Small Heat Shock Protein αB-crystallin Research Articles

Manual and automatic assignment of two different Aβ40 amyloid fibril polymorphs using MAS solid-state NMR spectroscopy

Amyloid fibrils from Alzheimer’s amyloid-beta peptides (Aβ) are found to be polymorphic. So far, 14 Aβ40 fibril structures have been determined. The mechanism of why one particular protein sequence adopts so many different three-dimensional structures is yet not understood. In this work, we describe the assignment of the NMR chemical shifts of two Alzheimer’s disease fibril polymorphs, P1 and P2, which are formed by the amyloid-beta peptide Aβ40. The assignment is based on 13C-detected 3D NCACX and NCOCX experiments MAS solid-state NMR experiments. The fibril samples are prepared using an extensive seeding protocol in the absence and presence of the small heat shock protein αB-crystallin. In addition to manual assignments, we obtain chemical shift assignments using the automation software ARTINA. We present an analysis of the secondary chemical shifts and a discussion on the differences between the manual and automated assignment strategies.

Pro-cathepsin D prevents aberrant protein aggregation dependent on endoplasmic reticulum protein CLN6

We previously expressed a chimeric protein in which the small heat-shock protein αB-crystallin (αBC) is fused at its N-terminus to the C-terminus of the first transmembrane segment of the endoplasmic reticulum (ER) protein mitsugumin 23 and confirmed its localization to the ER. Moreover, overexpression of this N-terminally modified αBC was shown to prevent the aggregation of the coexpressed R120G αBC variant, which is highly aggregation-prone and associated with the hereditary myopathy αB-crystallinopathy. To uncover a molecular mechanism by which the ER-anchored αBC negatively regulates the protein aggregation, we isolated proteins that bind to the ER-anchored αBC and identified the lysosomal protease cathepsin D (CTSD) as one such interacting protein. Proteolytically active CTSD is produced by multi-step processing of pro-cathepsin D (proCTSD), which is initially synthesized in the ER and delivered to lysosomes. When overexpressed, CTSD itself prevented the coexpressed R120G αBC variant from aggregating. This anti-aggregate activity was also elicited upon overexpression of the W383C CTSD variant, which is predominantly sequestered in the ER and consequently remains unprocessed, suggesting that proCTSD, rather than mature CTSD, serves to suppress the aggregation of the R120G αBC variant. Meanwhile, overexpression of the A58V CTSD variant, which is identical to wild-type CTSD except for the Ala58Val substitution within the pro-peptide, did not suppress the protein aggregation, indicating that the integrity of the pro-peptide is required for proCTSD to exert its anti-aggregate activity. Based on our previous finding that overexpression of the ER transmembrane protein CLN6 (ceroid-lipofuscinosis, neuronal 6), identified as an interacting protein of the ER-anchored αBC, prevents the R120G αBC variant from aggregating, the CLN6-proCTSD coupling was hypothesized to underpin the functionality of proCTSD within the ER. Indeed, CTSD, when overexpressed in CLN6-depleted cells, was unable to exert its anti-aggregate activity, supporting our view. Collectively, we show here that proCTSD prevents the protein aggregation through the functional association with CLN6 in the microenvironment surrounding the ER membrane, shedding light on a novel aspect of proCTSD and its potential involvement in CTSD-related disorders characterized by the accumulation of aberrant protein aggregates.

Modulation of Alzheimer's Disease Aβ40 Fibril Polymorphism by the Small Heat Shock Protein αB-Crystallin.

Deposition of amyloid plaques in the brains of Alzheimer's disease (AD) patients is a hallmark of the disease. AD plaques consist primarily of the beta-amyloid (Aβ) peptide but can contain other factors such as lipids, proteoglycans, and chaperones. So far, it is unclear how the cellular environment modulates fibril polymorphism and how differences in fibril structure affect cell viability. The small heat-shock protein (sHSP) alpha-B-Crystallin (αBC) is abundant in brains of AD patients, and colocalizes with Aβ amyloid plaques. Using solid-state NMR spectroscopy, we show that the Aβ40 fibril seed structure is not replicated in the presence of the sHSP. αBC prevents the generation of a compact fibril structure and leads to the formation of a new polymorph with a dynamic N-terminus. We find that the N-terminal fuzzy coat and the stability of the C-terminal residues in the Aβ40 fibril core affect the chemical and thermodynamic stability of the fibrils and influence their seeding capacity. We believe that our results yield a better understanding of how sHSP, such as αBC, that are part of the cellular environment, can affect fibril structures related to cell degeneration in amyloid diseases.

The small heat shock protein αB-Crystallin protects versus withaferin A-induced apoptosis and confers a more metastatic phenotype in cisplatin-resistant ovarian cancer cells.

Since a majority of ovarian tumors recur in a drug-resistant form leaving patients few treatment options, the goal of this study was to explore phenotypic and molecular characteristics of a cisplatin-resistant ovarian cancer cell line (OVCAR8R) as compared to its cisplatin-sensitive syngeneic counterpart (OVCAR8) and to explore the effectiveness of a novel chemotherapeutic, Withaferin A (WA). In addition to unique morphological characteristics, the small heat shock proteins (Hsps) αB-Crystallin (HspB5) and Hsp27 are constitutively expressed along with increased expression of vimentin in OVCAR8R cells, while OVCAR8 cells do not endogenously express these Hsps, supporting that Hsp overexpression may confer resistance to chemotherapy and promote more aggressive tumor types. WA increases apoptosis in a dose-dependent manner in OVCAR8 cells, while OVCAR8R cells remain more viable at comparable doses of WA coincident with the upregulation of αB-Crystallin. To determine the significance of αB-Crystallin in conferring a more aggressive phenotype, αB-Crystallin was silenced by CRISPR-Cas9 in OVCAR8R cells. The morphology of the OVCAR8R clones in which αB-Crystallin was silenced reverted to the morphology of the original cisplatin-sensitive OVCAR8 cells. Further, cisplatin-resistant OVCAR8R cells constitutively express higher levels of vimentin and migrate more readily than cisplatin-sensitive OVCAR8 and OVCAR8R cells in which αB-Crystallin was silenced. Transient overexpression of wildtype αB-Crystallin, but not a chaperone-defective-mutant, alters the morphology of these cells to closely resemble the cisplatin-resistant OVCAR8R cells and protects versus WA-induced apoptosis. Together, this research supports the potential effectiveness of WA as a therapy for ovarian cancer cells that have not yet acquired resistance to platinum-based therapies, and importantly, underscores that αB-Crystallin contributes to a more aggressive cellular phenotype and as such, may be a promising molecular target for a better clinical outcome.

Abstract 865: The small heat shock protein αB-crystallin protects versus withaferin A-induced apoptosis and confers a more metastatic phenotype in cisplatin-resistant ovarian cancer cells

Abstract A majority of ovarian tumors eventually recur in a drug-resistant form leaving patients few treatment options. The goal of this study was to explore the phenotypic and molecular characteristics of a cisplatin-resistant ovarian cancer cell line (OVCAR8R) as compared to its cisplatin-sensitive syngeneic counterpart (OVCAR8) and to explore the effectiveness of a novel therapeutic, Withaferin A (WA). In addition to unique morphological characteristics and increased expression of vimentin, the small heat shock proteins (Hsps) αB-Crystallin and Hsp27 are constitutively expressed in OVCAR8R cells while OVCAR8 cells do not express these Hsps, supporting that Hsp overexpression may confer resistance to chemotherapy and promote a more aggressive tumor type. WA treatment causes apoptosis in a dose-dependent manner in OVCAR8 cells, while the OVCAR8R cells remain viable at comparable doses of WA. αB-Crystallin was upregulated in a dose dependent manner in the WA-treated OVCAR8R cells. To determine the significance of αB-crystallin expression in conferring a more aggressive phenotype, αB-crystallin was silenced by CRISPR-Cas9 in OVCAR8R cells. Strikingly, the morphology of the OVCAR8R-αB-crystallin-knockout clones revert to the morphology of the cisplatin-sensitive OVCAR8 cell line. Molecular characterization reveals concurrent downregulation of Hsp27, and decreased expression of vimentin supporting that loss of αB-Crystallin is coincident with a less aggressive molecular phenotype. Upon challenge with WA, vimentin is downregulated in the αB-crystallin-null clones and in the cisplatin-sensitive OVCAR8 cells, but not in the OVCAR8R cells. Transient transfection of wildtype αB-Crystallin, but not a chaperone-defective mutant, alters the morphology of the OVCAR8 cells where the αB-Crystallin-knockout clones closely resemble the cisplatin-resistant OVCAR8R cells. Further, transient overexpression of wildtype αB-crystallin, but not a chaperone-defective-mutant, protects cells from WA-induced apoptosis as compared to GFP-transfected cells. Migration assays reveal that OVCAR8R cells have increased migration capability as compared to their cisplatin-sensitive counterparts and silencing of αB-Crystallin decreases migration of cisplatin-resistant cells. This research supports the potential effectiveness of WA as a therapy for ovarian cancer cells that acquire resistance to platinum-based therapies and underscores that αB-crystallin confers a more aggressive cellular phenotype and therefore, may be a promising molecular target for a better clinical outcome. Citation Format: Merideth K. Krevosky, Melissa M. Carmichael. The small heat shock protein αB-crystallin protects versus withaferin A-induced apoptosis and confers a more metastatic phenotype in cisplatin-resistant ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 865.

Small heat shock protein αB-crystallin potentiates Aβ neurotoxicity by hetero-oligomeric stabilization.

Amyloid plaque, a neuropathological hallmark of Alzheimer's disease (AD), is composed of neurotoxic amyloid-β (Aβ) peptides and other proteins, including the small heat shock protein αB-crystallin (also known as HSPB5). We investigated the cellular origin of αB-crystallin, its secretion properties and how it may affect formation of neurotoxic Aβ hetero-oligomers. Immunohistofluorescence, Confocal Microscopy, Immunogold electron microscopy, SDS-page and immunoblotting, electron paramagnetic resonance spectroscopy (EPR), quasi-elastic light scattering (QLS) spectroscopy, cell and organotypic slice culture, LDH and propidium iodide assays. Immunohistofluorescence analysis of human AD brain revealed that Aβ and αB-crystallin co-localize in extracellular dense core and diffuse plaques and in surrounding reactive astrocytes. Immunohistochemical and immunoblotting analyses showed that astrocytes secrete αB-crystallin in vitro via an exosomal pathway. Co-incubation of αB-crystallin and Aβ suppressed Aβ fibrillogenesis by sequestration in hetero-oligomeric complexes however, this resulted in αB-crystallin potentiated Aβ neurotoxicity in mouse primary neurons and organotypic rat hippocampus slice culture as revealed by LDH and Propidium Iodide assays. Moreover, αB-crystallin acutely potentiated Aβ-induced impairment of activity-dependent long-term potentiation (LTP) of rat Schaffer collateral-CA1 synaptic transmission. These results indicate that the small heat shock protein αB-crystallin is secreted by reactive astrocytes and potentiates Aβ neurotoxicity by stabilizing hetero-oligomers.

The binding of the small heat-shock protein αB-crystallin to fibrils of α-synuclein is driven by entropic forces

Molecular chaperones are key components of the cellular proteostasis network whose role includes the suppression of the formation and proliferation of pathogenic aggregates associated with neurodegenerative diseases. The molecular principles that allow chaperones to recognize misfolded and aggregated proteins remain, however, incompletely understood. To address this challenge, here we probe the thermodynamics and kinetics of the interactions between chaperones and protein aggregates under native solution conditions using a microfluidic platform. We focus on the binding between amyloid fibrils of α-synuclein, associated with Parkinson's disease, to the small heat-shock protein αB-crystallin, a chaperone widely involved in the cellular stress response. We find that αB-crystallin binds to α-synuclein fibrils with high nanomolar affinity and that the binding is driven by entropy rather than enthalpy. Measurements of the change in heat capacity indicate significant entropic gain originates from the disassembly of the oligomeric chaperones that function as an entropic buffer system. These results shed light on the functional roles of chaperone oligomerization and show that chaperones are stored as inactive complexes which are capable of releasing active subunits to target aberrant misfolded species.

Transmembrane protein 168 mutation reduces cardiomyocyte cell surface expression of Nav1.5 through αB-crystallin intracellular dynamics.

Transmembrane protein 168 (TMEM168) was found to be localized on the nuclear membrane. A heterozygous mutation (c.1616G>A, p. R539Q) in TMEM168 was identified in patients with Brugada syndrome. This mutation reduced expression of cardiomyocyte sodium channel Nav1.5 via Nedd4-2 E3 ubiquitin ligase-induced ubiquitination and degradation. However, the detailed molecular mechanism provoked by the TMEM168 mutant remains unclear. Here, we demonstrated that small heat shock protein αB-crystallin, which can bind to Nav1.5 and Nedd4-2 and interfere with the association of both proteins, was strongly recruited from the cell surface to the perinuclear region because of the much higher affinity of αB-crystallin with the TMEM168 mutant than with wild-type TMEM168. Following knockdown of αB-crystallin in HL-1 cardiomyocytes, the interaction of Nav1.5 with Nedd4-2 was increased, despite the reduced expression of Nav1.5. Moreover, reduction of Nav1.5 expression by αB-crystallin knockdown was rescued in the presence of a proteasome inhibitor MG-132, suggesting the importance of the αB-crystallin-modulated ubiquitin-proteasome system for the stability of Nav1.5 expression. Collectively, the balance of molecular interactions among Nav1.5, Nedd4-2 and αB-crystallin plays a role in the regulation of cardiomyocyte cell surface expression of Nav1.5, and the TMEM168 mutant disturbs this balance, resulting in a decrease in Nav1.5 expression.

Extracellular αB-crystallin modulates the inflammatory responses.

Neuroinflammation is considered a challenging clinical problem. Chronic inflammatory responses play important roles in the onset and progression of various neurodegenerative diseases, including multiple sclerosis (MS). Previous studies have shown that astrocytes express small heat shock protein αB-crystallin (CRYAB) which is capable of inhibiting inflammatory responses in astrocytes per se. However, the underlying mechanisms of CRYAB-induced modulation of neuroinflammation are still not fully understood. In the present study, we investigated the role of extracellular CRYAB in the interaction between microglia and astrocytes in the context of MS-associated neuroinflammation. We found that the expression of CRYAB was profoundly increased in EAE mice. CRYAB was preferentially expressed in astrocytes and could be secreted via exosomes. Levels of exosomal CRYAB secreted from astrocytes were markedly increased under stress conditions. Furthermore, incubation of immortalized astrocytes or microglia cell lines with CRYAB remarkably suppressed astrocytes and microglia-mediated inflammatory responses in both autocrine and paracrine manners. Our results reveal a novel function for extracellular CRYAB in the regulation of neuroinflammation. Targeting extracellular CRYAB-modulated neuroinflammation is a potential therapeutic intervention for MS.

Loss of αB-crystallin function in zebrafish reveals critical roles in the development of the lens and stress resistance of the heart

Genetic mutations in the human small heat shock protein αB-crystallin have been implicated in autosomal cataracts and skeletal myopathies, including heart muscle diseases (cardiomyopathy). Although these mutations lead to modulation of their chaperone activity in vitro, the in vivo functions of αB-crystallin in the maintenance of both lens transparency and muscle integrity remain unclear. This lack of information has hindered a mechanistic understanding of these diseases. To better define the functional roles of αB-crystallin, we generated loss-of-function zebrafish mutant lines by utilizing the CRISPR/Cas9 system to specifically disrupt the two αB-crystallin genes, αBa and αBb We observed lens abnormalities in the mutant lines of both genes, and the penetrance of the lens phenotype was higher in αBa than αBb mutants. This finding is in contrast with the lack of a phenotype previously reported in αB-crystallin knock-out mice and suggests that the elevated chaperone activity of the two zebrafish orthologs is critical for lens development. Besides its key role in the lens, we uncovered another critical role for αB-crystallin in providing stress tolerance to the heart. The αB-crystallin mutants exhibited hypersusceptibility to develop pericardial edema when challenged by crowding stress or exposed to elevated cortisol stress, both of which activate glucocorticoid receptor signaling. Our work illuminates the involvement of αB-crystallin in stress tolerance of the heart presumably through the proteostasis network and reinforces the critical role of the chaperone activity of αB-crystallin in the maintenance of lens transparency.

The small heat shock proteins αB-crystallin (HSPB5) and Hsp27 (HSPB1) inhibit the intracellular aggregation of α-synuclein.

Protein homeostasis, or proteostasis, is the process of maintaining the conformational and functional integrity of the proteome. Proteostasis is preserved in the face of stress by a complex network of cellular machinery, including the small heat shock molecular chaperone proteins (sHsps), which act to inhibit the aggregation and deposition of misfolded protein intermediates. Despite this, the pathogenesis of several neurodegenerative diseases has been inextricably linked with the amyloid fibrillar aggregation and deposition of α-synuclein (α-syn). The sHsps are potent inhibitors of α-syn aggregation in vitro. However, the limited availability of a robust, cell-based model of α-syn aggregation has, thus far, restricted evaluation of sHsp efficacy in the cellular context. As such, this work sought to establish a robust model of intracellular α-syn aggregation using Neuro-2a cells. Aggregation of α-syn was found to be sensitive to inhibition of autophagy and the proteasome, resulting in a significant increase in the proportion of cells containing α-syn inclusions. This model was then used to evaluate the capacity of the sHsps, αB-c and Hsp27, to prevent α-syn aggregation in cells. To do so, we used bicistronic expression plasmids to express the sHsps. Unlike traditional fluorescent fusion constructs, these bicistronic expression plasmids enable only individual transfected cells expressing the sHsps (via expression of the fluorescent reporter) to be analysed, but without the need to tag the sHsp, which can affect its oligomeric structure and chaperone activity. Overexpression of both αB-c and Hsp27 significantly reduced the intracellular aggregation of α-syn. Thus, these findings suggest that overexpressing or boosting the activity of sHsps may be a way of preventing amyloid fibrillar aggregation of α-syn in the context of neurodegenerative disease.

Phosphorylation of αB-crystallin supports reactive astrogliosis in demyelination

The small heat shock protein αB-crystallin (CRYAB) has been implicated in multiple sclerosis (MS) pathogenesis. Earlier studies have indicated that CRYAB inhibits inflammation and attenuates clinical disease when administered in the experimental autoimmune encephalomyelitis model of MS. In this study, we evaluated the role of CRYAB in primary demyelinating events. Using the cuprizone model of demyelination, a noninflammatory model that allows the analysis of glial responses in MS, we show that endogenous CRYAB expression is associated with increased severity of demyelination. Moreover, we demonstrate a strong correlation between the expression of CRYAB and the extent of reactive astrogliosis in demyelinating areas and in in vitro assays. In addition, we reveal that CRYAB is differentially phosphorylated in astrocytes in active demyelinating MS lesions, as well as in cuprizone-induced lesions, and that this phosphorylation is required for the reactive astrocyte response associated with demyelination. Furthermore, taking a proteomics approach to identify proteins that are bound by the phosphorylated forms of CRYAB in primary cultured astrocytes, we show that there is clear differential binding of protein targets due to the specific phosphorylation of CRYAB. Subsequent Ingenuity Pathway Analysis of these targets reveals implications for intracellular pathways and biological processes that could be affected by these modifications. Together, these findings demonstrate that astrocytes play a pivotal role in demyelination, making them a potential target for therapeutic intervention, and that phosphorylation of CRYAB is a key factor supporting the pathogenic response of astrocytes to oligodendrocyte injury.

Small Heat Shock Protein αB-Crystallin Controls Shape and Adhesion of Glioma and Myoblast Cells in the Absence of Stress.

Cell shape and adhesion and their proper controls are fundamental for all biological systems. Mesenchymal cells migrate at an average rate of 6 to 60 μm/hr, depending on the extracellular matrix environment and cell signaling. Myotubes, fully differentiated muscle cells, are specialized for power-generation and therefore lose motility. Cell spreading and stabilities of focal adhesion are regulated by the critical protein vinculin from immature myoblast to mature costamere of differentiated myotubes where myofibril Z-band linked to sarcolemma. The Z-band is constituted from microtubules, intermediate filaments, cell adhesion molecules and other adapter proteins that communicate with the outer environment. Mesenchymal cells, including myoblast cells, convert actomyosin contraction forces to tension through mechano-responsive adhesion assembly complexes as Z-band equivalents. There is growing evidence that microtubule dynamics are involved in the generation of contractile forces; however, the roles of microtubules in cell adhesion dynamics are not well determined. Here, we show for the first time that αB-crystallin, a molecular chaperon for tubulin/microtubules, is involved in cell shape determination. Moreover, knockdown of this molecule caused myoblasts and glioma cells to lose their ability for adhesion as they tended to behave like migratory cells. Surprisingly, αB-crystallin knockdown in both C6 glial cells and L6 myoblast permitted cells to migrate more rapidly (2.7 times faster for C6 and 1.3 times faster for L6 cells) than dermal fibroblast. On the other hand, overexpression of αB-crystallin in cells led to an immortal phenotype because of persistent adhesion. Position of matured focal adhesion as visualized by vinculin immuno-staining, stress fiber direction, length, and density were clearly αB-crystallin dependent. These results indicate that the small HSP αB-crystallin has important roles for cell adhesion, and thus microtubule dynamics are necessary for persistent adhesion.

Inhibition of the Expression of the Small Heat Shock Protein αB-Crystallin Inhibits Exosome Secretion in Human Retinal Pigment Epithelial Cells in Culture

Exosomes carry cell type-specific molecular cargo to extracellular destinations and therefore act as lateral vectors of intercellular communication and transfer of genetic information from one cell to the other. We have shown previously that the small heat shock protein αB-crystallin (αB) is exported out of the adult human retinal pigment epithelial cells (ARPE19) packaged in exosomes. Here, we demonstrate that inhibition of the expression of αB via shRNA inhibits exosome secretion from ARPE19 cells indicating that exosomal cargo may have a role in exosome biogenesis (synthesis and/or secretion). Sucrose density gradient fractionation of the culture medium and cellular extracts suggests continued synthesis of exosomes but an inhibition of exosome secretion. In cells where αB expression was inhibited, the distribution of CD63 (LAMP3), an exosome marker, is markedly altered from the normal dispersed pattern to a stacked perinuclear presence. Interestingly, the total anti-CD63(LAMP3) immunofluorescence in the native and αB-inhibited cells remains unchanged suggesting continued exosome synthesis under conditions of impaired exosome secretion. Importantly, inhibition of the expression of αB results in a phenotype of the RPE cell that contains an increased number of vacuoles and enlarged (fused) vesicles that show increased presence of CD63(LAMP3) and LAMP1 indicating enhancement of the endolysosomal compartment. This is further corroborated by increased Rab7 labeling of this compartment (RabGTPase 7 is known to be associated with late endosome maturation). These data collectively point to a regulatory role for αB in exosome biogenesis possibly via its involvement at a branch point in the endocytic pathway that facilitates secretion of exosomes.

AB-Crystallin Binds to Titin Ig Domains and Increases Stiffness of Skinned Cardiac Trabeculae

Abnormally stiff or compliant cardiac muscle is commonly observed following acute damage or disease. Cardiac stiffness is primarily modulated by the extracellular matrix protein collagen and intracellularly by the giant sarcomeric protein titin. However, it is not clear, particularly in the absence of fibrosis, how stiffness is altered in disease conditions. Recently, a mutation in the small heat shock protein αB-crystallin (R157H) was shown to cause inherited dilated cardiomyopathy. This abundant protein (3-5% of total soluble protein in the heart) is thought to bind titin and may regulate its stiffness. To test this, we measured the passive stiffness of skinned mouse trabeculae (with endogenous αB-crystallin extracted) by extending the sarcomere length from 2.0 to 2.6 µm in relaxing solution and measuring the resulting tension; addition of 1 mg/ml recombinant WT αB-crystallin significantly increased stiffness (linear Young's Modulus, extracted 31.2 ± 3.3; WT αB-crystallin 55.3 ± 8.7 mN/mm2). Passive stiffness at lower sarcomere lengths was increased more than at longer sarcomere lengths, indicating an increased contribution to force by titin relative to collagen. Interestingly, addition of the R157H mutant produced a significantly weaker effect. We show that αB-crystallin binds to fully folded titin Ig domains in vitro using native mass spectrometry and methyl trosy NMR. Our results indicate that αB-crystallin increases muscle stiffness not simply by stabilizing titin domains that have become unfolded during stretching, but by using a novel mechanism that is dissimilar to its regular function as a holdase chaperone. This work illustrates a further complexity in cardiac muscle regulation that may clarify disease pathogenesis and lead to additional therapeutic pathways.

αB‐crystallin and HSP27 in glial cells in tauopathies

Tauopathies are neurodegenerative diseases characterized by hyper-phosphorylated tau deposition in neurons and glial cells. Chaperones, such as small heat shock proteins αB-crystallin and HSP27 highly expressed in normal glial cells, have been postulated as putative molecules preventing abnormal deposition and folding in glial cells in tauopathies. The objective of this work was to assess the expression of αB-crystallin, phosphorylated αB-crystallin at Ser59 and HSP27 in glial cells with and without tau deposits in progressive supranuclear palsy, corticobasal degeneration (CBD), argyrophilic grain disease (AGD), Pick's disease (PiD), Alzheimer's disease, frontotemporal lobar degeneration associated with mutations in the tau gene (FTLD-tau), globular glial tauopathy (GGT) and tauopathy in the elderly. Immunohistochemistry, and double-labeling immunofluorescence and confocal microscopy have been used for this purpose. Increased expression of αB-crystallin and phosphorylated αB-crystallin at Ser59 occurs in a subpopulation of glial cells with and without hyper-phosphorylated tau deposition in all the analyzed tauopathies, but their expression in neurons is restricted to ballooned neurons in CBD, AGD and PiD. HSP27 barely co-localizes with tau and with phosphorylated αB-crystallin at Ser59, thus making the formation of active dimers operating as chaperones unlikely. Results suggest a limited function of αB-crystallin and HSP27 in preventing abnormal tau protein deposition in glial cells and neurons; in addition, the expression of αB-crystallin phosphorylated at Ser59 may act as a protective factor in glial cells.

Levels of oestrogen receptor, progesterone receptor and αB-crystallin in eutopic endometrium in relation to pregnancy in women with endometriosis

Endometriosis affects fertility in many women and may partly be due to decreased endometrial receptivity. Several mechanisms have been suggested, notably, progesterone resistance for which a number of candidate biomarkers have been suggested. Here we demonstrate aberrant levels of steroid hormone receptors and the small heat shock protein αB-crystallin in eutopic endometrial epithelium from 38 women with peritoneal endometriosis diagnosed during investigation for secondary infertility. Spontaneous pregnancies within 1 year after medical and surgical treatment for endometriosis were recorded and semi-quantitative immunohistochemistry data compared between women with endometriosis who did or did not become pregnant and healthy controls.Stronger immunostaining for ER-α was detected in luminal and glandular endometrial epithelium from women with endometriosis who did not become pregnant during the post-treatment observation period versus endometriosis patients who became pregnant and controls. Staining levels of PR and PR-B were lower in patients without subsequent pregnancies than in the two other groups. Endometrial levels of αB-crystallin in endometriosis patients similar to those in controls were strongly correlated with the chance of becoming pregnant, whereas higher or lower levels were not.

Dynamical structure of αB-crystallin

The human small heat-shock protein αB-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 αB-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 αB-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 αB-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 αB-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 αB-crystallin continues to represent a paradigm for dynamical biology.

Spherical nucleic acids for precision medicine

Spherical nucleic acids for precision medicine

Regulated structural transitions unleash the chaperone activity of αB-crystallin

The small heat shock protein αB-crystallin is an oligomeric molecular chaperone that binds aggregation-prone proteins. As a component of the proteostasis system, it is associated with cataract, neurodegenerative diseases, and myopathies. The structural determinants for the regulation of its chaperone function are still largely elusive. Combining different experimental approaches, we show that phosphorylation-induced destabilization of intersubunit interactions mediated by the N-terminal domain (NTD) results in the remodeling of the oligomer ensemble with an increase in smaller, activated species, predominantly 12-mers and 6-mers. Their 3D structures determined by cryo-electron microscopy and biochemical analyses reveal that the NTD in these species gains flexibility and solvent accessibility. These modulated properties are accompanied by an increase in chaperone activity in vivo and in vitro and a more efficient cooperation with the heat shock protein 70 system in client folding. Thus, the modulation of the structural flexibility of the NTD, as described here for phosphorylation, appears to regulate the chaperone activity of αB-crystallin rendering the NTD a conformational sensor for nonnative proteins.