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Mitochondrial Changes in β0-Thalassemia/Hb E Disease.

Khungwanmaythawee K, Sornjai W, Paemanee A, Jaratsittisin J, Fucharoen S, Svasti S, Lithanatudom P, Roytrakul S, Smith DR - PLoS ONE (2016)

Bottom Line: While a number of factors are known that moderate presentation, these factors do not account for the full spectrum of presentation.Differences in relative mitochondria number and gene expression were seen primarily in day 10 cells.The evidence presented here suggest that there are inherent differences in these cells as early as the erythroid progenitor cell stage, and that maximum deficit is seen coincident with high levels of globin gene expression.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biosciences, Mahidol University, Salaya campus, 25/25 Phuttamontol Sai 4, Salaya, Nakorn Pathom, 73170, Thailand.

ABSTRACT
The compound β°-thalassemia/Hb E hemoglobinopathy is characterized by an unusually large range of presentation from essentially asymptomatic to a severe transfusion dependent state. While a number of factors are known that moderate presentation, these factors do not account for the full spectrum of presentation. Mitochondria are subcellular organelles that are pivotal in a number of cellular processes including oxidative phosphorylation and apoptosis. A mitochondrial protein enriched proteome was determined and validated from erythroblasts from normal controls and β°-thalassemia/Hb E patients of different severities. Mitochondria were evaluated through the use of mitotracker staining, analysis of relative mitochondrial genome number and evaluation of mitochondrial gene expression in addition to assay of overall cellular redox status through the use of alamarBlue assays. Fifty differentially regulated mitochondrial proteins were identified. Mitotracker staining revealed significant differences in staining between normal control erythroblasts and those from β°-thalassemia/Hb E patients. Differences in relative mitochondria number and gene expression were seen primarily in day 10 cells. Significant differences were seen in redox status as evaluated by alamarBlue staining in newly isolated CD34+ cells. Mitochondria mediate oxidative phosphorylation and apoptosis, both of which are known to be dysregulated in differentiating erythrocytes from β°-thalassemia/Hb E patients. The evidence presented here suggest that there are inherent differences in these cells as early as the erythroid progenitor cell stage, and that maximum deficit is seen coincident with high levels of globin gene expression.

No MeSH data available.


Related in: MedlinePlus

The mitochondrial protein enriched proteome of erythroid cells.Mitochondrial protein enriched preparations from day 10 erythroid cells from normal controls and β°-thalassemia/Hb E patients (mild and severe) were subjected to GelC-MS/MS analysis. Venn diagrams of (a) total proteins identified and (b) mitochondrial proteins as identified by the Mitoproteome database and GoCat ontological analysis of (c) cellular processes and (d) functional categorization of the identified mitochondrial proteins and GoCat ontological analysis of (e) cellular processes and (f) functional categorization of the identified differentially regulated proteins.
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pone.0153831.g001: The mitochondrial protein enriched proteome of erythroid cells.Mitochondrial protein enriched preparations from day 10 erythroid cells from normal controls and β°-thalassemia/Hb E patients (mild and severe) were subjected to GelC-MS/MS analysis. Venn diagrams of (a) total proteins identified and (b) mitochondrial proteins as identified by the Mitoproteome database and GoCat ontological analysis of (c) cellular processes and (d) functional categorization of the identified mitochondrial proteins and GoCat ontological analysis of (e) cellular processes and (f) functional categorization of the identified differentially regulated proteins.

Mentions: To investigate the profiles of mitochondria associated proteins in β°-thalassemia/Hb E patients as compared to normal controls, mitochondria enriched proteins were obtained from day 10 erythroblasts of five normal controls, five severe and five mild β°-thalassaemia/Hb E patients, using anti-TOM22 microbeads to purify mitochondria followed by protein extraction. The protocol was shown to significantly enrich for mitochondrial proteins as shown by Western blotting for NADH:ubiquinone oxidoreductase subunit A9 (NDUFA9; Fig A in S1 File). The proteins were analyzed by a gel-enhanced liquid chromatography tandem mass spectroscopy (GeLC-MS/MS) [14]. Original gels are shown in Fig B in S1 File. Resultant analysis of the generated spectra identified 4392 peptides corresponding to 1837 proteins, of which some 1428 were not differentially expressed between samples (Fig 1a). Ontological analysis of these proteins for biological process and molecular function is shown in Fig 1c and 1d. The spectra were subsequently screened against the Mitoproteome database on 10th and 19th June 2013 which identified some 288 mitochondrial proteins, representing approximately 40% of the database annotated mitochondrial proteome. Of these 288 proteins, three proteins showed significantly increased expression in mild β°-thalassemia/Hb E patients only, while a further five proteins showed significantly increased expression in severe β°-thalassemia/Hb E patients only. A total of forty-two proteins were significantly up-regulated in both mild and severe β°-thalassemia/Hb E patients as compared to normal controls (Fig 1b and Table B in S1 File). The heat map is shown in Fig C in S1 File. Interestingly, no protein was detected as showing down-regulation in β°-thalassemia/Hb E erythroblasts as compared to normal control erythroblasts. To validate the proteomic data, western blot analysis of two proteins identified as differentially expressed was undertaken on samples from an independent cohort of patients and controls. The proteins selected, heat shock protein 60 (hsp60) and prohibitin2 are both well characterized mitochondrial chaperone proteins and both have been shown to have additional, albeit predominantly antagonistic, roles in the regulation of apoptosis (reviewed in [15]). As the proteome data showed discordant expression of these two proteins (hsp60 up in severe and prohibitin 2 down in severe) despite their similar chaperone functions their expression profile was considered to be important to validate. Both proteins showed results consistent with the proteome data (Fig 2).


Mitochondrial Changes in β0-Thalassemia/Hb E Disease.

Khungwanmaythawee K, Sornjai W, Paemanee A, Jaratsittisin J, Fucharoen S, Svasti S, Lithanatudom P, Roytrakul S, Smith DR - PLoS ONE (2016)

The mitochondrial protein enriched proteome of erythroid cells.Mitochondrial protein enriched preparations from day 10 erythroid cells from normal controls and β°-thalassemia/Hb E patients (mild and severe) were subjected to GelC-MS/MS analysis. Venn diagrams of (a) total proteins identified and (b) mitochondrial proteins as identified by the Mitoproteome database and GoCat ontological analysis of (c) cellular processes and (d) functional categorization of the identified mitochondrial proteins and GoCat ontological analysis of (e) cellular processes and (f) functional categorization of the identified differentially regulated proteins.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4836671&req=5

pone.0153831.g001: The mitochondrial protein enriched proteome of erythroid cells.Mitochondrial protein enriched preparations from day 10 erythroid cells from normal controls and β°-thalassemia/Hb E patients (mild and severe) were subjected to GelC-MS/MS analysis. Venn diagrams of (a) total proteins identified and (b) mitochondrial proteins as identified by the Mitoproteome database and GoCat ontological analysis of (c) cellular processes and (d) functional categorization of the identified mitochondrial proteins and GoCat ontological analysis of (e) cellular processes and (f) functional categorization of the identified differentially regulated proteins.
Mentions: To investigate the profiles of mitochondria associated proteins in β°-thalassemia/Hb E patients as compared to normal controls, mitochondria enriched proteins were obtained from day 10 erythroblasts of five normal controls, five severe and five mild β°-thalassaemia/Hb E patients, using anti-TOM22 microbeads to purify mitochondria followed by protein extraction. The protocol was shown to significantly enrich for mitochondrial proteins as shown by Western blotting for NADH:ubiquinone oxidoreductase subunit A9 (NDUFA9; Fig A in S1 File). The proteins were analyzed by a gel-enhanced liquid chromatography tandem mass spectroscopy (GeLC-MS/MS) [14]. Original gels are shown in Fig B in S1 File. Resultant analysis of the generated spectra identified 4392 peptides corresponding to 1837 proteins, of which some 1428 were not differentially expressed between samples (Fig 1a). Ontological analysis of these proteins for biological process and molecular function is shown in Fig 1c and 1d. The spectra were subsequently screened against the Mitoproteome database on 10th and 19th June 2013 which identified some 288 mitochondrial proteins, representing approximately 40% of the database annotated mitochondrial proteome. Of these 288 proteins, three proteins showed significantly increased expression in mild β°-thalassemia/Hb E patients only, while a further five proteins showed significantly increased expression in severe β°-thalassemia/Hb E patients only. A total of forty-two proteins were significantly up-regulated in both mild and severe β°-thalassemia/Hb E patients as compared to normal controls (Fig 1b and Table B in S1 File). The heat map is shown in Fig C in S1 File. Interestingly, no protein was detected as showing down-regulation in β°-thalassemia/Hb E erythroblasts as compared to normal control erythroblasts. To validate the proteomic data, western blot analysis of two proteins identified as differentially expressed was undertaken on samples from an independent cohort of patients and controls. The proteins selected, heat shock protein 60 (hsp60) and prohibitin2 are both well characterized mitochondrial chaperone proteins and both have been shown to have additional, albeit predominantly antagonistic, roles in the regulation of apoptosis (reviewed in [15]). As the proteome data showed discordant expression of these two proteins (hsp60 up in severe and prohibitin 2 down in severe) despite their similar chaperone functions their expression profile was considered to be important to validate. Both proteins showed results consistent with the proteome data (Fig 2).

Bottom Line: While a number of factors are known that moderate presentation, these factors do not account for the full spectrum of presentation.Differences in relative mitochondria number and gene expression were seen primarily in day 10 cells.The evidence presented here suggest that there are inherent differences in these cells as early as the erythroid progenitor cell stage, and that maximum deficit is seen coincident with high levels of globin gene expression.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biosciences, Mahidol University, Salaya campus, 25/25 Phuttamontol Sai 4, Salaya, Nakorn Pathom, 73170, Thailand.

ABSTRACT
The compound β°-thalassemia/Hb E hemoglobinopathy is characterized by an unusually large range of presentation from essentially asymptomatic to a severe transfusion dependent state. While a number of factors are known that moderate presentation, these factors do not account for the full spectrum of presentation. Mitochondria are subcellular organelles that are pivotal in a number of cellular processes including oxidative phosphorylation and apoptosis. A mitochondrial protein enriched proteome was determined and validated from erythroblasts from normal controls and β°-thalassemia/Hb E patients of different severities. Mitochondria were evaluated through the use of mitotracker staining, analysis of relative mitochondrial genome number and evaluation of mitochondrial gene expression in addition to assay of overall cellular redox status through the use of alamarBlue assays. Fifty differentially regulated mitochondrial proteins were identified. Mitotracker staining revealed significant differences in staining between normal control erythroblasts and those from β°-thalassemia/Hb E patients. Differences in relative mitochondria number and gene expression were seen primarily in day 10 cells. Significant differences were seen in redox status as evaluated by alamarBlue staining in newly isolated CD34+ cells. Mitochondria mediate oxidative phosphorylation and apoptosis, both of which are known to be dysregulated in differentiating erythrocytes from β°-thalassemia/Hb E patients. The evidence presented here suggest that there are inherent differences in these cells as early as the erythroid progenitor cell stage, and that maximum deficit is seen coincident with high levels of globin gene expression.

No MeSH data available.


Related in: MedlinePlus