Ultraviolet (UV) radiation-induced oxidation of tryptophan (Trp) to kynurenine (KN) (TRP > KN) in human γD-crystallins (HγD-Crys) promotes the conversion of proteins into partially unfolded species that act as important precursors for sequential large-scale aggregation. Herein, we report that lanosterol shows protective activity to the structure of the TRP > KN mutant HγD-Crys, particularly its N-terminal domain (N-td), by using all-atom molecular dynamics simulations. The Trp68 > KN mutation significantly destabilizes the originally highly stable \"Tyr55-Trp68-Tyr62\" cluster, thereby causing loop2, where the mutation occurs, to become very flexible. The large fluctuation of loop2 induces cracks, which appear on the protein surface, resulting in the intrusion of water molecules into the hydrophobic core of the N-td. This event eventually triggers the unfolding of the N-td. However, lanosterol can suppress the large fluctuation of loop2 to protect the structural stability of the mutant N-td, thus reducing the aggregation propensity of the TRP > KN mutant HγD-Crys. This structure protective activity of lanosterol arises from its capability to preferentially bind to the hydrophobic regions near loop2. Thus, lanosterol acts as a \"water blocker\" to prevent the invasion of solvent molecules into the hydrophobic core. These findings provide some valuable insights into the development of potential lanosterol-based drugs for cataract prevention and treatment.

Crystallin proteins undergo various posttranslational modifications with aging of eye lens. Oxidation of tryptophan (Trp) residues of a major γ-crystallin namely human γD-crystallin (HGD) was found to be inhibited by a naturally occurring flavonoid hesperetin at relatively low concentration mostly due to its antioxidant activity. Further the molecular interactions between HGD and hesperetin were elucidated on the basis of the quenching of Trp fluorescence of the protein by the flavonoid. Ground state complexation between HGD and hesperetin caused static quenching of the Trp fluorescence of HGD. Binding and quenching constants were in the order of (103- 104 M-1). Energy transfer from protein to hesperetin was suggested by FRET calculations. Thermodynamic parameters reveal significant hydrophobic association between the protein and hesperetin. Synchronous fluorescence and CD spectroscopic results had ruled out conformational changes in the protein due to binding of hesperetin. Docking studies suggested the proximity of hesperetin with Trp 42, which largely corroborates our experimental findings.

Single point mutants of human γS-crystallin cause dominant congenital cataracts, a recent one of which involves the substitution of highly conserved glycine at 57th position with a bulkier tryptophan. Our high-resolution 3D structure of this G57W mutant (abbreviated hereafter as γS-G57W), reported recently revealed site-specific structural perturbations with higher aggregation and lower stability compared to its wild-type; a structural feature associated with important functional and therapeutic consequences. In this communication, we report for the first time, residue resolved conformational dynamics in both γS-WT and γS-G57W using solution NMR spectroscopy, and suggest how these differences could crucially affect the biochemistry of the mutant. Guided by our critical structural investigations, extensive conformational dynamics and biophysical studies presented here show that loss of structural stability arises from enhanced dynamics in Greek key motif 2 inducing flexibility in the N-terminal domain as opposed to its structurally unperturbed C-terminal counterpart. NMR spectral density correlations and internal dynamics comparisons with the wild-type suggest that the overall thermodynamic instability propagates from the mutated N-terminal β4-β5 loop providing a residue level understanding of the structural changes associated with this early onset of lens opacification. Our results highlight the vital role of conserved Greek key motifs in conferring structural stability to crystallins and provide crucial molecular insights into crystallin aggregation in the eye lens, which triggers cataract formation in children. Overall, this critical study provides a residue level understanding of how conformational changes affect the structure and function of crystallins in particular and proteins in general, during health and disease.

Human γS-crystallin (CrygS) is an important component of the human eye lens nucleus and cortex. The mutation G57W in the molecule is reported to be associated with congenital cataract in children. We compare the conformational features and aggregation properties of the mutant protein G57W with the wild-type CrygS to understand how the structural changes in the mutant are related to the mechanism of opacification.
Wild-type and mutant proteins were cloned, expressed, and purified, and their structural properties were studied in solution. Conformational features and the structural stability of the proteins were compared in solution, using circular dichroism (CD) and fluorescence spectroscopic analysis, and the proteins\' tendencies to aggregate were compared using extrinsic spectral probes. In addition, we analyzed the proteins\' structural differences with extensive molecular modeling in silico.
CD and intrinsic fluorescence analysis suggested the secondary and tertiary structures of the mutant are slightly altered. Experiments using extrinsic spectral probes revealed that the compact close-packed structure is loosened somewhat, and the mutant tends to self-aggregate. Denaturation (both thermal and chemical) studies indicate that the replacement of glycine (G) in position 57 by tryptophan (W) lowered the structural stability of the molecule. Further, the mutant had a tendency to precipitate and scatters light more easily than the wild-type.
The replacement of glycine at position 57 by the tryptophan residue in human γS-crystallin weakens the stability of the mutant molecule and causes the molecule to self-aggregate, thus generating light-scattering particles. This set of changes in the mutant offers a molecular insight into the mechanism of opacification.

The lens protein, gamma B-crystallin, precipitates during cataract formation. As a recombinant protein, in aqueous solution, gamma B aggregates and precipitates upon heating, cooling, exposure to ultraviolet light, or refolding from a denatured state. We have studied soluble gamma B crystallin, as well as each of the above aggregated forms, to determine whether gamma B\'s polypeptide chain is differently organized in each form. For this purpose, we used : (a) Fourier Transform Infra Red (FTIR) spectroscopy in the horizontal attenuated total reflectance (HATR) mode, to examine changes in secondary structural content, and (b) transmission electron microscopy (TEM) to examine gross morphological differences. The peak of the gamma B FTIR amide I band shifts from approximately 1633 cm(-1) to approximately 1618 cm(-1) in heat-, UV- and refolding-induced gamma B precipitates, indicating that narrow beta sheets with fewer strands and higher strand twist angles are becoming reorganized into wider, more planar sheets containing larger numbers of shorter strands, with smaller twist angles. In contrast, in cold-induced precipitates, a loss of anti-parallel beta sheet content is observed. This difference is partly explained by the differential effects of temperature on different non-covalent interactions stabilizing protein structures. The native beta sheet content of gamma B crystallin (approximately 50%) is raised in heat- (approximately 60%) and refolding-induced (approximately 58%) precipitates, but lowered in cold- (approximately 41%), and UV-induced (approximately 44%) precipitates. Cold precipitates also display approximately 26% helical content. All four aggregates have distinctively different morphological characteristics; this appears to be in keeping with their distinctively different secondary structural contents.

The Arg14 to Cys (R14C) mutation in the human gammaD-crystallin (HGD) gene has been associated with a juvenile-onset hereditary cataract. We showed previously [Pande, A., et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 1993-1998] that rapid oxidation of Cys14 in the mutant leads to the formation of intermolecular, disulfide-cross-linked aggregates at physiological pH. Here we present a Raman spectroscopic analysis of R14C and HGD and show that R14C forms such aggregates even at pH 4.5. The lower pH enabled us to monitor the evolution of a variety of disulfide cross-links with distinct conformations around the CC-SS-CC dihedral angles. At least three cysteine residues are involved, forming protein-protein cross-links through disulfide-exchange reactions. From the pattern of the S-S and Trp Raman bands, we infer that Cys32 is likely to be involved in the cross-linking. The data suggest that protein precipitation in the mutant may not be the direct result of disulfide cross-linking, although such cross-linking is the initiating event. Thus, our Raman data not only enhance the understanding of the reactivity of Cys14 in the R14C mutant and the mechanism of opacity, but also shed light on the mechanism of oxidative degradation during long-term storage of thiol-containing pharmaceuticals.

The Pro23 to Thr (P23T) mutation in human gammaD-crystallin (HGD) shows several cataract phenotypes. We found earlier [A. Pande, O. Annunziata, N. Asherie, O. Ogun, G.B. Benedek, J. Pande, Decrease in protein solubility and cataract formation caused by the Pro23 to Thr mutation in human gamma D-crystallin, Biochemistry 44 (2005) 2491-2500] that the mutation dramatically lowers the solubility of P23T but the overall protein fold is maintained. Recently we observed that solutions of P23T showed liquid-liquid phase transition behavior similar to that of HGD but the liquid-protein crystal phase transition was altered, suggesting an asymmetric distribution of \"sticky\" patches on the protein surface [J.J. McManus, A. Lomakin, O. Ogun, A. Pande, M. Basan, J. Pande, G.B. Benedek, Altered phase diagram due to a single point mutation in human gammaD-crystallin, Proc. Natl. Acad. Sci. USA 104 (2007) 16856-16861]. Here we present high-resolution NMR studies of HGD and P23T in which we have made nearly complete backbone assignments. The data provide a structural basis for explaining the retrograde solubility of P23T by (a) identifying possible \"sticky\" patches on the surface of P23T and (b) highlighting their asymmetric distribution.

The human lens crystallin gene CRYGC T5P is associated with Coppock-like cataract and has a phenotype of a dust-like opacity of the fetal lens nucleus and deep cortical region. Previous in vitro mutation studies indicate that the protein has changed conformation, solubility, and stability, which may make it susceptible to aggregation, as seen in cataractous lens and cell culture expression. To investigate the mechanisms leading to these events, we studied protein-protein interactions using confocal fluorescence resonance energy transfer (FRET) microscopy. The method detects protein-protein interactions in the natural environment of living cells. Crystallin genes (CRYGC T5P, CRYGC, and CRYAA) were fused to either the green fluorescence protein (GFP) or red fluorescence protein (DsRED or RFP) vector. Each of the following GFP-RFP (donor-acceptor) plasmid pairs was cotransfected into HeLa cells: gammaC-gammaC, gammaC-gammaCT5P, gammaCT5P-gammaCT5P, alphaA-gammaC, and alphaA-gammaCT5P. After culture, confocal fluorescence cell images were taken. Protein-protein interactions in the form of net FRET were evaluated. The confocal fluorescence images show that cells expressing T5P gammaC-crystallin contain many protein aggregates, but cells co-expressing with either gammaC- or alphaA-crystallin reduce the aggregation considerably. FRET determination indicates that gammaCT5P-gammaCT5P shows less protein-protein interaction than either gammaC-gammaC or gammaC-gammaCT5P. Cotransfection with alphaA-crystallin (alphaA-gammaC or alphaA-T5PgammaC) increases nFRET compared with gammaC-gammaC or gammaC-T5PgammaC. Our results demonstrate that T5P gammaC-crystallin shows more protein aggregates and less protein-protein interaction than WT gammaC-crystallin. Chaperone alphaA-crystallin can rescue T5P gammaC-crystallin from aggregation through increased protein interaction. The formation of congenital cataract may be due to reduced protein-protein interactions and increased aggregation from an insufficient amount of alpha-crystallin for protection.

MIP26/AQP0 is the major lens fiber membrane protein and has been reported to interact with many other lens components including crystallins, lipid, and cytoskeletal proteins. Regarding crystallins, many previous reports indicate that MIP26/AQP0 interacts with either only alpha-crystallin or some specific gamma-crystallins. Considering the possibly important role of MIP26/AQP0 in the reduction of light scattering in the lenses, we have further investigated its interaction with crystallins using confocal fluorescence resonance energy transfer (FRET) microscopy. Specifically, we used MIP26 tagged with a green fluorescence protein (GFP) as a donor and a crystallin (alphaA-, alphaB-, betaB2-, or gammaC-crystallin) tagged with a red fluorescence protein (RFP) as an acceptor. The two plasmids were cotransfected to HeLa cells. After culture, laser scattering microscopy images were taken in each of the three channels: GFP, RFP, and FRET. The net FRET images were then obtained by removing the contribution of spectral bleed-through. The pixels of net FRET were normalized with those of GFP. The results show the presence of measurable interactions between MIP26 and all crystallins, with the extent of interactions decreasing from alphaA- and alphaB-crystallin to betaB2- and gammaC-crystallin. Competitive interaction study using untagged alphaA-crystallin shows decreased net FRET, indicating specificity of the interactions between MIP26 and alphaA-crystallin. We conclude that all crystallins interact with MIP26, the physiological significance of which may be a reduction in the difference of refractive index between membrane and cytoplasm.

A model of neoplastic transformation by the combination of SV40 large T antigen (LT), SV40 small T antigen (ST), oncogenic Ras, and human telomerase reverse trasncriptase subunit (hTERT) has become established and replicated in primary human fibroblasts, however, there is no report on human hepatocytes. Here we use cell transplantation model, and show that transplantation of human hepatocytes of HL-7702 and HL-7703 expressing Ha-RasV12 and SV40 LT into subrenal capsule of immunodeficient mice results in fully malignant tumors, in contrast to conventional subcutaneous injections where tumors fail to develop. In GM-847 cell study, we have found that hTERT is not required for tumorigenic growth in subrenal capsule transplantation, however, it is required in subcutaneous injection assay. These results demonstrate that Human hepatocytes can be transformed under kidney capsule by coexpressing SV40 LT and Ha-RasV12, neither hTERT nor protein phosphatase 2A (PP2A) inhibition are required for malignant transformation, a gene which increases cell survival in the subcutaneous injection model is not required for tumorigenic growth in subrenal capsule.