Probing the specific interactions of AlphaA- crystallin and its aging- and cataract-associated forms with lens cell membrane mimics

探讨 AlphaA-晶状体蛋白及其与衰老和白内障相关的形式与晶状体细胞膜模拟物的特定相互作用

• 批准号: 10667060
• 负责人: Jayanti Pande
• 金额: $ 23.46万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT ALBANY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10667060
• 关键词: Affect; Age; Aging; Binding; C-terminal; Cataract; Cell membrane; Characteristics; Cholesterol; Circular Dichroism; Client; Crystalline Lens; Crystallins; Disulfides; Equilibrium; Heat shock proteins; Human; Individual; Lens Fiber; Lens Opacities; Lipids; Liposomes; Literature; Measurement; Measures; Membrane; Membrane Lipids; Modification; Molecular; Molecular Chaperones; Molecular Weight; Nature; Phospholipids; Play; Population; Post-Translational Protein Processing; Process; Protein Conformation; Protein Family; Proteins; Raman Spectrum Analysis; Recombinants; Rest; Role; Senile Cataract; Senility; Spectroscopy, Fourier Transform Infrared; Sphingomyelins; Structure; Techniques; Testing; age related; alpha-Crystallins; biophysical techniques; deamidation; lens; lens transparency; member; monolayer; mutant; oxidation; prevent; protein aggregation

ABSTRACT The human lens contains high concentrations of the molecular chaperones, alpha-crystallins (ACs), that maintain lens transparency. AC consists of alphaA- (AAC) and alphaB- (ABC) crystallins, both distinct proteins, broadly similar in structure and function. Upon aging and in age-related cataract (ARC), AC is bound to the lens fiber cell membrane which leads to the loss of transparency. It is unclear which of the two proteins (AAC or ABC) binds to the membrane, or if both do, and how; but the binding does promote opacity. Therefore, to eventually delay or mitigate lens opacity it is important to first determine the molecular basis underlying such binding. AAC is primarily lens specific − unlike ABC − and is present at about three times higher concentration. There is more evidence implicating AAC than ABC in direct membrane binding – therefore AAC is our focus here. The lens membrane is unique in composition, and contains elevated levels of sphingomyelin (SM), dihydrosphingomyelin (DSM), and cholesterol. Aging- related protein modifications and membrane compositional changes appear to result in a significant increase in ACs binding to the membrane, especially in the population of AAC-containing intramolecular disulfides (S-S). This raises the question: Do the well-known aging-related AAC modifications, i.e., (i) truncation of C-terminal Ser (AAC-1-172), and (ii) deamidation of Asn 101 (AAC-N101D), or (iii) its “pre- senile” cataract-associated G98R mutant, show increased membrane-binding relative to AAC? To distinguish these processes, we hypothesize that AAC binds to SM (and DSM), aided by other phospholipids (PLs), and modulated by cholesterol in the human lens membrane. The disulfide-oxidized form of AAC may play a role in this process. For clearly defining the role of individual membrane-lipid components, we will use liposomes (membrane mimics with defined compositions) instead of human lens membranes, and recombinant human crystallins. For a comprehensive understanding of the binding process, we will use targeted biophysical techniques: FTIR (for SM, cholesterol, and protein); Raman (for −SH and SS bonds and bond strength); CD (for protein conformation); solution NMR (for residue-specific structural information), and Langmuir balance (to measure lipid-monolayer insertion by AAC). We propose the following specific aims: 1. Characterize AAC binding to the membrane and define the role of, (i) lipid composition, and (ii) oxidized AAC in membrane binding. 2. Determine if (i) membrane binding increases in age-modified AAC forms (AAC-1-172, & AAC-N101D), and (ii) the “pre-senile” G98R mutant; and if (iii) the oxidized age-modified AAC forms play a role in membrane binding.

抽象的 人透镜含有高浓度的分子伴侣，α-晶状体 （ACS），维持晶状体透明度。 AC由αA-（AAC）和Alphab-（ABC）结晶蛋白组成 不同的蛋白质，结构和功能广泛相似。衰老和年龄相关性白内障（ARC）时， AC与透镜纤维细胞膜结合，导致透明度的丧失。目前尚不清楚哪个 两种蛋白质（AAC或ABC）与膜结合，或者两者都会结合，以及如何结合；但是装订确实 促进不透明度。因此，为了最终延迟或减轻镜头不透明度，首先确定很重要 这种结合的分子基础。 AAC是特异性 - 与ABC-不同，并且存在 浓度高约三倍。有更多的证据暗示AAC，而不是直接ABC 膜结合 - 因此，AAC是我们在这里的重点。镜头膜在成分中是独一无二的，并且 含有升高的鞘磷脂（SM），二氢磷酸霉素（DSM）和胆固醇水平。老化- 相关的蛋白质修饰和膜组成变化似乎导致显着 AC与膜结合的增加，特别是在含AAC分子内的群体中 二硫化物（S-S）。这就提出了一个问题：是否著名的与衰老有关的AAC修改，即（i） C末端SER（AAC-172）的截断和（ii）ASN 101（AAC-N101D）的脱氨酸，或（iii）” 与白内障相关的G98R突变体相对于AAC？ 区分这些过程，我们假设AAC与SM（和DSM）结合，由其他 磷脂（PLS），并由人透镜膜中的胆固醇调节。 AAC的二硫化氧化形式可能在此过程中发挥作用。为了明确定义 单个膜脂质成分，我们将使用脂质体（膜模仿带有定义的膜 组成）代替人透镜膜和重组人类晶体。对于 对结合过程的全面了解，我们将使用有针对性的生物物理技术：FTIR （用于SM，胆固醇和蛋白质）；拉曼（用于-SH和SS键和键强）； CD（用于蛋白质） 构象）；解决方案NMR（用于退休特异性结构信息）和Langmuir平衡（ 通过AAC测量脂质单层插入）。我们提出以下具体目标： 1。表征AAC与膜结合并定义（i）脂质组成的作用，（ii） 在膜结合中氧化了AAC。 2。确定（i）膜结合在年龄变化中是否增加 AAC形式（AAC-172和AAC-N101D）和（ii）“临时” G98R突变体；如果（iii）氧化 年龄改性的AAC形式在膜结合中起作用。