Targeting spectrin redox switches to regulate the mechanoproperties of red blood cells.

红细胞（RBC）的机械特性是其生理学的基础。为了运输氧气，RBC需要在血管树中的血液动力学变化中存活，以动态地促进流动以降低血管阻力，并且在通过较窄血管期间变形。RBC机械特性主要由其细胞骨架的结构特征赋予，其主要由连接到锚蛋白和α-微管蛋白的节点的血影蛋白支架组成。氧化还原状态的变化和巯基靶向分子的处理降低了RBC的变形性，并影响血影蛋白细胞骨架的结构/稳定性。然而，氧化还原开关的结构特征、分子定位及其与红细胞机械特性、血液粘度和组织灌注的功能联系尚不清楚。我们假设血影蛋白细胞骨架上的半胱氨酸氧化还原开关（CRS）是红细胞中氧化还原信号传导的靶点，并调节红细胞的机械特性，从而有助于调节血液粘度、血流和组织灌注。因此，在本项目中，我们的目标是（1）。鉴定、实验验证和分析推定CRS在红细胞血影蛋白中的作用;（2.）分析CRS改性对RBC力学性能的影响;（3.）探讨血影蛋白CRS在体内红细胞生理学中的功能意义。首先，通过综合建模，我们将在原子水平上生成血影蛋白的结构模型，这将使我们能够识别和定位血影蛋白中假定的CRS，并研究它们的化学修饰如何影响血影蛋白的弹性性质。同时，将通过实验分析CSR修饰对细胞中血影蛋白的结构/功能特征的影响，包括稳定性、机械性质和蛋白质/蛋白质与其他细胞骨架蛋白的相互作用。第二，用生理刺激物处理RBC，诱导细胞内产生反应性物质（即，NO、O2-/H2 O2、硫化物/过硫化物/多硫化物），或细胞氧化还原状态的变化。然后，我们将分析血影蛋白CSR修饰，以及它们对RBC形态和RBC机械特性的影响，包括RBC变形性、渗透稳定性和血液粘度。第三，我们将分析（1）的影响。NO或硫化物的药理学应用，（2.）苯肼引起的氧化还原状态的变化，或（3.）小鼠红细胞内源性NO合成对血影蛋白CRS修饰的遗传调节，以及它们对红细胞功能特性、气体交换和组织灌注的影响。推定血影蛋白CRS的结构和功能表征将允许阐明氧化还原调节对RBC机械性质的作用，并且可以提供新的功能靶点来调节RBC功能、血流和组织灌注。

The mechanical properties of red blood cells (RBCs) are fundamental for their physiology. To transport oxygen, RBCs need to survive the hemodynamic changes in the vascular tree, to dynamically contribute to the flow for decreasing vascular resistance, and to deform during the passage through narrower vessels. RBC mechanoproperties are conferred mainly by the structural characteristics of their cytoskeleton, which mainly consists of a spectrin scaffold connected to nodes of ankyrin and alpha-tubulin. Changes in redox state and treatment with thiol-targeting molecules decrease deformability of RBCs, and affect the structure/stability of the spectrin cytoskeleton. However, structural characteristics, molecular localization of redox switches and their functional link to RBC mechanoproperties, blood viscosity and tissue perfusion are unknown. We hypothesize that cysteine redox switches (CRS) on the spectrin cytoskeleton are targeted by redox signaling in RBCs and modulate RBC mechanical properties, thus, contributing to the modulation of blood viscosity, blood flow, and tissue perfusion. Hence, in this project we aim to (1.) to identify, experimentally validate and analyze the effects of putative CRS in erythrocytic spectrin; (2.) to analyze the effects of CRS modification on RBC mechanical properties; (3.) to investigate the functional significance of spectrin CRS in the physiology of RBCs in vivo. First, by integrative modeling, we will generate a structural model of spectrin at the atomistic level, which will allow us to identify and localize putative CRS in spectrin and to investigate how their chemical modification impacts elastic properties of spectrin. In conjunction, the impact of CSR modifications on structure/functional characteristics of spectrin in cells, including stability, mechanical properties, and protein/protein interactions with other cytoskeletal proteins will be experimentally analyzed. Second, RBC will be treated with physiological stimuli inducing intracellular production of reactive species (i.e., NO, O2-/H2O2, sulfide/persulfide/polysulfide) under normoxia and hypoxia, or changes in the cellular redox state. We will then analyze spectrin CSR modification, and their effects on RBC morphology and RBC mechanical properties, including RBC deformability, osmotic stability, and blood viscosity. Third, we will analyze the effects of (1.) pharmacological application of NO or sulfide, (2.) changes in redox state by phenylhydrazine, or (3.) genetic modulation of endogenous NO synthesis in RBC in mice on spectrin CRS modification, and their effects on RBC functional properties, gas exchange, and tissue perfusion. Structural and functional characterization of putative spectrin CRS will allow to shed light on the role of redox regulation on RBC mechanical properties, and may provide novel functional targets to modulate RBC function, blood flow, and tissue perfusion.