Bakthisaran Raman

TEMPERATURE DEPENDENT CHAPERONE-LIKE ACTIVITY OF ALPHA-CRYSTALLIN

Alpha‐crystallin, a multimeric protein present in the eye lens, is known to have chaperone‐like activity in preventing the aggregation of enzymes and other crystallins. We have studied the chaperone‐like activity of this protein towards the aggregation of insulin B chain, induced by reducing the interchain disulphide bond with dithiothreitol. At room temperature, there is no detectable protection (at a 1:1 (w/w) ratio of insulin: α‐crystallin) against the aggregation of insulin B chain by α‐crystallin, whereas it completely prevents this aggregation at 40°C. We have monitored the temperature dependence of the protection of aggregation by α‐crystallin; the protection increases sharply above 30°C and reaches almost 100% by 41°C. Probing the hydrophobic surfaces of α‐crystallin with the hydrophobic fluorphore 8‐anilino‐1 naphthalene sulfonate suggests that the hydrophobic surfaces of α‐crystallin are exposed to a greater extent above 30°C. A complete prevention of the aggregation is achieved at 27.6°C by increasing the concentration of α‐crystallin by more than 8 fold. Similar temperature dependent chaperone‐like activity of α‐crystallin is observed towards the aggregation of zetacrystallin, an enzyme crystallin from guinea pig. We have earlier shown that α‐crystallin exposes hydrophobic surface(s) at temperatures above 30°C. These results support our earlier hypothesis [Raman, B. and Rao, Ch.M. (1994) J. Biol. Chem. 269, 27264–27268] that the chaperone‐like activity of α‐crystallin is more pronounced in its structurally perturbed state.

Metal Ion-dependent Effects of Clioquinol on the Fibril Growth of an Amyloid β Peptide

Although metal ions such as Cu2+, Zn2+, and Fe3+ are implicated to play a key role in Alzheimer disease, their role is rather complex, and comprehensive understanding is not yet obtained. We show that Cu2+ and Zn2+ but not Fe3+ renders the amyloid β peptide, Aβ1–40, nonfibrillogenic in nature. However, preformed fibrils of Aβ1–40 were stable when treated with these metal ions. Consequently, fibril growth of Aβ1–40 could be switched on/off by switching the molecule between its apo- and holo-forms. Clioquinol, a potential drug for Alzheimer disease, induced resumption of the Cu2+-suppressed but not the Zn2+-suppressed fibril growth of Aβ1–40. The observed synergistic effect of clioquinol and Zn2+ suggests that Zn2+-clioquinol complex effectively retards fibril growth. Thus, clioquinol has dual effects; although it disaggregates the metal ion-induced aggregates of Aβ1–40 through metal chelation, it further retards the fibril growth along with Zn2+. These results indicate the mechanism of metal ions in suppressing Aβ amyloid formation, as well as providing information toward the use of metal ion chelators, particularly clioquinol, as potential drugs for Alzheimer disease.

αB-crystallin, a small heat-shock protein, prevents the amyloid fibril growth of an amyloid β-peptide and β2-microglobulin

AlphaB-crystallin, a small heat-shock protein, exhibits molecular chaperone activity. We have studied the effect of alphaB-crystallin on the fibril growth of the Abeta (amyloid beta)-peptides Abeta-(1-40) and Abeta-(1-42). alphaB-crystallin, but not BSA or hen egg-white lysozyme, prevented the fibril growth of Abeta-(1-40), as revealed by thioflavin T binding, total internal reflection fluorescence microscopy and CD spectroscopy. Comparison of the activity of some mutants and chimaeric alpha-crystallins in preventing Abeta-(1-40) fibril growth with their previously reported chaperone ability in preventing dithiothreitol-induced aggregation of insulin suggests that there might be both common and distinct sites of interaction on alpha-crystallin involved in the prevention of amorphous aggregation of insulin and fibril growth of Abeta-(1-40). alphaB-crystallin also prevents the spontaneous fibril formation (without externally added seeds) of Abeta-(1-42), as well as the fibril growth of Abeta-(1-40) when seeded with the Abeta-(1-42) fibril seed. Sedimentation velocity measurements show that alphaB-crystallin does not form a stable complex with Abeta-(1-40). The mechanism by which it prevents the fibril growth differs from the known mechanism by which it prevents the amorphous aggregation of proteins. alphaB-crystallin binds to the amyloid fibrils of Abeta-(1-40), indicating that the preferential interaction of the chaperone with the fibril nucleus, which inhibits nucleation-dependent polymerization of amyloid fibrils, is the mechanism that is predominantly involved. We found that alphaB-crystallin prevents the fibril growth of beta2-microglobulin under acidic conditions. It also retards the depolymerization of beta2-microglobulin fibrils, indicating that it can interact with the fibrils. Our study sheds light on the role of small heat-shock proteins in protein conformational diseases, particularly in Alzheimers disease.

Molten-Globule State of Carbonic Anhydrase Binds to the Chaperone-like α-Crystallin

α-Crystallin, a multimeric protein, exhibits chaperone-like activity in preventing aggregation of several proteins. We have studied the chaperone-like activity of α-crystallin toward heat-induced aggregation of bovine and human carbonic anhydrase. Human carbonic anhydrase aggregates at 60°C, while bovine carbonic anhydrase does not aggregate significantly at this temperature. Removal of the enzyme-bound metal ion, Zn2+, by EDTA modulates the aggregation behavior of bovine carbonic anhydrase. Fluorescence and circular dichroism studies show that removal of the metal ion from the bovine carbonic anhydrase by a chelator such as EDTA enhances the propensity of the enzyme to adopt the molten-globule state. α-Crystallin binds to this state of the enzyme and prevents aggregation. Fluorescence and circular dichroism studies on the α-crystallin-enzyme complexes show that the enzymes in the complex are in the molten-globule state. These results are of relevance to the interaction of chaperones with the partially unfolded states of target proteins.

Mammalian Hsp22 is a heat-inducible small heat-shock protein with chaperone-like activity

A newly identified 22 kDa protein that interacts with Hsp27 (heat-shock protein 27) was shown to possess the characteristic alpha-crystallin domain, hence named Hsp22, and categorized as a member of the sHsp (small Hsp) family. Independent studies from different laboratories reported the protein with different names such as Hsp22, H11 kinase, E2IG1 and HspB8. We have identified, on the basis of the nucleotide sequence analysis, putative heat-shock factor 1 binding sites upstream of the Hsp22 translation start site. We demonstrate that indeed Hsp22 is heat-inducible. We show, in vitro, chaperone-like activity of Hsp22 in preventing dithiothreitol-induced aggregation of insulin and thermal aggregation of citrate synthase. We have cloned rat Hsp22, overexpressed and purified the protein to homogeneity and studied its structural and functional aspects. We find that Hsp22 fragments on storage. MS analysis of fragments suggests that the fragmentation might be due to the presence of labile peptide bonds. We have established conditions to improve its stability. Far-UV CD indicates a randomly coiled structure for Hsp22. Quaternary structure analyses by glycerol density-gradient centrifugation and gel filtration chromatography show that Hsp22 exists as a monomer in vitro, unlike other members of the sHsp family. Hsp22 exhibits significantly exposed hydrophobic surfaces as reported by bis-8-anilinonaphthalene-l-sulphonic acid fluorescence. We find that the chaperone-like activity is temperature dependent. Thus Hsp22 appears to be a true member of the sHsp family, which exists as a monomer in vitro and exhibits chaperone-like activity.

Refolding of Denatured and Denatured/Reduced Lysozyme at High Concentrations

Refolding of proteins at high concentrations often results in aggregation. To gain insight into the molecular aspects of refolding and to improve the yield of active protein, we have studied the refolding of lysozyme either from its denatured state or from its denatured/reduced state. Refolding of denatured lysozyme, even at 1 mg/ml, yields fully active enzyme without aggregation. However, refolding of denatured/reduced lysozyme into buffer that lacks thiol/disulfide reagents leads to aggregation. Thiol/disulfide redox reagents such as cysteine/cystine and reduced/oxidized glutathione facilitate the renaturation, with the yield depending on their absolute concentrations. We have obtained an ∼70% renaturation yield upon refolding of lysozyme at 150 μg/ml. The cysteine/cystine redox system is more efficient compared with the glutathione redox system. When lysozyme is refolded in the absence of redox reagents, a transient intermediate that has regained a significant amount of secondary structure is formed. The tryptophans in this intermediate are as exposed to water as in the fully unfolded protein. It shows increased exposure of hydrophobic surfaces compared with the native or completely unfolded enzyme. This aggregation-prone intermediate folds to active enzyme upon addition of oxidized glutathione before the aggregation process starts. These properties of the intermediate in the refolding pathway of lysozyme are similar to those proposed for the molten globule.

Interaction of human recombinant αA- and αB-crystallins with early and late unfolding intermediates of citrate synthase on its thermal denaturation

We have investigated the role of recombinant human αA‐ and αB‐crystallins in the heat‐induced inactivation and aggregation of citrate synthase. Homo‐multimers of both αA‐ and αB‐crystallins confer protection against heat‐induced inactivation in a concentration‐dependent manner and also prevent aggregation. Interaction of crystallins with early unfolding intermediates of citrate synthase reduces their partitioning into aggregation‐prone intermediates. This appears to result in enhanced population of early unfolding intermediates that can be reactivated by its substrate, oxaloacetate. Both these homo‐multimers do not form a stable complex with the early unfolding intermediates. However, they can form a soluble, stable complex with aggregation‐prone late unfolding intermediates. This soluble complex formation prevents aggregation. Thus, it appears that the chaperone activity of α‐crystallin involves both transient and stable interactions depending on the nature of intermediates on the unfolding pathway; one leads to reactivation of the enzyme activity while the other prevents aggregation.

Role of the conserved SRLFDQFFG region of alpha crystallin, a small heat shock protein: Effect on oligomeric size, subunit exchange and chaperone-like activity

Small heat shock proteins (sHsps) are necessary for several cellular functions and in stress tolerance. Most sHsps are oligomers; intersubunit interactions leading to changes in oligomeric structure and exposure of specific regions may modulate their functioning. Many sHsps, including αA- and αB-crystallin, contain a well conserved SRLFDQFFG sequence motif in the N-terminal region. Sequence-based prediction shows that it exhibits helical propensity with amphipathic character, suggesting that it plays a critical role in the structure and function of α-crystallins. In order to investigate the role of this motif in the structure and function of sHsps, we have made constructs deleting this sequence from αA- and αB-crystallin, overexpressed, purified, and studied these engineered proteins. Circular dichroism spectroscopic studies show changes in tertiary and secondary structure on deletion of the sequence. Glycerol density gradient centrifugation and dynamic light scattering studies show that the multimeric size of the mutant proteins is significantly reduced, indicating a role for this motif in higher order organization of the subunits. Both deletion mutants exhibit similar oligomeric size and increased chaperone-like activity. Urea-induced denaturation study shows that the SRLFDQFFG sequence contributes significantly to the structural stability. Fluorescence resonance energy transfer studies show that the rate of exchange of the subunits in the αAdel-crystallin oligomer is higher compared with that in the αA-crystallin oligomer, suggesting that this region contributes to the oligomer dynamics in addition to the higher order assembly and structural stability. Thus, our study shows that the SRLFDQFFG sequence is one of the critical motifs in structure-function regulation of αA- and αB-crystallin.

Structural perturbation of α-crystallin and its chaperone-like activity

Abstract α -Crystallin is a multimeric lenticular protein that has recently been shown to be expressed in several non-lenticular tissues as well. It is shown to prevent aggregation of non-native proteins as a molecular chaperone. By using a non-thermal aggregation model, we could show that this process is temperature-dependent. We investigated the chaperone-like activity of α -crystallin towards photo-induced aggregation of γ -crystallin, aggregation of insulin and on the refolding induced aggregation of β - and γ -crystallins. We observed that α -crystallin could prevent photo-aggregation of γ -crystallin and this chaperone-like activity of α -crystallin is enhanced several fold at temperatures above 30°C. This enhancement parallels the exposure of its hydrophobic surfaces as a function of temperature, probed using hydrophobic fluorescent probes such as pyrene and 8-anilinonaphthalene-1-sulfonate. We, therefore, concluded that α -crystallin prevents the aggregation of other proteins by providing appropriately placed hydrophobic surfaces; a structural transition above 30°C involving enhanced or re-organized hydrophobic surfaces of α -crystallin is important for its chaperone-like activity. We also addressed the issue of conformational aspects of target proteins and found that their aggregation prone molten globule states bind to α -crystallin. We trace these developments and discuss some new lines that suggest the role of tertiary structural aspects in the chaperone process.

Selective Cu2+ Binding, Redox Silencing, and Cytoprotective Effects of the Small Heat Shock Proteins αA- and αB-Crystallin

Oxidative stress and Cu(2+) have been implicated in several neurodegenerative diseases and in cataract. Oxidative stress, as well as Cu(2+), is also known to induce the expression of the small heat shock proteins alpha-crystallins. However, the role of alpha-crystallins in oxidative stress and in Cu(2+)-mediated processes is not clearly understood. We demonstrate using fluorescence and isothermal titration calorimetry that alpha-crystallins (alphaA- and alphaB-crystallin and its phosphorylation mimic, 3DalphaB-crystallin) bind Cu(2+) with close to picomolar range affinity. The presence of other tested divalent cations such as Zn(2+), Mg(2+), and Ca(2+) does not affect Cu(2+) binding, indicating selectivity of the Cu(2+)-binding site(s) in alpha-crystallins. Cu(2+) binding induces structural changes and increase in the hydrodynamic radii of alpha-crystallins. Cu(2+) binding increases the stability of alpha-crystallins towards guanidinium chloride-induced unfolding. Chaperone activity of alphaA-crystallin increases significantly upon Cu(2+) binding. Alpha-crystallins rescue amyloid beta peptide, Abeta(1-40), from Cu(2+)-induced aggregation in vitro. Alpha-crystallins inhibit Cu(2+)-induced oxidation of ascorbate and, hence, prevent the generation of reactive oxygen species. Interestingly, alpha-synuclein, a Cu(2+)-binding protein, does not inhibit this oxidation process significantly. We find that the Cu(2+)-sequestering (or redox-silencing) property of alpha-crystallins confers cytoprotection. To the best of our knowledge, this is the first study to reveal high affinity (close to picomolar) for Cu(2+) binding and redox silencing of Cu(2+) by any heat shock protein. Thus, our study ascribes a novel functional role to alpha-crystallins in Cu(2+) homeostasis and helps in understanding their protective role in neurodegenerative diseases and cataract.