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

Ponnikorn S, Panichakul T, Sresanga K, Wongborisuth C, Roytrakul S, Hongeng S, Tungpradabkul S - J Transl Med (2011)

Bottom Line: Ineffective erythropoiesis has been studied in human hematopoietic stem cells, however the distinct apoptotic mechanism was unclear.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand.

ABSTRACT

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

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Pie chart representing the characterization of identified phosphoproteins according to (A) biological processes, (B) molecular functions and (C) cellular localization.
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Figure 3: Pie chart representing the characterization of identified phosphoproteins according to (A) biological processes, (B) molecular functions and (C) cellular localization.

Mentions: After LC-MS/MS analysis of the isolated phosphopeptides, all MS/MS spectra were searched against human protein database. A total of 347 peptides from CD34+ cells were detected and found to correspond to 229 proteins which were encoded by 226 genes (additional file 1). Of the 347 unique peptide identifications, 204 phosphopeptides (58.79%) with 306 phosphorylation sites were identified. The specific amino acid residues of pSer:pThr:pTyr were represented as 67:31:2. The biological characterization of phosphoproteome in CD34+ cells could be classified according to biological process, molecular function and cellular localization (Figure 3). The subcellular protein localization for identified phosphoproteins was available and included several cellular compartments. As expected in an investigation of the proteome, we identified an abundance of cytosol (33%), nucleus (24%) and membrane proteins (23%). The gene ontology analysis of our phosphoproteome revealed several proteins involved in basic molecular functions such as transcription/translation factors (32%), DNA/RNA binding (19%) and catalytic activity (14%). In addition to the biological characterization, the proteins could be identified specifically in metabolic processes (35%) and signal transduction (16%). Moreover, these proteins were categorized by PANTHER cellular pathway classification together with Uniprot gene ontology to match with 5 cellular pathways by various relationships such as protein interactions, modifications and regulation of expressions. However, this does not mean that all protein interactions are directly involved with a certain cellular pathway, but it enables identification of those biochemical pathways that are possibly altered in the HbE/β-thalassemic stem cell. In this study, five selected signaling pathways were represented in Table 1 with protein expression ratios between patients and normal donors greater than 2:1. These proteins were categorized as hematopoietic related proteins and other cellular pathway proteins (Table 1 and additional file 2).


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

Ponnikorn S, Panichakul T, Sresanga K, Wongborisuth C, Roytrakul S, Hongeng S, Tungpradabkul S - J Transl Med (2011)

Pie chart representing the characterization of identified phosphoproteins according to (A) biological processes, (B) molecular functions and (C) cellular localization.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3142509&req=5

Figure 3: Pie chart representing the characterization of identified phosphoproteins according to (A) biological processes, (B) molecular functions and (C) cellular localization.
Mentions: After LC-MS/MS analysis of the isolated phosphopeptides, all MS/MS spectra were searched against human protein database. A total of 347 peptides from CD34+ cells were detected and found to correspond to 229 proteins which were encoded by 226 genes (additional file 1). Of the 347 unique peptide identifications, 204 phosphopeptides (58.79%) with 306 phosphorylation sites were identified. The specific amino acid residues of pSer:pThr:pTyr were represented as 67:31:2. The biological characterization of phosphoproteome in CD34+ cells could be classified according to biological process, molecular function and cellular localization (Figure 3). The subcellular protein localization for identified phosphoproteins was available and included several cellular compartments. As expected in an investigation of the proteome, we identified an abundance of cytosol (33%), nucleus (24%) and membrane proteins (23%). The gene ontology analysis of our phosphoproteome revealed several proteins involved in basic molecular functions such as transcription/translation factors (32%), DNA/RNA binding (19%) and catalytic activity (14%). In addition to the biological characterization, the proteins could be identified specifically in metabolic processes (35%) and signal transduction (16%). Moreover, these proteins were categorized by PANTHER cellular pathway classification together with Uniprot gene ontology to match with 5 cellular pathways by various relationships such as protein interactions, modifications and regulation of expressions. However, this does not mean that all protein interactions are directly involved with a certain cellular pathway, but it enables identification of those biochemical pathways that are possibly altered in the HbE/β-thalassemic stem cell. In this study, five selected signaling pathways were represented in Table 1 with protein expression ratios between patients and normal donors greater than 2:1. These proteins were categorized as hematopoietic related proteins and other cellular pathway proteins (Table 1 and additional file 2).

Bottom Line: Ineffective erythropoiesis has been studied in human hematopoietic stem cells, however the distinct apoptotic mechanism was unclear.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand.

ABSTRACT

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Show MeSH
Related in: MedlinePlus