RESUMO
Spherocytosis is one of the most common inherited disorders, yet presents with a wide range of clinical severity. While several genes have been found mutated in patients with spherocytosis, the molecular basis for the variability in severity of haemolytic anaemia is not entirely understood. To identify candidate proteins involved in haemolytic anaemia pathophysiology, we utilized a label-free comparative proteomic approach to detect differences in red blood cells (RBCs) from normal and ß-adducin (Add2) knock-out mice. We detected seven proteins that were decreased and 48 proteins that were increased in ß-adducin null RBC ghosts. Since haemolytic anaemias are characterized by reticulocytosis, we compared reticulocyte-enriched samples from phenylhydrazine-treated mice with mature RBCs from untreated mice. Among the 48 proteins increased in Add2 knockout RBCs, only 11 were also increased in reticulocytes. Of the proteins decreased in Add2 knockout RBCs, α-adducin showed the greatest intensity difference, followed by SLC9A1, the sodium-hydrogen exchanger previously termed NHE1. We verified these mass spectrometry results by immunoblot. This is the first example of SLC9A1deficiency in haemolytic anaemia and suggests new insights into the mechanisms leading to fragile RBCs.
Assuntos
Proteínas de Transporte de Cátions/deficiência , Eritrócitos/metabolismo , Proteínas dos Microfilamentos/deficiência , Animais , Proteínas Sanguíneas/metabolismo , Proteínas de Transporte de Cátions/sangue , Proteínas do Citoesqueleto , Membrana Eritrocítica/metabolismo , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/sangue , Proteômica/métodos , Reticulócitos/metabolismo , Trocador 1 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/sangueRESUMO
Hemolytic anemia is one of the most common inherited disorders. To identify candidate proteins involved in hemolytic anemia pathophysiology, we utilized a label-free comparative proteomic approach to detect differences in RBCs from normal and beta-adducin (Add2) knock-out mice. We detected 7 proteins that were decreased and 48 proteins that were increased in the beta-adducin knock-out RBC ghost. Since hemolytic anemias are characterized by reticulocytosis, we compared reticulocyte-enriched samples from phenylhydrazine-treated mice with mature RBCs from untreated mice. Label-free analysis identified 47 proteins that were increased in the reticulocyte-enriched samples and 21 proteins that were decreased. Among the proteins increased in Add2 knockout RBCs, only 11 were also found increased in reticulocytes. Among the proteins decreased in Add2 knockout RBCs, beta- and alpha-adducin showed the greatest intensity difference, followed by NHE-1 (Slc9a1), the sodium-hydrogen exchanger. We verified these mass spectrometry results by immunoblot. This is the first example of a deficiency of NHE-1 in hemolytic anemia and suggests new insights into the mechanisms leading to fragile RBCs. Our use of label-free comparative proteomics to make this discovery demonstrates the usefulness of this approach as opposed to metabolic or chemical isotopic labeling of mice.
Assuntos
Anemia Hemolítica/genética , Proteínas de Ligação a Calmodulina , Proteínas de Transporte de Cátions/genética , Eritrócitos/metabolismo , Fragilidade Osmótica/genética , Fenil-Hidrazinas/efeitos adversos , Isoformas de Proteínas/genética , Proteômica/métodos , Reticulócitos/metabolismo , Trocadores de Sódio-Hidrogênio/genética , Sequência de Aminoácidos , Anemia Hemolítica/induzido quimicamente , Anemia Hemolítica/metabolismo , Anemia Hemolítica/patologia , Animais , Western Blotting , Proteínas de Ligação a Calmodulina/deficiência , Proteínas de Ligação a Calmodulina/genética , Proteínas de Transporte de Cátions/deficiência , Modelos Animais de Doenças , Contagem de Eritrócitos , Membrana Eritrocítica/genética , Membrana Eritrocítica/metabolismo , Eritrócitos/citologia , Perfilação da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Fenil-Hidrazinas/farmacologia , Isoformas de Proteínas/metabolismo , Contagem de Reticulócitos , Reticulócitos/citologia , Trocador 1 de Sódio-Hidrogênio , Espectrometria de Massas em TandemRESUMO
The ultimate goal of most shotgun proteomic pipelines is the discovery of novel biomarkers to direct the development of quantitative diagnostics for the detection and treatment of disease. Differential comparisons of biological samples identify candidate peptides that can serve as proxys of candidate proteins. While these discovery approaches are robust and fairly comprehensive, they have relatively low throughput. When merged with targeted mass spectrometry, this pipeline can fuel hypothesis-driven studies and the development of novel diagnostics and therapeutics.
Assuntos
Biomarcadores/análise , Proteínas/análise , Proteômica/métodos , Perfilação da Expressão Gênica , Humanos , Isótopos/química , Espectrometria de Massas/métodos , Miocárdio/metabolismo , Peptídeos/química , Espectrometria de Massas em TandemRESUMO
Hematopoietic protein-1 (Hem-1) is a hematopoietic cell specific member of the WAVE (Wiskott-Aldrich syndrome verprolin-homologous protein) complex, which regulates filamentous actin (F-actin) polymerization in many cell types including immune cells. However, the roles of Hem-1 and the WAVE complex in erythrocyte biology are not known. In this study, we utilized mice lacking Hem-1 expression due to a non-coding point mutation in the Hem1 gene to show that absence of Hem-1 results in microcytic, hypochromic anemia characterized by abnormally shaped erythrocytes with aberrant F-actin foci and decreased lifespan. We find that Hem-1 and members of the associated WAVE complex are normally expressed in wildtype erythrocyte progenitors and mature erythrocytes. Using mass spectrometry and global proteomics, Coomassie staining, and immunoblotting, we find that the absence of Hem-1 results in decreased representation of essential erythrocyte membrane skeletal proteins including α- and ß- spectrin, dematin, p55, adducin, ankyrin, tropomodulin 1, band 3, and band 4.1. Hem1â»/â» erythrocytes exhibit increased protein kinase C-dependent phosphorylation of adducin at Ser724, which targets adducin family members for dissociation from spectrin and actin, and subsequent proteolysis. Increased adducin Ser724 phosphorylation in Hem1â»/â» erythrocytes correlates with decreased protein expression of the regulatory subunit of protein phosphatase 2A (PP2A), which is required for PP2A-dependent dephosphorylation of PKC targets. These results reveal a novel, critical role for Hem-1 in the homeostasis of structural proteins required for formation and stability of the actin membrane skeleton in erythrocytes.