Laboratory of Biochemistry and Vascular Biology, Center for Biologics Evaluation and Research, Food and Drug Administration (FDA), 10903 New Hampshire Avenue, Building 52/72, Room 4106, Silver Spring, MD, 20993, USA.
Hemoglobin Oxygen Therapeutics, Souderton, PA, USA.
Sci Rep. 2020 Aug 26;10(1):14218. doi: 10.1038/s41598-020-71096-6.
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 RBC-derived 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.
细胞内氧化应激和镰状血红蛋白 (HbS) 的氧化修饰在镰状细胞病 (SCD) 的发病机制中起作用。最近，我们报道了 Hb 依赖性氧化应激诱导转基因 SCD 小鼠的 Hb 和红细胞 (RBC) 膜蛋白的翻译后修饰 (PTM)。为了确定这些蛋白质修饰的机制基础，我们在体外观察了从 RBC 获得的细胞内 Hb 以及来自 23 名 SCD 患者（HbSS）的 RBC 衍生的微颗粒 (MP) 中发生的氧化变化，其中 11 名患者正在接受羟基脲 (HU) 治疗，12 名患者已停止 HU 治疗，还有 5 名种族匹配的对照者。我们使用基于质谱的蛋白质组学技术来描述这些患者（n = 4）和另一组在开始 HU 治疗前和治疗期间进行研究的患者（n = 2）的这些氧化 PTM。总的来说，这些数据表明，与对照组相比，在 SCD 患者中，参与 MPs 形成的带-3 及其相互作用网络表现出更多的蛋白质磷酸化和泛素化。HU 治疗将这些氧化 PTM 逆转回对照组的水平。这些 PTM 也通过正交免疫沉淀实验得到了证实。此外，我们观察到了反映氧化应激的特异性标志物，包括βCys93 的不可逆氧化和 Hb βLys145（和βLys96）的泛素化。总体而言，这些研究强烈表明，广泛的红细胞膜蛋白磷酸化和泛素化参与了 SCD 的发病机制，并进一步深入了解 HU 治疗的多方面影响。
