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

摘要 人的透镜含有高浓度的分子伴侣，α-晶体蛋白 (ACs)保持透镜透明度。AC由alphaA-（AAC）和alphaB-（ABC）晶体蛋白组成， 不同的蛋白质，在结构和功能上大致相似。在衰老和年龄相关性白内障（ARC）中， AC与透镜纤维细胞膜结合，导致透明度丧失。目前尚不清楚哪些 两种蛋白质（AAC或ABC）中的一种结合到膜上，或者如果两者都结合，以及如何结合;但是结合不 促进不透明。因此，为了最终延迟或减轻透镜不透明度，重要的是首先确定 这种结合的分子基础。AAC主要是透镜特异性的-与ABC不同-并且存在于 浓度高出三倍有更多的证据表明，AAC比ABC直接 膜结合-因此AAC是我们的重点。该透镜膜在组成上是独特的， 含有高水平的鞘磷脂（SM）、二氢鞘磷脂（DSM）和胆固醇。老化- 相关的蛋白质修饰和膜组成的变化似乎导致了显着的 与膜结合的AC增加，特别是在含有AAC的分子内 二硫化物（S-S）。这就提出了一个问题：众所周知的与衰老有关的AAC修饰，即，（一） C-末端Ser的截短（AAC-1-172），和（ii）Asn 101的脱酰胺（AAC-N101 D），或（iii）其“前 老年性白内障相关的G98 R突变体，显示增加膜结合相对AAC？到 区分这些过程，我们假设AAC结合SM（和DSM），在其他辅助下， 磷脂（PL），并由人透镜膜中的胆固醇调节。的 二硫化物氧化形式的AAC可能在这一过程中发挥作用。明确界定了 单独的膜脂质组分，我们将使用脂质体（具有限定的 组合物）代替人透镜膜，以及重组人晶状体蛋白。用于 为了全面了解结合过程，我们将使用有针对性的生物物理技术：FTIR (for SM、胆固醇和蛋白质）;拉曼（用于−SH和SS键以及键强度）; CD（用于蛋白质 构象）;溶液NMR（用于残基特异性结构信息）和朗缪尔平衡（以 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与膜的结合，并确定（i）脂质组成和（ii）的作用 膜结合中的氧化AAC。2.确定（i）老化修饰的细胞膜结合是否增加 AAC形式（AAC-1-172，& AAC-N101 D），和（ii）“老年前期”G98 R突变体;和如果（iii）氧化的 老化修饰的AAC形式在膜结合中起作用。

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.