Pharmaco-Proteomic Study of Hydroxyurea-Induced Modifications in the Sickle Red Blood Cell Membrane Proteome

Abstract

Hydroxyurea (HU) is an effective oral drug for the management of homozygous sickle cell anemia (SS) in part because it increases fetal hemoglobin (HbF) levels within sickle red blood cells (RBCs) and thus reduces sickling. However, results from the Multicenter Study of HU suggested that clinical symptoms often improved before a significant increase in HbF levels occurred. This indicated that HU may be acting through the modification of additional cellular mechanisms that are yet to be identified. Hence, in this study, we focused on the analysis of the sickle RBC membrane proteome +/− HU treatment. 2D-DIGE (Two Dimensional Difference In-Gel Electrophoresis) technology and tandem mass spectrometry has been used to determine quantitative differences between sickle cell membrane proteins in the presence and absence of a clinically relevant concentration of HU. In vitro protein profiling of 13 sickle RBC membrane samples +/− 50 μM HU identified 10 statistically significant protein spots. Of these, the most remarkable class of proteins to show a statistically significant increase was the antioxidant enzymes—catalase, thioredoxin peroxidase and biliverdin reductase and the chaperonin containing TCP1 complex assisting in the folding of RBC cytoskeletal proteins. Interestingly, catalase immunoblots showed an increase in the acidic forms of the enzyme within sickle RBC membranes on incubation with 50 μM HU. We further identified this modification in catalase to be phosphorylation and demonstrated that HU exposed SS RBC membranes showed a 2-fold increase in tyrosine phosphorylation of catalase as compared to counterparts not exposed to HU. These results present an attractive model for HU-induced post-translational modification and potential activation of catalase in mature sickle RBCs. These findings also identify protein targets of HU other than fetal hemoglobin and enhance the understanding of the drug mechanism.

Monoclonal Antibody against α-Actinin 4 from Human Umbilical Vein Endothelial Cells Inhibits Endothelium-Dependent Vasorelaxation

“…Among the antibodies generated, the membrane antigens to the C-7 antibody in biotinylated HUVECs were Ig heavy chain (VHDJ region) and chaperonin-containing T-complex polypeptide 1, as well as α-actinin 4. Similar to this result, a number of reports showed that these proteins are present on the cell membranes of a variety of cells [36,38,39,40]. These results imply that the proteins identified in this study can be classified as membrane proteins of HUVECs and these methods, e.g., antibody neutralization assay and immunoprecipitation-associated proteomics, are useful for the discovery of functional molecules on the cell membrane.…”

In vivo pharmaco-proteomic analysis of hydroxyurea induced changes in the sickle red blood cell membrane proteome

“…For example, glyceraldehyde-3-phosphate dehydrogenase was identified in spots 20 through 30 with all spots demonstrating a significant increase suggesting HU-induced increase in expression of the protein during erythropoiesis. Of these proteins, p55 was the only common HU target identified in our present in vivo and previous in vitro analyses of HU- induced changes [7]. The only significant decrease was observed in chaperonin containing TCP1 subunit 2, a component of a large multiprotein TCP1 ring complex involved in the proper folding of actin and tubulin in an ATP-dependent way [14].…”

“…Through this in vitro study, we further demonstrated that 50 μM HU exposed sickle RBC membranes showed a 2-fold increase in tyrosine phosphorylation of catalase as compared to counterparts not exposed to HU [7]. The in vitro protein profiling system allowed us to look at the same sickle RBC membrane sample from individual SS patients with and without HU exposure to identify dose-dependent proteomic changes in vitro , which is difficult to achieve in an in vivo clinical setting.…”

The Application of Proteomics to PK-PD Modeling and Simulation

“…Ghatpande et al [38] reported in vitro protein profiling using twodimensional difference gel electrophoresis (2D-DIGE) on sickle red blood cell (RBC) membranes and identified a significant increase in predominantly anti-oxidant enzyme, protein repair and degradation components. The in vitro protein profiling system allowed to look at the same sickle RBC membrane from individual sickle cell anemia (SS) patients with and without drug of hydroxyurea (HU) exposure to identify dose dependent proteomic changes in vitro, which is difficult to achieve in an in vivo clinical setting.…”