2D DIGE based proteomics study of erythrocyte cytosol in sickle cell disease: Altered proteostasis and oxidative stress

Abstract

Sickle cell disease (SCD) is a hemolytic disorder caused by a mutation in beta-globin gene and affects millions of people worldwide. Though clinical manifestations of the disease are quite heterogeneous, many of them occur due to erythrocyte sickling at reduced oxygen concentration and vascular occlusion mediated via blood cell adhesion to the vessel wall. We have followed proteomic approach to resolve the differentially regulated proteins of erythrocyte cytosol. The deregulated proteins mainly fall in the group of chaperone proteins such as heat shock protein 70, alpha hemoglobin stabilizing protein, and redox regulators such as aldehyde dehydrogenase and peroxiredoxin-2 proteoforms. Proteasomal subunits are found to be upregulated and phospho-catalase level also got altered. Severe oxidative stress inside erythrocyte is evident from the ROS analysis and Oxyblot(TM) experiments. Peroxiredoxin-2 shows significant dimerization in the SCD patients, a hallmark of oxidative stress inside erythrocytes. One interesting fact is that most of the differentially regulated proteins are also common for hemoglobinopathies such as Eβ thalassemia. These could provide important clues in understanding the pathophysiology of SCD and lead us to better patient management in the future.

Localizing the chaperone activity of erythroid spectrin

“…Human blood samples were collected from healthy volunteers with proper informed consent. Blood samples were obtained from Ramkrishna Mission Seva Pratisthan Hospital, Kolkata, India, with informed written consent of the patients following the guidelines of the Institutional Ethical Committee as elaborated earlier (Basu et al, 2013;Chakrabarti, Bhattacharya, Deb, & Chakraborty, 2013;Mishra et al, 2017).…”

Erythrocyte and platelet proteomics in hematological disorders

“…This resulted in a 20-fold increase of visual detection of protein spots in a Coomassie-stained 17 cm 2D gel (418 spots vs. <20) and using SYPRO RUBY protein stain, they could detect more than 600 protein spots in a 2D gel. Following the novel hemoglobin depletion method, we could study differential protein expression in case of HbE␤ thalassemia [19], SCD [51], and hereditary spherocytosis [12]. Combining a hexapeptide-based ligand library (ProteoMiner beads) technique with 2DE as well as nano-LC-MS/MS techniques, researchers were able to identify an impressive 1578 cytosolic proteins, which included a number of new globin chains not identified before [52].…”

“…Their data indicate that HSP27 and HSP70 play a novel role as erythrocytes membrane protein protectors and as possibly new markers of severity of erythrocyte membrane damage during acute vasoocclusive crises. Very recently our group for the first time has reported differentially regulated proteins of erythrocyte cytosol using proteomics approach [51]. The deregulated proteins mainly consist of chaperone proteins such as HSP70, alpha-hemoglobin-stabilizing protein, and redox regulators such as aldehyde dehydrogenase and Prdx2 proteoforms.…”

Platelet proteomics in thalassemia: Factors responsible for hypercoagulation

“…The resulting Cy3 (primary) and Cy5 (secondary) gel images were processed and analyzed using differential in gel analysis (DIA) module of the DeCyder 6.5 software (Amersham Biosciences) and protein spots that exhibited ࣙ± 1.5fold changes in protein content at least in three DIGE experiments were considered. The gels were poststained with Sypro Ruby (Molecular Probes) for identification of protein spots by methods described earlier [13,14].…”