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SYMPOSIUM: PROGRESS IN PHARMACOGENETICS AND ITS PROMISE FOR MEDICINE

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

Swati S. Ghatpande^*, Pankaj K. Choudhary, Charles T. Quinn and Steven R. Goodman^*,,1

^* Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, Texas 75083-0688; Department of Mathematical Sciences and Statistics, University of Texas at Dallas, Richardson, Texas 75083-0688; Department of Pediatrics, Division of Pediatric Hematology-Oncology, University of Texas Southwestern Medical Center at Dallas and Children’s Medical Center of Dallas, Dallas, Texas 75390-9063; and Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390

To whom requests for reprints should be addressed at ¹ SUNY Upstate Medical University, 1258 Weiskotten Hall, 750 E. Adams Street, Syracuse, NY 13210-2311. E-mail: goodmans{at}upstate.edu

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 anti-oxidant enzymes—catalase, thioredoxin peroxidase and biliver-din 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.

Key Words: sickle cell disease • hydroxyurea • 2D-DIGE • mass spectrometry • catalase • red blood cell