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In-Depth, Label-Free Analysis of the Erythrocyte Cytoplasmic Proteome in Diamond Blackfan Anemia Identifies a Unique Inflammatory Signature.

Pesciotta EN, Lam HS, Kossenkov A, Ge J, Showe LC, Mason PJ, Bessler M, Speicher DW - PLoS ONE (2015)

Bottom Line: Label-free quantitation and statistical analysis identified 29 proteins with significantly altered abundance levels in DBA patients compared to matched healthy control donors.Proteins that were significantly increased in DBA erythrocyte cytoplasms included three proteasome subunit beta proteins that make up the immunoproteasome and proteins induced by interferon-γ such as n-myc interactor and interferon-induced 35 kDa protein [NMI and IFI35 respectively].These results show that erythrocytes in DBA patients are intrinsically different from those in healthy controls which may be due to an inflammatory response resulting from the inherent molecular defect of ribosomal protein haploinsufficiency or changes in the bone marrow microenvironment that leads to red cell aplasia in DBA patients.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology, Department of Pediatrics, Children's Hospital of Philadelphia, University of Pennsylvania, 3615 Civic Center Blvd Philadelphia, PA, 19104, United States of America; Center for Systems and Computational Biology and Molecular and Cellular Oncogenesis Program, The Wistar Institute, 3601 Spruce St. Philadelphia, PA, 19104, United States of America.

ABSTRACT
Diamond Blackfan Anemia (DBA) is a rare, congenital erythrocyte aplasia that is usually caused by haploinsufficiency of ribosomal proteins due to diverse mutations in one of several ribosomal genes. A striking feature of this disease is that a range of different mutations in ribosomal proteins results in similar disease phenotypes primarily characterized by erythrocyte abnormalities and macrocytic anemia, while most other cell types in the body are minimally affected. Previously, we analyzed the erythrocyte membrane proteomes of several DBA patients and identified several proteins that are not typically associated with this cell type and that suggested inflammatory mechanisms contribute to the pathogenesis of DBA. In this study, we evaluated the erythrocyte cytosolic proteome of DBA patients through in-depth analysis of hemoglobin-depleted erythrocyte cytosols. Simple, reproducible, hemoglobin depletion using nickel columns enabled in-depth analysis of over 1000 cytosolic erythrocyte proteins with only moderate total analysis time per proteome. Label-free quantitation and statistical analysis identified 29 proteins with significantly altered abundance levels in DBA patients compared to matched healthy control donors. Proteins that were significantly increased in DBA erythrocyte cytoplasms included three proteasome subunit beta proteins that make up the immunoproteasome and proteins induced by interferon-γ such as n-myc interactor and interferon-induced 35 kDa protein [NMI and IFI35 respectively]. Pathway analysis confirmed the presence of an inflammatory signature in erythrocytes of DBA patients and predicted key upstream regulators including mitogen activated kinase 1, interferon-γ, tumor suppressor p53, and tumor necrosis factor. These results show that erythrocytes in DBA patients are intrinsically different from those in healthy controls which may be due to an inflammatory response resulting from the inherent molecular defect of ribosomal protein haploinsufficiency or changes in the bone marrow microenvironment that leads to red cell aplasia in DBA patients.

No MeSH data available.


Related in: MedlinePlus

Heatmap of significantly changed proteins.Protein intensity z-scores of significantly changed proteins listed in Table 2. Red denotes high relative protein intensity while blue indicates low intensity with black dots signifying zero values that were imputed for statistical analysis.
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pone.0140036.g006: Heatmap of significantly changed proteins.Protein intensity z-scores of significantly changed proteins listed in Table 2. Red denotes high relative protein intensity while blue indicates low intensity with black dots signifying zero values that were imputed for statistical analysis.

Mentions: Statistical analysis was performed to identify proteins that were significantly changed in the DBA patient cohort. Proteins were considered significantly changed if the difference was significant as determined by the student t-test (p < 0.01) and greater than 3-fold (Table 2). Based on these criteria, far more proteins were significantly increased in DBA patients (24 proteins) than showed a decrease (5 proteins) as demonstrated by the heatmap of protein intensity z-scores (Fig 6). Western blots of six proteins that were increased in DBA patients (VCL, NMI, PSMB8, ACTN4, GARS, and GBP1) and one protein that was decreased in DBA patients (TFG) were performed to validate the proteomic label-free data analysis (Fig 7A). Overall, there was good agreement between the western results and label-free quantitation. To further evaluate the consistency between western results and protein quantitation, densitometric values from immunoblots of VCL were compared to LC-MS protein intensities (Fig 7B). A strong positive correlation was observed with an r2 of 0.82 (p = 0.001) (Fig 7B). Finally, fluorescence microscopy of cells stained for VCL and erythrocyte membrane protein band 3 confirmed that these proteins could be detected in cells with the expected biconcave shape of erythrocytes (Fig 7C). Similarly, ACTN4 and GBP1 were visualized by immunofluorescence within the cytosol of patient D3, although the signals were weak presumably due to poor antibodies and low protein abundance, respectively (data not shown).


In-Depth, Label-Free Analysis of the Erythrocyte Cytoplasmic Proteome in Diamond Blackfan Anemia Identifies a Unique Inflammatory Signature.

Pesciotta EN, Lam HS, Kossenkov A, Ge J, Showe LC, Mason PJ, Bessler M, Speicher DW - PLoS ONE (2015)

Heatmap of significantly changed proteins.Protein intensity z-scores of significantly changed proteins listed in Table 2. Red denotes high relative protein intensity while blue indicates low intensity with black dots signifying zero values that were imputed for statistical analysis.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4608755&req=5

pone.0140036.g006: Heatmap of significantly changed proteins.Protein intensity z-scores of significantly changed proteins listed in Table 2. Red denotes high relative protein intensity while blue indicates low intensity with black dots signifying zero values that were imputed for statistical analysis.
Mentions: Statistical analysis was performed to identify proteins that were significantly changed in the DBA patient cohort. Proteins were considered significantly changed if the difference was significant as determined by the student t-test (p < 0.01) and greater than 3-fold (Table 2). Based on these criteria, far more proteins were significantly increased in DBA patients (24 proteins) than showed a decrease (5 proteins) as demonstrated by the heatmap of protein intensity z-scores (Fig 6). Western blots of six proteins that were increased in DBA patients (VCL, NMI, PSMB8, ACTN4, GARS, and GBP1) and one protein that was decreased in DBA patients (TFG) were performed to validate the proteomic label-free data analysis (Fig 7A). Overall, there was good agreement between the western results and label-free quantitation. To further evaluate the consistency between western results and protein quantitation, densitometric values from immunoblots of VCL were compared to LC-MS protein intensities (Fig 7B). A strong positive correlation was observed with an r2 of 0.82 (p = 0.001) (Fig 7B). Finally, fluorescence microscopy of cells stained for VCL and erythrocyte membrane protein band 3 confirmed that these proteins could be detected in cells with the expected biconcave shape of erythrocytes (Fig 7C). Similarly, ACTN4 and GBP1 were visualized by immunofluorescence within the cytosol of patient D3, although the signals were weak presumably due to poor antibodies and low protein abundance, respectively (data not shown).

Bottom Line: Label-free quantitation and statistical analysis identified 29 proteins with significantly altered abundance levels in DBA patients compared to matched healthy control donors.Proteins that were significantly increased in DBA erythrocyte cytoplasms included three proteasome subunit beta proteins that make up the immunoproteasome and proteins induced by interferon-γ such as n-myc interactor and interferon-induced 35 kDa protein [NMI and IFI35 respectively].These results show that erythrocytes in DBA patients are intrinsically different from those in healthy controls which may be due to an inflammatory response resulting from the inherent molecular defect of ribosomal protein haploinsufficiency or changes in the bone marrow microenvironment that leads to red cell aplasia in DBA patients.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology, Department of Pediatrics, Children's Hospital of Philadelphia, University of Pennsylvania, 3615 Civic Center Blvd Philadelphia, PA, 19104, United States of America; Center for Systems and Computational Biology and Molecular and Cellular Oncogenesis Program, The Wistar Institute, 3601 Spruce St. Philadelphia, PA, 19104, United States of America.

ABSTRACT
Diamond Blackfan Anemia (DBA) is a rare, congenital erythrocyte aplasia that is usually caused by haploinsufficiency of ribosomal proteins due to diverse mutations in one of several ribosomal genes. A striking feature of this disease is that a range of different mutations in ribosomal proteins results in similar disease phenotypes primarily characterized by erythrocyte abnormalities and macrocytic anemia, while most other cell types in the body are minimally affected. Previously, we analyzed the erythrocyte membrane proteomes of several DBA patients and identified several proteins that are not typically associated with this cell type and that suggested inflammatory mechanisms contribute to the pathogenesis of DBA. In this study, we evaluated the erythrocyte cytosolic proteome of DBA patients through in-depth analysis of hemoglobin-depleted erythrocyte cytosols. Simple, reproducible, hemoglobin depletion using nickel columns enabled in-depth analysis of over 1000 cytosolic erythrocyte proteins with only moderate total analysis time per proteome. Label-free quantitation and statistical analysis identified 29 proteins with significantly altered abundance levels in DBA patients compared to matched healthy control donors. Proteins that were significantly increased in DBA erythrocyte cytoplasms included three proteasome subunit beta proteins that make up the immunoproteasome and proteins induced by interferon-γ such as n-myc interactor and interferon-induced 35 kDa protein [NMI and IFI35 respectively]. Pathway analysis confirmed the presence of an inflammatory signature in erythrocytes of DBA patients and predicted key upstream regulators including mitogen activated kinase 1, interferon-γ, tumor suppressor p53, and tumor necrosis factor. These results show that erythrocytes in DBA patients are intrinsically different from those in healthy controls which may be due to an inflammatory response resulting from the inherent molecular defect of ribosomal protein haploinsufficiency or changes in the bone marrow microenvironment that leads to red cell aplasia in DBA patients.

No MeSH data available.


Related in: MedlinePlus