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In vivo, small heat-shock proteins (sHsps) are key players in maintaining a healthy proteome. αB-crystallin (αB-c) or HspB5 is one of the most widespread and populous of the ten human sHsps. Intracellularly, αB-c acts via its molecular chaperone action as the first line of defence in preventing target protein unfolding and aggregation under conditions of cellular stress. In this review, we explore how the structure of αB-c confers its function and interactions within its oligomeric self, with other sHsps, and with aggregation-prone target proteins. Firstly, the interaction between the two highly conserved regions of αB-c, the central α-crystallin domain and the C-terminal IXI motif and how this regulates αB-c chaperone activity are explored. Secondly, subunit exchange is rationalised as an integral structural and functional feature of αB-c. Thirdly, it is argued that monomeric αB-c may be its most chaperone-species active, but at the cost of increased hydrophobicity and instability. Fourthly, the reasons why hetero-oligomerisation of αB-c with other sHsps is important in regulating cellular proteostasis are examined. Finally, the interaction of αB-c with aggregation-prone, partially folded target proteins is discussed. Overall, this paper highlights the remarkably diverse capabilities of αB-c as a caretaker of the cell. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12192-020-01098-w) contains supplementary material, which is available to authorized users.

αB-crystallin (CRYAB) is an important actor in the immediate cell stabilizing response following mechanical stress in skeletal muscle. Yet, only little is known regarding myofiber type-specific stress responses of CRYAB. We investigated whether the phosphorylation of CRYAB at serine 59 (pCRYABSer59) and its cytoskeleton association are influenced by varying load-intensity and -volume in a fiber type-specific manner. Male subjects were assigned to 1, 5, and 10 sets of different acute resistance exercise protocols: hypertrophy (HYP), maximum strength (MAX), strength endurance (SE), low intensity (LI), and three sets of maximum eccentric resistance exercise (ECC). Skeletal muscle biopsies were taken at baseline and 30 min after exercise. Western blot revealed an increase inpCRYABSer59only following 5 and 10 sets in groups HYP, MAX, SE, and LI as well as following 3 sets in the ECC group. In type I fibers, immunohistochemistry determined increasedpCRYABSer59in all groups. In type II fibers,pCRYABSer59only increased in MAX and ECC groups, with the increase in type II fibers exceeding that of type I fibers in ECC. Association of CRYAB andpCRYABSer59with the cytoskeleton reflected the fiber type-specific phosphorylation pattern. Phosphorylation of CRYAB and its association with the cytoskeleton in type I and II myofibers is highly specific in terms of loading intensity and volume. Most likely, this is based on specific recruitment patterns of the different myofiber entities due to the different resistance exercise loadings. We conclude thatpCRYABSer59indicates contraction-induced mechanical stress exposure of single myofibers in consequence of resistance exercise.NEW & NOTEWORTHY We determined that the phosphorylation of αB-crystallin at serine 59 (pCRYABSer59) after resistance exercise differs between myofiber types in a load- and intensity-dependent manner. The determination ofpCRYABSer59could serve as a marker indirectly indicating contractile involvement and applied mechanical stress on individual fibers. By that, it is possible to retrospectively assess the impact of resistance exercise loading on skeletal muscle fiber entities.

The assembly of amyloidogenic peptides and proteins such as the β-amyloid peptide (Aβ), α-synuclein, huntingtin, tau, and islet amyloid polypeptide (IAPP) into amyloid fibrils and oligomers is directly linked to amyloid diseases, such as Alzheimer’s, Parkinson’s, and Huntington’s diseases, frontotemporal dementias, and type II diabetes. Although amyloid oligomers have emerged as especially important in amyloid diseases, high-resolution structures of the oligomers formed by full-length amyloidogenic peptides and proteins have remained elusive. Investigations of oligomers assembled from fragments or stabilized β-hairpin segments of amyloidogenic peptides and proteins have allowed investigators to illuminate some of the structural, biophysical, and biological properties of amyloid oligomers. Here, we summarize recent advances in the application of these peptide model systems to investigate and understand the structures, biological properties, and biophysical properties of amyloid oligomers.

AlphaB crystallin (αB-crystallin) is a key protein for maintaining the long-term transparency of the eye lens. In the eye lens, αB-crystallin is a “dynamical” oligomer regulated by subunit exchange between the oligomers. To elucidate the unsettled mechanism of subunit exchange in αB-crystallin oligomers, the study was carried out at two different protein concentrations, 28.5 mg/mL (dense sample) and 0.45 mg/mL (dilute sample), through inverse contrast matching small-angle neutron scattering. Interestingly, the exchange rate of the dense sample was the same as that of the dilute sample. From analytical ultracentrifuge measurements, the coexistence of small molecular weight components and oligomers was detected, regardless of the protein concentration. The model proposed that subunit exchange could proceed through the assistance of monomers and other small oligomers; the key mechanism is attaching/detaching monomers and other small oligomers to/from oligomers. Moreover, this model successfully reproduced the experimental results for both dense and dilute solutions. It is concluded that the monomer and other small oligomers attaching/detaching mainly regulates the subunit exchange in αB-crystallin oligomer.

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.

In recent years, significant advancements have been made in the understanding of the population distributions and dynamic oligomeric states of the molecular chaperone αB-crystallin and its core domain variants. In this work, we provide solution-phase evidence of the polydispersity of αB- crystallin using microfluidic methods, used for separating the oligomeric species present in solution according to their different electrophoretic mobilities on-chip in a matter of seconds. We, in particular demonstrate that microfluidic high-field electrophoresis and diffusion can detect the oligomerisation of these highly dynamic molecular chaperones and characterise the dominant oligomeric species present. We thereby provide a robust microfluidic method for characterising the individual species within complex protein mixtures of biological relevance.

Physico-chemical properties of G154S, R157H and A171T mutants of αB-crystallin (HspB5) associated with congenital human diseases including certain myopathies and cataract were investigated. Oligomers formed by G154S and A171T mutants have the size and apparent molecular weight indistinguishable from those of the wild-type HspB5, whereas the size of oligomers formed by R157H mutant is slightly smaller. All mutants are less thermostable and start to aggregate at a lower temperature than the wild-type protein. All mutants effectively interact with a triple phosphomimicking mutant of HspB1 and form large heterooligomeric complexes of similar composition. All mutants interact with HspB6 forming heterooligomeric complexes with size and composition dependent on the molar ratio of two proteins. The wild-type HspB5 and its G154S and A171T mutants form only high molecular weight (300–450 kDa) heterooligomeric complexes with HspB6, whereas the R157H mutant forms both high and low (∼120 kDa) molecular weight complexes. The wild-type HspB5 and its G154S and A171T mutants form two types of heterooligomers with HspB4, whereas R157H mutant effectively forms only one type of heterooligomers with HspB4. G154S and A171T mutants have lower chaperone-like activity than the wild-type protein when subfragment S1 of myosin or βL-crystallin are used as a model substrates. With these substrates, the R157H mutant shows equal or higher chaperone activity than the wild-type HspB5. We hypothesize that the mutations in the C-terminal region modulate the binding of the IP(I/V) motif to the core α-crystallin domain. The R157H mutation is located in the immediate proximity of this motif. Such modulation could cause altered interaction of HspB5 with partners and substrates and eventually lead to pathological processes.

Physico-chemical properties of three cataract-associated missense mutants of αB-crystallin (HspB5) (R11H, P20S, R56W) were analyzed. The oligomers formed by the R11H mutant were smaller, whereas the oligomers of the P20S and R56W mutants were larger than those of the wild-type protein. The P20S mutant possessed lower thermal stability than the wild-type HspB5 or two other HspB5 mutants. All HspB5 mutants were able to form heterooligomeric complexes with αA-crystallin (HspB4), a genuine component of eye lens. However, the P20S and R56W mutants were less effective in the formation of these complexes and properties of heterooligomeric complexes formed by these mutants and HspB4 and analyzed by ion-exchange chromatography were different from those formed by the wild-type HspB5 and HspB4. All HspB5 variants also heterooligomerized with another partner protein, HspB6. Specifically for the P20S mutant forming two distinct sizes of homooligomers, only the smaller homooligomer population was able to interact with HspB6. P20S and R56W mutants possessed lower chaperone-like activity than the wild-type HspB5 when UV-irradiated βL-crystallin was used as a model substrate. Importantly, all three mutations are localized in three earlier postulated short α-helical regions present in the N-terminal domain of αB-crystallin. These observations suggest an important structural and functional role of these regions. Correspondingly, therein localized mutations ultimately result in clinically relevant cataracts.

Introduction: αB-crystallin (CryAB) is a chaperone protein that plays a pivotal role in the maintenance of the structural and mechanical integrity of the sarcomere. Mutation in αB-crystallin (CryAB R120G) causes sarcomere disorganization leading to protein aggregation. Accumulation of toxic protein aggregates causes cardiac contractile dysfunction and perturbed mitochondrial spatial organization. However, the role of mitochondria in the pathogenesis of protein aggregation-induced cardiomyopathies remain obscure. Objective: In this study, we investigated the role of mitochondrial dynamics and function in the development of R120G αB-crystallin induced cardiac proteotoxicity. Methods and Results: CryAB R120G Tg hearts showed accumulation of toxic protein aggregates resulting in disruption of the sarcomere structure, development of cardiomyocyte hypertrophy, and contractile dysfunction. Hearts obtained from CryAB R120G Tg mice showed increased expression and localization of Drp1 on the mitochondrial membrane. Alteration in the expression of mitochondrial dynamics regulatory proteins was observed at two months age when these mice exhibited normal cardiac morphometry and systolic function. Adenoviral-mediated expression of CryAB R120G in cardiomyocytes also showed increased mitochondrial fission, expression dependent inhibition of mitochondrial respiration and activation of cellular toxicity. Inhibition mitochondrial fission by Drp1 partial knockdown in CryABR120G expressing cardiomyocytes significantly decreased protein aggregation and improved mitochondrial respiration. Conclusion: Drp1-dependent excessive mitochondrial fission results in defects in mitochondrial respiration and accumulation of protein aggregates in CryABR120G expressing cardiomyocytes. Therefore, our study shows that maladaptive aberrant mitochondrial fission causes CryABR120G -induced mitochondrial dysfunction and cardiac proteotoxicity-associated cellular dysfunction.

Silicone oil is a chemically inert and biocompatible material. However, the use of silicone oil as an adjunct for internal tamponade in the treatment of retinal detachment is accompanied by the sequelae of cataract, the molecular mechanism of which has been a mystery for scientists. In this study, we focused on the influence of silicone oil on the aggregating behaviors of one of the important proteins in the eyes, αB-crystallin (CRYAB). We found that silicone oil could promote the amyloid-like aggregation of CRYAB, verified using atomic force microscopy (AFM), transmission electron microscopy (TEM), laser granularity and number analysis (Nanosight), and turbidity measurements. Furthermore, fluorescent experiments using Thioflavin T (ThT), Congo red, and 1-anilinonaphthalene-8-sulfonic acid (ANS) suggested formation of a β-sheet structure in CRYAB in the presence of silicone oil, which was also confirmed by far-UV circular dichroism (CD) spectroscopy. These findings provide a direct evidence of the changes in the secondary structures of CRYAB protein and amyloid aggregation behavior upon adding silicone oil in the solution, and could be helpful for understanding the molecular mechanisms of the cataract formed in silicone oil-filled eyes.

The small heat shock protein αB-Crystallin (CryAB, HspB5) and SH2 domain-containing tyrosine phosphatase 2 (Shp2) are important molecules in heart response to pathophysiological stress. Here we show that CryAB interacts with and potentially regulates Shp2 catalytic activity in stretched cardiomyocytes. Such an interaction requires CryAB oligomer to attenuate Shp2 activation. Stretched cardiomyocytes show a robust CryAB/Shp2 association accompanied by a reduction in the Shp2 phosphatase activity. Accordingly, CryAB knock-down in cardiomyocytes enhances Shp2 activity induced by mechanical stress. These results revealed a new role for CryAB, as a modulator of Shp2 phosphatase activity during a functionally relevant stimulus in cardiomyocytes.

Background: Compromised protein quality control causes the accumulation of misfolded proteins and intracellular aggregates, contributing to cardiac disease and heart failure. An unbiased genome-wide short hairpin RNA screen was performed to seek novel genes that could prevent or clear proteotoxic aggregates in a model of desmin-related cardiomyopathy, caused by the R120G mutation of αB-crystallin (CryAB R120G ). Among a total of 236 hits, we validated a candidate gene named ubiquitin-conjugating E2 enzyme variant 1 (ube2v1). Ube2v1 expression is detectable in the heart and the literature states that it must bind to ube2n to form a heterodimer. The heterodimer then catalyzes the synthesis of Lys63-linked poly-ubiquitin chains, participating in DNA repair and the damage response. Ube2v1 protein levels were increased in CryAB R120G transgenic hearts. However, the function of ube2v1 in cardiac proteotoxicity is completely unknown. To assess whether and how ube2v1 impacts cardiac protein aggregation catalyzed by cardiomyocyte-specific expression of mutated CryAB. Methods and Results: Neonatal rat ventricular cardiomyocytes (NRVMs) were infected with adenoviruses expressing either wild-type CryAB or CryAB R120G . Subsequently, loss- and gain-of-function experiments were performed using ube2v1 siRNA or recombinant adenovirus mediated ube2v1 overexpression. Knockdown of ube2v1 decreased aggregate accumulation and attenuated cytotoxicity caused by CryAB R120G expression, while overexpressing ube2v1 enhanced aggregate formation. Ubiquitin proteasome system (UPS) function was analyzed using a UPS reporter protein consisting of a short degron, CL1, fused to the COOH-terminus of green fluorescent protein (GFPu). Knockdown of ube2v1 improved proteasomal function and promoted the degradation of insoluble ubiquitinated proteins in CryAB R120G cardiomyocytes, but did not alter autophagic flux. Conclusions: Inhibition of ube2v1 improves CryAB R120G -induced aggregate formation through enhancing proteasome activity. It provides a novel therapeutic target to treat cardiac proteinopathies.

Amyloid formation is implicated in a range of neurodegenerative conditions including Alzheimer’s and Parkinson’s diseases. The small heat-shock protein αB-crystallin (αBC) is associated with both, and directly inhibits amyloid formation in vitro and its toxicity in cells. Studying the mechanism of aggregation inhibition is challenging owing to sample heterogeneity and the dynamic nature of the process. Here, by means of NMR spectroscopy and chemical kinetics, we establish the mechanism by which the protein α-lactalbumin aggregates and forms amyloid, and how this is inhibited by αBC. In particular, we characterise the lifetime of the unstable aggregation nucleus, and determine that this species is specifically destabilised by αBC. This mechanism allows the chaperone to delay the onset of aggregation, although it is overwhelmed on longer timescales. The methodology we present provides a mechanistic understanding of how αBC reduces the toxicity of amyloids, and is widely applicable to other complex mixtures.

One of the major reasons for age-related cataract formation is an accumulation of insoluble lens proteins. In particular, higher-order α-crystallin aggregates, comprising αA and αB subunits, are insolubilized by the build up of various post-translational modifications over time. Although we previously found an exceptional amount of Asp96 isomerization in αB-crystallin from aged human lens, the biological effect remains unknown. To approximate the effect of Asp 96 isomerization in αB-crystallin, here residues 93-103 of αB-crystallin were chemically synthesized as peptides in which l -α-Asp was replaced with l -β-Asp, d -α-Asp, or d -β-Asp. The resulting peptides were then compared in a biological assay. The results showed that isomerization of Asp 96 altered both the local structure of peptide and its stability against enzymatic digestion. In addition, the synthesized peptides decreased the insoluble fraction of heated α-crystallin. The d -β-Asp-containing peptide further decreased heat-induced precipitation of α-crystallin, and a chaperone assay based on heated alcohol dehydrogenase implied differential interaction of the peptides with substrate depending on the Asp isomer present in each. Our results suggest that the formation of Asp isomers is likely to affect the higher-order oligomer structure of α-crystallin and thereby its chaperone functions in aged lens.

Transparency in the eye lens is maintained via specific, functional interactions among the structural βγ- and chaperone α-crystallins. Here we report the structure and α-crystallin binding interface of the G18V variant of human γS-crystallin (γS-G18V), which is linked to hereditary childhood-onset cortical cataract. Comparison of the solution NMR structures of wild-type and G18V γS-crystallin, both presented here, reveal that the increased aggregation propensity of γS-G18V results from neither global misfolding nor the solvent exposure of a hydrophobic residue, but instead involves backbone rearrangement within the N-terminal domain. αB-crystallin binds more strongly to the variant, via a well-defined interaction surface that represents the first such interface directly observed between a variant structural crystallin and α-crystallin. In the context of the αB-crystallin structure and the finding that it forms heterogeneous multimers, our structural studies suggest a potential mechanism for cataract formation via the depletion of the finite αB-crystallin population of the lens.

Mutations in cardiac Na + channel Na v 1.5 cause cardiac arrhythmias and sudden death. The cardiac Na + channel functions as a protein complex, however, its complete components remain to be fully elucidated. A yeast two-hybrid screen identified a new candidate Na v 1.5-interacting protein, αB-crystallin. GST-pull-down, co-immunoprecipitation and immunostaining analyses validated the interaction between Na v 1.5 and αB-crystallin. Overexpression of αB-crystallin significantly increased peak sodium current ( I Na ) density, and the underlying molecular mechanism is the increased cell surface expression level of Na v 1.5 via reduced internalization of cell surface Na v 1.5 and ubiquitination of Na v 1.5. Knockout of αB-crystallin expression significantly decreased the cell-surface expression level of Na v 1.5. αB-crystallin interacted with Nedd4-2, however, a catalytically inactive Nedd4-2-C801S mutant reduced the interaction between αB-crystallin and Nedd4-2 and also blocked the increase of peak I Na density by αB-crystallin. Na v 1.5 mutation V1980A at the interaction site for Nedd4-2 eliminated the effect of αB-crystallin on reduction of Na v 1.5 ubiquitination and increases of I Na density. The data suggest that the interactions between αB-crystallin and functionally active Nedd4-2 and between αB-crystallin and Na v 1.5 are critical to increased I Na density by αB-crystallin. Multiple mutations in αB-crystallin have been associated with human diseases. Two mutations, R109H and R151X, eliminated the effect of αB-crystallin on I Na . This study identifies αB-crystallin as a new binding partner for Na v 1.5. αB-crystallin interacts with Na v 1.5 and increases I Na by modulating the expression level and internalization of cell surface Na v 1.5 and ubiquitination of Na v 1.5, which requires the protein-protein interactions between αB-crystallin and Na v 1.5 and between αB-crystallin and functionally active Nedd4-2.

We established in vivo and in vitro models to investigate the role of αB-Crystallin (CryAB) and assess the ability of aspirin (ASA) to protect the myocardium during prolonged heat stress. Thirty-day-old chickens were divided into three groups (n = 90): heat stress (HS, 40±1 °C); ASA(−)HS(+), 1 mg/kg ASA orally 2 h before heat stress; and ASA(+)HS(−), pretreated with aspirin, no heat stress (25 °C). Hearts were excised after 0, 1, 2, 3, 5, 7, 10, 15 and 24 h. Heat stress increased body temperature, though the ASA(−)HS(+) group had significantly higher temperatures than the ASA(+)HS(+) group at all time points. Compared to ASA(+)HS(+), the ASA(−)HS(+) group displayed increased sensitivity to heat stress. Pathological analysis revealed the ASA (+)HS(+) myocardium showed less severe changes (narrowed, chaotic fibers; fewer necrotic cells) than the ASA(−)HS(+) group (bleeding and extensive cell death). In vitro, ASA-pretreatment significantly increased primary chicken myocardial cell survival during heat stress. ELISAs indicated ASA induced CryAB in vivo to protect against heat stress-induced myocardial damage, but ASA did not induce CryAB in primary chicken myocardial cells. The mechanisms by which ASA induces the expression of CryAB in vivo and protects the myocardium during heat stress merit further research.

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.

SummaryWe report structural models for the most abundant oligomers populated by the polydisperse molecular chaperone αB-crystallin. Subunit connectivity is determined by using restraints obtained from nuclear magnetic resonance spectroscopy and mass spectrometry measurements, enabling the construction of various oligomeric models. These candidate structures are filtered according to their correspondence with ion-mobility spectrometry data and cross-validated by using electron microscopy. The ensuing best-fit structures reveal the polyhedral architecture of αB-crystallin oligomers, and provide a rationale for their polydispersity and facile interconversion.

Peripapillary glial cells (PPGCs) are a peculiar macroglia in avian species, located in the central retina adjacent to the optic nerve head. PPGCs have a similar shape and orientation to Müller cells, which traverse the entire layer of the retina; however, there are differences in protein expression between the two cell types. In the present study, we first demonstrated that PPGCs expressed αB-crystallin, which is not expressed in Müller cells, during retinal development. αB-crystallin was first faintly expressed in PPGCs of the E5 retina, adjacent to the optic nerve head. Further, αB-crystallin was exclusively expressed in PPGCs up to E14. The shape of these cells was bipolar with vitread and ventricular processes. The vitread processes of αB-crystallin+ PPGCs became finer at E18. Double labeling analysis clearly demonstrated that only vimentin+ or GFAP+ astrocytes were located in the optic nerve head and were demarcated from the retina by αB-crystallin+ PPGCs. Furthermore, we determined that αB-crystallin+ PPGCs, with a number of processes, completely wrapped the optic nerve head and were densely located in the junction of the optic nerve head and the retina in a whole mount preparation and in vertical-sectioned retinae. The results of present study, together with reports that retinal astrocytes migrate from the optic nerve head, suggest that PPGCs prevent astrocytes from migrating into the retina in avian species.

Human gamma-crystallins are long-lived, unusually stable proteins of the eye lens exhibiting duplicated, double Greek key domains. The lens also contains high concentrations of the small heat shock chaperone alpha-crystallin, which suppresses aggregation of model substrates in vitro. Mature-onset cataract is believed to represent an aggregated state of partially unfolded and covalently damaged crystallins. Nonetheless, the lack of cell or tissue culture for anucleate lens fibers and the insoluble state of cataract proteins have made it difficult to identify the conformation of the human gamma-crystallin substrate species recognized by human alpha-crystallin. The three major human lens monomeric gamma-crystallins, gammaD, gammaC, and gammaS, all refold in vitro in the absence of chaperones, on dilution from denaturant into buffer. However, off-pathway aggregation of the partially folded intermediates competes with productive refolding. Incubation with human alphaB-crystallin chaperone during refolding suppressed the aggregation pathways of the three human gamma-crystallin proteins. The chaperone did not dissociate or refold the aggregated chains under these conditions. The alphaB-crystallin oligomers formed long-lived stable complexes with their gammaD-crystallin substrates. Using alpha-crystallin chaperone variants lacking tryptophans, we obtained fluorescence spectra of the chaperone-substrate complex. Binding of substrate gamma-crystallins with two or three of the four buried tryptophans replaced by phenylalanines showed that the bound substrate remained in a partially folded state with neither domain native-like. These in vitro results provide support for protein unfolding/protein aggregation models for cataract, with alpha-crystallin suppressing aggregation of damaged or unfolded proteins through early adulthood but becoming saturated with advancing age.

Changes in retinal crystallin gene expression have been implicated in the development of myopia in animal models. We therefore investigated the expression of alphaB-crystallin (cryab) in the chicken retina during periods of increased ocular growth induced by form-deprivation and negative lens-wear, and during periods of decreased ocular growth induced by diffuser removal from previously form-deprived eyes, and plus lens-wear. Cryab RNA transcript levels in the chicken retina were measured using semi-quantitative real-time RT-PCR, at times between 1 h and 10 days after the fitting of diffusers or negative lenses, and at times between 1 h and 3 days following the removal of diffusers from previously form-deprived eyes, or the addition of plus lenses. Changes in expression for each condition at each time-point are analysed relative to expression in retinas from age-matched untreated control birds. No change in relative expression of cryab RNA transcript was detected 1 h after fitting diffusers to induce form-deprivation myopia. A transient increase in cryab RNA transcript expression was detected around 1 day later (p = 0.02), but expression returned to control levels after three days. After 7 (p = 0.005) and 10 (p = 0.001) days, retinal cryab RNA transcript expression progressively increased relative to controls. After removal of the diffusers, to initiate recovery, cryab RNA transcript expression remained elevated, with only a slight return to control levels. During the development of lens-induced myopia, no changes in cryab RNA transcript expression relative to controls were seen on day 1, but increases were seen at 10 days (p = 0.004). No significant changes in retinal cryab RNA transcript expression were seen in response to plus lenses compared to either contralateral control values (MANOVA; F = 0.60, p = 0.48) or age-matched untreated values (MANOVA; F = 4.10, p = 0.08). Changes in retinal cryab RNA transcript expression were not systematically related to changes in the rate of eye growth. The role of the transient increase in cryab expression observed after 1 day of form-deprivation, which was not seen after fitting negative lenses, is unclear. The later increases in relative cryab expression seen during the development of form-deprivation and lens-induced myopia occur too late to have a major role in the differential regulation of eye growth between experimental and control eyes. Given that cryab is a member of the small heat shock protein family, the later increases may reflect the emergence of cell damage related to high myopic pathology in the experimentally enlarged eyes and retina.

The response to hydration of the internal protein dynamics was studied by the means of solid state NMR relaxation and magic angle spinning exchange techniques. Two proteins, lysozyme from bacteriophage T4 and human alphaB-crystallin were used as exemplars. The relaxation rates R1 and R1rho of 13C and 15N nuclei were measured as a function of a hydration level of the proteins in the range 0-0.6 g of water/g of protein. Both proteins were totally 15N-enriched with natural 13C abundance. The relaxation rates were measured for different spectral bands (peaks) that enabled the characterization of the dynamics separately for the backbone, side chains, and CH3 and NH3+ groups. The data obtained allowed a comparative analysis of the hydration response of the protein dynamics in different frequency ranges and different sites in the protein for two different proteins and two magnetic nuclei. The most important result is a demonstration of a qualitatively different response to hydration of the internal dynamics in different frequency ranges. The amplitude of the fast (nanosecond time scale) motion gradually increases with increasing hydration, whereas that of the slow (microsecond time scale) motion increases only until the hydration level 0.2-0.3 g of water/g of protein and then shows almost no hydration dependence. The reason for such a difference is discussed in terms of the different physical natures of these two dynamic processes. Backbone and side chain nuclei show the same features of the response of dynamics with hydration despite the fact that the backbone motional amplitudes are much smaller than those of side chains. Although T4 lysozyme and alphaB-crystallin possess rather different structural and biochemical properties, both proteins show qualitatively very similar hydration responses. In addition to the internal motions, exchange NMR data enabled the identification of one more type of motion in the millisecond to second time scale that appears only at high hydration levels. This motion was attributed to the restricted librations of the protein as a whole.

The comparison of the chaperone-like activity of native and covalently cross-linked human alphaB-crystallins has confirmed the important role of the subunit mobility in the chaperoning mechanism. Our data clearly demonstrate that the chaperone-like activity of alpha-crystallin is not only a surface phenomenon as was suggested by some researchers.

International audience; Small heat-shock proteins, including αB-crystallin (αB), play an important part in protein homeostasis, because their ATP-independent chaperone activity inhibits uncontrolled protein aggregation. Mechanistic details of human αB, particularly in its client-bound state, have been elusive so far, owing to the high molecular weight and the heterogeneity of these complexes. Here we provide structural insights into this highly dynamic assembly and show, by using state-of-the-art NMR spectroscopy, that the αB complex is assembled from asymmetric building blocks. Interaction studies demonstrated that the fibril-forming Alzheimer's disease Aβ1-40 peptide preferentially binds to a hydrophobic edge of the central β-sandwich of αB. In contrast, the amorphously aggregating client lysozyme is captured by the partially disordered N-terminal domain of αB. We suggest that αB uses its inherent structural plasticity to expose distinct binding interfaces and thus interact with a wide range of structurally variable clients.

Nature Communications 5: Article number: 5519 (2014); Published: 16 October 2014; Updated: 23 March 2015 An image in Supplementary Fig. 11a in this Article, representing the negative control, was inadvertently duplicated from the ‘myc-FAK’-transfected group. The correct version of the figure appearsbelow.

Alpha-crystallin is a member of the family of small heat-shock proteins (sHSP) and is composed of two subunits, alphaA-crystallin and alphaB-crystallin, which exhibit molecular chaperone-like properties. In a previous study, we found that residues 70-88 in alphaA-crystallin can function like a molecular chaperone by preventing the aggregation and precipitation of denaturing substrate proteins [Sharma, K. K., et al. (2000) J. Biol. Chem. 275, 3767-3771]. In this study, we show that the complementary sequence in alphaB-crystallin, residues 73-92 (DRFSVNLDVKHFSPEELKVK), is the functional chaperone site of alphaB-crystallin. Like the mini-alphaA-crystallin chaperone, the mini-alphaB-crystallin chaperone interacts with 1,1'-bi(4-anilino) naphthalene-5,5'-disulphonic acid (bis-ANS) and also possesses significant beta-sheet and random coil structure. Deletion of four residues (DRFS) from the N-terminus or deletion of C-terminus LKVK residues from the 73-92 peptide abolishes the chaperone-like activity against denaturing alcohol dehydrogenase. However, removal of DRFS or HFSPEELKVK is necessary to completely abolish the antiaggregation property of the peptide in insulin reduction assay. Substitution of Asp at a site corresponding to D80 in alphaB-crystallin with d-Asp or beta-Asp results in a significant loss of chaperone-like activity. Kynurenine modification of His in the peptide abolishes the antiaggregation property of the mini-chaperone. These data suggest that the 73-92 region in alphaB-crystallin is one of the substrate binding sites during chaperone activity.

Three-dimensional models for alphaB-crystallin and its complex with zinc were obtained by molecular homology modeling and quantum mechanical calculations in order to explain the effect of the metal on the chaperone-like activity of alphaB-crystallin. In fact, measurements of the chaperone-like activity of alphaB-crystallin revealed that it is significantly increased in presence of the zinc. The theoretical models allowed us to estimate the increased exposition of hydrophobic residues caused by the presence of zinc, suggesting a relationship between structural changes and the increased chaperone-like activity.

Previously, using the peptide scan method, we have determined that residues 42-57 and 60-71 in alphaB-crystallin (TSLSPFYLRPPSFLRA, named recognition sequence 1 or RS-1, and WFDTGLSEMRLE, named recognition sequence 2 or RS-2) are involved in interaction with alphaA-crystallin. To understand the significance of the RS-2 region in interactions between alphaA- and alphaB-crystallins, W60R, F61N, and S66G mutants of alphaB-crystallin were made and tested for their ability to interact with alphaA-crystallin. W60R and S66G mutations increased the oligomeric size of alphaB-crystallin by 1.6- and 2.7-fold respectively, whereas the F61N mutation had no effect. The tryptophan fluorescence intensity of alphaBS66G was 1.5-fold higher than that for the wild type. The intrinsic fluorescence of alphaBF61N was marginally lower than that of alphaB, whereas the fluorescence intensity of alphaBW60R decreased by 40% compared with that of alphaB. The relative availability of hydrophobic sites in the mutants was in the following order: alphaBS66G >> alphaB = alphaBF61N = alphaBW60R. The far-UV CD profiles for the wild type and alphaB-crystallin mutants indicated no significant changes in their secondary structures, except for alphaBS66G, which showed an increase in alpha-helical content. The near-UV CD profiles of alphaBW60R and alphaBF61N were nearly similar to that of wild type alphaB. On the other hand, alphaBS66G beyond 270 nm exhibited a signature completely different from that of wild type alphaB. Mutations did not alter the chaperone-like activity of these proteins. The W60R mutation did not affect the rate of subunit exchange between alphaB- and alphaA-crystallins. On the other hand, the S66G mutation increased the subunit exchange rate by 100%, whereas the F61N mutation decreased the rate of subunit exchange between alphaB- and alphaA-crystallins by 36%. Our results establish the importance of residues 60-71 in oligomerization of alphaB-crystallin and subunit interaction between alphaB- and alphaA-crystallins.

Recent studies implicate hyperglycemia as a cause of vascular complications in diabetes. Our study confirmed that high concentration of glucose (30 mM) induces apoptosis in cultures of human umbilical vein endothelial cells. After 5 days of culture TUNEL positive cells in high concentration of glucose were nearly 63% higher when compared to normal concentration of glucose (5 mM). Transfection of pcDNA3-rat alphaB-crystallin into these cells inhibited high glucose-induced apoptosis by approximately 36%, such an effect was not observed when cells were transfected with an empty vector. AlphaB-crystallin transfection inhibited by about 35% of high glucose induced activation of caspase-3. High concentration of glucose enhanced formation of reactive oxygen species (ROS) in these cells but this was significantly (p0.001) curtailed by transfection of alphaB-crystallin. Results of our study indicate that alphaB-crystallin effectively inhibits both ROS formation and apoptosis in cultured vascular endothelial cells and provide a basis for future therapeutic interventions in diabetic vascular complications.

Digital three-dimensional (3-D) functional reconstructions of inner medullary nephrons were performed. Antibodies against aquaporins (AQP)-1 and -2 and the chloride channel ClC-K1 identified descending thin limbs (DTLs), collecting ducts (CDs), and ascending thin limbs (ATLs), respectively, through indirect immunofluorescence. Tubules were labeled in transverse sections and assembled into 3-D arrays, permitting individual tubule or combined surface representations to depths of 3.3 mm to be viewed in an interactive digital model. Surface representations of 75 tubules positioned near the central region of the inner medulla were reconstructed. In most DTL segments that form loops below 1 mm from the inner medullary base, AQP1 expression begins at the base, becomes intermittent for variable lengths, and continues nearly midway to the loop. The terminal DTL segment exhibiting undetectable AQP1 represents nearly 60% of the distance from the medullary base to the tip of the loop. AQP1 expression was entirely undetectable in shorter long-looped DTLs. ClC-K1 is expressed continuously along the terminal portion of all DTLs reconstructed here, beginning with a prebend region approximately 164 microm before the bend in all tubules and continuing through the entire ascent of the ATLs to the base of the inner medulla. CDs express AQP2 continuously and extensive branching patterns are illustrated. 3-D functional reconstruction of inner medullary nephrons is capable of showing axial distribution of membrane proteins in tubules of the inner medulla and can contribute to further development and refinement of models that attempt to elucidate the concentrating mechanism.

Lens alpha-crystallin, alpha A- and alpha B-crystallin, and Hsp27 are members of the small heat shock protein family. Both alpha A- and alpha B-crystallin are expressed in the lens and serve as structural proteins and as chaperones, but alpha B-crystallin is also expressed in nonlenticular organs where Hsp27, rather than alpha A-crystallin, is expressed along with alpha B-crystallin. It is not known what additional function Hsp27 has besides as a heat shock protein, but it may serve, as alpha A-crystallin does in the lens, to stabilize alpha B-crystallin. In this study, we investigate aspects on conformation and thermal stability for the mixture of Hsp27 and alpha B-crystallin. Size exclusion chromatography, circular dichroism (CD), and light scattering measurements indicated that Hsp27 prevented alpha B-crystallin from heat-induced structural changes and high molecular weight (HMW) aggregation. The results indicate that Hsp27 indeed promotes stability of alpha B-crystallin.

Several small heat shock proteins contain a well conserved α-crystallin domain, flanked by an N-terminal domain and a C-terminal extension, both of which vary in length and sequence. The structural and functional role of the C-terminal extension of small heat shock proteins, particularly of αA- and αB-crystallins, is not well understood. We have swapped the C-terminal extensions between αA- and αB-crystallins and generated two novel chimeric proteins, αABc and αBAc. We have investigated the domain-swapped chimeras for structural and functional alterations. We have used thermal and non-thermal models of protein aggregation and found that the chimeric αB with the C-terminal extension of αA-crystallin, αBAc, exhibits dramatically enhanced chaperone-like activity. Interestingly, however, the chimeric αA with the C-terminal extension of αB-crystallin, αABc, has almost lost its activity. Pyrene solubilization and bis-1-anilino-8-naphthalenesulfonate binding studies show that αBAc exhibits more solvent-exposed hydrophobic pockets than αA, αB, or αABc. Significant tertiary structural changes are revealed by tryptophan fluorescence and near-UV CD studies upon swapping the C-terminal extensions. The far-UV CD spectrum of αBAc differs from that of αB-crystallin whereas that of αABc overlaps with that of αA-crystallin. Gel filtration chromatography shows alteration in the size of the proteins upon swapping the C-terminal extensions. Our study demonstrates that the unstructured C-terminal extensions play a crucial role in the structure and chaperone activity, in addition to generally believed electrostatic "solubilizer" function.

A major stress protein, α-crystallin, functions as a chaperone. Site-directed mutagenesis has been used to identify regions of the protein necessary for chaperone function. In this work we have taken some of the previously described mutants produced and assessed their chaperone function by both a traditional heat-induced aggregation method at elevated temperature and using enzyme methods at 37 °C. In general the different assays gave parallel results indicating that the same property is being measured. Discrepancies were explicable by the heat lability of some mutants. Most mutants had full chaperone function showing the robust nature of α-crystallin. A mutant corresponding to a minor component of rodent αA-crystallin, αAins-crystallin, had decreased chaperone function. Decreased chaperone function was also found for human Ser139 Arg, Thr144Arg, Ser59Ala mutants of αB-crystallin and double mutants Ser45Ala/Ser59Ala, Lys103 Leu/His104Ile, and Glu110His/His111Glu. A mutant Phe27Arg that was the subject of previous controversy was shown to be fully active at physiological temperatures.

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.

Changes in αB-crystallin content in adult rat soleus and extensor digitorum longus (EDL) were examined after 8 wk of 3,5,3′-triiodothyronine (T3) and propylthiouracil (PTU) treatments. Cellular distributions of αB-crystallin expression related to fiber type, and distribution shifts with these treatments were also examined in detail from the gray level of reactivity to specific anti-αB-crystallin antibody. αB-crystallin content in both soleus and EDL muscles was significantly decreased after T3, and that in EDL was significantly increased over twofold after PTU treatment. In both control soleus and EDL muscles, the gray level of type I fibers was higher than that of type II fibers. αB-crystallin expression among type II subtypes was muscle specific; the order was type I > IIa > IIx > IIb in control EDL muscle and type IIx ≥ IIa in soleus muscle. The relation was basically unchanged in both muscles after T3treatment and was, in particular, well maintained in EDL muscle. Under hypothyroidism conditions with PTU, the mean αB-crystallin levels of type IIa and IIx fibers were significantly lower than levels under control conditions. Thus the relation between fiber type and the expression manner of stress protein αB-crystallin is muscle specific and also is well regulated under thyroid hormone, especially in fast EDL muscle.

alpha-Crystallin, a heteromultimeric protein made up of alphaA- and alphaB-crystallins, functions as a molecular chaperone in preventing the aggregation of proteins. We have shown earlier that structural perturbation of alpha-crystallin can enhance its chaperone-like activity severalfold. The two subunits of alpha-crystallin have extensive sequence homology and individually display chaperone-like activity. We have investigated the chaperone-like activity of alphaA- and alphaB-crystallin homoaggregates against thermal and nonthermal modes of aggregation. We find that, against a nonthermal mode of aggregation, alphaB-crystallin shows significant protective ability even at subphysiological temperatures, at which alphaA-crystallin or heteromultimeric alpha-crystallin exhibit very little chaperone-like activity. Interestingly, differences in the protective ability of these homoaggregates against the thermal aggregation of beta(L)-crystallin is negligible. To investigate this differential behavior, we have monitored the temperature-dependent structural changes in both the proteins using fluorescence and circular dichroism spectroscopy. Intrinsic tryptophan fluorescence quench-ing by acrylamide shows that the tryptophans in alphaB-crystallin are more accessible than the lone tryptophan in alphaA-crystallin even at 25 degrees C. Protein-bound 8-anilinonaphthalene-1-sulfonate fluorescence demonstrates the higher solvent accessibility of hydrophobic surfaces on alphaB-crystallin. Circular dichroism studies show some tertiary structural changes in alphaA-crystallin above 50 degrees C. alphaB-crystallin, on the other hand, shows significant alteration of tertiary structure by 45 degrees C. Our study demonstrates that despite a high degree of sequence homology and their generally accepted structural similarity, alphaB-crystallin is much more sensitive to temperature-dependent structural perturbation than alphaA- or alpha-crystallin and shows differences in its chaperone-like properties. These differences appear to be relevant to temperature-dependent enhancement of chaperone-like activity of alpha-crystallin and indicate different roles for the two proteins both in alpha-crystallin heteroaggregate and as separate proteins under stress conditions.

The major lens protein alpha-crystallin is composed of two related types of subunits, alphaA- and alphaB-crystallin, of which the former is essentially lens-restricted, while the latter also occurs in various other tissues. With regard to their respective chaperone capacities, it has been reported that homomultimeric alphaA-crystallin complexes perform better in preventing thermal aggregation of proteins, while alphaB-crystallin complexes protect more efficiently against reduction-induced aggregation of proteins. Here, we demonstrate that this seeming discrepancy is solved when the reduction assay is performed at increasing temperatures: above 50 degrees C alphaA- performs better than alphaB-crystallin also in this assay. This inversion in protective capacity might relate to the greater resistance of alphaA-crystallin to heat denaturation. Infrared spectroscopy, however, revealed that this is not due to a higher thermostability of alphaA-crystallin's secondary structure. Also the accessible hydrophobic surfaces do not account for the chaperoning differences of alphaA- and alphaB-crystallin, since regardless of the experimental temperature alphaB-crystallin displays a higher hydrophobicity. It is argued that the greater complex stability of alphaA-crystallin, as evident upon urea denaturation, and the higher chaperone capacity of alphaB-crystallin at physiological temperatures reflect the evolutionary compromise to obtain an optimal functioning of heteromeric alpha-crystallin as a lens protein.

Lens alpha-crystallin subunits alphaA and alphaB are differentially expressed and have a 3-to-1 ratio in most mammalian lenses by intermolecular exchange. The biological significance of this composition and the mechanism of exchange are not clear. Preparations of human recombinant alphaA- and alphaB-crystallins provide a good system in which to study this phenomenon. Both recombinant alphaA- and alphaB-crystallins are folded and aggregated to the size of the native alpha-crystallin. During incubation together, they undergo an intermolecular exchange as shown by native isoelectric focusing. Circular dichroism measurements indicate that the protein with a 3-to-1 ratio of alphaA- and alphaB-crystallins has the same secondary structure but somewhat different tertiary structures after exchange: the near-UV CD increases after exchange. The resulting hybrid aggregate is more stable than the individual homogeneous aggregates: at 62 degrees C, alphaB-crystallin is more susceptible to aggregation and displays a greater light scattering than alphaA-crystallin. This heat-induced aggregation of alphaB-crystallin, however, was suppressed by intermolecular exchange with alphaA-crystallin. These phenomena are also observed by fast performance liquid chromatography gel filtration patterns. The protein structure of alphaB-crystallin is stabilized by intermolecular exchange with alphaA-crystallin.

alpha-Crystallin, the predominant eye lens protein with sequence homology to small heat shock proteins, acts like a molecular chaperone by suppressing the aggregation of damaged crystallins and proteins. To gain an insight into the amino acid sequences in alpha-crystallin involved in chaperone-like function, we used a cleavable, fluorescent, photoactive, crosslinking agent, sulfosuccinimidyl-2 (7-azido-4-methylcoumarin-3-acetamido)-ethyl-1,3' dithiopropionate (SAED), to derivatize yeast alcohol dehydrogenase (ADH) and allowed it to complex with bovine alpha-crystallin at 48 degrees C. The complex was photolyzed and reduced with DTT and the subunits of alpha-crystallin, alpha A- and alpha B-, were separated. Fluorescence analysis showed that both alpha A- and alpha B-crystallins interacted with ADH during chaperone-like function. Tryptic digestion, amino acid sequencing, and mass spectral analysis of alpha B-crystallin revealed that APSWIDTGLSEMR (57-69) and VLGDVIEVHGKHEER (93-107) sequences were involved in binding with ADH.

Photoincorporation of the fluorescent probe 4,4'-bis(1-anilino-8-naphthalene sulfonic acid) (bis-ANS) can be used to locate solvent-exposed hydrophobic regions in proteins. We show that bis-ANS is specifically incorporated into the putative N-terminal domain of alpha B-crystallin. This incorporation diminishes the chaperone-like activity of alpha B-crystallin, suggesting that hydrophobic surfaces in the N-terminal domain are involved in the binding of unfolding proteins.

The molecular chaperone αB-crystallin, the major player in maintaining the transparency of the eye lens, prevents stress-damaged and aging lens proteins from aggregation. In nonlenticular cells, it is involved in various neurological diseases, diabetes, and cancer. Given its structural plasticity and dynamics, structure analysis of αB-crystallin presented hitherto a formidable challenge. Here we present a pseudoatomic model of a 24-meric αB-crystallin assembly obtained by a triple hybrid approach combining data from cryoelectron microscopy, NMR spectroscopy, and structural modeling. The model, confirmed by cross-linking and mass spectrometry, shows that the subunits interact within the oligomer in different, defined conformations. We further present the molecular architectures of additional well-defined αB-crystallin assemblies with larger or smaller numbers of subunits, provide the mechanism how “heterogeneity” is achieved by a small set of defined structural variations, and analyze the factors modulating the oligomer equilibrium of αB-crystallin and thus its chaperone activity.

Chaperone-like activities of α-crystallin, αB-crystallin and proline were studied using a test system based on aggregation of UV-irradiated glycogen phosphorylase b (Ph b ) from rabbit skeletal muscle. The biphasic character of the dependence of the initial rate of aggregation ( v agg ) of UV-irradiated Ph b on the concentration of α-crystallin or αB-crystallin is indicative of the existence of two types of chaperone–protein substrate complexes differing significantly in affinity between the components of the complex. The dependence of v agg on the proline concentration is sigmoid (Hill coefficient is equal to 1.6) suggesting that the positive cooperative interactions between the proline molecules bound on the surface of the protein particles occur. When studying the combined suppressive action of α-crystallin and proline on aggregation of UV-irradiated Ph b , a slight antagonism between proline used at a fixed concentration (0.15 M) and α-crystallin was observed. At higher concentration of proline (0.5 M) each chaperone acts independent of one another.