Purpose Cataracts can be broadly divided into two types: congenital cataracts

Purpose Cataracts can be broadly divided into two types: congenital cataracts and age-related cataracts. transfected in HeLa cells, and changes of proteinCprotein interactions were analyzed by the luciferase assay. Results Bioinformatics prediction suggested that G91 mutation alters both the predicted secondary structure and hydrophobic character of A3-crystallin, while deamidation Melanotan II only exhibits minimal effects. Mammalian two-hybrid results indicated that both G91 mutation and Q85/Q180 deamidation could significantly decrease the conversation of the A3-crystallin homodimer. Conclusion Our results provided evidence that both mutations involved in congenital cataracts and deamidation in aged lenses commonly altered proteinCprotein conversation between human lens A3-crystallins, which may lead to protein insolubilization and contribute to cataracts. Introduction According to the World Health Business, a cataract is usually defined as clouding of the Melanotan II lens of the eye which impedes the passage of light [1]. Worldwide, it is estimated that nearly half of all cases of blindness are caused by cataracts. Cataracts can be broadly divided into two types: congenital cataracts and age-related cataracts. Although most cataract cases are associated with the aging process, congenital cataracts are the leading cause of visual disability in children [2]. Crystallins are major component proteins Melanotan II in intact lens. -Crystallin is one of the three main lens crystallin components (-, -, and -crystallin) and is further subdivided into acidic (A1-, A2-, A3-, and A4-crystallin) and basic (B1-, B2-, and B3-crystallin) groups, which can form homo- or hetero-oligomers in the lens [3]. Previous reports have indicated that mutations in various crystallin genes are deleterious factors contributing to the loss of protein stability and lens transparency [4,5]. For example, since 1998, three kinds of congenital mutation in A3-crystallin gene have been recognized in seven cataract pedigrees. The A3-crystallin gene, located at 17q11C12, encodes two crystallin proteins (A3- and A1-crystallin) from a single mRNA. The A1-crystallin protein lacks the NH2-terminal 17 AA due to an alternate translation initiation site. These congenital mutations include a three base pair deletion (G91) and splice site mutations (IVS3+1G>C and IVS3+1G>A). Interestingly, even though ethnical backgrounds of patients are diverse, including India, Brazil, China, the UK, Switzerland, and Australia, five of them manifest the G91 mutation, which indicated the functional importance of this site [2,5-9]. On the other hand, proteomics analysis of post-translational modifications in young and aged lenses have recognized considerable modification sites in human crystallins [10,11]. Among the several potential post-modifications, deamidation is the most abundant modification in the lens and is significantly increased in aged and cataractous lenses [11]. These post-translational modifications were hypothesized to relate with the age-dependent loss of crystallin solubility. For example, among those previously recognized deamidation sites in A3-crystallin, Takata et al. [12-14] provided evidence that deamidation at Q85 and Q180 destabilizes A3-crystallin homodimer and disrupts conversation with other -crystallin subunits. From the functional viewpoint, proper folding and normal proteinCprotein interactions are two key aspects for ensuring crystallin’s cellular function. Therefore, to elucidate the functional effects of G91 mutation and the deamidation of A3-crystallin, we predicted the folding characteristics using bioinformatics analysis and further investigated into their homodimer formation by a mammalian two-hybrid system. The two deamidation sites, Q85 and Q180, were used as representative samples Rabbit Polyclonal to NXPH4 in this study because Q85 is located in the NH2-terminal domain name and Q180 is located in the COOH-terminal domain name. However, both sites are located in the crucial interface. Our results indicated that both mutations involved in congenital cataracts and deamidation in aged lenses generally alter proteinCprotein conversation in human lens A3-crystallin, which potentially contributes to decreased protein solubility and formation of cataract. Methods Protein secondary structure prediction The secondary structures of wild type and A3-crystallin mutants were predicted by the widely used SSpro8 algorithm around the Scrape server [15]. Based on recurrent neural networks and PSI-BLAST-derived profiles, SSpro8 predicts protein secondary structures according to the DSSP classification [16]. Protein hydrophobicity analysis We used Kyte-Doolittle hydrophobicity plots to detect the potential effects of protein mutants. Hydrophobicity for both wild type and mutants was calculated in a windows size of five, which is good for obtaining hydrophilic regions. Regions with values below zero are hydrophilic in character. Mammalian two-hybrid system The mammalian two-hybrid assay kit, obtained from Stratagene (La Jolla, CA), was used in this study. In this.