Congenital microcornea-cataract syndrome-causing mutation X253R increases ßB1-crystallin hydrophobicity to promote aggregate formation.

The high solubility and lifelong stability of crystallins are crucial to the maintenance of lens transparency and optical properties. Numerous crystallin mutations have been linked to congenital cataract, which is one of the leading causes of newborn blindness. Besides cataract, several crystallin mutations have also been linked to syndromes such as congenital microcornea-cataract syndrome (CMCC). However, the molecular mechanism of CMCC caused by crystallin mutations remains elusive. In the present study, we investigated the mechanism of CMCC caused by the X253R mutation in ßB1-crystallin. The exogenously expressed X253R proteins were prone to form p62-negative aggregates in HeLa cells, strongly inhibited cell proliferation and induced cell apoptosis. The intracellular X253R aggregates could be successfully redissolved by lanosterol but not cholesterol. The extra 26 residues at the C-terminus of ßB1-crystallin introduced by the X253R mutation had little impact on ßB1-crystallin structure and stability, but increased ßB1-crystallin hydrophobicity and decreased its solubility. Interestingly, the X253R mutant fully abolished the aggregatory propensity of ßB1- and ßA3/ßB1-crystallins at high temperatures, suggesting that X253R was an aggregation-inhibition mutation of ß-crystallin homomers and heteromers in dilute solutions. Our results suggest that an increase in hydrophobicity and a decrease in solubility might be responsible for cataractogenesis induced by the X253R mutation, while the cytotoxic effect of X253R aggregates might contribute to the defects in ocular development. Our results also highlight that, at least in some cases, the aggregatory propensity in dilute solutions could not fully mimic the behaviours of mutated proteins in the crowded cytoplasm of the cells.

The importance of the last strand at the C-terminus in ßB2-crystallin stability and assembly.

Congenital cataract is the leading cause of childhood blindness worldwide. Investigations of the effects of inherited mutations on protein structure and function not only help us to understand the molecular mechanisms underlying congenital hereditary cataract, but also facilitate the study of complicated cataract and non-lens abnormities caused by lens-specific genes. In this research, we studied the effects of the V187M, V187E and R188H mutations on ßB2-crystallin structure and stability using a combination of biophysical, cellular and molecular dynamic simulation analysis. Both V187 and R188 are located at the last strand of ßB2-crystallin Greek-key motif 4. All of the three mutations promoted ßB2-crystallin aggregation in vitro and at the cellular level. These three mutations affected ßB2-crystallin quite differentially: V187M influenced the hydrophobic core of the C-terminal domain, V187E was a Greek-key motif breaker with the disruption of the backbone H-bonding network, while R188H perturbed the dynamic oligomeric equilibrium by dissociating the dimer and stabilizing the tetramer. Our results highlighted the importance of the last strand in the structural integrity, folding, assembly and stability of ß-crystallins. More importantly, we proposed that the perturbation of the dynamic equilibrium between ß-crystallin oligomers was an important mechanism of congenital hereditary cataract. The selective stabilization of one specific high-order oligomer by mutations might also be deleterious to the stability and folding of the ß-crystalllin homomers and heteromers. The long-term structural stability and functional maintenance of ß-crystallins are achieved by the precisely regulated oligomeric equilibrium.

The N-terminal extension of ßB1-crystallin chaperones ß-crystallin folding and cooperates with αA-crystallin.

ß/γ-Crystallins are the major structural proteins in mammalian lens. The N-terminal truncation of ßB1-crystallin has been associated with the regulation of ß-crystallin size distributions in human lens. Herein we studied the roles of ßB1 N-terminal extension in protein structure and folding by constructing five N-terminal truncated forms. The truncations did not affect the secondary and tertiary structures of the main body as well as stability against denaturation. Truncations with more than 28 residues off the N-terminus promoted the dissociation of the dimeric ßB1 into monomers in diluted solutions. Interestingly, the N-terminal extension facilitated ßB1 to adopt the correct folding pathway, while truncated proteins were prone to undergo the misfolding/aggregation pathway during kinetic refolding. The N-terminal extension of ßB1 acted as an intramolecular chaperone (IMC) to regulate the kinetic partitioning between folding and misfolding. The IMC function of the N-terminal extension was also critical to the correct refolding of ß-crystallin heteromer and the action of the lens-specific molecular chaperone αA-crystallin. The cooperation between IMC and molecular chaperones produced a much stronger chaperoning effect than if they acted separately. To our knowledge, this is the first report showing the cooperation between IMC and molecular chaperones.

The benefits of being ß-crystallin heteromers: ßB1-crystallin protects ßA3-crystallin against aggregation during co-refolding.

ß-Crystallins are the major structural proteins in mammalian lens, and their stability is critical in maintaining the transparency and refraction index of the lens. Among the seven ß-crystallins, ßA3-crystallin and ßB1-crystallin, an acidic and a basic ß-crystallin, respectively, can form heteromers in vivo. However, the physiological roles of the heteromer have not been fully elucidated. In this research, we studied whether the basic ß-crystallin facilitates the folding of acidic ß-crystallin. Equilibrium folding studies revealed that the ßA3-crystallin and ßB1-crystallin homomers and the ßA3/ßB1-crystallin heteromer all undergo similar five-state folding pathways which include one dimeric and two monomeric intermediates. ßA3-Crystallin was found to be the most unstable among the three proteins, and the transition curve of ßA3/ßB1-crystallin was close to that of ßB1-crystallin. The dimeric intermediate may be a critical determinant in the aggregation process and thus is crucial to the lifelong stability of the ß-crystallins. A comparison of the Gibbs free energy of the equilibrium folding suggested that the formation of heteromer contributed to the stabilization of the dimer interface. On the other hand, ßA3-crystallin, the only protein whose refolding is challenged by serious aggregation, can be protected by ßB1-crystallin in a dose-dependent manner during the kinetic co-refolding. However, the protection is not observed in the presence of the pre-existed well-folded ßB1-crystallin. These findings suggested that the formation of ß-crystallin heteromers not only stabilizes the unstable acidic ß-crystallin but also protects them against aggregation during refolding from the stress-denatured states.

Cataract-causing mutation S228P promotes ßB1-crystallin aggregation and degradation by separating two interacting loops in C-terminal domain.

ß/γ-Crystallins are predominant structural proteins in the cytoplasm of lens fiber cells and share a similar fold composing of four Greek-key motifs divided into two domains. Numerous cataract-causing mutations have been identified in various ß/γ-crystallins, but the mechanisms underlying cataract caused by most mutations remains uncharacterized. The S228P mutation in ßB1-crystallin has been linked to autosomal dominant congenital nuclear cataract. Here we found that the S228P mutant was prone to aggregate and degrade in both of the human and E. coli cells. The intracellular S228P aggregates could be redissolved by lanosterol. The S228P mutation modified the refolding pathway of ßB1-crystallin by affecting the formation of the dimeric intermediate but not the monomeric intermediate. Compared with native ßB1-crystallin, the refolded S228P protein had less packed structures, unquenched Trp fluorophores and increased hydrophobic exposure. The refolded S228P protein was prone to aggregate at the physiological temperature and decreased the protective effect of ßB1-crystallin on ßA3-crystallin. Molecular dynamic simulation studies indicated that the mutation decreased the subunit binding energy and modified the distribution of surface electrostatic potentials. More importantly, the mutation separated two interacting loops in the C-terminal domain, which shielded the hydrophobic core from solvent in native ßB1-crystallin. These two interacting loops are highly conserved in both of the N- and C-terminal domains of all ß/γ-crystallins. We propose that these two interacting loops play an important role in the folding and structural stability of ß/γ-crystallin domains by protecting the hydrophobic core from solvent access.