Effect of Sorbitol on Alpha-Crystallin Structure and Function.

Loss of eye lens transparency due to cataract is the leading cause of blindness all over the world. While aggregation of lens crystallins is the most common endpoint in various types of cataracts, chaperone-like activity (CLA) of α-crystallin preventing protein aggregation is considered to be important for maintaining the eye lens transparency. Osmotic stress due to increased accumulation of sorbitol under hyperglycemic conditions is believed to be one of the mechanisms for diabetic cataract. In addition, compromised CLA of α-crystallin in diabetic cataract has been reported. However, the effect of sorbitol on the structure and function of α-crystallin has not been elucidated yet. Hence, in the present exploratory study, we described the effect of varying concentrations of sorbitol on the structure and function of α-crystallin. Alpha-crystallin purified from the rat lens was incubated with varying concentrations of sorbitol in the dark under sterile conditions for up to 5 days. At the end of incubation, structural properties and CLA were evaluated by spectroscopic methods. Interestingly, different concentrations of sorbitol showed contrasting results: at lower concentrations (5 and 50 mM) there was a decrease in CLA and subtle alterations in secondary and tertiary structure but not at higher concentrations (500 mM). Though, these results shed a light on the effect of sorbitol on α-crystallin structure-function, further studies are required to understand the mechanism of the observed effects and their implication to cataractogenesis.

Aggregation of ß-crystallin through covalent binding to 1,2-naphthoquinone is rescued by α-crystallin chaperone.

Cataract induced by exposure to naphthalene is thought to mainly involve its metabolic activation, forming 1,2-naphthoquinone (1,2-NQ), which can modify proteins through chemical modifications. In the present study, we examined the effect of 1,2-NQ on aggregation of crystallins (cry) associated with cataract. Incubation of bovine ß-cry with 1,2-NQ caused covalent modification of ß-cry at Cys117 and Lys125 accompanied by reduction in its thiol content, resulting in a concentration- and temperature-dependent aggregation of ß-cry, whereas only little aggregation of α-cry induced by 1,2-NQ was seen. Interestingly, addition of α-cry to the reaction mixture of ß-cry and 1,2-NQ markedly blocked ß-cry aggregation induced by 1,2-NQ in a concentration-dependent manner. These results suggest that ß-cry predominantly undergoes chemical modification by 1,2-NQ, causing its aggregation, which is suppressed by the chaperone-like protein, α-cry. This ß-cry aggregation may be, at least in part, involved in the induction of cataract caused by 1,2-NQ.

Targeting the molecular mechanisms of ischemic damage: Protective effects of alpha-crystallin-B.

AIMS: Molecular chaperones constitute protectors of intracellular protein integrity and seem to confer short-term defence against various cell insults. Myocardial damage is associated to a loss of protective chaperones. Ischemic post-conditioning (IPost-Co) is a procedure that seems to protect against reperfusion injury. However, little is known on alpha-crystallin-B-chain (cryab/HspB5) evolution in IPost-Co. Here we have investigated cryab in myocardial ischemia and IPost-Co. METHODS AND RESULTS: Pigs underwent closed-chest 1.5h mid-left anterior descending (LAD) balloon occlusion and were either sacrificed without reperfusion (I;N=10), subjected to 2.5h of reperfusion and sacrificed (I/R; N=5); or subjected to IPost-Co before reperfusion and sacrificed 2.5h afterwards (IPost-Co; N=5). A sham-operated group was included (N=6). Proteomic analysis (2-D-electrophoresis/MALDI-TOF/TOF) revealed cryab as a single spot (20kDa; pI7.6). Myocardial cryab-20-protein and cryab-gene expression levels were decreased after ischemia and I/R(P<0.05). After IPost-Co, cryab-20-protein and cryab-gene expression levels were similar to those found in the heart of sham-operated animals (P<0.05). There was a direct correlation between LVEF-improvement after IPost-Co and myocardial cryab-20-protein levels. In a mice proof-of-principle study, cryab-20-peptide was synthesized and administered 1h before LAD-ligation and ECG-proven MI. A 59% reduction in infarct size was achieved in cryab-20-treated animals (P<0.05). CONCLUSIONS: Ischemia and reperfusion induce a decrease in myocardial cryab-20-protein levels together with a clinical impairment of cardiac function. IPost-Co induces a clinical improvement of cardiac function and a preservation of cryab-20 levels. Intervention studies on a mice-MI model showed that cryab-20-peptide administration reduces infarct size. All together our results show a significant cardioprotective effect of cryab.

Quantification of anti-aggregation activity of UV-irradiated α-crystallin.

Ultraviolet radiation is a risk factor for cataractogenesis. It is believed that enhanced rates of lens opacification and cataract formation are the results of gradual loss of chaperone-like efficiency of α-crystallin upon exposure to UV light. To characterize chaperone-like activity of α-crystallin damaged by UV irradiation, a test system based on dithiothreitol-induced aggregation of holo-α-lactalbumin from bovine milk was used. The adsorption capacity of α-crystallin (AC0) with respect to the target protein (α-lactalbumin) was used as a measure of anti-aggregation activity of α-crystallin. The data on SDS-PAGE testify that UV irradiation of α-crystallin results in covalent cross-linking of subunits in α-crystallin oligomers. The dependence of AC0 value on the irradiation dose was compared with the UV-induced diminution of the portion of native α-crystallin estimated from the data on differential scanning calorimetry. On the basis of such comparison a conclusion has been made that the loss in chaperone-like activity is mainly due to UV-induced denaturation of α-crystallin subunits. Cross-linking of remaining native subunits leads to an additional decrease in anti-aggregation activity.

Thermal denaturation and aggregation of apoform of glycogen phosphorylase b. Effect of crowding agents and chaperones.

The effect of protein and chemical chaperones and crowders on thermal stability and aggregation of apoform of rabbit muscle glycogen phosphorylase b (apoPhb) has been studied at 37°C. Proline suppressed heat-induced loss in ability of apoPhb to reconstitution at 37°C, whereas α-crystallin did not reveal a protective action. To compare the antiaggregation activity of intact and crosslinked α-crystallins, an adsorption capacity (AC) of a protein chaperone with respect to a target protein was estimated. This parameter is a measure of the antiaggregation activity. Crosslinking of α-crystallin results in 11-fold decrease in the initial AC. The nonlinear character of the relative initial rate of apoPhb aggregation versus the [intact α-crystallin]/[apoPhb] ratio plot is indicative of the decrease in the AC of α-crystallin with increasing the [α-crystallin]/[apoPhb] ratio and can be interpreted as an evidence for dynamic chaperone structure and polydispersity of α-crystallin-target protein complexes. As for chemical chaperones, a semisaturation concentration of the latter was used as a characteristic of the antiaggregation activity. A decrease in the semisaturation concentration for proline was observed in the presence of the crowders (polyethylene glycol and Ficoll-70).

Latency-associated protein Acr1 impairs dendritic cell maturation and functionality: a possible mechanism of immune evasion by Mycobacterium tuberculosis.

Mycobacterium tuberculosis (M. tuberculosis) in latently infected individuals survives and thwarts the attempts of eradication by the immune system. During latency, Acr1 is predominantly expressed by the bacterium. However, whether M. tuberculosis exploits its Acr1 in impairing the host immunity remains widely unexplored. Hence, currently we have investigated the role of Acr1 in influencing the differentiation and function of dendritic cells (DCs), which play a cardinal role in innate and adaptive immunity. Therefore, for the first time, we have revealed a novel mechanism of mycobacterial Acr1 in inhibiting the maturation and differentiation of DCs by inducing tolerogenic phenotype by modulating the expression of PD-L1; Tim-3; indoleamine 2, 3-dioxygenase (IDO); and interleukin 10. Furthermore, Acr1 interferes in the differentiation of DCs by targeting STAT-6 and STAT-3 pathways. Continuous activation of STAT-3 inhibited the translocation of NF-κB in Acr1-treated DCs. Furthermore, Acr1 also augmented the induction of regulatory T cells. These DCs displayed decline in their antigen uptake capacity and reduced ability to help T cells. Interestingly, M. tuberculosis exhibited better survival in Acr1-treated DCs. Thus, this study provides a crucial insight into a strategy adopted by M. tuberculosis to survive in the host by impairing the function of DCs.

α-Crystallin protects RGC survival and inhibits microglial activation after optic nerve crush.

AIMS: Activation of retinal microglial cells (RMCs) is known to contribute to retinal ganglion cell (RGC) death after optic nerve injury. The purpose of this study was to investigate the effects of intravenous injection of α-crystallin on RGC survival and RMC activation in a rat model of optic nerve crush. MAIN METHODS: RGCs were retrogradely labeled with fluorogold. Rats were intravenously injected with normal saline or α-crystallin (0.05g/kg, 0.5g/kg, and 5 g/kg) at 2, 4, 6, 8, 10, and 12 days after the optic nerve crush. Activated RMCs were characterized using immunofluorescence labeling with CD11b, and TNF-α and iNOS expression was detected using immunoblot analyses. We analyzed the morphology and numbers of RGC and RMC 2 and 4 weeks after injury using fluorescence and confocal microscopy. KEY FINDINGS: The number of RGCs decreased after optic nerve injury, accompanied by significantly increased numbers of activated RMCs. Intravenous injection of α-crystallin decreased the number of RMCs, and enhanced the number of RGCs compared to saline injection. α-Crystallin administration inhibited TNF-α and iNOS protein expression induced by optic nerve injury. SIGNIFICANCE: Our results suggest that α-crystallin promotes RGC survival and inhibits RMC activation. Intravenous injection of α-crystallin could be a possible strategy for the treatment of optic nerve injury.

Effect of crowding and chaperones on self-association, aggregation and reconstitution of apophosphorylase b.

It was shown that the rate of reconstruction of muscle glycogen phosphorylase b (Phb) from apoenzyme and pyridoxal 5'-phosphate decreased under crowding conditions. The effect of crowding was counteracted by chaperones (α-crystallin and proline). Sedimentation analysis shows that crowding stimulates the formation of high-molecular-weight associates at 25 °C, whereas chaperones stabilize small oligomers. The study of the kinetics of apoPhb aggregation at 37 °C showed that the anti-aggregation activity of chaperones decreased under crowding conditions. When studying the sedimentation behavior of the mixture of apoPhb and α-crystallin, the complexes between unfolded apoPhb and dissociated forms of α-crystallin were observed. It is assumed that these complexes are responsible for realization of the chaperone-like activity of α-crystallin under crowding conditions.

Alpha-crystallin promotes rat retinal neurite growth on myelin substrates in vitro.

BACKGROUND: Myelin-associated molecules are major impediments to axon regeneration after optic nerve injury. Intravitreal injection of α-crystallin can protect axons from optic nerve degeneration after crushing in rats. OBJECTIVES: Our purpose was to investigate whether α-crystallin could counteract the inhibitory effect of myelin and promote neurite growth. METHODS: Newborn rat retinal neurons were cultured on myelin-coated dishes with DMEM containing α-crystallin (10(-4) g/l) or bovine serum albumin. The density of neurons with neurites and the length of the longest neurite of the cells were analyzed on days 1, 3 and 5. RESULTS: Cultures containing α-crystallin had significantly higher neurite-containing cell densities, and the neurites were significantly longer compared with cultures containing bovine serum albumin. These findings indicated that α-crystallin could counteract the effect of myelin inhibitory factors and stimulate neurite growth.

In vitro renaturation of alkaline family G/11 xylanase via a folding intermediate: alpha-crystallin facilitates refolding in an ATP-independent manner.

In this study, alkaliphilic family G/11 xylanase from alkali-tolerant filamentous fungi Penicillium citrinum MTCC 6489 was used as a model system to gain insight into the molecular aspects of unfolding/refolding of alkaliphilic glycosyl hydrolase protein family. The intrinsic protein fluorescence suggested a putative intermediate state of protein in presence of 2 M guanidium hydrochloride (GdmCl) with an emission maximum of 353 nm. Here we studied the refolding of GdmCl-denatured alkaline xylanase in the presence and the absence of a multimeric chaperone protein alpha-crystallin to elucidate the molecular mechanism of intramolecular interactions of the alkaliphilic xylanase protein that dictates its extremophilic character. Our results, based on intrinsic tryptophan fluorescence and hydrophobic fluorophore 8-anilino-1- naphthalene sulfonate-binding studies, suggest that alpha-crystallin formed a complex with a putative molten globule-like intermediate in the refolding pathway of xylanase in an ATP-independent manner. A 2 M GdmCl is sufficient to denature alkaline xylanase completely. The hydrodynamic radius (R(H)) of a native alkaline xylanase is 4.0, which becomes 5.0 in the presence of 2 M GdmCl whereas in presence of the higher concentration of GdmCl R(H) value was shifted to 100, indicating the aggregation of denatured xylanase. The alpha-crystallin.xylanase complex exhibited the recovery of functional activity with the extent of approximately 43%. Addition of ATP to the complex did not show any significant effect on activity recovery of the denatured protein.

Mechanism of suppression of dithiothreitol-induced aggregation of bovine alpha-lactalbumin by alpha-crystallin.

The kinetics of dithiothreitol (DTT)-induced aggregation of alpha-lactalbumin from bovine milk has been studied using dynamic light-scattering technique. Analysis of the distribution of the particles formed in the solution of alpha-lactalbumin after the addition of DTT by size showed that the initial stage of the aggregation process was the stage of formation of the start aggregates with the hydrodynamic radius (R(h)) of 80-100nm. Further growth of the protein aggregates proceeds as a result of sticking of the start aggregates. Suppression of alpha-lactalbumin aggregation by alpha-crystallin is mainly due to the increase in the duration of the lag period on the kinetic curves of aggregation. It is assumed that the initially formed complexes of unfolded alpha-lactalbumin with alpha-crystallin were transformed to the primary clusters prone to aggregation as a result of the redistribution of the denatured protein molecules on the surface of the alpha-crystallin particles.

Effect of alpha-crystallin on thermostability of mitochondrial aspartate aminotransferase.

Effect of alpha-crystallin on thermal inactivation, denaturation and aggregation of aspartate aminotransferase from pig heart mitochondria (mAAT) has been in the focus of this study. Acceleration of heat-induced inactivation of mAAT was demonstrated in the presence of alpha-crystallin. According to the data of differential scanning calorimetry, alpha-crystallin induces destabilization of the mAAT molecule. The size of protein aggregates formed at heating of mAAT at a constant rate (1 degree C/min) has been defined by dynamic light scattering. The obtained data show that aggregation of mAAT in the presence of alpha-crystallin proceeds in the regime of reaction-limited cluster-cluster aggregation.

alpha-Crystallin downregulates the expression of TNF-alpha and iNOS by activated rat retinal microglia in vitro and in vivo.

PURPOSE: Inhibition of microglial activation has become an important strategy to attenuate neurotoxic damage to the central nervous system. We evaluated the effects of alpha-crystallin on the production of cytokines in lipopolysaccharides (LPS) and optic nerve injury-activated retinal microglia. METHODS: Microglia were collected from retinas of newborn rats, cultured and treated with LPS in vitro. Microglia were also activated by an optic nerve crush in vivo. Pretreatments with and without alpha-crystallin were performed in cultured cells, and by intravitreal injection in adult rats. Expression of tumor necrosis factor-alpha (TNF-alpha), nitric oxide (NO) and inducible NOS synthase (iNOS) were measured by RT-PCR, ELISA, Western blot and the nitrate reductase method. RESULTS: Activated microglia significantly upregulated TNF-alpha and iNOS mRNA expression and protein production in vitro. An optic nerve crush also increased expression of retinal iNOS and TNF-alpha protein. Treatment with alpha-crystallin in vitro and in vivo downregulated their expression. CONCLUSION: The protective effect of alpha-crystallin may be due to its effect on microglia via a downregulation in the expression and release of 2 key immune regulatory and inflammatory molecules: TNF-alpha and iNOS.

Study of kinetics of thermal aggregation of mitochondrial aspartate aminotransferase by dynamic light scattering: protective effect of alpha-crystallin.

Thermal aggregation of aspartate aminotransferase from pig heart mitochondria (mAAT) has been studied at various temperatures and various protein concentrations by dynamic light scattering. The character of the dependence of protein aggregate size on time indicates that aggregation of mAAT proceeds in the regime of diffusion-limited cluster-cluster aggregation. Suppression of mAAT aggregation by alpha-crystallin is due to transition of the aggregation process into the regime of reaction-limited cluster-cluster aggregation. Realization of this regime of aggregation means that the sticking probability for the colliding particles is less than unity.

Alpha-crystallin protected axons from optic nerve degeneration after crushing in rats.

In mature mammals, optic nerve injury results in apoptosis of retinal ganglion cells. The literature confirms that lens injury enhances retinal ganglion cells survival, but the mechanism is not very clear. Using silver staining method and computer image analysis techniques, the effect of alpha-crystallin, a major component of the lens in the survival of retinal ganglion cell axons, was investigated in vivo after intravitreal injections. The results showed that enhanced survival of axotomized axons was observed beyond the crush site after a single intravitreal administration of alpha-crystallin at the time of axotomy. Axonal density of the retinal ganglion cell was significantly greater than in the untreated controls until 2 weeks after injection. This effect declined by 4 weeks after injection but survival of axons remained greater than controls. These findings indicate that alpha-crystallin plays a key role in protecting axons after optic nerve injury.

Effect of alpha-crystallin on thermal aggregation of glycogen phosphorylase b from rabbit skeletal muscle.

Thermal aggregation of rabbit skeletal muscle glycogen phosphorylase b (Phb) has been investigated using dynamic light scattering under conditions of a constant rate of temperature increase (1 K/min). The linear behavior of the dependence of the hydrodynamic radius on temperature for Phb aggregation is consistent with the idea that thermal aggregation of proteins proceeds in the kinetic regime wherein the rate of aggregation is limited by diffusion of the interacting particles (the regime of "diffusion-limited cluster-cluster aggregation"). In the presence of alpha-crystallin, a protein exhibiting chaperone-like activity, the dependence of the hydrodynamic radius on temperature follows the exponential law; this suggests that the aggregation process proceeds in the kinetic regime where the sticking probability for colliding particles becomes lower than unity (the regime of "reaction-limited cluster-cluster aggregation"). Based on analysis of the ratio between the light scattering intensity and the hydrodynamic radius of Phb aggregates, it has been concluded that the addition of alpha-crystallin results in formation of smaller size starting aggregates. The data on differential scanning calorimetry indicate that alpha-crystallin interacts with the intermediates of the unfolding process of the Phb molecule. The proposed scheme of thermal denaturation and aggregation of Phb includes the stage of reversible dissociation of dimers of Phb into monomers, the stage of the formation of the starting aggregates from the denatured monomers of Phb, and the stage of the sticking of the starting aggregates and higher order aggregates. Dissociation of Phb dimer into monomers at elevated temperatures has been confirmed by analytical ultracentrifugation.

Revival of glutathione reductase in human cataractous and clear lens extracts by thioredoxin and thioredoxin reductase, in conjunction with alpha-crystallin or thioltransferase.

Glutathione reductase (GR) plays a key role in maintaining thiol groups in the lens, and its activity decreases with aging and cataract formation. Mammalian thioredoxin (Trx) and thioredoxin reductase (TrxR), or the Trx/TrxR system, participates in the repair of oxidatively damaged lens proteins and enzymes. Alpha-crystallin, a molecular chaperone, prevents the aggregation of partially denatured proteins under various stress conditions. Thioltransferase (TTase, or glutaredoxin) can maintain the homeostasis of lens protein thiols thus protecting against oxidative stress. We investigated whether the Trx/TrxR system can revive GR activity in both the cortex and nucleus of human cataract and clear aged lenses and whether alpha-crystallin and TTase can help this effect. The GR activity in the cortex and nucleus of the cataractous lenses was significantly lower than that of the aged clear lenses. The highest activity in the cortex was observed in the clear aged lenses. The combination of Trx and TrxR revived the activity of GR from both the cortex and nucleus of aged clear lenses. However, in cataract lenses (grade II and grade IV), there was a statistically significant recovery of GR activity in the cortex, but not in the nucleus. No recovery was observed when Trx or TrxR were used separately. Alpha-crystallin successfully revived GR activity in the cortex of cataract grade II lenses, but not in the nucleus. The combination of alpha-crystallin and Trx/TrxR gave a further increase of activity. TTase alone revived some of the GR activity but together with the Trx/TrxR system gave no statistically significant enhancement of GR activity. These results indicate that both disulfide bond formation and protein unfolding are responsible for GR inactivation.

Chaperone-mediated inhibition of tubulin self-assembly.

Molecular chaperones are known to play an important role in facilitating the proper folding of many newly synthesized proteins. Here, we have shown that chaperone proteins exhibit another unique property to inhibit tubulin self-assembly efficiently. Chaperones tested include alpha-crystallin from bovine eye lenses, HSP16.3, HSP70 from Mycobacterium tuberculosis and alpha (s)-casein from milk. All of them inhibit polymerization in a dose-dependent manner independent of assembly inducers used. The critical concentration of MTP polymerization increases with increasing concentration of HSP16.3. Increase in chaperone concentration lowers the extent of polymerization and increases the lag time of self-assembly reaction. Although the addition of a chaperone at the early stage of elongation phase shows no effect on polymerization, the same concentration of chaperone inhibits polymerization completely when added before the initiation of polymerization. Bindings of HSP16.3 and alpha (s)-casein to tubulin have been confirmed using isothermal titration calorimetry. Affinity constants of tubulin are 5.3 xx 10(4) and 9.8 xx 10(5) M(-1) for HSP16.3 and alpha (s)-casein, respectively. Thermodynamic parameters indicate favourable entropy and enthalpy changes for both chaperones-tubulin interactions. Positive entropy change suggests that the interaction is hydrophobic in nature and desolvation occurring during formation of tubulin-chaperone complex. On the basis of thermodynamic data and observations made upon addition of chaperone at early elongation phase or before the initiation of polymerization, we hypothesize that chaperones bind tubulin at the protein-protein interaction site involved in the nucleation phase of self-assembly.

Thioredoxin, thioredoxin reductase, and alpha-crystallin revive inactivated glyceraldehyde 3-phosphate dehydrogenase in human aged and cataract lens extracts.

PURPOSE: To investigate whether mammalian thioredoxin (Trx) and thioredoxin reductase (TrxR), with or without alpha-crystallin can revive inactivated glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in both the cortex and nucleus of human aged clear and cataract lenses. METHODS: The lens cortex (including capsule-epithelium) and the nucleus were separated from human aged clear and cataract lenses (grade II and grade IV) with similar average age. The activity of GAPDH in the water-soluble fraction after incubation with or without Trx or/and TrxR for 60 min at 30 degrees C was measured spectrophotometrically. In addition, the effect of a combination of Trx/TrxR and bovine lens alpha-crystallin was investigated. RESULTS: GAPDH activity was lower in the nucleus of clear lenses than in the cortex, and considerably diminished in the cataractous lenses, particularly in the nucleus of cataract lenses grade IV. Trx and TrxR were able to revive the activity of GAPDH markedly in both the cortex and nucleus of the clear and cataract lenses. The percentage increase of activity in the cortex of the clear lenses was less than that of the nucleus in the presence of Trx and TrxR, whereas it was opposite in the cataract lenses. The revival of activity in both the cortex and nucleus from the cataract lenses grade II was higher than that of the grade IV. Moreover, Trx alone, but not TrxR, efficiently enhanced GAPDH activity. The combination of Trx and TrxR had greater effect than that of either alone. In addition, alpha(L)-crystallin enhanced the activity in the cortex of cataract grade II with Trx and TrxR present. However, it failed to provide a statistically significant increase of activity in the nucleus. CONCLUSIONS: This is the first evidence to show that mammalian Trx and TrxR are able to revive inactivated GAPDH in human aged clear and cataract lenses, and alpha-crystallin helped this effect. The inactivation of GAPDH during aging and cataract development must be caused in part by disulphide formation and in part by unfolding, and can be recovered by reducing agents and a molecular chaperone.

Small heat shock proteins inhibit amyloid-beta protein aggregation and cerebrovascular amyloid-beta protein toxicity.

Small heat shock proteins Hsp20 and HspB2/B3 co-localize with Abeta deposition in senile plaques and cerebral amyloid angiopathy in Alzheimer's disease brains, respectively. It was the aim of our study to investigate if these and other sHsps bind to wild-type Abeta1-42 or the more toxic Abeta1-40 carrying the 'Dutch' mutation (22Glu-->Gln) (D-Abeta1-40), affect Abeta aggregation and thereby influence Abeta cytotoxicity. Binding affinity between sHsps and Abeta was investigated by surface plasmon resonance. Abeta aggregation was studied by using circular dichroism spectroscopy and electron microscopy. Furthermore, we used cultured cerebrovascular cells to investigate the effects of sHsps on Abeta-mediated cytotoxicity. Hsp20, Hsp27 and alphaB-crystallin, but not HspB2/B3, bound to Abeta (both D-Abeta1-40 and Abeta1-42) and reduced or completely inhibited aggregation of D-Abeta1-40 into mature fibrils but did not affect Abeta1-42 aggregation. Furthermore, these sHsps were effective inhibitors of the cerebrovascular toxicity of Abeta (both D-Abeta1-40 and Abeta1-42) in vitro. Binding affinity of the sHsps to D-Abeta1-40 correlated to the degree of inhibition of Abeta-mediated cytotoxicity and the potential to reduce Abeta beta-sheet and fibril formation. With Abeta1-42, a similar correlation between binding affinity and cytotoxicity was observed, but not with its aggregation state. In conclusion, sHsps may regulate Abeta aggregation and serve as antagonists of the biological action of Abeta, but the extent of their interaction depends on the type of sHsp and Abeta peptide.