Nano-assemblies enhance chaperone activity, stability, and delivery of alpha B-crystallin-D3 (αB-D3)

Crystallin proteins are the major structural constituents of the eye lens that control lens transparency and refractivity [1,2]. In fact, alterations in the molecular structure of crystallins in the lens is known to be a key player in the formation of cataracts or loss of lens transparency [1,[3], [4], [5], [6]]. They are also present in numerous cell types and tissues including those of the brain, retina, and defects in the crystallin protein structure and/or expression in these tissues are associated with various degenerative diseases [7]. Three major classes of crystallins alpha-, beta-, and gamma- are predominantly present in the lens fiber cells of vertebrates [8]. Of these, alpha crystallin exists in two forms consisting of A and B subunits that associate to form oligomeric complexes of variable molecular weights [1]. The αB-crystallins belong to the family of small heat shock proteins (sHSP) with chaperone activity and appear early in the lens epithelium during embryonic development [1,[9], [10], [11]]. Various studies indicated that the absence of αB-crystallins in mutant mouse models leads to several eye complications including cataracts, retinal cell damage, endophthalmitis, and uveitis [12]. Also, upregulation of αB-crystallin in several different models of diabetes strongly suggests that they may have a protective role in this disease [12]. In addition to a possible neuroprotective role, αB-crystallin is thought to control vascular permeability, neovascularization, and angiogenesis in the oxygen induced retinopathy (OIR) and choroidal neovascularization (CNV) models, possibly through modulation of vascular endothelial growth factor (VEGF) [13].

In addition to ocular tissues, αB-crystallin is expressed in other organs. This αB-crystallin expression has been observed to be associated with cells or tissues which experience protein misfolding and in aggregation-associated diseases like Parkinson's disease [14], multiple sclerosis [15], and Alzheimer's [16,17]. Chaperone function of αB-crystallin by itself has been well established in various in vitro protein aggregation models where it has been shown to protect proteins from aggregating under reducing conditions [18] and heat stress [[18], [19], [20]]. Furthermore, it reduces amyloid fibril formation under in vitro stress conditions [21,22,23]. The ability of αB-crystallin to protect the activity of enzymes such as glucose-6-phosphate dehydrogenase, and glutathione reductase has been reported [24,25]. Thus, these studies clearly indicate the therapeutic potential of αB-crystallin in treating various protein aggregation associated diseases.

The αB-crystallin protein undergoes post-translation modification, predominantly phosphorylation, at serine residues positioned at 19, 45, and 59 [26,27]. A phosphorylation-mimicking αB-crystallin-D3 (αB-D3; also referred to as 3D-αB, αB-STD) mutant with three aspartates in place of serine at positions 19, 45, and 59 display higher chaperone activity when compared to the wild type protein [26,28]. Since oligomeric forms of αB-crystallin are known to exhibit chaperone activity and are likely involved in prevention of protein aggregation under various stress and disease conditions [12,18,29], administration of oligomeric αB-crystallin variants such as αB-D3 in stable and active slow-release nano- and micro- sized formulations may be of therapeutic value in treating eye diseases. Our prior analysis indicated that persistence of intravitreally injected drugs increases with an increase in drug molecular weight [30]. Thus, the purpose of this study was to develop oligomeric nano- and micro- sized supramolecular assemblies of αB-D3 and assess their stability, activity, and intracellular delivery. Assemblies of αB-D3 were created by controlled addition of zinc chloride (ZnCl2) and were assessed for stability using circular dichroism and fluorescence spectroscopy. Additionally, the influence of assembly size on chaperone function was assessed by measuring the ability of the assemblies to prevent β-mercaptoethanol induced insulin aggregation and loss of activity. Further, intracellular delivery of assemblies in a human retinal pigment epithelial (ARPE-19) cell line was monitored using fluorometry and microscopy. The potential applications of αB-D3 nano-assemblies include treatment of protein aggregation diseases, prolongation of vitreal persistence, and prevention of pharmaceutical protein aggregation.

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