced transitions of cytochrome oxidase from the oxidized to the peroxy (P) and from the peroxy to the oxoferryl state are not associated with any significant proton release or uptake. The results point to the following mechanism of O−
2 generation and protonation states of the cytochrome oxidase compounds P and F:
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