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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.

HSPB5 (heat shock protein B5), also known as αB-crystallin, is one of the most widespread and populous of the ten human small heat shock proteins (sHsps). Over the past decades, extensive research has been conducted on HSPB5. However, few studies have statistically analyzed these publications. Herein, we conducted a bibliometric analysis to track the global research trend and current development status of HSPB5 research from the Web of Science Core Collection (WoSCC) database between 1985 and 2020. Our results demonstrate that 1220 original articles cited 54,778 times in 391 scholarly journals were published. Visualization analyses reveal that the Journal of Biological Chemistry was the most influential journal with 85 articles. The USA dominated this field with 520 publications (42.62%), followed by Japan with 149 publications (12.21%), and Kato contributed the largest number of publications. Most related publications were published in journals focusing on biochemistry molecular biology, cell biology, neurosciences neurology, and ophthalmology. In addition, keyword co-occurrence analyses identify three predominant research topics: expression of HSPB5, chaperone studies for HSPB5, and pathological studies of HSPB5. This study provides valuable guidance for researchers and leads to collaborative opportunities between diverse research interests to be integrated for HSPB5 research. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12192-021-01220-6.

Background and Aims Renal replacement therapy by peritoneal dialysis (PD) is limited in use and duration by progressive impairment of peritoneal membrane integrity and homeostasis. Preservation of peritoneal membrane integrity during chronic PD remains an urgent but long-unmet medical need. PD therapy failure results from peritoneal fibrosis and angiogenesis caused by hypertonic PD fluid (PDF)-induced mesothelial cytotoxicity. The incompletely defined pathophysiological mechanisms involved confound informed selection of therapeutic targets. Addition of cytoprotective agents to PDF have been shown to counteract pathophysiological mechanisms induced by current PDF. Lithium is a well described inhibitor of glycogen synthase kinase 3β and has recently been shown to also have nephroprotective effects in low doses. Here, we aim to characterize icodextrin-based, PDF-induced cellular injury with a combined omics approach and to investigate the effects of LiCl on the PD-induced observed molecular perturbations. Method To investigate mechanisms of acute cellular damage by PDF we chose an in vitro model of primary omental-derived peritoneal mesothelial cells with direct exposure to icodextrin-based PDF, followed by short-term or extended recovery for detection of short-term and long-term changes in transcriptome, proteome, and cell injury. 0, 2.5 or 10 mM LiCl were added to the PDF. In-vitro findings were validated in peritoneal biopsies (n=41) from pediatric PD and CDK5 patients or healthy controls and peritoneal effluents from adult and pediatric PD patients (n=27) or ascites samples (n=4) as control. For in-vivo experiments, healthy and uremic mice (C57/Bl6, female) were chronically exposed to PD-fluid without or with the addition of 5 mM LiCl via an implanted catheter. In-vivo overexpression of CRYAB was induced by i.p. injection of an adenoviral vector. All animal experiments and use of patient samples were approved by the local ethics committees and performed according to animal protection laws or the Declaration of Helsinki, respectively. Results LiCl significantly improved mesothelial cell survival in a dose-dependent manner. Combined transcriptomic and proteomic characterization of icodextrin-based PDF-induced mesothelial cell injury identified αB-crystallin as the mesothelial cell protein most significantly and consistently counter-regulated by LiCl. In-vitro and in-vivo overexpression of αB-crystallin triggered a fibrotic phenotype and PDF-like upregulation of vascular endothelial growth factor (VEGF), CD31-positive cells, and TGFβ-independent activation of TGFβ-regulated targets. In contrast, αB-crystallin knock-down decreased VEGF expression and early mesothelial-to-mesenchymal transition (MMT). LiCl reduced VEGF release and counteracted fibrosis- and angiogenesis-associated processes. αB-crystallin in patient-derived mesothelial cells was specifically upregulated in response to PDF and increased in peritoneal mesothelial cells from pediatric PD patient biopsies, correlating with markers of angiogenesis and fibrosis. Conclusion The cytoprotective effects of LiCl-supplemented PDF may be explained by counter-regulation of PD-induced angiogenesis via the novel target αB-crystallin. Reduction of mesothelial cell damage, peritoneal fibrosis and VEGF suggests therapeutic potential of this intervention. Repurposing LiCl as a cytoprotective PDF additive may offer a translatable therapeutic strategy to combat peritoneal membrane deterioration during PD therapy. Further study of LiCl-supplemented PDF is merited as a realistic approach to improving treatment longevity and patient outcomes during PD treatment.

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.

Background The mutated α‐B‐Crystallin (CryAB R120G ) mouse model of desmin‐related myopathy (DRM) shows an age‐dependent onset of pathologic cardiac remodeling and progression of heart failure. CryAB R120G expression in cardiomyocytes affects the mitochondrial spatial organization within the myofibrils, but the molecular perturbation within the mitochondria in the relation of the overall course of the proteotoxic disease remains unclear. Methods and Results CryAB R120G mice show an accumulation of electron‐dense aggregates and myofibrillar degeneration associated with the development of cardiac dysfunction. Though extensive studies demonstrated that these altered ultrastructural changes cause cardiac contractility impairment, the molecular mechanism of cardiomyocyte death remains elusive. Here, we explore early pathological processes within the mitochondria contributing to the contractile dysfunction and determine the pathogenic basis for the heart failure observed in the CryAB R120G mice. In the present study, we report that the CryAB R120G mice transgenic hearts undergo altered mitochondrial dynamics associated with increased level of dynamin‐related protein 1 and decreased level of optic atrophy type 1 as well as mitofusin 1 over the disease process. In association with these changes, an altered level of the components of mitochondrial oxidative phosphorylation and pyruvate dehydrogenase complex regulatory proteins occurs before the manifestation of pathologic adverse remodeling in the CryAB R120G hearts. Mitochondria isolated from CryAB R120G transgenic hearts without visible pathology show decreased electron transport chain complex activities and mitochondrial respiration. Taken together, we demonstrated the involvement of mitochondria in the pathologic remodeling and progression of DRM‐associated cellular dysfunction. Conclusions Mitochondrial dysfunction in the form of altered mitochondrial dynamics, oxidative phosphorylation and pyruvate dehydrogenase complex proteins level, abnormal electron transport chain complex activities, and mitochondrial respiration are evident on the CryAB R120G hearts before the onset of detectable pathologies and development of cardiac contractile dysfunction.

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.

Patients with rheumatoid arthritis (RA) frequently suffer from muscle weakness. We examined whether eccentric training prevents skeletal muscle weakness in adjuvant-induced arthritis (AIA) rat, a widely used animal model for RA. AIA was induced in the knees of Wistar rats by injection of complete Freund’s adjuvant. To induce eccentric contractions (ECCs), neuromuscular electrical stimulation (45 V) was applied to the plantar flexor muscles simultaneously with forced dorsiflexion of the ankle joint (0–40°) and was given every 6 s. ECC exercise was applied every other day for a total of 11 sessions and consisted of 4 sets of 5 contractions. There was a significant reduction in in vitro maximum Ca2+-activated force in skinned fibers in gastrocnemius muscle from AIA rats. These changes were associated with reduced expression levels of contractile proteins (i.e., myosin and actin), increased levels of inflammation redox stress-related biomarkers (i.e., TNF-α, malondialdehyde-protein adducts, NADPH oxidase 2, and neuronal nitric oxide synthase), and autolyzed active calpain-1 in AIA muscles. ECC training markedly enhanced the steady-state levels of αB-crystallin, a small heat shock protein, and its binding to the myofibrils and prevented the AIA-induced myofibrillar dysfunction, reduction in contractile proteins, and inflammation-oxidative stress insults. Our findings demonstrate that ECC training preserves myofibrillar function without muscle damage in AIA rats, which is at least partially attributable to the protective effect of αB-crystallin on the myofibrils against oxidative stress-mediated protein degeneration. Thus ECC training can be a safe and effective intervention, counteracting the loss of muscle strength in RA patients. NEW & NOTEWORTHY Eccentric contractions (ECCs) are regarded as an effective way to increase muscle strength. No studies, however, assess safety and effectiveness of ECC training on muscle weakness associated with rheumatoid arthritis. Here, we used adjuvant-induced arthritis (AIA) rats to demonstrate that ECC training prevents intrinsic contractile dysfunction without muscle damage in AIA rats, which may be attributed to the protective effect of αB-crystallin on the myofibrils against inflammation-oxidative stress insults.

Background Mutations in αB‐crystallin result in proteotoxic cardiomyopathy with desmin mislocalization to protein aggregates. Intermittent fasting (IF) is a novel approach to activate transcription factor EB (TFEB), a master regulator of the autophagy‐lysosomal pathway, in the myocardium. We tested whether TFEB activation can be harnessed to treat advanced proteotoxic cardiomyopathy. Methods and Results Mice overexpressing the R120G mutant of αB‐crystallin in cardiomyocytes (Myh6‐CryABR120G) were subjected to IF or ad‐lib feeding, or transduced with adeno‐associated virus–TFEB or adeno‐associated virus—green fluorescent protein after development of advanced proteotoxic cardiomyopathy. Adeno‐associated virus–short hairpin RNA–mediated knockdown of TFEB and HSPB8 was performed simultaneously with IF. Myh6‐CryABR120G mice demonstrated impaired autophagic flux, reduced lysosome abundance, and mammalian target of rapamycin activation in the myocardium. IF resulted in mammalian target of rapamycin inhibition and nuclear translocation of TFEB with restored lysosome abundance and autophagic flux; and reduced aggregates with normalized desmin localization. IF also attenuated left ventricular dilation and myocardial hypertrophy, increased percentage fractional shortening, and increased survival. Adeno‐associated virus–TFEB transduction was sufficient to rescue cardiomyopathic manifestations, and resulted in reduced aggregates and normalized desmin localization in Myh6‐CryABR120G mice. CryABR120G‐expressing hearts demonstrated increased interaction of desmin with αB‐crystallin and reduced interaction with chaperone protein, HSPB8, compared with wild type, which was reversed by both IF and TFEB transduction. TFEB stimulated autophagic flux to remove protein aggregates and transcriptionally upregulated HSPB8, to restore normal desmin localization in CryABR120G‐expressing cardiomyocytes. Short hairpin RNA–mediated knockdown of TFEB and HSPB8 abrogated IF effects, in vivo. Conclusions IF and TFEB activation are clinically relevant therapeutic strategies to rescue advanced R120G αB‐crystallin mutant‐induced cardiomyopathy by normalizing desmin localization via autophagy‐dependent and autophagy‐independent mechanisms., See Editorial by Mukai et al

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.

International trading markets of meat require the animal’s age information to prevent cross-contamination of ineligible meat products. Individual livestock age is either evaluated from physiological features or verified by breeding history. However, it remains impossible to perform age verification on meat when a suspicion of error occurred in the importing country. To investigate an age-related protein in skeletal muscle of livestock, we compared protein expression among chicken pectoralis major of different ages. Results indicated that the level of expression of chicken HSPB1, one of the small heat shock proteins, was increased in aged muscles. On the other hand, other heat shock proteins, heat shock factors, and myosin heavy chain isoform did not change the expression levels in aged chicken muscle. In addition, we identified that αB-crystallin interacted with HSPB1 in aged chicken muscle. These results suggest that HSPB1 protein forms complexes with αB-crystallin in aged chicken muscle and suppose to become the candidate of age-related bio-marker for verifying the age of chicken meat.

Small heat-shock proteins (sHsps), such as αB-crystallin, are one of the major classes of molecular chaperone proteins. In vivo, under conditions of cellular stress, sHsps are the principal defence proteins that prevent large-scale protein aggregation. Progress in determining the structure of sHsps has been significant recently, particularly in relation to the conserved, central and β-sheet structured α-crystallin domain (ACD). However, an understanding of the structure and functional roles of the N- and C-terminal flanking regions has proved elusive mainly because of their unstructured and dynamic nature. In this paper, we propose functional roles for both flanking regions, based around three properties: (i) they act in a localised crowding manner to regulate interactions with target proteins during chaperone action, (ii) they protect the ACD from deleterious amyloid fibril formation and (iii) the flexibility of these regions, particularly at the extreme C-terminus in mammalian sHsps, provides solubility for sHsps under chaperone and non-chaperone conditions. In the eye lens, these properties are highly relevant as the crystallin proteins, in particular the two sHsps αA- and αB-crystallin, are present at very high concentrations.

The human small heat-shock protein alphaB-crystallin is an extremely difficult molecule to study, with its inherent structural dynamics posing unique challenges to all biophysical and structural biology techniques. Here we highlight how the polydispersity and quaternary dynamics of alphaB-crystallin are intrinsically inter-twined, and how this can impact on measurements of the oligomeric distribution. We show that, in spite of these difficulties, considerable understanding of the varied fluctuations alphaB-crystallin undergoes at equilibrium has emerged in the last few years. By reporting on data obtained from a variety of biophysical techniques, we demonstrate how the alphaB-crystallin solution ensemble is governed by molecular motions of varying amplitude and time-scales spanning several orders of magnitude. We describe how these diverse measurements are being used to construct an integrated view of the dynamical structure of alphaB-crystallin, and highlight areas that require further interrogation. With its study motivating the refinement of experimental techniques, and the development of new approaches to combine the hybrid datasets, we conclude that alphaB-crystallin continues to represent a paradigm for dynamical biology.

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.

Contains fulltext : 125273pub.pdf (Publisher’s version ) (Closed access) alphaB-crystallin is regarded as a biomarker for triple-negative and/or basal-like breast cancer. In normal breast cells, overexpression of alphaB-crystallin leads to neoplastic-like changes, which likely relate to enhanced expression of phosphorylated ERK1/2 (pERK1/2). In this study, we investigated whether alphaB-crystallin expression is correlated to pERK1/2 expression in breast cancer. In a balanced tissue microarray the expression of alphaB-crystallin and pERK1/2 kinase were determined immunohistochemically, together with the triple-negativity and basal-like markers CK5/6 and SMA and the signaling molecules pAKT, pmTOR, EGFR, and IGF-1R. alphaB-crystallin expression significantly correlated with triple negativity and basal-like markers CK5/6 and SMA (Pearson Chi-square test p=0.004, p=0.001, and p

The major len protein αB-crystallin (αB) is an intracellular chaperone. It belongs to the family of small heat shock proteins (sHsps) which plays a critical role in maintaining protein homeostasis and preventing protein aggregation, especially under stress conditions. Dysfunction of αB is closely related to cataract, and many neurodegenerative diseases including Alzheimer's, Parkinson's, and Creutzfeldt-Jakob disease. Due to the extremely heterogeneous and polydispersed nature of αB, it remains unclear how αB self-assemblies and prevents its client proteins from aggregation. In this minireview, we summarize the structural studies of αB in self-assembly, chaperoning client proteins and amyloid aggregation. We also mention the recent progress in identification of small molecules preventing αB aggregation for potential cataract treatment. This review highlights the polymorphic structures of αB under different conditions and its wide-spectrum chaperone activities, and sheds light on understanding the complex relationship among αB, client proteins and the related diseases.

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.

Recent structure analyses of αB-crystallin have proposed some models of the N-terminal domain and the manner of oligomerization, whereas the effects of the significantly high content of Pro residues at the N-terminal domain remain unclear. We report the properties of a novel P39R mutant of αB-crystallin. The content of α-helix was increased, and the molecular size of the P39R mutant was larger than that of wild-type αB-crystallin. A slight loss of chaperone-like activity was observed using alcohol dehydrogenase (ADH), while a significant increase was detected by insulin assay. The Pro residue at the N-terminal domain of αB-crystallin is important for oligomerization and function.

Tsang J Y S, Lai M W H, Wong K H Y, Chan S-K, Lam C C F, Tsang A K H, Yu A M C, Tan P-H & Tse G M (2012) Histopathology 61, 378–386 αB-crystallin is a useful marker for triple negative and basal breast cancers Aims: Basal-like breast cancers (BLBCs), a breast cancer subtype with triple-negative status, pose significant problems in clinical management because of their aggressive behaviour. Recently, an association between αΒ-crystallin expression and BLBCs has been suggested, and we therefore investigated whether αΒ-crystallin could be a putative marker allowing BLBCs to be identified more accurately. Methods and results: We evaluated the expression of αB-crystallin and other biomarkers in 395 cases of breast carcinoma by immunohistochemistry, analysed the correlation of their expression with different breast cancer subtypes, and compared their sensitivity as well as specificity in identifying BLBCs. αΒ-crystallin expression was found to be correlated positively with basal markers and histological subtypes associated with BLBCs. A significant positive correlation of αΒ-crystallin expression was also found with triple-negative breast cancers (TNBC) (C = 0.409, P

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.