Post-translational modification as a response to cellular stress induced by hemoglobin oxidation in sickle cell disease

Abstract

Intracellular oxidative stress and oxidative modification of sickle hemoglobin (HbS) play a role in sickle cell disease (SCD) pathogenesis. Recently, we reported that Hb-dependent oxidative stress induced post-translational modifications (PTMs) of Hb and red blood cell (RBC) membrane proteins of transgenic SCD mice. To identify the mechanistic basis of these protein modifications, we followed in vitro oxidative changes occurring in intracellular Hb obtained from RBCs and RBCderived microparticles (MPs) from the blood of 23 SCD patients (HbSS) of which 11 were on, and 12, off hydroxyurea (HU) treatment, and 5 ethnic matched controls. We used mass spectrometry-based proteomics to characterize these oxidative PTMs on a cross-sectional group of these patients (n = 4) and a separate subgroup of patients (n = 2) studied prior to initiation and during HU treatment. Collectively, these data indicated that band-3 and its interaction network involved in MPs formation exhibited more protein phosphorylation and ubiquitination in SCD patients than in controls. HU treatment reversed these oxidative PTMs back to level observed in controls. These PTMs were also confirmed using orthogonal immunoprecipitation experiments. Moreover, we observed specific markers reflective of oxidative stress, including irreversible oxidation of βCys93 and ubiquitination of Hb βLys145 (and βLys96). Overall, these studies strongly suggest that extensive erythrocyte membrane protein phosphorylation and ubiquitination are involved in SCD pathogenesis and provide further insight into the multifaceted effects of HU treatment. Sickle cell disease (SCD) has long been recognized as a "molecular disease" due to the substitution of valine for glutamic acid at position 6 of the β globin chain. Replacing a negatively charged glutamic acid with valine, causes inter-β subunit hydrophobic packing between positionally equivalent amino acids 1,2. This leads to hemoglobin (Hb) polymerization during deoxygenation causing long Hb fiber formation, RBC deformation (sickling), membrane instability and cellular rupture (hemolysis). The consequential hemolytic anemia, release of Hb and RBC-derived microparticles (MPs) appear to collectively trigger an endothelial inflammatory response 3,4. An additional consequence is the interaction of sickled RBCs with leukocytes and platelets with consequent adhesion to the damaged endothelium leading to vaso-occlusive events and cumulative organ damage 2. During the RBC lifetime, these circulating cells are exposed to a variety of internal and external oxidative stresses, despite the presence of antioxidant systems that maintain Hb in the reduced functional form 5. Hb autoxidation and impaired antioxidant capacity favor a pro-oxidative milieu in sickle RBCs compromising the redox state of RBCs and impairing metabolic processes 6-8. For instance, ferrous (Fe 2+) HbS autoxidizes to the non-functional ferric (Fe 3+) (metHb) species faster than normal Hb (HbA), despite the presence of reducing enzymes, causing metHb accumulation inside the ...

The Providence Mutation (βK82D) in Human Hemoglobin Substantially Reduces βCysteine 93 Oxidation and Oxidative Stress in Endothelial Cells

“…However, there was 1.5 to 2.0-fold increases at the low and middle [H 2 O 2 ] and 1.0-fold at extreme [H 2 O 2 ], in HbS over HbA [ 16 ]. Overall, these data support many of our past and current lab studies that show HbS is oxidatively less stable than wild-type HbA [ 18 , 19 , 20 ].…”

Sickle Cell Disease: Role of Oxidative Stress and Antioxidant Therapy

“…In RBCs from SCD patients, several proteins undergo oxidative-mediated PTPM, including HbS itself and several cytoskeletal proteins. Moreover, revealed in RBCs from transgenic SCD mice, these irreversible PTPM detected in HbS were found in β chain and included the irreversible oxidation of Cys93 and the ubiquitination of Lys96 and Lys145 [ 64 , 65 ]. HbS ubiquitination has been attributed to the accumulation of oxidatively damaged HbS molecules in RBCs as well as in MP and could be due to the likely redox imbalance-dependent proteasomal inhibition in SCD [ 64 ].…”

“…HbS ubiquitination has been attributed to the accumulation of oxidatively damaged HbS molecules in RBCs as well as in MP and could be due to the likely redox imbalance-dependent proteasomal inhibition in SCD [ 64 ]. A recent analysis of RBCs and MP proteome showed, in addition to higher amounts of protein carbonyl groups, increased phosphorylation and ubiquitination of cytoskeletal proteins from patients SCD with respect to control cells [ 65 ]. Interestingly, these PTPM have been identified in band 3, spectrin, ankyrin, carbonic anhydrase, and band 4.1 and were significantly reduced after the treatment in vivo with hydroxyurea [ 65 ].…”

“…A recent analysis of RBCs and MP proteome showed, in addition to higher amounts of protein carbonyl groups, increased phosphorylation and ubiquitination of cytoskeletal proteins from patients SCD with respect to control cells [ 65 ]. Interestingly, these PTPM have been identified in band 3, spectrin, ankyrin, carbonic anhydrase, and band 4.1 and were significantly reduced after the treatment in vivo with hydroxyurea [ 65 ].…”

Sickle cell vaso-occlusion: The dialectic between red cells and white cells

“…Control of the intracellular oxidative stress generated in SCD may diminish membrane instability 223 and cellular activation 49 and consequently vaso-occlusive processes. L-glutamine supplementation was found to ameliorate the redox potential of SRBC 224,225 and was approved by the FDA in 2017 for use in treating SCD in the form of Endari L-glutamine oral powder following findings of a randomized, double-blind, placebo-controlled, multicenter clinical trial that showed that twice-daily administration of this amino acid reduced the frequency of sickle cell crises and hospitalization; 226 however, hurdles in the initiation and adherence to L-glutamine have been reported.…”