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

Abstract

BackgroundHemoglobin 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.MethodsThe 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.ResultsA 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.ConclusionsOur 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.

Enhancement of β-Globin Gene Expression in Thalassemic IVS2-654 Induced Pluripotent Stem Cell-Derived Erythroid Cells by Modified U7 snRNA

“…Moreover, the apoptotic effector protein, activated Caspase 3, was also significantly expressed in a synthetic erythropoiesis model of b-thalassemia derived by knocking down b-globin gene of normal CD34 1 cells [65]. The supporting evidence by phosphoproteomic analysis from hemoglobin E/b-thalassemia CD34 1 cells showed the various apoptosis-related protein upregulations including apoptosis-inducing factor, mitochondria associated 1 (AIFM1) [66]. Therefore, in this study we demonstrated that the apoptosis related-genes, CASP3 (caspase 3) and AFFM1, were highly expressed in patient erythroblasts and decreased in the corrected cells suggesting that the apoptosis cascade process might be less active as a consequential result of the partial restoration of correctly spliced b-globin mRNA.…”

Analysis of protein profiling studies of β‐thalassemia/Hb E disease

“…A total of seven studies have investigated differential protein expression in a range of patient specimens (Table ). Most of the studies have investigated differences between normal controls and β‐thalassemia/Hb E patients [, ], while in one study four cohorts (Normal control, β‐thalassemia/Hb E non‐splenectomized, β‐thalassemia/Hb E splenectomized and β‐thalassemia) were investigated .…”

“…additionally compared between mild and severe forms of β‐thalassemia/Hb E . The studies variously analyzed platelets , erythrocytes , platelet‐free plasma derived microparticles , plasma , erythroid progenitor cells and bone marrow derived CD34+ cells . The predominant separation methodology employed was two‐dimensional gel electrophoresis (2‐DE; ) which was used by five studies , although one study additionally employed two‐dimensional difference in gel electrophoresis (2D‐DIGE), a technique solely employed by Weeraphan et al.…”

“…. One study utilized immobilized metal affinity chromatography (IMAC) to select phosphoproteins with no further protein separation .…”

Phosphoproteomics: Detection, Identification and Importance of Protein Phosphorylation

“…Ineffective erythropoiesis in human hematopoietic stem cells has been implicated in Hemoglobin E/beta-thalassemia. Ponnikorn et al compared the phosphoprofiling of human hematopoietic cells between healthy donors and Hemoglobin E/beta-thalassemia patients (Ponnikorn et al, 2011). They enriched the phoshoproteins by IMAC, followed by LC-MS/MS for identification, and found 229 differentially phosphorylated proteins.…”