The Structure and Stability of the Disulfide-Linked γS-Crystallin Dimer Provide Insight into Oxidation Products Associated with Lens Cataract Formation

Thorn, David C.; Grosas, Aidan B.; Mabbitt, Peter D.; Ray, Nicholas J.; Jackson, Colin J.; Carver, John A.

doi:10.1016/j.jmb.2018.12.005

Cited by 39 publications

(66 citation statements)

References 72 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…2 and Table 1). This is consistent with literature values of 2.0-2.5 nm for monomeric γS (30,31). At a higher concentration of 5 mg/mL, similar Rh was detected for the main peak, but with a greater DLS intensity plus an additional species >100 nm ( Fig.…”

Section: Resultssupporting

confidence: 92%

“…Proteins were heated at 70 °C (i.e. the approximate midpoint of thermal unfolding (30)) and changes in turbidity measured. An aliquot of 100 uL of each crystallin was diluted to 24 uM using 100 mM NaPi (pH 7.4), 100 mM KCl, 1 mM EDTA, 2.5 mM TCEP and heated to 70 °C in Peltier Thermal Cycler-200 (MJ Research, CA).…”

Section: Size-exclusion Chromatography and Multi-angle Light Scatterimentioning

confidence: 99%

See 1 more Smart Citation

Accumulative deamidation of human lens protein γS-crystallin leads to partially unfolded intermediates with enhanced aggregation propensity

Vetter

Thorn

Wheeler

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

Age-related cataract is a major cause of blindness worldwide. Yet, the molecular mechanisms whereby large, light scattering aggregates form is poorly understood, because of the complexity of the aggregates isolated from human lenses. The predominant proteins in the lens are structural proteins called crystallins. The γS-crystallin is heavily modified in cataractous lenses by deamidation, which introduces a negative charge at labile asparagine residues. The effects of deamidation at asparagines, N14, N76, and N143, were mimicked by replacing the asparagine with aspartate using site-directed mutagenesis. The effects of these surface deamidations on the stability, unfolding, and aggregation properties of γS were determined using dynamic light scattering, chemical and thermal-denaturation, and hydrogen-deuterium exchange with mass spectrometry. We found that a small population of all the deamidation mimics aggregated directly into large light scattering bodies with a radius greater than 10 nm that contributed 14-60% of the total scattering intensity compared to 7% for WT under the same conditions. A possible mechanism was identified under partially denaturing conditions, where deamidation led to significantly more rapid unfolding and aggregation particularly for N76D compared to WT. The triple mutant was further destabilized, reflecting the enhanced aggregation properties of N14D and N143D. Thus, the effects of deamidation were both site-specific and cumulative. αA-crystallin was ineffective at acting as a chaperone to prevent the aggregation of destabilized, deamidated γS. It is concluded that surface deamidations, while causing minimal structural disruption individually, progressively destabilize crystallin proteins, leading to their unfolding and precipitation in aged and cataractous lenses. Abbreviations List:Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), dithiothreitol (DTT), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), guanidine hydrochloride (GuHCl), Dynamic, static, and multi-angle light scattering (DLS, SLS, and MALS), hydrogen deuterium exchange (H/D), mass spectrometry (MS), wild type (WT), triple mutant (TM), N-terminal domain (N-td), C-terminal domain (C-td). IntroductionIn mammals, the eye lens plays a crucial role in vision via transmitting, refracting and focusing light onto the retina. Lens functionality is maintained via a high concentration of crystallin proteins (up to 300-500 mg/mL in the center of the lens) that are arranged in a supramolecular array with short-range order (1). The lens is a unique organ in that it has no blood supply and there is no protein turnover in its fiber cells because they lack the organelles for protein synthesis and degradation. As a result, crystallins are long-lived proteins (2).Cataract arises from opacification and concomitant light scattering associated with the unfolding, aggregation and precipitation of crystallins. Cataract is the major cause of blindness in the world and is the leading cause of low vision in the United States (3)...

show abstract

Section: Resultssupporting

confidence: 92%

Section: Size-exclusion Chromatography and Multi-angle Light Scatterimentioning

confidence: 99%

Accumulative deamidation of human lens protein γS-crystallin leads to partially unfolded intermediates with enhanced aggregation propensity

Vetter

Thorn

Wheeler

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…The former is consistent with literature R h values of 2.0-2.5 nm for monomeric γS. 39,40 At a higher concentration of 5 mg/ml, a similar R h was detected for the main peak with significantly less %Pd (9%), but a greater amount of the larger species was detected plus an additional species with a R h of >100 nm ( Figure 2, Table 1).…”

Section: Aggregation Of Human Wild Type and Deamidated γS Under Natsupporting

confidence: 91%

“…This intermolecular crosslink via C24 was previously identified in disulfide-linked dimers of WT γS that had enhanced aggregation properties compared with monomeric γS. 39 Taken together, these results imply that deamidation of γS promotes the formation of intermolecular disulfide linkages that, in turn, facilitates its aggregation. WT and TM γS were next incubated at 65 C with and without the presence of a 2:1 mass ratio of the molecular chaperone αA-crystallin (αA) to probe differences in chaperone ability introduced by γS surface deamidations ( Figure 7b).…”

Section: Thermally-induced Aggregation Of Wild Type and Deamidated γSsupporting

confidence: 57%

Cumulative deamidations of the major lens protein γS‐crystallin increase its aggregation during unfolding and oxidation

et al. 2020

Self Cite

View full text Add to dashboard Cite

Age‐related lens cataract is the major cause of blindness worldwide. The mechanisms whereby crystallins, the predominant lens proteins, assemble into large aggregates that scatter light within the lens, and cause cataract, are poorly understood. Due to the lack of protein turnover in the lens, crystallins are long‐lived. A major crystallin, γS, is heavily modified by deamidation, in particular at surface‐exposed N14, N76, and N143 to introduce negative charges. In this present study, deamidated γS was mimicked by mutation with aspartate at these sites and the effect on biophysical properties of γS was assessed via dynamic light scattering, chemical and thermal denaturation, hydrogen‐deuterium exchange, and susceptibility to disulfide cross‐linking. Compared with wild type γS, a small population of each deamidated mutant aggregated rapidly into large, light‐scattering species that contributed significantly to the total scattering. Under partially denaturing conditions in guanidine hydrochloride or elevated temperature, deamidation led to more rapid unfolding and aggregation and increased susceptibility to oxidation. The triple mutant was further destabilized, suggesting that the effects of deamidation were cumulative. Molecular dynamics simulations predicted that deamidation augments the conformational dynamics of γS. We suggest that these perturbations disrupt the native disulfide arrangement of γS and promote the formation of disulfide‐linked aggregates. The lens‐specific chaperone αA‐crystallin was poor at preventing the aggregation of the triple mutant. It is concluded that surface deamidations cause minimal structural disruption individually, but cumulatively they progressively destabilize γS‐crystallin leading to unfolding and aggregation, as occurs in aged and cataractous lenses.

show abstract

“…This is illustrated by the amorphous aggregation of γ-crystallins, known to cause cataract. Reduced antioxidant capacity induces the formation of an intramolecular disulfide bond between C32 and C41, which is both necessary and sufficient to induce irreversible aggregation through the destabilization of the N-terminus [82,83]. This is especially interesting in an age-related context since the reducing capacity of the eye diminishes with age [84].…”

Section: Cysteine Oxidation-driven Protein Aggregation In Diseasementioning

confidence: 99%

Cross-talk between redox signalling and protein aggregation

Dam

Dansen

2020

Biochemical Society Transactions

View full text Add to dashboard Cite

It is well established that both an increase in reactive oxygen species (ROS: i.e. O2•−, H2O2 and OH•), as well as protein aggregation, accompany ageing and proteinopathies such as Parkinson's and Alzheimer's disease. However, it is far from clear whether there is a causal relation between the two. This review describes how protein aggregation can be affected both by redox signalling (downstream of H2O2), as well as by ROS-induced damage, and aims to give an overview of the current knowledge of how redox signalling affects protein aggregation and vice versa. Redox signalling has been shown to play roles in almost every step of protein aggregation and amyloid formation, from aggregation initiation to the rapid oligomerization of large amyloids, which tend to be less toxic than oligomeric prefibrillar aggregates. We explore the hypothesis that age-associated elevated ROS production could be part of a redox signalling-dependent-stress response in an attempt to curb protein aggregation and minimize toxicity.

show abstract

The Structure and Stability of the Disulfide-Linked γS-Crystallin Dimer Provide Insight into Oxidation Products Associated with Lens Cataract Formation

Cited by 39 publications

References 72 publications

Accumulative deamidation of human lens protein γS-crystallin leads to partially unfolded intermediates with enhanced aggregation propensity

Accumulative deamidation of human lens protein γS-crystallin leads to partially unfolded intermediates with enhanced aggregation propensity

Cumulative deamidations of the major lens protein γS‐crystallin increase its aggregation during unfolding and oxidation

Cross-talk between redox signalling and protein aggregation

Contact Info

Product

Resources

About