Modulation of Alzheimer's Disease Aß40 Fibril Polymorphism by the Small Heat Shock Protein αB-Crystallin.

Deposition of amyloid plaques in the brains of Alzheimer's disease (AD) patients is a hallmark of the disease. AD plaques consist primarily of the beta-amyloid (Aß) peptide but can contain other factors such as lipids, proteoglycans, and chaperones. So far, it is unclear how the cellular environment modulates fibril polymorphism and how differences in fibril structure affect cell viability. The small heat-shock protein (sHSP) alpha-B-Crystallin (αBC) is abundant in brains of AD patients, and colocalizes with Aß amyloid plaques. Using solid-state NMR spectroscopy, we show that the Aß40 fibril seed structure is not replicated in the presence of the sHSP. αBC prevents the generation of a compact fibril structure and leads to the formation of a new polymorph with a dynamic N-terminus. We find that the N-terminal fuzzy coat and the stability of the C-terminal residues in the Aß40 fibril core affect the chemical and thermodynamic stability of the fibrils and influence their seeding capacity. We believe that our results yield a better understanding of how sHSP, such as αBC, that are part of the cellular environment, can affect fibril structures related to cell degeneration in amyloid diseases.

Unraveling the impact of the p.R107L mutation on the structure and function of human αB-Crystallin: Implications for cataract formation.

To date, several pathogenic mutations have been identified in the primary structure of human α-Crystallin, frequently involving the substitution of arginine with a different amino acid. These mutations can lead to the incidence of cataracts and myopathy. Recently, an important cataract-associated mutation has been reported in the functional α-Crystallin domain (ACD) of human αB-Crystallin protein, where arginine 107 (R107) is replaced by a leucine. In this study, we investigated the structure, chaperone function, stability, oligomerization, and amyloidogenic properties of the p.R107L human αB-Crystallin using a number of different techniques. Our results suggest that the p.R107L mutation can cause significant changes in the secondary, tertiary, and quaternary structures of αB-Crystallin. This cataractogenic mutation led to the formation of protein oligomers with larger sizes than the wild-type protein and reduced the chemical and thermal stability of the mutant chaperone. Both fluorescence and microscopic assessments indicated that this mutation significantly altered the amyloidogenic properties of human αB-Crystallin. Furthermore, the mutant protein indicated an attenuated in vitro chaperone activity. The molecular dynamics (MD) simulation confirmed the experimental results and indicated that p.R107L mutation could alter the proper conformation of human αB-Crystallin dimers. In summary, our results indicated that the p.R107L mutation could promote the formation of larger oligomers, diminish the stability and chaperone activity of human αB-Crystallin, and these changes, in turn, can play a crucial role in the development of cataract disorder.

Exploring the significance of potassium homeostasis in copper ion binding to human αB-Crystallin.

αB-Crystallin (αB-Cry) is a small heat shock protein known for its protective role, with an adaptable structure that responds to environmental changes through oligomeric dynamics. Cu(II) ions are crucial for cellular processes but excessive amounts are linked to diseases like cataracts and neurodegeneration. This study investigated how optimal and detrimental Cu(II) concentrations affect αB-Cry oligomers and their chaperone activity, within the potassium-regulated ionic-strength environment. Techniques including isothermal titration calorimetry, differential scanning calorimetry, fluorescence spectroscopy, inductively coupled plasma atomic emission spectroscopy, cyclic voltammetry, dynamic light scattering, circular dichroism, and MTT assay were employed and complemented by computational methods. Results showed that potassium ions affected αB-Cry's structure, promoting Cu(II) binding at multiple sites and scavenging ability, and inhibiting ion redox reactions. Low concentrations of Cu(II), through modifications of oligomeric interfaces, induce regulation of surface charge and hydrophobicity, resulting in an increase in chaperone activity. Subunit dynamics were regulated, maintaining stable interfaces, thereby inhibiting further aggregation and allowing the functional reversion to oligomers after stress. High Cu(II) disrupted charge/hydrophobicity balance, sewing sizable oligomers together through subunit-subunit interactions, suppressing oligomer dissociation, and reducing chaperone efficiency. This study offers insights into how Cu(II) and potassium ions influence αB-Cry, advancing our understanding of Cu(II)-related diseases.

Unveiling the structural and functional consequences of the p.D109G pathogenic mutation in human αB-Crystallin responsible for restrictive cardiomyopathy and skeletal myopathy.

αB-Crystallin (αB-Cry) is expressed in many tissues, and mutations in this protein are linked to various diseases, including cataracts, Alzheimer's disease, Parkinson's disease, and several types of myopathies and cardiomyopathies. The p.D109G mutation, which substitutes a conserved aspartate residue involved in the interchain salt bridges, with glycine leads to the development of both restrictive cardiomyopathy (RCM) and skeletal myopathy. In this study, we generated this mutation in the α-Cry domain (ACD) which is crucial for forming the active chaperone dimeric state, using site-directed mutagenesis. After inducing expression in the bacterial host, we purified the mutant and wild-type recombinant proteins using anion exchange chromatography. Various spectroscopic evaluations revealed significant changes in the secondary, tertiary, and quaternary structures of human αB-Cry caused by this mutation. Furthermore, this pathogenic mutation led to the formation of protein oligomers with larger sizes than those of the wild-type protein counterpart. The mutant protein also exhibited increased chaperone activity and decreased chemical, thermal, and proteolytic stability. Atomic force microscopy (AFM), transmission electron microscopy (TEM), and fluorescence microscopy (FM) demonstrated that p.D109G mutant protein is more prone to forming amyloid aggregates. The misfolding associated with the p.D109G mutation may result in abnormal interactions of human αB-Cry with its natural partners (e.g., desmin), leading to the formation of protein aggregates. These aggregates can interfere with normal cellular processes and may contribute to muscle cell dysfunction and damage, resulting in the pathogenic involvement of the p.D109G mutant protein in restrictive cardiomyopathy and skeletal myopathy.

Investigating the role of double mutations R12C/P20R, and R12C/R69C on structure, chaperone-like activity, and amyloidogenic properties of human αB-crystallin.

α-crystallin is a structurally essential small heat shock protein (sHSP) with a chaperone-like activity which maintains transparency of the lenticular tissues during a period of time that is as long as human life. α-crystallin is a multimeric protein consisting of αA and αB subunits, with 57 % homology. The CRYAB gene on chromosome 11 encodes human αB-crystallin (αB-Cry), which contains 175 amino acid residues. In the current study, the cataractogenic mutations R12C, P20R, R69C, and double mutations R12C/P20R and R12C/P20R were embedded into the human CRYAB gene. Following successful expression in the prokaryotic system and purification, a number of spectroscopic techniques, gel electrophoresis, dynamic light scattering (DLS), and transmission electron microscopy (TEM) were applied to assess the role of these mutations on the structure, amyloidogenicity, and biological function of human αB-Cry. The created mutations caused significant changes in the structure, and oligomeric state of human αB-Cry. These mutations, particularly R12C, R12C/P20R, and R12C/R69C, dramatically enhanced the tendency of this protein for the amyloid fibril formation and reduced its chaperone-like activity. Since double mutations R12C/P20R and R12C/P20R were able to intensely change the protein's structure and chaperone function, it can be suggested that they may play a destructive role in a cumulative manner. Our findings indicated that the simultaneous presence of two pathogenic mutations may have a cumulative destructive impacts on the structure and function of human αB-Cry and this observation is likely related to the disease severity of the mutated proteins.

The Monomeric α-Crystallin Domain of the Small Heat-shock Proteins αB-crystallin and Hsp27 Binds Amyloid Fibril Ends.

Small heat-shock proteins (sHSPs) are ubiquitously expressed molecular chaperones present in all kingdoms of life that inhibit protein misfolding and aggregation. Despite their importance in proteostasis, the structure-function relationships of sHSPs remain elusive. Human sHSPs are characterised by a central, highly conserved α-crystallin domain (ACD) and variable-length N- and C-terminal regions. The ACD forms antiparallel homodimers via an extended ß-strand, creating a shared ß-sheet at the dimer interface. The N- and C-terminal regions mediate formation of higher order oligomers that are thought to act as storage forms for chaperone-active dimers. We investigated the interactions of the ACD of two human sHSPs, αB-crystallin (αB-C) and Hsp27, with apolipoprotein C-II amyloid fibrils using analytical ultracentrifugation and nuclear magnetic resonance spectroscopy. The ACD was found to interact transiently with amyloid fibrils to inhibit fibril elongation and naturally occurring fibril end-to-end joining. This interaction was sensitive to the concentration of fibril ends indicating a 'fibril-capping' interaction. Furthermore, resonances arising from the ACD monomer were attenuated to a greater extent than those of the ACD dimer in the presence of fibrils, suggesting that the monomer may bind fibrils. This hypothesis was supported by mutagenesis studies in which disulfide cross-linked ACD dimers formed by both αB-C and Hsp27 were less effective at inhibiting amyloid fibril elongation and fibril end-to-end joining than ACD constructs lacking disulfide cross-linking. Our results indicate that sHSP monomers inhibit amyloid fibril elongation, highlighting the importance of the dynamic oligomeric nature of sHSPs for client binding.

Increased chaperone activity of human α|B-crystallin with incomplete oxidation as a new defense mechanism against oxidative stress.

Previous research has shown that production of the high levels of oxidants overwhelms the body's antioxidant defense system during diabetes mellitus. Under this circumstance, ocular lens proteins are one of the main molecular targets for oxidative damage. In the present study, the individual effect of partial and extensive oxidation on the structure and function of human αB-crystallin was investigated using electrophoresis and various spectroscopic methods. The results of our study suggested that widespread oxidation causes loss of the chaperone activity of this protein, while partial oxidation significantly enhances this activity. Our studies also suggested that partial and extensive oxidation induces the formation of different structures in this protein. In fact, the chaperone-active and chaperone-inactive states of this protein are respectively associated with a minor and extensive structural alteration. Moreover, the oligomeric size distribution shows an inverse relationship with the chaperone activity of this protein. Increasing the chaperone activity of this protein during partial oxidation may be a natural defense mechanism to overcome the damages caused by oxidative stress, especially in diabetes and other pathological diseases.

Deletion of Specific Conserved Motifs from the N-Terminal Domain of αB-Crystallin Results in the Activation of Chaperone Functions.

Smaller oligomeric chaperones of α-crystallins (αA- and αB-) have received increasing attention due to their improved therapeutic potential in preventing protein aggregating diseases. Our previous study suggested that deleting 54-61 residues from the N-terminal domain (NTD) of αB-crystallin (αBΔ54-61) decreases the oligomer size and increases the chaperone function. Several studies have also suggested that NTD plays a significant role in protein oligomerization and chaperone function. The current study was undertaken to assess the effect of deleting conserved 21-28 residues from the activated αBΔ54-61 (to get αBΔ21-28, Δ54-61) on the structure-function of recombinant αBΔ21-28, Δ54-61. The αBΔ21-28, Δ54-61 mutant shows an 80% reduction in oligomer size and 3- to 25-fold increases in chaperone activity against model substrates when compared to αB-WT. Additionally, the αB∆21-28, ∆54-61 was found to prevent ß-amyloid (Aß1-42) fibril formation in vitro and suppressed Aß1-42-induced cytotoxicity in ARPE-19 cells in a more effective manner than seen with αB-WT or αB∆54-61. Cytotoxicity and reactive oxygen species (ROS) detection studies with sodium iodate (SI) showed that the double mutant protein has higher anti-apoptotic and anti-oxidative activities than the wild-type or αB∆54-61 in oxidatively stressed cells. Our study shows that the residues 21-28 and 54-61 in αB-crystallin contribute to the oligomerization and modulate chaperone function. The deletion of conserved 21-28 residues further potentiates the activated αBΔ54-61. We propose that increased substrate affinity, altered subunit structure, and assembly leading to smaller oligomers could be the causative factors for the increased chaperone activity of αBΔ21-28, Δ54-61.

Structural and functional studies of D109A human αB-crystallin contributing to the development of cataract and cardiomyopathy diseases.

αB-crystallin (heat shock protein ß5/HSPB5) is a member of the family of small heat shock proteins that is expressed in various organs of the human body including eye lenses and muscles. Therefore, mutations in the gene of this protein (CRYAB) might have many pathological consequences. A new mutation has recently been discovered in the α-crystallin domain of this chaperone protein which replaces aspartate 109 with alanine (D109A). This mutation can cause myofibrillar myopathy (MFM), cataracts, and cardiomyopathy. In the current study, several spectroscopic and microscopic analyses, as well as gel electrophoresis assessment were applied to elucidate the pathogenic contribution of human αB-crystallin bearing D109A mutation in development of eye lens cataract and myopathies. The protein oligomerization, chaperone-like activity and chemical/thermal stabilities of the mutant and wild-type protein were also investigated in the comparative assessments. Our results suggested that the D109A mutation has a significant impact on the important features of human αB-crystallin, including its structure, size of the protein oligomers, tendency to form amyloid fibrils, stability, and chaperone-like activity. Given the importance of aspartate 109 in maintaining the proper structure of the α-crystallin domain, its role in the dimerization and chaperone-like activity, as well as preserving protein stability through the formation of salt bridges; mutation at this important site might have critical consequences and can explain the genesis of myopathy and cataract disorders. Also, the formation of large light-scattering aggregates and disruption of the chaperone-like activity by D109A mutation might be considered as important contributing factors in development of the eye lens opacity.

Effect of Structural Changes Induced by Deletion of 54FLRAPSWF61 Sequence in αB-crystallin on Chaperone Function and Anti-Apoptotic Activity.

Previously, we showed that the removal of the 54-61 residues from αB-crystallin (αBΔ54-61) results in a fifty percent reduction in the oligomeric mass and a ten-fold increase in chaperone-like activity. In this study, we investigated the oligomeric organization changes in the deletion mutant contributing to the increased chaperone activity and evaluated the cytoprotection properties of the mutant protein using ARPE-19 cells. Trypsin digestion studies revealed that additional tryptic cleavage sites become susceptible in the deletion mutant than in the wild-type protein, suggesting a different subunit organization in the oligomer of the mutant protein. Static and dynamic light scattering analyses of chaperone-substrate complexes showed that the deletion mutant has more significant interaction with the substrates than wild-type protein, resulting in increased binding of the unfolding proteins. Cytotoxicity studies carried out with ARPE-19 cells showed an enhancement in anti-apoptotic activity in αBΔ54-61 as compared with the wild-type protein. The improved anti-apoptotic activity of the mutant is also supported by reduced caspase activation and normalization of the apoptotic cascade components level in cells treated with the deletion mutant. Our study suggests that altered oligomeric assembly with increased substrate affinity could be the basis for the enhanced chaperone function of the αBΔ54-61 protein.

Analysis of amyloid-like secondary structure in the Cryab-R120G knock-in mouse model of hereditary cataracts by two-dimensional infrared spectroscopy.

αB-crystallin is a small heat shock protein that forms a heterooligomeric complex with αA-crystallin in the ocular lens. It is also widely distributed in tissues throughout the body and has been linked with neurodegenerative diseases such as Alzheimer's, where it is associated with amyloid fibrils. Crystallins can form amorphous aggregates in cataracts as well as more structured amyloid-like fibrils. The arginine 120 to glycine (R120G) mutation in αB-crystallin (Cryab-R120G) results in high molecular weight crystallin protein aggregates and loss of the chaperone activity of the protein in vitro, and it is associated with human hereditary cataracts and myopathy. Characterizing the amorphous (unstructured) versus the highly ordered (amyloid fibril) nature of crystallin aggregates is important in understanding their role in disease and important to developing pharmacological treatments for cataracts. We investigated protein secondary structure in wild-type (WT) and Cryab-R120G knock-in mutant mouse lenses using two-dimensional infrared (2DIR) spectroscopy, which has been used to detect amyloid-like fibrils in human lenses and measure UV radiation-induced changes in porcine lenses. Our goal was to compare the aggregated proteins in this mouse lens model to human lenses and evaluate the protein structural relevance of the Cryab-R120G knock-in mouse model to general age-related cataract disease. In the 2DIR spectra, amide I diagonal peak frequencies were red-shifted to smaller wavenumbers in mutant mouse lenses as compared to WT mouse lenses, consistent with an increase in ordered secondary structure. The cross peak frequency and intensity indicated the presence of amyloid in the mutant mouse lenses. While the diagonal and cross peak changes in location and intensity from the 2DIR spectra indicated significant structural differences between the wild type and mutant mouse lenses, these differences were smaller than those found in human lenses; thus, the Cryab-R120G knock-in mouse lenses contain less amyloid-like secondary structure than human lenses. The results of the 2DIR spectroscopy study confirm the presence of amyloid-like secondary structure in Cryab-R120G knock-in mice with cataracts and support the use of this model to study age-related cataract.

The biochemical association between R157H mutation in human αB-crystallin and development of cardiomyopathy: Structural and functional analyses of the mutant protein.

In human αB-crystallin or HspB5, the substitution of arginine residue at position 157 with histidine has been reported to cause cardiomyopathy. In this study, the impact of R157H mutation on the structure, stability and functional properties of human αB-crystallin was investigated using a variety of spectroscopic techniques and microscopic analyses. Our spectroscopic analyses revealed that this mutation has a negligible impact on the secondary and tertiary structures of HspB5 but its quaternary structure underwent fundamental changes. Although the chemical stability of the mutant protein remained largely unchanged, the differential scanning calorimetry (DSC) measurement suggested that its thermal stability was reduced. As examined with transmission electron microscopy, αB-crystallin and its mutant indicated a similar tendency for the amyloid fibril formation under thermochemical stress. Dynamic light scattering (DLS) analysis suggested important changes in the quaternary (oligomeric) structures of the mutant protein as compared with the native protein counterpart. Also, the mutant protein indicated an improved chaperone-like activity under in vitro assessment. In a pH-dependent manner, the side chains of arginine and histidine have different capabilities for establishing hydrogen bonds and electrostatic interaction (salt bridge) and this variation may be sufficient to produce the larger changes that ultimately alter the interaction of this protein with other target proteins. Overall, the pathogenic contribution of this mutation in cardiomyopathy can be explained by its role in quaternary structure/stability alteration of the mutated protein.

The engineered expression of secreted HSPB5-Fc in CHO cells exhibits cytoprotection in vitro.

BACKGROUND: HSPB5 is an ATP-independent molecular chaperone that is induced by heat shock or other proteotoxic stresses. HSPB5 is cytoprotective against stress both intracellularly and extracellularly. It acts as a potential therapeutic candidate in ischemia-reperfusion and neurodegenerative diseases. RESULTS: In this paper, we constructed a recombinant plasmid that expresses and extracellularly secrets a HSPB5-Fc fusion protein (sHSPB5-Fc) at 0.42 µg/ml in CHO-K1 cells. This sHSPB5-Fc protein contains a Fc-tag at the C-terminal extension of HSPB5, facilitating protein-affinity purification. Our study shows that sHSPB5-Fc inhibits heat-induced aggregation of citrate synthase in a time and dose dependent manner in vitro. Administration of sHSPB5-Fc protects lens epithelial cells against cisplatin- or UVB-induced cell apoptosis. It also decreases GFP-Httex1-Q74 insolubility, and reduces the size and cytotoxicity of GFP-Httex1-Q74 aggregates in PC-12 cells. CONCLUSION: This recombinant sHSPB5-Fc exhibits chaperone activity to protect cells against proteotoxicity.

Single-molecule fluorescence-based approach reveals novel mechanistic insights into human small heat shock protein chaperone function.

Small heat shock proteins (sHsps) are a family of ubiquitous intracellular molecular chaperones; some sHsp family members are upregulated under stress conditions and play a vital role in protein homeostasis (proteostasis). It is commonly accepted that these chaperones work by trapping misfolded proteins to prevent their aggregation; however, fundamental questions regarding the molecular mechanism by which sHsps interact with misfolded proteins remain unanswered. The dynamic and polydisperse nature of sHsp oligomers has made studying them challenging using traditional biochemical approaches. Therefore, we have utilized a single-molecule fluorescence-based approach to observe the chaperone action of human alphaB-crystallin (αBc, HSPB5). Using this approach we have, for the first time, determined the stoichiometries of complexes formed between αBc and a model client protein, chloride intracellular channel 1. By examining the dispersity and stoichiometries of these complexes over time, and in response to different concentrations of αBc, we have uncovered unique and important insights into a two-step mechanism by which αBc interacts with misfolded client proteins to prevent their aggregation.

Transcriptomic analysis reveals the role of a peptide derived from CRYAB on the CoCl2-induced hypoxic HL-1 cardiomyocytes.

Acute myocardial infarction (AMI) is a life-threatening disease that often results in heart failure. CRYAB, a small heat shock protein, has been shown to have cardioprotective effects against oxidative stress-induced apoptosis in AMI. Previously, we purified a peptide derived from CRYAB (LEDQFFGEH), which we named PDFC. In this study, we determined the function of PDFC on HL-1 cardiomyocytes and explored the mechanism underlying its function. A hypoxic myocardiocyte cell line was generated by stimulation of HL-1 mouse cardiac muscle cells with different concentrations of CoCl2. Then, the hypoxic HL-1 cells were treated with the synthetic PDFC peptide, and cell proliferation, migration, and apoptosis were assessed to examine the effects of PDFC on HL-1 and hypoxic HL-1 cells. To examine the mechanism underlying the effects of PDFC on hypoxic cells, PDFC-treated hypoxic HL-1 cells were submitted for deep RNA sequencing. Finally, several differentially expressed genes in different pathways were selected for confirmation by RT-qPCR. Hypoxic myocardiocytes were generated by stimulating HL-1 cells with 800 µM CoCl2 for 24 h, which significantly upregulated HIF-1α. PDFC at 200 µg/ml showed the most positive effects on cell viability. Although hypoxic HL-1 cells and PDFC-treated hypoxic HL-1 cells both showed lower viability and migration and higher levels of apoptosis than untreated HL-1 cells, compared to hypoxic HL-1 cells, PDFC-treated hypoxic HL-1 cells showed higher viability and migration and lower apoptosis. The deep sequencing showed that 812 genes were upregulated and 1946 genes were downregulated. Among these differentially expressed genes, 699 of the upregulated genes and 1488 of the downregulated genes were protein-coding genes. Gene ontology and pathway enrichment analysis showed that the downregulated genes were dominant and that the PI3K-Akt pathway was located in the center of the network. A protein-protein interaction network was constructed, and 892 nodes were determined. In PDFC-treated hypoxic HL-1 cells, Fn1, Pik3r5, and Creb5 were downregulated, while Insr, Bcl2, Mapk14, and Pten were upregulated when compared to the levels in hypoxic HL-1 cells. In conclusion, this study reveals the significant bioactive effect of the CRYAB-derived peptide, PDFC on cardiomyocytes and the underlying mechanism.

Alpha-B-Crystallin Effect on Mature Amyloid Fibrils: Different Degradation Mechanisms and Changes in Cytotoxicity.

Given the ability of molecular chaperones and chaperone-like proteins to inhibit the formation of pathological amyloid fibrils, the chaperone-based therapy of amyloidosis has recently been proposed. However, since these diseases are often diagnosed at the stages when a large amount of amyloids is already accumulated in the patient's body, in this work we pay attention to the undeservedly poorly studied problem of chaperone and chaperone-like proteins' effect on mature amyloid fibrils. We showed that a heat shock protein alpha-B-crystallin, which is capable of inhibiting fibrillogenesis and is found in large quantities as a part of amyloid plaques, can induce degradation of mature amyloids by two different mechanisms. Under physiological conditions, alpha-B-crystallin induces fluffing and unweaving of amyloid fibrils, which leads to a partial decrease in their structural ordering without lowering their stability and can increase their cytotoxicity. We found a higher correlation between the rate and effectiveness of amyloids degradation with the size of fibrils clusters rather than with amino acid sequence of amyloidogenic protein. Some external effects (such as an increase in medium acidity) can lead to a change in the mechanism of fibrils degradation induced by alpha-B-crystallin: amyloid fibers are fragmented without changing their secondary structure and properties. According to recent data, fibrils cutting can lead to the generation of seeds for new bona fide amyloid fibrils and accelerate the accumulation of amyloids, as well as enhance the ability of fibrils to disrupt membranes and to reduce cell viability. Our results emphasize the need to test the chaperone effect not only on fibrillogenesis, but also on the mature amyloid fibrils, including stress conditions, in order to avoid undesirable disease progression during chaperone-based therapy.

Transient elevation of temperature promotes cross-linking of α-crystallin-client proteins through formation of advanced glycation endproducts: A potential role in presbyopia and cataracts.

The chaperone activity of α-crystallin is important for maintaining the transparency of the human lens. αB-crystallin (αBC) is a long-lived protein in the lens that accumulates chemical modifications during aging. The formation of advanced glycation end products (AGEs) through glycation is one such modification. αBC is a small heat shock protein that exhibits chaperone activity. We have previously shown that αBC-client protein complexes can undergo AGE-mediated interprotein cross-linking. Here, we demonstrate that short-term (1 h) exposure to elevated temperatures and methylglyoxal (MGO) during the chaperoning of client proteins by αBC promotes AGE-mediated interprotein cross-linking. Liquid chromatography/mass spectrometry (LC-MS/MS) analyses revealed the rapid formation of AGEs by MGO. Interestingly, we found that despite protein cross-linking, the chaperone activity of αBC increased during the transient elevation of temperature in the presence of MGO. Together, these results imply that transient and subtle elevation of temperature in the lens of the eye can promote protein cross-linking through AGEs, and if this phenomenon recurs over a period of many years, it could lead to early onset of presbyopia and age-related cataracts.

The Aggregation of αB-Crystallin under Crowding Conditions Is Prevented by αA-Crystallin: Implications for α-Crystallin Stability and Lens Transparency.

One of the most crowded biological environments is the eye lens which contains a high concentration of crystallin proteins. The molecular chaperones αB-crystallin (αBc) with its lens partner αA-crystallin (αAc) prevent deleterious crystallin aggregation and cataract formation. However, some forms of cataract are associated with structural alteration and dysfunction of αBc. While many studies have investigated the structure and function of αBc under dilute in vitro conditions, the effect of crowding on these aspects is not well understood despite its in vivo relevance. The structure and chaperone ability of αBc under conditions that mimic the crowded lens environment were investigated using the polysaccharide Ficoll 400 and bovine γ-crystallin as crowding agents and a variety of biophysical methods, principally contrast variation small-angle neutron scattering. Under crowding conditions, αBc unfolds, increases its size/oligomeric state, decreases its thermal stability and chaperone ability, and forms kinetically distinct amorphous and fibrillar aggregates. However, the presence of αAc stabilizes αBc against aggregation. These observations provide a rationale, at the molecular level, for the aggregation of αBc in the crowded lens, a process that exhibits structural and functional similarities to the aggregation of cataract-associated αBc mutants R120G and D109A under dilute conditions. Strategies that maintain or restore αBc stability, as αAc does, may provide therapeutic avenues for the treatment of cataract.

Chaperone-Like Activity of HSPB5: The Effects of Quaternary Structure Dynamics and Crowding.

Small heat-shock proteins (sHSPs) are ATP-independent molecular chaperones that interact with partially unfolded proteins, preventing their aberrant aggregation, thereby exhibiting a chaperone-like activity. Dynamics of the quaternary structure plays an important role in the chaperone-like activity of sHSPs. However, relationship between the dynamic structure of sHSPs and their chaperone-like activity remains insufficiently characterized. Many factors (temperature, ions, a target protein, crowding etc.) affect the structure and activity of sHSPs. The least studied is an effect of crowding on sHSPs activity. In this work the chaperone-like activity of HSPB5 was quantitatively characterized by dynamic light scattering using two test systems, namely test systems based on heat-induced aggregation of muscle glycogen phosphorylase b (Phb) at 48 °C and dithiothreitol-induced aggregation of α-lactalbumin at 37 °C. Analytical ultracentrifugation was used to control the oligomeric state of HSPB5 and target proteins. The possible anti-aggregation functioning of suboligomeric forms of HSPB5 is discussed. The effect of crowding on HSPB5 anti-aggregation activity was characterized using Phb as a target protein. The duration of the nucleation stage was shown to decrease with simultaneous increase in the relative rate of aggregation of Phb in the presence of HSPB5 under crowded conditions. Crowding may subtly modulate sHSPs activity.

A novel dominant mutation in CRYAB gene leading to a severe phenotype with childhood onset.

BACKGROUND: αB-crystallin is a promiscuous protein involved in numerous cell functions. Mutations in CRYAB have been found in patients with different pathological phenotypes that are not properly understood. Patients can present different diseases like cataracts, muscle weakness, myopathy, cardiomyopathy, respiratory insufficiency or dysphagia, but also a variable combination of these pathologies has been found. These mutations can show either autosomal dominant or recessive mode of inheritance and variable penetrance and expressivity. This is the first report of congenital cataracts and myopathy described in childhood due to a CRYAB mutation with autosomal dominant mode of inheritance. METHODS: The whole exome sequence was subjected to phenotype-driven analysis and a novel variant in CRYAB was detected: c.514delG, p.(Ala172ProfsTer14). The mutation was located in the C-terminal domain of the protein, which is essential for chaperone activity. The deduced protein was analyzed searching for alterations of the relevant physico-chemical properties described for this domain. A muscle biopsy was also tested for CRYAB with immunohistochemical and histoenzymatic techniques. RESULTS: CRYAB displayed a mild immunoreactivity in the subsarcolemmal compartment with no pathological sarcoplasmic accumulation. It agrees with an alteration of the physico-chemical properties predicted for the C-terminal domain: hydrophobicity, stiffness, and isomerization. CONCLUSIONS: The described mutation leads to elongation of the protein at the carboxi-terminal domain (CTD) with altered properties, which are essential for solubility and activity. It suggests that can be the cause of the severe conditions observed in this patient.