The role of αB-crystallin in skeletal and cardiac muscle tissues

All organisms and cells respond to various stress conditions such as environmental, metabolic, or pathophysiological stress by generally upregulating, among others, the expression and/or activation of a group of proteins called heat shock proteins (HSPs). Among the HSPs, special attention has been devoted to the mutations affecting the function of the αB-crystallin (HSPB5), a small heat shock protein (sHsp) playing a critical role in the modulation of several cellular processes related to survival and stress recovery, such as protein degradation, cytoskeletal stabilization, and apoptosis. Because of the emerging role in general health and disease conditions, the main objective of this mini-review is to provide a brief account on the role of HSPB5 in mammalian muscle physiopathology. Here, we report the current known state of the regulation and localization of HSPB5 in skeletal and cardiac tissue, making also a critical summary of all human HSPB5 mutations known to be strictly associated to specific skeletal and cardiac diseases, such as desmin-related myopathies (DRM), dilated (DCM) and restrictive (RCM) cardiomyopathy. Finally, pointing to putative strategies for HSPB5-based therapy to prevent or counteract these forms of human muscular disorders.     

Differential expression of αB-crystallin causes maturationdependent susceptibility of oligodendrocytes to oxidative stress

Oligodendrocyte precursor cells (OPCs) are most susceptible to oxidative stress in the brain. However, the cause of differences in susceptibility to oxidative stress between OPCs and mature oligodendrocytes (mOLs) remains unclear. Recently, we identified in vivo that αB-crystallin (aBC) is expressed in mOLs but not in OPCs. Therefore, we examined in the present study whether aBC expression could affect cell survival under oxidative stress induced by hydrogen peroxide using primary cultures of OPCs and mOLs from neonatal rat brains. Expression of aBC was greater in mOLs than in OPCs, and the survival rate of mOLs was significantly higher than that of OPCs under oxidative stress. Suppression of aBC by siRNA transfection resulted in a decrease in the survival rate of mOLs under oxidative stress. These data suggest that higher susceptibility of OPCs than mOLs to oxidative stress is due, at least in part, to low levels of aBC expression. [BMB Reports 2013; 46(10): 501-506]     

The role of ecto-5′-nucleotidase/CD73 in glioma cell line proliferation

The antioxidant activity of β-carotene and oxygenated carotenoids lutein, canthaxanthin, and astaxanthin was investigated during spontaneous and peroxyl-radical-induced cholesterol oxidation. Cholesterol oxidation, measured as generation of 7-keto-cholesterol (7-KC), was evaluated in a heterogeneous solution with cholesterol, AAPH, and carotenoids solubilized in tetrahydrofuran and in water, and in a homogeneous solution of chlorobenzene, with AIBN as a prooxidant. The formation of 7-KC was dependent on temperature and on cholesterol and prooxidant concentrations. All the carotenoids tested, exhibited significant antioxidant activity by inhibiting spontaneous, AAPH- and AIBN-induced formation of 7-KC, although the overall order of efficacy of these compounds was astaxanthin > canthaxanthin > lutein = β-carotene. The finding that carotenoids exert protective effects on spontaneous and free radical-induced cholesterol oxidation may have important beneficial effects on human health, by limiting the formation of atheroma and by inhibiting cholesterol oxidation in food processing or storage.     

Concentration dependence of chaperone-like activities of α-crystallin, αB-crystallin and proline

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 (Phb) from rabbit skeletal muscle. The biphasic character of the dependence of the initial rate of aggregation (v(agg)) of UV-irradiated Phb 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 Phb, a slight antagonism between proline used at a fixed concentration (0.15M) and α-crystallin was observed. At higher concentration of proline (0.5M) each chaperone acts independent of one another.     

Reduction of oxidative stress and ornithine decarboxylase expression in a human prostate cancer cell line PC-3 by a combined treatment with α-tocopherol and naringenin

Amino Acids - Differentiation of a human aggressive PC-3 cancer cell line was obtained, in a previous investigation, by the synergic effect of α-tocopherol (α-TOC) and naringenin (NG)....     

The effects of estrogen receptors α- and β-specific agonists and antagonists on cell proliferation and energy metabolism in human bone cell line

In cultured human osteoblasts estradiol-17β (E2) modulated DNA synthesis, the specific activity of creatine kinase BB (CK), 12 and 15 lipoxygenase (LO) mRNA expression and formation of 12- and 15-hydroxyeicosatetraenoic acid (HETE). We now investigate the response of human bone cell line (SaOS2) to phytoestrogens and estrogen receptors (ER)-specific agonists and antagonists. Treatment of SaSO2 with E2, 2,3-bis (4-hydroxyphenyl)-propionitrile (DPN; ERβ-specific agonist), 4,4',4″-[4-propyl-(1H)-pyrazol-1,3,5-triyl] tris-phenol (PPT; ERα-specific agonist), biochainin A (BA), daidzein (D), genistein (G) and raloxifene (Ral) showed increased DNA synthesis and CK. Ral inhibited completely all stimulations except DPN and to some extent D. The ERα-specific antagonist methyl-piperidino-pyrazole (MPP) and the ERβ-specific antagonist 4-[2-phenyl-5,7-bis (tri-fluoro-methyl) pyrazolo [1,5-a]pyrimidin-3-yl] phenol (PTHPP) inhibited DNA synthesis, CK and reactive oxygen species (ROS) formation induced by estrogens according to their receptors affinity. The LO inhibitor baicaleine inhibited only E2, DPN and G's effects. E2 and Ral unlike all other compounds had no effect on ERα mRNA expression, while ERβ mRNA expression was stimulated by all compounds. All compounds modulated the expression of 12LO and 15LO mRNA, except E2, PPT and Ral for 12LO, and 12- and 15-HETE productions and stimulated ROS formation which was inhibited by NADPH oxidase inhibitors diphenyleneiodonium chloride (DPI) and N-acetyl cysteine and the estrogen inhibitor ICI. DPI did not affect hormonal-induced DNA and CK. In conclusion, we provide evidence for the separation of mediation via ERα and ERβ pathways in the effects of estrogenic compounds on osteoblasts, but the role of LO/HETE/ROS is unclear.     

Multiple aggregates and aggresomes of C-terminal truncated human αA-crystallins in mammalian cells and protection by αB-crystallin

Background

Cleavage of 11 (αA162), 5 (αA168) and 1 (αA172) residues from the C-terminus of αA-crystallin creates structurally and functionally different proteins. The formation of these post-translationally modified αA-crystallins is enhanced in diabetes. In the present study, the fate of the truncated αA-crystallins expressed in living mammalian cells in the presence and absence of native αA- or αB-crystallin has been studied by laser scanning confocal microscopy (LSM).

Methodology/Principal Findings

YFP tagged αAwt, αA162, αA168 and αA172, were individually transfected or co-transfected with CFP tagged αAwt or αBwt, expressed in HeLa cells and studied by LSM. Difference in protein aggregation was not caused by different level of α-crystallin expression because Western blotting results showed nearly same level of expression of the various α-crystallins. The FRET-acceptor photo-bleaching protocol was followed to study in situ protein-protein interaction. αA172 interacted with αAwt and αBwt better than αA168 and αA162, interaction of αBwt being two-fold stronger than that of αAwt. Furthermore, aggresomes were detected in cells individually expressing αA162 and αA168 constructs and co-expression with αBwt significantly sequestered the aggresomes. There was no sequestration of aggresomes with αAwt co-expression with the truncated constructs, αA162 and αA168. Double immunocytochemistry technique was used for co-localization of γ-tubulin with αA-crystallin to demonstrate the perinuclear aggregates were aggresomes.

Conclusions/Significance

αA172 showed the strongest interaction with both αAwt and αBwt. Native αB-crystallin provided protection to partially unfolded truncated αA-crystallins whereas native αA-crystallin did not. Aggresomes were detected in cells expressing αA162 and αA168 and αBwt co-expression with these constructs diminished the aggresome formation. Co-localization of γ-tubulin in perinuclear aggregates validates for aggresomes.     

Thermal response in murine L929 cells lacking αB-crystallin expression and αB-crystallin expressing L929 transfectants

We investigated the role of B-crystallin expression in the development of thermotolerance in murine L929 cells. An initial heat-shock of 10 min at 45°C induced thermotolerance in these cells to a heat challenge at 45°C administered 24 h later. The thermotolerance ratio at 10^–1 isosurvival was 1.7. Expression of B-crystallin gene was not detected during the 24 h incubation at 37°C following heat shock by either northern or western blots. In contrast, inducible HSP70 synthesis was observed during this time period. Thus, this cell line provided an unique system in which to examine the effects of transfected B-crystallin on thermoresistance and thermotolerance. Cells stably transfected with B-crystallin under the control of an inducible promoter did not show a significant increase in the ability to develop thermotolerance. However, a stably transfected L929 clone expressing high levels of constitutive B-crystallin exhibited an approximately 50% increase in thermal resistance over parental and control cells. Though expression of B-crystallin is not requisite for the development of thermotolerance in L929 cells, overexpression of transfected B-crystallin can contribute to increased thermoresistance.     

Is hypothermia a stress condition in HepG2 cells? Expression and localization of Hsp70 in human hepatoma cell line

To understand hypothermia as a stress condition we determined the expression and localization of Hsp70 under hyperthermic and hypothermic stress in human hepatoma HepG2 cells. Western blot analysis indicates that there was a statistically significant increase of Hsp70 expression under thermal stresses. Immunohistochemically, the distribution of inducible Hsp70 in stressed cells showed a granular pattern mostly in the cytoplasm. At subcellular level, Hsp70 was localized in the nucleus, vacuoles, cytoskeletal components and dispersed throughout the cytoplasm. Accumulation of Hsp70 in cells under hypothermia could be related to restitution of cell equilibrium modified by this thermal stress condition. The protective effect of hypothermia could be associated with promotion of Hsp expression. We suggest that hypothermia is a stress capable of inducing Hsp70 expression in human HepG2 cells.     

O-GlcNAcylation of αB-crystallin regulates its stress-induced translocation and cytoprotection

Under normal conditions, the ubiquitously expressed αB-crystallin functions as a chaperone. αB-crystallin has been implicated in a variety of pathologies, consistent with a build-up of protein aggregates, such as neuromuscular disorders, myofibrillar myopathies, and cardiomyopathies. αB-crystallins’ cardioprotection is partially attributed to its translocation and binding to cytoskeletal elements in response to stress. The triggers for this translocation are not clearly understood. In the heart, αB-crystallin undergoes at least three significant post-translational modifications: phosphorylation at ser-45 and 59 and O-GlcNAcylation (O-linked attachment of the monosaccharide β-N-acetyl-glucosamine) at thr-170. Whether phosphorylation status drives translocation remains controversial. Therefore, we evaluated the role of αB-crystallins’ O-GlcNAcylation in its stress-induced translocation and cytoprotection in cardiomyocytes under stress. Immunoblotting and precipitation experiments with anti-O-GlcNAc antibody (CTD110.6) and glycoprotein staining (Pro-Q Emerald) both demonstrate robust stress-induced O-GlcNAcylation of αB-crystallin. A non-O-GlcNAcylatable αB-crystallin mutant (αB-T170A) showed diminished translocation in response to heat shock and robust phosphorylation at both ser-45 and ser-59. Cell survival assays show a loss of overexpression-associated cytoprotection with the non-glycosylatable mutant to multiple stresses. While ectopic expression of wild-type αB-crystallin strongly stabilized ZsProSensor, a fusion protein rapidly degraded by the proteasome, the non-O-GlcNAcylatable version did not. Therefore, we believe the O-GlcNAcylation of αB-crystallin is a dynamic and important regulator of both its localization and function.     

Structural and mechanistic implications of metal binding in the small heat-shock protein αB-crystallin

The human small heat-shock protein αB-crystallin (αB) rescues misfolded proteins from irreversible aggregation during cellular stress. Binding of Cu(II) was shown to modulate the oligomeric architecture and the chaperone activity of αB. However, the mechanistic basis of this stimulation is so far not understood. We provide here first structural insights into this Cu(II)-mediated modulation of chaperone function using NMR spectroscopy and other biophysical approaches. We show that the α-crystallin domain is the elementary Cu(II)-binding unit specifically coordinating one Cu(II) ion with picomolar binding affinity. Putative Cu(II) ligands are His(83), His(104), His(111), and Asp(109) at the dimer interface. These loop residues are conserved among different metazoans, but also for human αA-crystallin, HSP20, and HSP27. The involvement of Asp(109) has direct implications for dimer stability, because this residue forms a salt bridge with the disease-related Arg(120) of the neighboring monomer. Furthermore, we observe structural reorganization of strands β2-β3 triggered by Cu(II) binding. This N-terminal region is known to mediate both the intermolecular arrangement in αB oligomers and the binding of client proteins. In the presence of Cu(II), the size and the heterogeneity of αB multimers are increased. At the same time, Cu(II) increases the chaperone activity of αB toward the lens-specific protein β(L)-crystallin. We therefore suggest that Cu(II) binding unblocks potential client binding sites and alters quaternary dynamics of both the dimeric building block as well as the higher order assemblies of αB.     

Stability of coral–endosymbiont associations during and after a thermal stress event in the southern Great Barrier Reef

Shifts in the community of symbiotic dinoflagellates to those that are better suited to the prevailing environmental condition may provide reef-building corals with a rapid mechanism by which to adapt to changes in the environment. In this study, the dominant Symbiodinium in 10 coral species in the southern Great Barrier Reef was monitored over a 1-year period in 2002 that coincided with a thermal stress event. Molecular genetic profiling of Symbiodinium communities using single strand conformational polymorphism of the large subunit rDNA and denaturing gradient gel electrophoresis of the internal transcribed spacer 2 region did not detect any changes in the communities during and after this thermal-stress event. Coral colonies of seven species bleached but recovered with their original symbionts. This study suggests that the shuffling or switching of symbionts in response to thermal stress may be restricted to certain coral species and is probably not a universal feature of the coral–symbiont relationship. Communicated by Biology Editor Dr. Clay Cook     

Structure,chaperone activity,and aggregation of wild-type and R12C mutant αB-crystallins in the presence of thermal stress and calcium ion – Implications for role of calcium in cataract pathogenesis

The current study was performed with the aim to evaluate the chaperoning ability, structural features, and aggregation propensity of wild-type and R12C mutant αB-crystallins (αB-Cry) under thermal stress and in the presence of calcium ion. The results of different spectroscopic analyses suggest that wild-type and mutant αB-Cry have dissimilar secondary and tertiary structures. Moreover, αB-Cry indicates slightly improved chaperone activity upon the R12C mutation. Thermal stress and calcium, respectively, enhance and reduce the extent of solvent-exposed hydrophobic surfaces accompanying formation of ordered and non-ordered aggregate entities in both proteins. Compared to the wild-type protein, the R12C mutant counterpart shows significant resistance against thermal and calcium-induced aggregation. In addition, in the presence of calcium, significant structural variation was accompanied by reduction in the solvent-exposed hydrophobic patches and attenuation of chaperone activity in both proteins. Additionally, gel mobility shift assay indicates the intrinsic propensity of R12C mutant αB-Cry for disulfide bridge-mediated protein dimerization. Overall, the results of this study are of high significance for understanding the molecular details of different factors that are involved in the pathomechanism of cataract disorders.     

Phosphorylation of Ser45 and Ser59 of αB-crystallin and p38/extracellular regulated kinase activity determine αB-crystallin-mediated protection of rat brain astrocytes from C2-ceramide- and staurosporine-induced cell death

We previously demonstrated that αB-crystallin and protease-activated receptor (PAR) are involved in protection of astrocytes against C2-ceramide- and staurosporine-induced cell death [Li et al. (2009) J. Neurochem.110, 1433-1444]. Here, we further investigated the mechanism of cytoprotection by αB-crystallin. Our current data revealed that after down-regulation of αB-crystallin by siRNA, cell death caused by C2-ceramide and staurosporine is increased. Furthermore, we investigated the mechanism of cytoprotection of astrocytes by intracellular αB-crystallin. Application of specific inhibitors of p38 and extracellular regulated kinase (ERK) abrogates the protection of astrocytes by over-expression of αB-crystallin. Thus, p38 and ERK contribute to protective processes by αB-crystallin. To reveal the molecular mechanism of αB-crystallin-mediated cytoprotection, we mimicked phosphorylation or unphosphorylation of αB-crystallin. In these experiments, we found that the phosphorylation of αB-crystallin at Ser45 and Ser59 is required for protection. Ser19 phosphorylation of αB-crystallin does not contribute to protection. Moreover, we detected that PAR-2 activation increases the phosphorylation level of αB-crystallin at Ser59, but does not affect the expression level of αB-crystallin. Thus, endogenous αB-crystallin has protective capacity employing a mechanism, which involves regulation of the phosphorylation status of αB-crystallin and p38 and ERK activity. Moreover, we report that PAR-2 activation evokes the phosphorylation of αB-crystallin to increase astrocytes survival.     

Cell kinetic status of mouse lens epithelial cells lacking αA- and αB-crystallin

alphaA- and alphaB-crystallins are small heat shock proteins and molecular chaperones that are known to prevent non-specific aggregation of denaturing proteins. Recent work indicates that alphaA-/- lens epithelial cells grow at a slower rate than wild-type cells, and cultured alphaB-/- cells demonstrate increased hyperproliferation and genomic instability, suggesting that these proteins may exert a direct effect on the cell cycle kinetics, and influence cell proliferation. However, the cell cycle parameters of alphaA/alphaBKO (double knockout) cells have not been analyzed. Here we investigate the cell cycle kinetics of synchronized mouse lens epithelial cultures derived from wild-type and alphaA/alphaB double knockout (alphaA/alphaBKO) mice using BrdU labeling of proliferating cells, and flow cytometric analysis. We also provide data on the changing pattern of expression of HSP25, a small heat shock protein in alphaA/alphaBKO and wild-type cells during the cell cycle. Using serum starvation to synchronize cells in the quiescent G0 phase, and restimulation with serum followed by BrdU labeling and flow cytometry, the data indicated that as compared to wild-type cells, a <50% smaller fraction of the alphaA/alphaBKO cells entered the DNA synthetic S phase of the cell cycle. Furthermore, there was a delay in cell cycle transit through S phase in alphaA/alphaBKO cells, suggesting that although capable of entering S phase, the alphaA/alphaBKO cells are blocked in G1 phase, and are delayed in their cell cycle progression. Immunoblot analysis with antibodies to the small heat shock protein HSP25 indicated that although HSP25 increased in G1 phase of wild-type cells, and remained elevated on further progression through the cell cycle, HSP25 accumulation was delayed to S phase in alphaA/alphaBKO cells. These data can be interpreted to indicate that mouse lens epithelial cell progression through the cell cycle is significantly affected by expression of alphaA and alphaB-crystallin.     

Effects of decorin on the expression of α-smooth muscle actin in a human myofibroblast cell line

Myofibroblasts are metabolically and morphologically distinctive fibroblasts expressing α-smooth muscle actin (α-SMA), and their activation plays a key role in development of the fibrotic response. In an activated state, myofibroblasts cease to proliferate and start to synthesize large amounts of extracellular component proteins. The expression of α-SMA correlates with the activation of myofibroblasts. Decorin, a member of the small leucine-rich proteoglycan gene family, has been implicated in the negative control of cell proliferation primarily by upregulating the expression of p21, a potent inhibitor of cyclin-dependent kinase. In order to examine the effect of decorin on myofibroblast cell growth, we rendered a human lung myofibroblast cell line, MRC-5, quiescent by either cell–cell contact or serum starvation, and examined the relationship between decorin and α-SMA expression in these cells. The expression of decorin in cells made quiescent by serum starvation was lower than that in cells made quiescent by cell–cell contact. In contrast, the expression of α-SMA in cells made quiescent by cell–cell contact was lower than that in cells made quiescent by serum starvation. Furthermore, forced expression of decorin was accompanied by a suppression of α-SMA expression, whereas knocking down of decorin expression by RNA interference increased the expression of α-SMA.     

Exploring the thermal stability and activity of α-chymotrypsin in the presence of spermine

Polyamines such as spermine can have interaction with protein. The aim of the present study was to investigate how spermine could influence the structure, thermal stability, and the activity of α-chymotrypsin. Kinetics, thermodynamics, molecular dynamics (MD), and docking simulations studies were conducted to investigate the effect of spermine on the activity and structure of α-Chymotrypsin (α-Chy) in 50 mM Tris–HCl buffer, with the pH 8, using different spectroscopic techniques as well as molecular docking and MD simulations. The stability and activity of α-Chy were increased in the presence of spermine. The results of the kinetic study showed that the activity of spermine was increased. Enzyme activation was accompanied by changes on the α-Chy conformation. Fluorescence intensity changes showed dynamic quenching during spermine binding. The fluorescence quenching of the α-Chy suggested the more polar location of Trp residues. Near-UV and Far-UV circular dichroism studies also demonstrated the transfer of Trp, Phe, and Tyr residues to a more flexible environment. The increase in the absorption of α-Chy in the presence of spermine was as a result of the formation of spermine–α-Chy complex. Molecular docking results revealed the presence of one binding site with a negative value for the Gibbs free energy of the binding of spermine to α-Chy. Docking study also revealed that van der Waals interactions and hydrogen bonds played a major role in stabilizing the complex.