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).

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.

Conserved core tryptophans of FnII domains are crucial for the membranolytic and chaperone-like activities of bovine seminal plasma protein PDC-109.

The fibronectin type II (FnII) domain, present in diverse vertebrate proteins, plays crucial roles in several fundamental biological processes. PDC-109, the major bovine seminal plasma protein, contains two FnII domains that bind to choline phospholipids on sperm plasma membrane and induce lipid efflux crucial for successful fertilization. PDC-109 also exhibits chaperone-like activity and protects other proteins against various types of stress.

Clusterin: full-length protein and one of its chains show opposing effects on cellular lipid accumulation.

Proteins, made up of either single or multiple chains, are designed to carry out specific biological functions. We found an interesting example of a two-chain protein where administration of one of its chains leads to a diametrically opposite outcome than that reported for the full-length protein. Clusterin is a highly glycosylated protein consisting of two chains, α- and β-clusterin.

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.

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.

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).

The extracellular chaperone haptoglobin prevents serum fatty acid-promoted amyloid fibril formation of β2-microglobulin, resistance to lysosomal degradation, and cytotoxicity.

Fibril formation of β2-microglobulin and associated inflammation occur in patients on long term dialysis. We show that the plasma protein haptoglobin prevents the fatty acid-promoted de novo fibril formation of β2-microglobulin even at substoichiometric concentration. The fibrils are cytotoxic, and haptoglobin abolishes the cytotoxicity by preventing fibril formation.

Structural aspects and chaperone activity of human HspB3: role of the "C-terminal extension".

HspB3, an as yet uncharacterized sHsp, is present in muscle, brain, heart, and in fetal tissues. A point mutation correlates with the development of axonal motor neuropathy. We purified recombinant human HspB3.

HspB2/myotonic dystrophy protein kinase binding protein (MKBP) as a novel molecular chaperone: structural and functional aspects.

The small heat shock protein, human HspB2, also known as Myotonic Dystrophy Kinase Binding Protein (MKBP), specifically associates with and activates Myotonic Dystrophy Protein Kinase (DMPK), a serine/threonine protein kinase that plays an important role in maintaining muscle structure and function. The structure and function of HspB2 are not well understood. We have cloned and expressed the protein in E.

Inhibition of Cu2+-mediated generation of reactive oxygen species by the small heat shock protein αB-crystallin: the relative contributions of the N- and C-terminal domains.

Oxidative stress, Cu(2+) homeostasis, and small heat shock proteins (sHsp's) have important implications in several neurodegenerative diseases. The ubiquitous sHsp αB-crystallin is an oligomeric protein that binds Cu(2+). We have investigated the relative contributions of the N- and C-terminal (C-TDαB-crystallin) domains of αB-crystallin to its Cu(2+)-binding and redox-attenuation properties and mapped the Cu(2+)-binding regions.

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.

Selective Cu2+ binding, redox silencing, and cytoprotective effects of the small heat shock proteins alphaA- and alphaB-crystallin.

Oxidative stress and Cu(2+) have been implicated in several neurodegenerative diseases and in cataract. Oxidative stress, as well as Cu(2+), is also known to induce the expression of the small heat shock proteins alpha-crystallins. However, the role of alpha-crystallins in oxidative stress and in Cu(2+)-mediated processes is not clearly understood.

Effect of phosphorylation on alpha B-crystallin: differences in stability, subunit exchange and chaperone activity of homo and mixed oligomers of alpha B-crystallin and its phosphorylation-mimicking mutant.

Phosphorylation appears to be one of the modulators of chaperone functions of small heat shock proteins. However, the role of phosphorylation is not completely understood. We have investigated the structural and functional consequences of a phosphorylation-mimicking mutation in alpha B-crystallin, a small heat shock protein with chaperone activity.

Mixed oligomer formation between human alphaA-crystallin and its cataract-causing G98R mutant: structural, stability and functional differences.

Mutation of the glycine 98 residue to arginine in alphaA-crystallin has been shown to cause presenile cataract in an Indian family. Our earlier study showed that the mutant protein exhibits folding defects that lead to aggregation and inclusion body formation in Escherichia coli. Despite the presence of a normal copy, the pathology is seen in the heterozygous individuals.

Association of alphaB-crystallin, a small heat shock protein, with actin: role in modulating actin filament dynamics in vivo.

Disruption of cytoskeletal assembly is one of the early effects of any stress that can ultimately lead to cell death. Stabilization of cytoskeletal assembly, therefore, is a critical event that regulates cell survival under stress. alphaB-crystallin, a small heat shock protein, has been shown to associate with cytoskeletal proteins under normal and stress conditions.

The cataract-causing mutation G98R in human alphaA-crystallin leads to folding defects and loss of chaperone activity.

Purpose: The objective of this study is to understand the molecular basis of cataract that develops due to the mutation of the glycine-98 residue to arginine in alphaA-crystallin.

Methods: The glycine-98 residue was mutated to arginine by site-directed mutagenesis. The expression, structural and chaperone properties and thermal stability of the mutant, G98RalphaA-crystallin have been studied.

Fibrillogenic and non-fibrillogenic ensembles of SDS-bound human alpha-synuclein.

Fibril formation of alpha-synuclein is associated with several neurodegenerative diseases, including Parkinson's disease in humans. The anionic detergent sodium dodecyl sulfate (SDS) can accelerate the fibril formation in vitro. However, the molecular basis of this acceleration is not clear.

Interaction of mammalian Hsp22 with lipid membranes.

Hsp22/HspB8 is a member of the small heat-shock protein family, whose function is not yet completely understood. Our immunolocalization studies in a human neuroblastoma cell line, SK-N-SH, using confocal microscopy show that a significant fraction of Hsp22 is localized to the plasma membrane. We therefore investigated its interactions with lipid vesicles in vitro.

AlphaB-crystallin, a small heat-shock protein, prevents the amyloid fibril growth of an amyloid beta-peptide and beta2-microglobulin.

AlphaB-crystallin, a small heat-shock protein, exhibits molecular chaperone activity. We have studied the effect of alphaB-crystallin on the fibril growth of the Abeta (amyloid beta)-peptides Abeta-(1-40) and Abeta-(1-42). alphaB-crystallin, but not BSA or hen egg-white lysozyme, prevented the fibril growth of Abeta-(1-40), as revealed by thioflavin T binding, total internal reflection fluorescence microscopy and CD spectroscopy.

Oligomeric Hsp33 with enhanced chaperone activity: gel filtration, cross-linking, and small angle x-ray scattering (SAXS) analysis.

Hsp33, an Escherichia coli cytosolic chaperone, is inactive under normal conditions but becomes active upon oxidative stress. It was previously shown to dimerize upon activation in a concentration- and temperature-dependent manner. This dimer was thought to bind to aggregation-prone target proteins, preventing their aggregation.

The IXI/V motif in the C-terminal extension of alpha-crystallins: alternative interactions and oligomeric assemblies.

Purpose: Alpha-crystallin, a hetero-oligomer of alphaA- and alphaB-crystallin, is involved in maintaining eye lens transparency, primarily by its structural packing and chaperone activity. alphaA- and alphaB-crystallin share significant sequence homology, which is almost exclusively restricted to the central, conserved "alphaA-crystallin domain". The flanking N-terminal domain and C-terminal extension are highly variable both in sequence and length.

Mammalian Hsp22 is a heat-inducible small heat-shock protein with chaperone-like activity.

A newly identified 22 kDa protein that interacts with Hsp27 (heat-shock protein 27) was shown to possess the characteristic alpha-crystallin domain, hence named Hsp22, and categorized as a member of the sHsp (small Hsp) family. Independent studies from different laboratories reported the protein with different names such as Hsp22, H11 kinase, E2IG1 and HspB8. We have identified, on the basis of the nucleotide sequence analysis, putative heat-shock factor 1 binding sites upstream of the Hsp22 translation start site.

Role of the conserved SRLFDQFFG region of alpha-crystallin, a small heat shock protein. Effect on oligomeric size, subunit exchange, and chaperone-like activity.

Small heat shock proteins (sHsps) are necessary for several cellular functions and in stress tolerance. Most sHsps are oligomers; intersubunit interactions leading to changes in oligomeric structure and exposure of specific regions may modulate their functioning. Many sHsps, including alpha A- and alpha B-crystallin, contain a well conserved SRLFDQFFG sequence motif in the N-terminal region.

Structural perturbation and enhancement of the chaperone-like activity of alpha-crystallin by arginine hydrochloride.

Structural perturbation of alpha-crystallin is shown to enhance its molecular chaperone-like activity in preventing aggregation of target proteins. We demonstrate that arginine, a biologically compatible molecule that is known to bind to the peptide backbone and negatively charged side-chains, increases the chaperone-like activity of calf eye lens alpha-crystallin as well as recombinant human alphaA- and alphaB-crystallins. Arginine-induced increase in the chaperone activity is more pronounced for alphaB-crystallin than for alphaA-crystallin.