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.

C-reactive protein (CRP) 3 is a major human acute phase reactant composed of five identical subunits (1, 2). Accumulating evidence demonstrates that the actions of CRP depend on conformation and localization (3-5). CRP is primarily produced by the liver and circulates in the blood as a pentamer but is induced to dissociate into subunits (monomeric CRP, mCRP) upon interaction with the microenvironment at inflammatory loci (6 -18). mCRP exhibits markedly enhanced activities and recognizes an expanded list of binding partners. Following reduction of the intra-subunit disulfide bond, mCRP can be further activated (19). The degradation of mCRP, on the other hand, would generate bioactive fragments showing anti-inflammatory actions (20 -23). The differential contributions of these CRP conformations at distinct locations may therefore account for the intense controversies regarding the function of CRP in animal models and in clinical studies (3-5). mCRP appears to be the major conformation of CRP that regulates local inflammation (3-5), yet how it acts remains incompletely understood. In particular, though the markedly enhanced binding capability of mCRP underlies its actions, little is known through which sites mCRP recognizes diverse ligands. Consequently, no means is available to specifically modulate the actions of mCRP, which is necessary for clarifying the exact contributions of different CRP conformations in vitro and in vivo. The current study was designed to identify the recognition site of mCRP for ligand binding. The results unexpectedly demonstrated cholesterol binding sequence (CBS) as a versatile motif that mediates the interactions of mCRP with different types of ligands, including two newly identified herein. We further showed that synthetic CBS peptide was able to inhibit the proinflammatory actions of mCRP both in vitro and in vivo. Hence, optimized CBS peptide may be developed as a potential inhibitor of mCRP. Experimental ProceduresReagents-Human native CRP (purity Ͼ 99%) purified from ascites was purchased from the BindingSite (Birmingham, UK; catalogue number: BP300.X). mCRP and acylated Cys-mutated mCRP was prepared as described (19,24). Proteins were dialyzed to remove NaN 3 , and passed through Detoxi-Gel Columns (Thermo Fisher Scientific, Rockford, IL; catalogue number: 20344) to remove endotoxin when necessary. CRP peptides (purity Ͼ 98%) were synthesized by Science Peptide Biological Technology (Shanghai, China). Lyophilized peptides were reconstituted aseptically with DMSO at 40 mg/ml and stored at Ϫ20°C in aliquots or kept at 4°C for a maximum of 1 week.

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Congenital cataract is one of the leading causes of human blindness. In this study, we identified a novel, heterozygous c.385G