Lanosterol reverses protein aggregation in cataracts.

The human lens is comprised largely of crystallin proteins assembled into a highly ordered, interactive macro-structure essential for lens transparency and refractive index. Any disruption of intra- or inter-protein interactions will alter this delicate structure, exposing hydrophobic surfaces, with consequent protein aggregation and cataract formation. Cataracts are the most common cause of blindness worldwide, affecting tens of millions of people, and currently the only treatment is surgical removal of cataractous lenses. The precise mechanisms by which lens proteins both prevent aggregation and maintain lens transparency are largely unknown. Lanosterol is an amphipathic molecule enriched in the lens. It is synthesized by lanosterol synthase (LSS) in a key cyclization reaction of a cholesterol synthesis pathway. Here we identify two distinct homozygous LSS missense mutations (W581R and G588S) in two families with extensive congenital cataracts. Both of these mutations affect highly conserved amino acid residues and impair key catalytic functions of LSS. Engineered expression of wild-type, but not mutant, LSS prevents intracellular protein aggregation of various cataract-causing mutant crystallins. Treatment by lanosterol, but not cholesterol, significantly decreased preformed protein aggregates both in vitro and in cell-transfection experiments. We further show that lanosterol treatment could reduce cataract severity and increase transparency in dissected rabbit cataractous lenses in vitro and cataract severity in vivo in dogs. Our study identifies lanosterol as a key molecule in the prevention of lens protein aggregation and points to a novel strategy for cataract prevention and treatment.

Membrane insertion of αA-crystallin is oligomer-size dependent.

Vertebrate lens is one of the tissues with the highest soluble protein concentration. The predominant soluble proteins in lens fiber cells are crystallins, and among them, α-crystallins belong to the small heat shock protein family with chaperone-like activity. Although α-crystallins are highly soluble in waters, α-crystallins have been detected in the membrane-bound fraction of lens, which will increase in the aged or cataractous lens. In this research, we found αA-crystallin exhibited a complex thermal transition with remarkable changes in secondary and quaternary structures. Treatment of αA-crystallin at high temperatures induced larger oliogomers with higher hydrophobic exposure. Both heat-treated and untreated αA-crystallin could insert into lipid monolayer directly as revealed by monolayer surface pressure experiments. Heat-treatment facilitated the membrane insertion of αA-crystallin and increased the membrane-bound fraction in the cells. The membrane-binding ability of αA-crystallin could be altered by cataract-causing mutations R116C, R116H and Y118D. Our results suggested that the irreversible changes in oligomer size induced by various stresses might promote the membrane association of αA-crystallin and therefore might play a role in aged cataract. Alternations in the membrane binding ability of α-crystallins might be important to the understanding of both aged and congenital cataracts.

Cataract-causing mutation R233H affects the stabilities of ßB1- and ßA3/ßB1-crystallins with different pH-dependence.

Disease-causing mutations can be stabilizing or destabilizing. Missense mutations of structural residues are generally destabilizing, while stabilizing mutations are usually linked to alterations in protein functions. Stabilizing mutations are rarely identified in mutations linked to congenital cataract, a disease caused by the opacification of the lens. In this research, we found that R233H mutation had little impact on ßB1-crystallin structure, solubility and thermal stability under neutral solution pH conditions. The mutation increased ßB1 stability against guanidine hydrochloride-induced denaturation, suggesting that Arg233 might be a functional residue. Further analysis indicated that the R233H mutation did not affect the formation of ßA3/ßB1 heteromer, but significantly reduced heteromer stability against heat- and guanidine hydrochloride-induced denaturation. The R233H mutation negatively affected the thermal stabilities and aggregatory propensities of ßB1 and ßA3/ßB1 with different pH-dependence, implying that the protonation of His side chains during acidification played a regulatory role in crystallin stability and aggregation. Molecular dynamic simulations indicated that Arg233 is one of the residues forming an inter-subunit ion-pairing network with intrinsically dynamic nature. Based on these observations, we proposed that the highly dynamic ion-pairing network contributed to the tradeoff among ßB1 solubility, stability, aggregatory propensity and function of protecting ßA3.