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Amyloid fiber formation in human ?D-Crystallin induced by UV-B photodamage.

ABSTRACT: ?D-Crystallin is an abundant structural protein of the lens that is found in native and modified forms in cataractous aggregates. We establish that UV-B irradiation of ?D-Crystallin leads to structurally specific modifications and precipitation via two mechanisms: amorphous aggregates and amyloid fibers. UV-B radiation causes cleavage of the backbone, in large measure near the interdomain interface, where side chain oxidations are also concentrated. 2D IR spectroscopy and expressed protein ligation localize fiber formation exclusively to the C-terminal domain of ?D-Crystallin. The native ?-sandwich domains are not retained upon precipitation by either mechanism. The similarities between the amyloid forming pathways when induced by either UV-B radiation or low pH suggest that the propensity for the C-terminal ?-sandwich domain to form amyloid ?-sheets determines the misfolding pathway independent of the mechanism of denaturation.

SUBMITTER: Moran SD

PROVIDER: S-EPMC3859806 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Amyloid fiber formation in human γD-Crystallin induced by UV-B photodamage.

Moran Sean D SD Zhang Tianqi O TO Decatur Sean M SM Zanni Martin T MT

Biochemistry 20130829 36

γD-Crystallin is an abundant structural protein of the lens that is found in native and modified forms in cataractous aggregates. We establish that UV-B irradiation of γD-Crystallin leads to structurally specific modifications and precipitation via two mechanisms: amorphous aggregates and amyloid fibers. UV-B radiation causes cleavage of the backbone, in large measure near the interdomain interface, where side chain oxidations are also concentrated. 2D IR spectroscopy and expressed protein ligat ...[more]

PMID: 23957864

Similar Datasets

Mechanism of human γD-crystallin protein aggregation in UV-C light.

Project description:PurposeTo characterize intermediate aggregate species on the aggregation pathway of γD-crystallin protein in ultraviolet (UV)-C light.MethodsThe kinetics of γD-crystallin protein aggregation was studied with reversed-phase high-performance liquid chromatography (RP-HPLC) sedimentation assay, ThT binding assay, and light scattering. We used analytical ultracentrifugation to recognize intermediate aggregate species and characterized them with Fourier transform infrared spectroscopy (FTIR). Quantification of free sulfhydryl groups in an ongoing aggregation reaction was achieved by using Ellman's assay.ResultsNegligible lag phase was found in the aggregation kinetic experiments of the γD-crystallin protein. Dimer, tetramer, octamer, and higher oligomer intermediates were formed on the aggregation pathway. The protein changes its conformation to form intermediate aggregate species. FTIR and trypsin digestion indicated structural differences between the protein monomer, intermediate aggregate species, and fibrils. Ellman's assay revealed that disulfide bonds were formed in the protein monomers and aggregates during the aggregation process.ConclusionsThis study showed that various intermediate and structurally different aggregate species are formed on the aggregation pathway of γD-crystallin protein in UV-C light.

| S-EPMC8254662 | biostudies-literature

The mechanism for thermal-enhanced chaperone-like activity of α-crystallin against UV irradiation-induced aggregation of γD-crystallin.

Project description:Exposure to solar UV irradiation damages γ-crystallin, leading to cataract formation via aggregation. α-Crystallin, as a small heat shock protein, efficiently suppresses this irreversible aggregation by selectively binding the denatured γ-crystallin monomer. In this study, liquid chromatography tandem mass spectrometry was used to evaluate UV-325 nm irradiation-induced photodamage of human γD-crystallin in the presence of bovine α-crystallin, atomic force microscope (AFM) and dynamic light scattering (DLS) techniques were used to detect the quaternary structure changes of the α-crystallin oligomer, and Fourier transform infrared spectroscopy and temperature-jump nanosecond time-resolved IR absorbance difference spectroscopy were used to probe the secondary structure changes of bovine α-crystallin. We find that the thermal-induced subunit dissociation of the α-crystallin oligomer involves the breaking of hydrogen bonds at the dimeric interface, leading to three different spectral components at varied temperature regions as resolved from temperature-dependent IR spectra. Under UV-325 nm irradiation, unfolded γD-crystallin binds to the dissociated α-crystallin subunit to form an αγ-complex, then follows the reassociation of the αγ-complex to the partially dissociated α-crystallin oligomer. This prevents the aggregation of denatured γD-crystallin. The formation of the γD-bound α-crystallin oligomer is further confirmed by AFM and DLS analysis, which reveals an obvious size expansion in the reassociated αγ-oligomers. In addition, UV-325 nm irradiation causes a peptide bond cleavage of γD-crystallin at Ala158 in the presence of α-crystallin. Our results suggest a very effective protection mechanism for subunits dissociated from α-crystallin oligomers against UV irradiation-induced aggregation of γD-crystallin, at the expense of a loss of a short C-terminal peptide in γD-crystallin.

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Tryptophan cluster protects human γD-crystallin from ultraviolet radiation-induced photoaggregation in vitro.

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Acetylation of Gly1 and Lys2 promotes aggregation of human γD-crystallin.

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Group II archaeal chaperonin recognition of partially folded human γD-crystallin mutants.

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| S-EPMC4093946 | biostudies-literature

Disordered region encodes α-crystallin chaperone activity toward lens client γD-crystallin.

Project description:Small heat-shock proteins (sHSPs) are a widely expressed family of ATP-independent molecular chaperones that are among the first responders to cellular stress. Mechanisms by which sHSPs delay aggregation of client proteins remain undefined. sHSPs have high intrinsic disorder content of up to ~60% and assemble into large, polydisperse homo- and hetero-oligomers, making them challenging structural and biochemical targets. Two sHSPs, HSPB4 and HSPB5, are present at millimolar concentrations in eye lens, where they are responsible for maintaining lens transparency over the lifetime of an organism. Together, HSPB4 and HSPB5 compose the hetero-oligomeric chaperone known as α-crystallin. To identify the determinants of sHSP function, we compared the effectiveness of HSPB4 and HSPB5 homo-oligomers and HSPB4/HSPB5 hetero-oligomers in delaying the aggregation of the lens protein γD-crystallin. In chimeric versions of HSPB4 and HSPB5, chaperone activity tracked with the identity of the 60-residue disordered N-terminal regions (NTR). A short 10-residue stretch in the middle of the NTR ("Critical sequence") contains three residues that are responsible for high HSPB5 chaperone activity toward γD-crystallin. These residues affect structure and dynamics throughout the NTR. Abundant interactions involving the NTR Critical sequence reveal it to be a hub for a network of interactions within oligomers. We propose a model whereby the NTR critical sequence influences local structure and NTR dynamics that modulate accessibility of the NTR, which in turn modulates chaperone activity.

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Project description:Excessive UV-B exposure is well known to be a risk factor for corneal phototoxicity including direct DNA damage and disturbances in the antioxidant balance. Here, we showed a successful synthesis of a water-soluble and biocompatible small molecule DHPM 1 with dihydropyrimidinthione skeleton, which could effectively protect human corneal epithelial (HCE-2) cells from UV-B damage. In separate experiments, DHPM 1 absorbed UV-B rays and exhibited scavenging activity against intracellular ROS induced by UV-B radiation, thereby reducing the levels of DNA fragmentation. Additionally, UV-B exposure increased the expression of cleaved caspase-3, as well as the ratio of Bax/Bcl-2 at protein levels, while pretreatment with DHPM 1 significantly reversed these changes. To the best of our knowledge, this is the first report of a study based on dihydropyrimidinthione derivatives to develop a promising eye drops, which may well find extensive applications in UV-B caused corneal damage.

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UV-B induced fibrillization of crystallin protein mixtures.

Project description:Environmental factors, mainly oxidative stress and exposure to sunlight, induce the oxidation, cross-linking, cleavage, and deamination of crystallin proteins, resulting in their aggregation and, ultimately, cataract formation. Various denaturants have been used to initiate the aggregation of crystallin proteins in vitro. All of these regimens, however, are obviously far from replicating conditions that exist in vivo that lead to cataract formation. In fact, it is our supposition that only UV-B radiation may mimic the observed in vivo cause of crystallin alteration leading to cataract formation. This means of inducing cataract formation may provide the most appropriate in vitro platform for in-depth study of the fundamental cataractous fibril properties and allow for testing of possible treatment strategies. Herein, we showed that cataractous fibrils can be formed using UV-B radiation from ?:?:? crystallin protein mixtures. Characterization of the properties of formed aggregates confirmed the development of amyloid-like fibrils, which are in cross-?-pattern and possibly in anti-parallel ?-sheet arrangement. Furthermore, we were also able to confirm that the presence of the molecular chaperone, ?-crystallin, was able to inhibit fibril formation, as observed for 'naturally' occurring fibrils. Finally, the time-dependent fibrillation profile was found to be similar to the gradual formation of age-related nuclear cataracts. This data provided evidence for the initiation of fibril formation from physiologically relevant crystallin mixtures using UV-B radiation, and that the formed fibrils had several traits similar to that expected from cataracts developing in vivo.

| S-EPMC5444657 | biostudies-literature

Assessing the Structures and Interactions of γD-Crystallin Deamidation Variants.

Project description:Cataracts involve the deposition of the crystallin proteins in the vertebrate eye lens, causing opacification and blindness. They are associated with either genetic mutation or protein damage that accumulates over the lifetime of the organism. Deamidation of Asn residues in several different crystallins has been observed and is frequently invoked as a cause of cataract. Here, we investigated the properties of Asp variants, deamidation products of γD-crystallin, by solution NMR, X-ray crystallography, and other biophysical techniques. No substantive structural or stability changes were noted for all seven Asn to Asp γD-crystallins. Importantly, no changes in diffusion interaction behavior could be detected. Our combined experimental results demonstrate that introduction of single Asp residues on the surface of γD-crystallin by deamidation is unlikely to be the driver of cataract formation in the eye lens.

| S-EPMC7935750 | biostudies-literature

Contributions of aromatic pairs to the folding and stability of long-lived human γD-crystallin.

Project description:Human γD-crystallin (HγD-Crys) is a highly stable protein that remains folded in the eye lens for the majority of an individual's lifetime. HγD-Crys exhibits two homologous crystallin domains, each containing two Greek key motifs with eight β-strands. Six aromatic pairs (four Tyr/Tyr, one Tyr/Phe and one Phe/Phe) are present in the β-hairpin sequences of the Greek keys. Ultraviolet damage to the aromatic residues in lens crystallins may contribute to the genesis of cataract. Mutant proteins with these aromatic residues substituted with alanines were constructed and expressed in E. coli. All mutant proteins except F115A and F117A had lower thermal stability than the WT protein. In equilibrium experiments in guanidine hydrochloride (GuHCl), all mutant proteins had lower thermodynamic stability than the WT protein. N-terminal domain (N-td) substitutions shifted the N-td transition to lower GuHCl concentration, but the C-terminal domain (C-td) transition remained unaffected. C-td substitutions led to a more cooperative unfolding/refolding process, with both the N-td and C-td transitions shifted to lower GuHCl concentration. The aromatic pairs conserved for each Greek key motif (Greek key pairs) had larger contributions to both thermal stability and thermodynamic stability than the other pairs. Aromatic-aromatic interaction was estimated as 1.5-2.0 kcal/mol. In kinetic experiments, N-td substitutions accelerated the early phase of unfolding, while C-td substitutions accelerated the late phase, suggesting independent domain unfolding. Only substitutions of the second Greek key pair of each crystallin domain slowed refolding. The second Greek keys may provide nucleation sites during the folding of the double-Greek-key crystallin domains.

| S-EPMC3064831 | biostudies-literature

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