Phosphoproteomic analysis of apoptotic hematopoietic stem cells from hemoglobin E/β-thalassemia

<p>Abstract</p> <p>Background</p> <p>Hemoglobin E/β-thalassemia is particularly common in Southeast Asia and has variable symptoms ranging from mild to severe anemia. Previous investigations demonstrated the remarkable symptoms of β-thalassemia in terms of the acceleration of apoptotic cell death. Ineffective erythropoiesis has been studied in human hematopoietic stem cells, however the distinct apoptotic mechanism was unclear.</p> <p>Methods</p> <p>The phosphoproteome of bone marrow HSCs/CD34⁺cells from HbE/β-thalassemic patients was analyzed using IMAC phosphoprotein isolation followed by LC-MS/MS detection. Decyder MS software was used to quantitate differentially expressed proteins in 3 patients and 2 normal donors. The differentially expressed proteins from HSCs/CD34⁺cells were compared with HbE/β-thalassemia and normal HSCs.</p> <p>Results</p> <p>A significant change in abundance of 229 phosphoproteins was demonstrated. Importantly, the analysis of the candidate proteins revealed a high abundance of proteins that are commonly found in apoptotic cells including cytochrome C, caspase 6 and apoptosis inducing factors. Moreover, in the HSCs patients a significant increase was observed in a specific type of phosphoserine/threonine binding protein, which is known to act as an important signal mediator for the regulation of cell survival and apoptosis in HbE/β-thalassemia.</p> <p>Conclusions</p> <p>Our study used a novel method to investigate proteins that influence a particular pathway in a given disease or physiological condition. Ultimately, phosphoproteome profiling in HbE/β-thalassemic stem cells is an effective method to further investigate the cell death mechanism of ineffective erythropoiesis in β-thalassemia. Our report provides a comprehensive phosphoproteome, an important resource for the study of ineffective erythropoiesis and developing therapies for HbE/β-thalassemia.</p

Anti-oxidative properties of MK571 in erythrocytes of beta thalassemia/Hb E patients.

<p>(A) MK571 reduced H₂O₂-induced free radical production. Erythrocytes loaded with DCFH-DA were incubated for 1 h with DMSO (basal), DMSO plus MK571 (basal + MK571), H₂O₂ (10 mM), H₂O₂ plus MK571 (50 µM) or H₂O₂ plus MK571 and GlyH-101 (50 µM). Reactive oxygen species were measured by flow cytometry analysis at excitation wavelength of 490 nm and emission wavelength of 530 nm. Data were expressed as means ± S.E. *, p<0.05; **, p<0.001 compared with H₂O₂-treated group (n = 12). (B) Effect of MK571 on intracellular glutathione levels. Erythrocytes were treated for 1 h without (basal) or with H₂O₂ (10 mM) in the presence or absence of MK571 (50 µM) or MK571 plus GlyH-101 (50 µM) before measurements of GSH levels. Data were expressed as means ± S.E. NS, non-statistical significant difference from basal control; *, p<0.01 compared with basal control; #, p<0.05; ##, p<0.01 compared with H₂O₂-treated group (n = 8).</p

Suppression of erythroid development <it>in vitro</it> by <it>Plasmodium vivax</it>

<p>Abstract</p> <p>Background</p> <p>Severe anaemia due to dyserythropoiesis has been documented in patients infected with <it>Plasmodium vivax,</it> however the mechanism responsible for anaemia in vivax malaria is poorly understood. In order to better understand the role of <it>P. vivax</it> infection in anaemia the inhibition of erythropoiesis using haematopoietic stem cells was investigated.</p> <p>Methods</p> <p>Haematopoietic stem cells/CD34⁺ cells, isolated from normal human cord blood were used to generate growing erythroid cells. Exposure of CD34⁺ cells and growing erythroid cells to <it>P. vivax</it> parasites either from intact or lysed infected erythrocytes (IE) was examined for the effect on inhibition of cell development compared with untreated controls.</p> <p>Results</p> <p>Both lysed and intact infected erythrocytes significantly inhibited erythroid growth. The reduction of erythroid growth did not differ significantly between exposure to intact and lysed IE and the mean growth relative to unexposed controls was 59.4 ± 5.2 for lysed IE and 57 ± 8.5% for intact IE. Interestingly, CD34⁺ cells/erythroid progenitor cells were susceptible to the inhibitory effect of <it>P. vivax</it> on cell expansion. Exposure to <it>P. vivax</it> also inhibited erythroid development, as determined by the reduced expression of glycophorin A (28.1%) and CD 71 (43.9%). Moreover, vivax parasites perturbed the division of erythroid cells, as measured by the Cytokinesis Block Proliferation Index, which was reduced to 1.35 ± 0.05 (<it>P</it>-value < 0.01) from a value of 2.08 ± 0.07 in controls. Neither TNF-a nor IFN-g was detected in the culture medium of erythroid cells treated with <it>P. vivax,</it> indicating that impaired erythropoiesis was independent of these cytokines.</p> <p>Conclusions</p> <p>This study shows for the first time that <it>P. vivax</it> parasites inhibit erythroid development leading to ineffective erythropoiesis and highlights the potential of <it>P. vivax</it> to cause severe anaemia.</p