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Small heat shock proteins (sHSPs) play a central role in protein homeostasis under conditions of stress by binding partly unfolded, aggregate‐prone proteins and keeping them soluble. Like many sHSPs, the widely expressed human sHSP, αB‐crystallin (‘αB’), forms large polydisperse multimeric assemblies. Molecular interactions involved in both sHSP function and oligomer formation remain to be delineated. A growing database of structural information reveals that a central conserved α‐crystallin domain (ACD) forms dimeric building blocks, while flanking N‐ and C‐termini direct the formation of larger sHSP oligomers. The most commonly observed inter‐subunit interaction involves a highly conserved C‐terminal ‘IxI/V’ motif and a groove in the ACD that is also implicated in client binding. To investigate the inherent properties of this interaction, peptides mimicking the IxI/V motif of αB and other human sHSPs were tested for binding to dimeric αB‐ACD. IxI‐mimicking peptides bind the isolated ACD at 22°C in a manner similar to interactions observed in the oligomer at low temperature, confirming these interactions are likely to exist in functional αB oligomers. Cytoprotective small heat‐shock proteins display an intrinsic affinity for their C‐termini in solution, providing candidate binding sites for both sHSP function and oligomerization.

Cataracts reduce vision in 50% of individuals over 70 years of age and are a common form of blindness worldwide. Cataracts are caused when damage to the major lens crystallin proteins causes their misfolding and aggregation into insoluble amyloids. Using a thermal stability assay, we identified a class of molecules that bind α-crystallins (cryAA and cryAB) and reversed their aggregation in vitro. The most promising compound improved lens transparency in the R49C cryAA and R120G cryAB mouse models of hereditary cataract. It also partially restored protein solubility in the lenses of aged mice in vivo and in human lenses ex vivo. These findings suggest an approach to treating cataracts by stabilizing α-crystallins.

The αA-crystallin protein plays a vital role in maintaining the refractive index and transparency of the eye lens. Significant clinical studies have emerged as the αA-crystallin is prone to aggregation, resulting in the formation of cataracts with varied etiologies due to mutations. This work aims to comprehend the structural and functional role of cataract-causing mutations in αA-crystallin, particularly at N-Terminal and α-Crystallin Domains, using in-silico approaches including molecular dynamics simulation. About 19 mutants of αA-crystallin along with native structure were simulated for 100 ns and the post-simulations analyses reveal pronounced dynamics of αA-crystallin due to the enhanced structure flexibility as its native compactness was lost and is witnessed mainly by the mutants R12L, R21L, R21Q, R54L, R65Q, R116C and R116H. It is observed that αA-crystallin discloses the NTD motions as the dominant one and the same was endorsed by the linear variation between Rg and the center-of-mass of αA-crystallin. Interestingly, such enhanced dynamics of αA-crystallin mutants associated with the structure flexibility is internally modulated by the dynamic exchange of secondary structure elements β-sheets and coils (R2 = 0.619) during simulation. Besides, the observed pronounced dynamics of dimer interface region (β3-L6-β4 segment) of ACD along with CTD dynamics also gains importance. Particularly, the highly dynamic mutants are also characterized by enhanced non-covalent and hydrophobic interactions which renders detrimental effects towards its stability, and favours possible protein unfolding mechanisms. Overall, this study highlights the mutation-mediated structural distortions in αA-crystallin and demands the need for further potential development of inhibitors against cataract formation.

αA-Crystallin (αA) and αB-crystallin (αB) are small heat shock proteins responsible for the maintenance of transparency in the lens. In non-lenticular tissues, αB is involved in both maintenance of the cytoskeleton and suppression of neurodegeneration amongst other roles. Despite their importance in maintaining cellular health, modifications and mutations to αA and αB appear to play a role in disease states such as cataract and myopathies. The list of modifications that have been reported is extensive and include oxidation, disulphide bond formation, C- and N-terminal truncation, acetylation, carboxymethylation, carboxyethylation, carbamylation, deamidation, phosphorylation and methylation. Such modifications, notably phosphorylation, are alleged to cause changes to chaperone activity by inducing substructural changes and altering subunit exchange dynamics. Although the effect modification has on the activities of αA and αB is contentious, it has been proposed that these changes are responsible for the induction of hyperactivity and are thereby indirectly responsible for protein deposition characteristic of many diseases associated with αA and αB. This review compiles all reported sites of αA and αB modifications, and investigates the role phosphorylation, in particular, plays in cellular processes.

We previously demonstrated that deletion of the 54 FLRAPSW 61 sequence, containing the key phosphorylation site serine 59 (S59), resulted in a two-fold reduction in oligomeric mass and a ten-fold enhancement of αB-crystallin’s chaperone activity. This study examined whether targeted deletion (ΔS59) or phosphomimetic substitution (S59D) could replicate these effects. Using MALS analysis, we found that the average oligomeric mass decreased from 579 kDa in the wild type (αB-WT) to 556 kDa in αB-ΔS59 and 434 kDa in αB-S59D. Interestingly, the αB-S59A variant had an increased mass of 611 kDa. All variants retained their chaperone function, but their efficiencies varied significantly. Specifically, αB-S59D formed smaller, more polydisperse complexes that effectively suppressed aggregation when interacting with rapidly aggregating substrates. In contrast, αB-ΔS59 and αB-S59A created stable complexes with lysozyme, reducing precipitation and aggregate size. Zeta potential measurements revealed distinct surface charge profiles among the variants; however, no clear correlation was observed between these charges and their chaperone efficiency. Additionally, cytotoxicity assays conducted on ARPE-19 cells under oxidative stress showed that all S59 variants exhibited comparable protective effects against cell death relative to αB-WT. These results indicate that while S59 is not essential for oligomer formation or chaperone function, it plays a crucial role in modulating oligomer size and interactions with various substrates. Notably, the effects of αB-S59D were measurable but did not replicate the enhanced functionality observed with the complete deletion of the 54–61 motif, reinforcing the significance of the N-terminal region.

As a molecular chaperone and activator of Toll-like receptor 2-mediated protective responses by microglia and macrophages, the small heat shock protein alpha B-crystallin (HspB5) exerts therapeutic effects in different animal models for neuroinflammation, including the model for multiple sclerosis (MS). Yet, HspB5 can also stimulate human antigen-specific memory T cells to release IFN-γ, a cytokine with well-documented detrimental effects during MS. In this study, we explored in a Phase IIa randomized clinical trial the therapeutic application of HspB5 in relapsing-remitting MS (RR-MS), using intravenous doses sufficient to support its protective effects, but too low to trigger pathogenic memory T-cell responses. These sub-immunogenic doses were selected based on in vitro analysis of the dose-response profile of human T cells and macrophages to HspB5, and on the immunological effects of HspB5 in healthy humans as established in a preparatory Phase I study. In a 48-week randomized, placebo-controlled, double-blind Phase IIa trial, three bimonthly intravenous injections of 7.5, 12.5 or 17.5 mg HspB5 were found to be safe and well tolerated in RR-MS patients. While predefined clinical endpoints did not differ significantly between the relatively small groups of MS patients treated with either HspB5 or placebo, repeated administration especially of the lower doses of HspB5 led to a progressive decline in MS lesion activity as monitored by magnetic resonance imaging (MRI), which was not seen in the placebo group. Exploratory linear regression analysis revealed this decline to be significant in the combined group receiving either of the two lower doses, and to result in a 76% reduction in both number and total volumes of active MRI lesions at 9 months into the study. These data provide the first indication for clinical benefit resulting from intervention in RR-MS with HspB5. ClinicalTrials.gov Phase I: NCT02442557; Phase IIa: NCT02442570.

Interaction among crystallins is required for the maintenance of lens transparency. Deamidation is one of the most common post-translational modifications in crystallins, which results in incorrect interaction and leads to aggregate formation. Various studies have established interaction among the α- and β-crystallins. Here, we investigated the effects of the deamidation of αA- and αB-crystallins on their interaction with βA3-crystallin using surface plasmon resonance (SPR) and fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET) methods. SPR analysis confirmed adherence of WT αA- and WT αB-crystallins and their deamidated mutants with βA3-crystallin. The deamidated mutants of αA-crystallin (αA N101D and αA N123D) displayed lower adherence propensity for βA3-crystallin relative to the binding affinity shown by WT αA-crystallin. Among αB-crystallin mutants, αB N78D displayed higher adherence propensity whereas αB N146D mutant showed slightly lower binding affinity for βA3-crystallin relative to that shown by WT αB-crystallin. Under the in vivo condition (FLIM-FRET), both αA-deamidated mutants (αA N101D and αA N123D) exhibited strong interaction with βA3-crystallin (32±4% and 36±4% FRET efficiencies, respectively) compared to WT αA-crystallin (18±4%). Similarly, the αB N78D and αB N146D mutants showed strong interaction (36±4% and 22±4% FRET efficiencies, respectively) with βA3-crystallin compared to 18±4% FRET efficiency of WT αB-crystallin. Further, FLIM-FRET analysis of the C-terminal domain (CTE), N-terminal domain (NTD), and core domain (CD) of αA- and αB-crystallins with βA3-crystallin suggested that interaction sites most likely reside in the αA CTE and αB NTD regions, respectively, as these domains showed the highest FRET efficiencies. Overall, results suggest that similar to WT αA- and WTαB-crystallins, the deamidated mutants showed strong interactionfor βA3-crystallin. Variable in vitro and in vivo interactions are most likely due to the mutant's large size oligomers, reduced hydrophobicity, and altered structures. Together, the results suggest that deamidation of α-crystallin may facilitate greater interaction and the formation of large oligomers with other crystallins, and this may contribute to the cataractogenic mechanism.

CRYβA1-ΔG91 (βA3ΔG91) is a mutational hotspot in CRYβA1, which causes autosomal dominant congenital nuclear cataract in humans and mice. Previous in-vitro studies of recombinant βA3ΔG91 showed defective folding, decreased solubility, and aberrant oligomerization of βA3ΔG91 with other crystallins. Emerging evidence demonstrates an association between autophagy and βA3ΔG91-induced congenital cataracts. To gain further understanding of the molecular mechanism of congenital cataract development in βA3ΔG91 mice, we examined the βA3ΔG91- vs WT- lenses for complete gene profiling, lens epithelial cell (LEC) proliferation and migration, and lens epithelial-fiber cell differentiation. We also determined the changes in crystallin proteomic profiles in water-soluble, water-insoluble-urea-soluble, and water-insoluble-urea-insoluble fractions. Our results show that relative to WT lenses, the βA3ΔG91 lenses showed: (A) downregulation of genes associated with LECs proliferation and migration (B) abnormal suture line pattern, (C) significant reduction in proliferation and migration of LECs, (D) abnormal F-actin distribution, (E) increased high molecular weight (HMW) peak, and (F) insolubilization and degradation of crystallins and other lens proteins. Together, these defects contribute to the formation of the lens opacity in βA3ΔG91 mice lenses.

α-Crystallin (αABc) is a major protein comprised of αA-crystallin (αAc) and αB-crystallin (αBc) that is found in the human eye lens and works as a molecular chaperone by preventing the aggregation of proteins and providing tolerance to stress. However, with age and cataract formation, the concentration of αABc in the eye lens cytoplasm decreases, with a corresponding increase in the membrane-bound αABc. This study uses the electron paramagnetic resonance (EPR) spin-labeling method to investigate the role of cholesterol (Chol) and Chol bilayer domains (CBDs) in the binding of αAc, αBc, and αABc to the Chol/model of human lens-lipid (Chol/MHLL) membranes. The maximum percentage of membrane surface occupied (MMSO) by αAc, αBc, and αABc to Chol/MHLL membranes at a mixing ratio of 0 followed the trends: MMSO (αAc) > MMSO (αBc) ≈ MMSO (αABc), indicating that a higher amount of αAc binds to these membranes compared to αBc and αABc. However, with an increase in the Chol concentration in the Chol/MHLL membranes, the MMSO by αAc, αBc, and αABc decreases until it is completely diminished at a mixing ratio of 1.5. The Ka of αAc, αBc, and αABc to Chol/MHLL membranes at a mixing ratio of 0 followed the trend: Ka (αBc) ≈ Ka (αABc) > Ka (αAc), but it was close to zero with the diminished binding at a Chol/MHLL mixing ratio of 1.5. The mobility near the membrane headgroup regions decreased with αAc, αBc, and αABc binding, and the Chol antagonized the capacity of the αAc, αBc, and αABc to decrease mobility near the headgroup regions. No significant change in membrane order near the headgroup regions was observed, with an increase in αAc, αBc, and αABc concentrations. Our results show that αAc, αBc, and αABc bind differently with Chol/MHLL membranes at mixing ratios of 0 and 0.5, decreasing the mobility and increasing hydrophobicity near the membrane headgroup region, likely forming the hydrophobic barrier for the passage of polar and ionic molecules, including antioxidants (glutathione), creating an oxidative environment inside the lens, leading to the development of cataracts. However, all binding was completely diminished at a mixing ratio of 1.5, indicating that high Chol and CBDs inhibit the binding of αAc, αBc, and αABc to membranes, preventing the formation of hydrophobic barriers and likely protecting against cataract formation.

Human αB-crystallin is a small heat shock protein that functions as a chaperone and anti-apoptotic protein to maintain cellular protein integrity. A specific mutation (p.R163C) in the C-terminal domain has been linked to dilated cardiomyopathy (DCM). However, the impact of this mutation on the protein’s structure, activity, stability, and amyloidogenic properties remains unclear. Here, we introduced the mutation, expressed and purified the protein, and used spectroscopic and microscopic techniques to conduct a comprehensive investigation of the mutant protein. The p.R163C mutation in αB-crystallin induces subtle changes in its secondary and tertiary structures, resulting in a slight increase in the distance and angle between monomer units within the dimer. The mutation causes the protein to form larger oligomers with increased chaperone activity, which may protect against cell death but could also lead to excessive client protein sequestration or coaggregation, potentially causing cytotoxicity. Accompanied by these alterations, the chemical and thermal stability of the mutant protein decrease, the resistance of the protein to enzymatic digestion increases, and finally, the propensity of the p.R163C mutated protein to form amyloid fibrils elevates. The substitution of the conserved arginine at position 163 with cysteine likely impacts the ability of the mutated protein to interact with cardiac muscle proteins. Collectively, these structural and functional modifications in the mutated protein may perturb cellular homeostasis and contribute to the onset of DCM.