Edathara C. Abraham

α-Crystallin chaperone function in diabetic rat and human lenses

This study focussed on the effect of diabetes on the chaperone function of α-crystallin. The authors relied on diabetic rats with a wide range of plasma glucose levels and non-diabetic control rats to establish a possible relationship between severity of diabetes and α-crystallin chaperone activity. In addition, 52–56 and 63–69 year-old diabetic and non-diabetic human lenses were used to show whether diabetes affects α-crystallin chaperone activity in human lenses. Correlation between plasma glucose levels and loss of chaperone activity of the αL-crystallin fraction in diabetic rats indicated good correlation. The glycemic threshold, reported before for cataract development in diabetic rats, seems to be valid for the chaperone activity loss as well. Analysis of the human lens αL-crystallin showed lower chaperone activity in all the diabetic lenses than in the age-matched control lenses. In the 63–69 age group, the loss in chaperone activity due to diabetes was significantly larger than in the 52–56 age group suggesting a dominant effect of duration of diabetes.

Inflammatory cytokines, interleukin-1 beta and tumor necrosis factor-alpha, upregulated in glioblastoma multiforme, raise the levels of CRYAB in exosomes secreted by U373 glioma cells

In the brain, levels of inflammatory cytokines, interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α), are elevated under traumatic brain injury, neuroinflammatory conditions and glioblastoma multiforme (GBM). In GBM, the levels of small heat shock protein, CRYAB (HspB5) are also reported to be elevated, where it has been shown to exert anti-apoptotic activity. Interestingly, CRYAB is secreted via exosomes by various cells. In order to understand the relation between inflammatory cytokines and CRYAB, U373 glioma cells, were stimulated with proinflammatory cytokines, IL-1β and TNF-α, and their effect on CRYAB levels in cells and secreted exosomes was studied. Our results show that U373 cells produce and secrete CRYAB via exosomes and that stimulation with IL-1β and TNF-α significantly increase the levels of CRYAB in not only the cells but also in the secreted exosomes. In addition, cytokine stimulation of U373 cells brings about changes in the secreted exosomal proteome, many of which are involved in cancer progression.

Substituted hydrophobic and hydrophilic residues at methionine-68 influence the chaperone-like function of αB-crystallin

Amino acid residues 57–69 in αB-crystallin have been implicated as a target protein binding site. Moreover, a direct correlation between the extent of α-crystallin hydrophobicity and chaperone-like activity has been demonstrated. The purpose of this study was to mutate a moderately hydrophobic residue Met-68 (M-68) in the above region to strongly hydrophobic and hydrophilic residues and show whether chaperoning ability is affected with or without structural changes. Mutation of M-68 to Val, Ile or Thr did not result in significant changes in molecular mass and secondary and tertiary structures. However, the Val and Ile mutants showed significant improvement and the Thr mutant showed substantial loss in chaperone activity. Differences in chaperone function in the absence of any structural changes confirmed that the hydrophobicity or hydrophilicity of the substituted amino acid in the putative target protein binding site was the only contributing factor.

Thermally induced disintegration of the oligomeric structure of alphaB-crystallin mutant F28S is associated with diminished chaperone activity.

AbstractαB-crystallin, a member of the small heat-shock protein (hsp) family of proteins, is able to function as a molecular chaperone by protecting other proteins from stress-induced aggregation by recognizing and binding to partially unfolded species of damaged proteins. The present work has investigated the role of phenylalanine-28 (F28) of the 22RLFDQFF28 region of αB-crystallin in maintaining chaperone function and oligomeric structure under physiological condition and under thermal stress. Bovine αB-crystallin was cloned for the first time and the cDNA sequence revealed greater than 90% homology to that of human, rat and mouse αB-crystallins. F28 was mutated to a serine followed by expression of the mutant F28S and the wild-type αB (αB-wt) in E. coli and subsequent purification of the protein by size-exclusion chromatography. Secondary and tertiary structure analyses showed some structural changes in the mutant. Chaperone activity and oligomeric size of the mutant was unchanged at 37°C whereas at 58°C the chaperone activity was significantly decreased and the oligomeric size ranged from low molecular weight to high molecular weight showing disintegration of the oligomeric structure. The data support the idea that the participation of large oligomeric structure rather than smaller units is required to have optimal chaperone activity and the hydrophobic F28 residue is needed for maintaining the native oligomeric structure under thermal stress.

C-Terminal truncation affects subunit exchange of human αA-crystallin with αB-crystallin

In human lenses, C-terminal cleavage of αA-crystallin at residues 172,168, and 162 have been reported. The effect of C-terminal truncation of αA-crystallin on subunit exchange and heterooligomer formation with αB-crystallin and homooligomer formation with native αA-crystallin is not known. We have conducted fluorescence resonance energy transfer studies which have shown that the rates of subunit exchange of αA1–172 and αA1–168 with αB-wt were two-fold lower than for αA-wt interacting with αB-wt. The subunit exchange rate between αA1–162 and αB-wt was six-fold lower. These data suggest that cleavage of the C-terminal residues could significantly affect heterooligomerization. On the other hand, the subunit exchange rates between αA-wt and the truncated αA-crystallins were either unchanged or only slightly decreased, which suggest that homooligomerization may not be significantly influenced by C-terminal truncation. The main conclusion from this study is that cleavage of C-terminal residues of αA-crystallin including the nine residues of the flexible tail is expected to significantly affect the formation of heteroaggregates. Reconstitution experiments showed that the presence of an intact C-terminus is essential for the formation of fully integrated heteroaggregates with equal proportion of αA and αB subunits.

Interaction of C-Terminal Truncated Human αA-Crystallins with Target Proteins

Background Significant portion of αA-crystallin in human lenses exists as C-terminal residues cleaved at residues 172, 168, and 162. Chaperone activity, determined with alcohol dehydrogenase (ADH) and βL-crystallin as target proteins, was increased in αA1–172 and decreased in αA1–168 and αA1–162. The purpose of this study was to show whether the absence of the C-terminal residues influences protein-protein interactions with target proteins. Methodology/Principal Findings Our hypothesis is that the chaperone-target protein binding kinetics, otherwise termed subunit exchange rates, are expected to reflect the changes in chaperone activity. To study this, we have relied on fluorescence resonance energy transfer (FRET) utilizing amine specific and cysteine specific fluorescent probes. The subunit exchange rate (k) for ADH and αA1–172 was nearly the same as that of ADH and αA-wt, αA1–168 had lower and αA1–162 had the lowest k values. When βL-crystallin was used as the target protein, αA1–172 had slightly higher k value than αA-wt and αA1–168 and αA1–162 had lower k values. As expected from earlier studies, the chaperone activity of αA1–172 was slightly better than that of αA-wt, the chaperone activity of αA1–168 was similar to that of αA-wt and αA1–162 had substantially decreased chaperone activity. Conclusions/Significance Cleavage of eleven C-terminal residues including Arg-163 and the C-terminal flexible arm significantly affects the interaction with target proteins. The predominantly hydrophilic flexible arm appears to be needed to keep the chaperone-target protein complex soluble.

Congenital Cataract Causing Mutants of αA-Crystallin/sHSP Form Aggregates and Aggresomes Degraded through Ubiquitin-Proteasome Pathway

Background Mutations of human αA-crystallin cause congenital cataract by protein aggregation. How mutations of αA-crystallin cause disease pathogenesis through protein aggregation is not well understood. To better understand the cellular events leading to protein aggregation, we transfected cataract causing mutants, R12C, R21L, R21W, R49C, R54C, R116C and R116H, of human αA-crystallin in HeLa cells and examined the formation of intracellular protein aggregates and aggresomes by confocal microscopy. Methodology/Principal Findings YFP-tagged human αA-wild-type (αA-wt) was sub-cloned and the mutants were generated by site-directed mutagenesis. The αA-wt and the mutants were individually transfected or co-transfected with CFP-tagged αA-wt or αB-wild-type (αB-wt) in HeLa cells. Overexpression of these mutants forms multiple small dispersed cytoplasmic aggregates as well as aggresomes. Co-expression of αB-wt with these mutants significantly inhibited protein aggregates where as co-expression with αA-wt enhanced protein aggregates which seems to be due to co-aggregation of the mutants with αA-wt. Aggresomes were validated by double immunofluorescence by co-localization of γ-tubulin, a centrosome marker protein with αA-crystallin. Furthermore, increased ubiquitination was detected in R21W, R116C and R116H as assessed by western blot analyses. Immunostaining with an ubiquitin antibody revealed that ubiquitin inclusions in the perinuclear regions were evident only in R116C transfected cells. Pulse chase assay, after cycloheximide treatment, suggested that R116C degraded faster than the wild-type control. Conclusions/Significance Mutants of αA-crystallin form aggregates and aggresomes. Co-expression of αA-wt with the mutants increased aggregates and co-expression of αB-wt with the mutants significantly decreased the aggregates. The mutant, R116C protein degraded faster than wild-type control and increased ubiquitination was evident in R116C expressing cells.

Multiple Aggregates and Aggresomes of C-Terminal Truncated Human αA-Crystallins in Mammalian Cells and Protection by αB-Crystallin

Background Cleavage of 11 (αA162), 5 (αA168) and 1 (αA172) residues from the C-terminus of αA-crystallin creates structurally and functionally different proteins. The formation of these post-translationally modified αA-crystallins is enhanced in diabetes. In the present study, the fate of the truncated αA-crystallins expressed in living mammalian cells in the presence and absence of native αA- or αB-crystallin has been studied by laser scanning confocal microscopy (LSM). Methodology/Principal Findings YFP tagged αAwt, αA162, αA168 and αA172, were individually transfected or co-transfected with CFP tagged αAwt or αBwt, expressed in HeLa cells and studied by LSM. Difference in protein aggregation was not caused by different level of α-crystallin expression because Western blotting results showed nearly same level of expression of the various α-crystallins. The FRET-acceptor photo-bleaching protocol was followed to study in situ protein-protein interaction. αA172 interacted with αAwt and αBwt better than αA168 and αA162, interaction of αBwt being two-fold stronger than that of αAwt. Furthermore, aggresomes were detected in cells individually expressing αA162 and αA168 constructs and co-expression with αBwt significantly sequestered the aggresomes. There was no sequestration of aggresomes with αAwt co-expression with the truncated constructs, αA162 and αA168. Double immunocytochemistry technique was used for co-localization of γ-tubulin with αA-crystallin to demonstrate the perinuclear aggregates were aggresomes. Conclusions/Significance αA172 showed the strongest interaction with both αAwt and αBwt. Native αB-crystallin provided protection to partially unfolded truncated αA-crystallins whereas native αA-crystallin did not. Aggresomes were detected in cells expressing αA162 and αA168 and αBwt co-expression with these constructs diminished the aggresome formation. Co-localization of γ-tubulin in perinuclear aggregates validates for aggresomes.

Mutants of human αB-crystallin cause enhanced protein aggregation and apoptosis in mammalian cells: influence of co-expression of HspB1.

Mutations of human αB-crystallin cause congenital cataract and cardio-myopathy by protein aggregation and cell death. How mutations of αB-crystallin become pathogenic is poorly understood. To better understand the cellular events related to protein aggregation and cell death, we transfected cataract and cardio-myopathy causing mutants, R11H, P20S, R56W, D109H, R120G, D140N, G154S, R157H and A171T in HeLa cells and assessed protein aggregation and apoptosis by laser scanning confocal microspy (LSCM) and flow cytometry. Cells individually transfected with the mutants, D109H, R120G, D140N and R157H significantly showed more aggregates. Cells overexpressed with HspB1 (Hsp27) significantly sequestered aggregates in all mutants and suppressed apoptosis in mutants, P20S, D109H and A171T. Significant increases of apoptotic cells as stained with Annexin V were observed in mutants, D109H and A171T transfected cells. Cells positive for active caspase-3 was increased in the mutant, D109H. Thus the previously recognized anti-apoptotic functions of αB-crystallin were compromised in these mutants.

Effect of Oxidation of αA- and αB-Crystallins on their Structure, Oligomerization and Chaperone Function

The purpose of this study was to investigate the effect of metal-catalyzed oxidation by H2O2 on the structure, oligomerization, and chaperone function of αA- and αB-crystallins. Recombinant αA-and αB-crystallins were prepared by expressing them in E. coli and purifying by size-exclusion chromatography. They were incubated with 1.5 mM H2O2 and 0.1 mM FeCl3 at 37 ∘C for 24 hrs and the reaction was stopped by adding catalase. Structural changes due to oxidation were ascertained by circular dichroism (CD) measurements and chaperone activity was assayed with alcohol dehydrogenase (ADH) and insulin as target proteins. The oligomeric nature of the oxidized proteins was assessed by molecular sieve HPLC. The secondary structure of the oxidized αA- and αB-crystallins has been substantially altered due to significant increase in random coils, in addition to decrease in β-sheet or α-helix contents. The tertiary structure also showed significant changes indicative of different mode of folding of the secondary structural elements. Chaperone function was significantly compromised as supported by nearly 50% loss in chaperone activity. Oxidation also resulted in the formation of higher molecular weight (HMW) proteins as well as lower molecular weight (LMW) proteins. Thus, oxidation leads to disintegration of the oligomeric structure of αA- and αB-crystallins. Chaperone activity of the HMW fraction is normal whereas the LMW fraction lacks any chaperone activity. So, it appears that the formation of the LMW proteins is the primary cause of the chaperone activity loss due to oxidation.