Protein Aggregation and Cataract: Role of Age-Related Modifications and Mutations in α-Crystallins.

* The article is published as a part of the Special Issue "Protein Misfolding and Aggregation in Cataract Disorders" (Vol. 87, No. 2). ** To whom correspondence should be addressed. Cataract is a major cause of blindness. Due to the lack of protein turnover, lens proteins accumulate age-related and environmental modifications that alter their native conformation, leading to the formation of aggregation-prone intermediates, as well as insoluble and light-scattering aggregates, thus compromising lens transparency. The lens protein, α-crystallin, is a molecular chaperone that prevents protein aggregation, thereby maintaining lens transparency. However, mutations or post-translational modifications, such as oxidation, deamidation, truncation and crosslinking, can render α-crystallins ineffective and lead to the disease exacerbation. Here, we describe such mutations and alterations, as well as their consequences. Age-related modifications in α-crystallins affect their structure, oligomerization, and chaperone function. Mutations in α-crystallins can lead to the aggregation/intracellular inclusions attributable to the perturbation of structure and oligomeric assembly and resulting in the rearrangement of aggregation-prone regions. Such rearrangements can lead to the exposure of hitherto buried aggregation-prone regions, thereby populating aggregation-prone state(s) and facilitating amorphous/amyloid aggregation and/or inappropriate interactions with cellular components. Investigations of the mutation-induced changes in the structure, oligomer assembly, aggregation mechanisms, and interactomes of α-crystallins will be useful in fighting protein aggregation-related diseases.

Nucleosomal association and altered interactome underlie the mechanism of cataract caused by the R54C mutation of αA-crystallin.

BACKGROUND: αA-crystallin plays an important role in eye lens development. Its N-terminal domain is implicated in several important biological functions. Mutations in certain conserved arginine residues in the N-terminal region of αA-crystallin lead to cataract with characteristic cytoplasmic/nuclear aggregation of the mutant protein. In this study, we attempt to gain mechanistic insights into the congenital cataract caused by the R54C mutation in human αA-crystallin. METHODS: We used several spectroscopic techniques to investigate the structure and function of the wild-type and R54CαA-crystallin. Immunoprecipitation, chromatin-enrichment followed by western blotting, immunofluorescence and cell-viability assay were performed to study the interaction partners, chromatin-association, stress-like response and cell-death caused by the mutant. RESULTS: Although R54CαA-crystallin exhibited slight changes in quaternary structure, its chaperone-like activity was comparable to that of wild-type. When expressed in lens epithelial cells, R54CαA-crystallin exhibited a speckled appearance in the nucleus rather than cytoplasmic localization. R54CαA-crystallin triggered a stress-like response, resulting in nuclear translocation of αB-crystallin, disassembly of cytoskeletal elements and activation of caspase 3, leading to apoptosis. Analysis of the "interactome" revealed an increase in interaction of the mutant protein with nucleosomal histones, and its association with chromatin. CONCLUSIONS: The study shows that alteration of "interactome" and nucleosomal association, rather than loss of chaperone-like activity, is the molecular basis of cataract caused by the R54C mutation in αA-crystallin. GENERAL SIGNIFICANCE: The study provides a novel mechanism of cataract caused by a mutant of αA-crystallin, and sheds light on the possible mechanism of stress and cell death caused by such nuclear inclusions.

Phosphorylation of αB-crystallin: Role in stress, aging and patho-physiological conditions.

BACKGROUND: αB-crystallin, once thought to be a lenticular protein, is ubiquitous and has critical roles in several cellular processes that are modulated by phosphorylation. Serine residues 19, 45 and 59 of αB-crystallin undergo phosphorylation. Phosphorylation of S45 is mediated by p44/42 MAP kinase, whereas S59 phosphorylation is mediated by MAPKAP kinase-2. Pathway involved in S19 phosphorylation is not known. SCOPE OF REVIEW: The review highlights the role of phosphorylation in (i) oligomeric structure, stability and chaperone activity, (ii) cellular processes such as apoptosis, myogenic differentiation, cell cycle regulation and angiogenesis, and (iii) aging, stress, cardiomyopathy-causing αB-crystallin mutants, and in other diseases. MAJOR CONCLUSIONS: Depending on the context and extent of phosphorylation, αB-crystallin seems to confer beneficial or deleterious effects. Phosphorylation alters structure, stability, size distribution and dynamics of the oligomeric assembly, thus modulating chaperone activity and various cellular processes. Phosphorylated αB-crystallin has a tendency to partition to the cytoskeleton and hence to the insoluble fraction. Low levels of phosphorylation appear to be protective, while hyperphosphorylation has negative implications. Mutations in αB-crystallin, such as R120G, Q151X and 464delCT, associated with inherited myofibrillar myopathy lead to hyperphosphorylation and intracellular inclusions. An ongoing study in our laboratory with phosphorylation-mimicking mutants indicates that phosphorylation of R120GαB-crystallin increases its propensity to aggregate. GENERAL SIGNIFICANCE: Phosphorylation of αB-crystallin has dual role that manifests either beneficial or deleterious consequences depending on the extent of phosphorylation and interaction with cytoskeleton. Considering that disease-causing mutants of αB-crystallin are hyperphosphorylated, moderation of phosphorylation may be a useful strategy in disease management. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.

Small heat shock proteins: Role in cellular functions and pathology.

Small heat shock proteins (sHsps) are conserved across species and are important in stress tolerance. Many sHsps exhibit chaperone-like activity in preventing aggregation of target proteins, keeping them in a folding-competent state and refolding them by themselves or in concert with other ATP-dependent chaperones. Mutations in human sHsps result in myopathies, neuropathies and cataract. Their expression is modulated in diseases such as Alzheimer's, Parkinson's and cancer. Their ability to bind Cu2+, and suppress generation of reactive oxygen species (ROS) may have implications in Cu2+-homeostasis and neurodegenerative diseases. Circulating αB-crystallin and Hsp27 in the plasma may exhibit immunomodulatory and anti-inflammatory functions. αB-crystallin and Hsp20 exhitbit anti-platelet aggregation: these beneficial effects indicate their use as potential therapeutic agents. sHsps have roles in differentiation, proteasomal degradation, autophagy and development. sHsps exhibit a robust anti-apoptotic property, involving several stages of mitochondrial-mediated, extrinsic apoptotic as well as pro-survival pathways. Dynamic N- and C-termini and oligomeric assemblies of αB-crystallin and Hsp27 are important factors for their functions. We propose a "dynamic partitioning hypothesis" for the promiscuous interactions and pleotropic functions exhibited by sHsps. Stress tolerance and anti-apoptotic properties of sHsps have both beneficial and deleterious consequences in human health and diseases. Conditional and targeted modulation of their expression and/or activity could be used as strategies in treating several human disorders. The review attempts to provide a critical overview of sHsps and their divergent roles in cellular processes particularly in the context of human health and disease.

Hsp27 suppresses the Cu(2+)-induced amyloidogenicity, redox activity, and cytotoxicity of α-synuclein by metal ion stripping.

Aberrant copper homeostasis and oxidative stress have critical roles in several neurodegenerative diseases. Expression of heat-shock protein 27 (Hsp27) is elevated under oxidative stress as well as upon treatment with Cu(2+), and elevated levels of Hsp27 are found in the brains of patients with Alzheimer and Parkinson diseases. We demonstrate, using steady-state and time-resolved fluorescence spectroscopy as well as isothermal titration calorimetry studies, that Hsp27 binds Cu(2+) with high affinity (Kd ~10(-11) M). Treating IMR-32 human neuroblastoma cells with Cu(2+) leads to upregulation of endogenous Hsp27. Further, overexpression of Hsp27 in IMR-32 human neuroblastoma cells confers cytoprotection against Cu(2+)-induced cell death. Hsp27 prevents the deleterious interaction of Cu(2+) with α-synuclein, the protein involved in Parkinson disease and synucleinopathies. Hsp27 attenuates Cu(2+)- or Cu(2+)-α-synuclein-mediated generation of reactive oxygen species and confers cytoprotection on IMR-32 cells as well as on mouse primary neural precursor cells. Hsp27 prevents Cu(2+)-ascorbate or Cu(2+)-α-synuclein-ascorbate treatment-induced increase in mitochondrial superoxide level and mitochondrial disorganization in IMR-32 cells. Hsp27 dislodges the α-synuclein-bound Cu(2+) and prevents the Cu(2+)-mediated amyloidogenesis of α-synuclein. Our findings that Hsp27 binds Cu(2+) with high affinity leading to beneficial effects and that Hsp27 can dislodge Cu(2+) from α-synuclein, preventing amyloid fibril formation, indicate potential therapeutic strategies for neurodegenerative diseases involving aberrant Cu(2+) homeostasis.

Synergistic effects of metal ion and the pre-senile cataract-causing G98R alphaA-crystallin: self-aggregation propensities and chaperone activity.

PURPOSE: alphaA- and alphaB-crystallins are abundantly present in the eye lens, belong to the small heat shock protein family, and exhibit molecular chaperone activity. They are also known to interact with metal ions such as Cu(2+), and their metal-binding modulates the structure and chaperone function. Unlike other point mutations in alphaA-crystallin that cause congenital cataracts, the G98R mutation causes pre-senile cataract. We have investigated the effect of Cu(2+) on the structure and function of G98R alphaA-crystallin. METHODS: Fluorescence spectroscopy and isothermal titration calorimetry were used to study Cu(2+) binding to alphaA- and G98R alphaA-crystallin. Circular dichroism spectroscopy was used to study secondary and tertiary structures, and dynamic light scattering was used to determine the hydrodynamic radii of the proteins. Chaperone activity and self-aggregation of the wild type and the mutant protein in the absence and the presence of the metal ions was monitored using light scattering. RESULTS: Our fluorescence quenching and isothermal titration calorimetric studies show that like alphaA-crystallin, G98R alphaA-crystallin binds Cu(2+) with picomolar range affinity. Further, both wild type and mutant alphaA-crystallin inhibit Cu(2+)-induced generation of reactive oxygen species with similar efficiency. However, G98R alphaA-crystallin undergoes pronounced self-aggregation above a certain concentration of Cu(2+) (above subunit to Cu(2+) molar ratio of 1:3 in HEPES-NaOH buffer, pH 7.4). At concentrations of Cu(2+) below this ratio, G98R alphaA-crystallin is more susceptible to Cu(2+)-induced tertiary and quaternary structural changes than alphaA-crystallin. Interestingly, Cu(2+) binding increases the chaperone-like activity of alphaA-crystallin toward the aggregation of citrate synthase at 43 degrees C while it decreases the chaperone-like activity of G98R alphaA-crystallin. Mixed oligomer formation between the wild type and the mutant subunits modulates the Cu(2+)-induced effect on the self-aggregation propensity. Other heavy metal ions, namely Cd(2+) and Zn(2+) but not Ca(2+), also promote the self-aggregation of G98R alphaA-crystallin and decrease its chaperone-like activity. CONCLUSIONS: Our study demonstrates that unlike wild type alphaA-crystallin, G98R alphaA-crystallin and its mixed oligomers with wild type protein are vulnerable to heavy metal ions. Our study provides insight into aspects of how environmental factors could augment phenotype(s) in certain genetically predisposed conditions.