Puttur Santhoshkumar

Inhibition of amyloid fibrillogenesis and toxicity by a peptide chaperone

Aggregation of proteins in tissues is associated with several diseases, including Alzheimers disease. It is characterized by the accumulation of amyloid β peptide (Aβ) in the extracellular spaces of the brain cells, resulting in neuronal death and other pathological changes. α-Crystallin, a small heat-shock protein in lens, and a peptide chaperone having the functional site sequence DFVIFLDVKHFSPEDLTVK of αA-crystallin may inhibit Aβ fibrillogenesis and toxicity. The peptide chaperone (mini-αA-crystallin), having an Aβ interacting domain and a complex solubilizing domain, was shown in previous studies to prevent aggregation of several proteins under denaturing conditions. In this in vitro study, using transmission electron microscopy and thioflavin T binding assay, we show that mini-αA-crystallin arrests the fibril formation of Aβ peptides. Mini-αA-crystallin also suppresses the toxic action of Aβ on rat pheochromocytoma (PC12) cells. The wide chaperoning capability of the peptide and its ability to inhibit amyloid fibril formation and suppress toxicity suggest that mini-αA-crystallin may serve as a universal chaperone in controlling diseases of protein aggregation, including Alzheimers disease. (Mol Cell Biochem 267: 147–155, 2004)

Significance of Interactions of Low Molecular Weight Crystallin Fragments in Lens Aging and Cataract Formation

Analysis of aged and cataract lenses shows the presence of increased amounts of crystallin fragments in the high molecular weight aggregates of water-soluble and water-insoluble fractions. However, the significance of accumulation and interaction of low molecular weight crystallin fragments in aging and cataract development is not clearly understood. In this study, 23 low molecular mass (<3.5-kDa) peptides in the urea-soluble fractions of young, aged, and aged cataract human lenses were identified by mass spectroscopy. Two peptides, αB-(1–18) (MDIAIHHPWIRRPFFPFH) and βA3/A1-(59–74) (SD(N)AYHIERLMSFRPIC), present in aged and cataract lens but not young lens, and a third peptide, γS-(167–178) (SPAVQSFRRIVE) present in all three lens groups were synthesized to study the effects of interaction of these peptides with intact α-, β-, and γ-crystallins and alcohol dehydrogenase, a protein used in aggregation studies. Interaction of αB-(1–18) and βA3/A1-(59–74) peptides increased the scattering of light by β- and γ-crystallin and alcohol dehydrogenase. The ability of α-crystallin subunits to function as molecular chaperones was significantly reduced by interaction with αB-(1–18) and βA3/A1-(59–74) peptides, whereas γS peptide had no effect on chaperone-like activity of α-crystallin. The βA3/A1-(59–74 peptide caused a 5.64-fold increase in αB-crystallin oligomeric mass and partial precipitation. Replacing hydrophobic residues in αB-(1–18) and βA3/A1-(59–74) peptides abolished their ability to induce crystallin aggregation and light scattering. Our study suggests that interaction of crystallin-derived peptides with intact crystallins could be a key event in age-related protein aggregation in lens and cataractogenesis.

Chaperone Peptides of α-Crystallin Inhibit Epithelial Cell Apoptosis, Protein Insolubilization, and Opacification in Experimental Cataracts

Background: Peptides derived from the core domain of human α-crystallin act as molecular chaperones. Results: Chaperone peptides of α-crystallin inhibit stress-induced apoptosis in cultured cells and prevent experimental cataracts in rats. Conclusion: Chaperone peptides of α-crystallin are anti-apoptotic and retain biological activity when injected into animals. Significance: α-Crystallin peptides could be used as therapeutic agents to inhibit protein aggregation and apoptosis in diseases. α-Crystallin is a member of the small heat-shock protein (sHSP) family and consists of two subunits, αA and αB. Both αA- and αB-crystallin act as chaperones and anti-apoptotic proteins. Previous studies have identified the peptide 70KFVIFLDVKHFSPEDLTVK88 in αA-crystallin and the peptide 73DRFSVNLDVKHFSPEELKVK92 in αB-crystallin as mini-chaperones. In the human lens, lysine 70 (Lys70) of αA and Lys92 of αB (in the mini-chaperone sequences) are acetylated. In this study, we investigated the cellular effects of the unmodified and acetyl mini-chaperones. The αA- and αB-crystallin peptides inhibited stress-induced aggregation of four client proteins, and the αA-acetyl peptide was more effective than the native peptide against three of the client proteins. Both the acetyl and native crystallin peptides inhibited stress-induced apoptosis in two mammalian cell types, and this property was directly related to the inhibition of cytochrome c release from mitochondria and the activity of caspase-3 and -9. In organ-cultured rat lenses, the peptides inhibited calcimycin-induced epithelial cell apoptosis. Intraperitoneal injection of the peptides inhibited cataract development in selenite-treated rats, which was accompanied by inhibition of oxidative stress, protein insolubilization, and caspase activity in the lens. These inhibitory effects were more pronounced for acetyl peptides than native peptides. A scrambled αA-crystallin peptide produced no such effects. The results suggest that the α-crystallin chaperone peptides could be used as therapeutic agents to treat cataracts and diseases in which protein aggregation and apoptosis are contributing factors.

Acetylation of αA-crystallin in the human lens: effects on structure and chaperone function.

α-Crystallin is a major protein in the human lens that is perceived to help to maintain the transparency of the lens through its chaperone function. In this study, we demonstrate that many lens proteins including αA-crystallin are acetylated in vivo. We found that K70 and K99 in αA-crystallin and, K92 and K166 in αB-crystallin are acetylated in the human lens. To determine the effect of acetylation on the chaperone function and structural changes, αA-crystallin was acetylated using acetic anhydride. The resulting protein showed strong immunoreactivity against a N(ε)-acetyllysine antibody, which was directly related to the degree of acetylation. When compared to the unmodified protein, the chaperone function of the in vitro acetylated αA-crystallin was higher against three of the four different client proteins tested. Because a lysine (residue 70; K70) in αA-crystallin is acetylated in vivo, we generated a protein with an acetylation mimic, replacing Lys70 with glutamine (K70Q). The K70Q mutant protein showed increased chaperone function against three client proteins compared to the Wt protein but decreased chaperone function against γ-crystallin. The acetylated protein displayed higher surface hydrophobicity and tryptophan fluorescence, had altered secondary and tertiary structures and displayed decreased thermodynamic stability. Together, our data suggest that acetylation of αA-crystallin occurs in the human lens and that it affects the chaperone function of the protein.

αA-Crystallin Peptide 66SDRDKFVIFLDVKHF80 Accumulating in Aging Lens Impairs the Function of α-Crystallin and Induces Lens Protein Aggregation

Background The eye lens is composed of fiber cells that are filled with α-, β- and γ-crystallins. The primary function of crystallins is to maintain the clarity of the lens through ordered interactions as well as through the chaperone-like function of α-crystallin. With aging, the chaperone function of α-crystallin decreases, with the concomitant accumulation of water-insoluble, light-scattering oligomers and crystallin-derived peptides. The role of crystallin-derived peptides in age-related lens protein aggregation and insolubilization is not understood. Methodology/Principal Findings We found that αA-crystallin-derived peptide, 66 SDRDKFVIFLDVKHF 80, which accumulates in the aging lens, can inhibit the chaperone activity of α-crystallin and cause aggregation and precipitation of lens crystallins. Age-related change in the concentration of αA-(66-80) peptide was estimated by mass spectrometry. The interaction of the peptide with native crystallin was studied by multi-angle light scattering and fluorescence methods. High molar ratios of peptide-to-crystallin were favourable for aggregation and precipitation. Time-lapse recordings showed that, in the presence of αA-(66-80) peptide, α-crystallin aggregates and functions as a nucleus for protein aggregation, attracting aggregation of additional α-, β- and γ-crystallins. Additionally, the αA-(66-80) peptide shares the principal properties of amyloid peptides, such as β-sheet structure and fibril formation. Conclusions/Significance These results suggest that crystallin-derived peptides such as αA-(66-80), generated in vivo, can induce age-related lens changes by disrupting the structure and organization of crystallins, leading to their insolubilization. The accumulation of such peptides in aging lenses may explain a novel mechanism for age-related crystallin aggregation and cataractogenesis.

Identification of a region in alcohol dehydrogenase that binds to α-crystallin during chaperone action

alpha-Crystallin, the major eye lens protein and a member of the small heat-shock protein family, has been shown to protect the aggregation of several proteins and enzymes under denaturing conditions. The region(s) in the denaturing proteins that interact with alpha-crystallin during chaperone action has not been identified. Determination of these sites would explain the wide chaperoning action (promiscuity) of alpha-crystallin. In the present study, using two different methods, we have identified a sequence in yeast alcohol dehydrogenase (ADH) that binds to alpha-crystallin during chaperone-like action. The first method involved the incubation of alpha-crystallin with ADH peptides at 48 degrees C for 1 h followed by separation and analysis of bound peptides. In the second method, alpha-crystallin was first derivatized with a photoactive trifunctional cross-linker, sulfosuccinimidyl-2[6-(biotinamido)-2-(p-azidobenzamido)-hexanoamido]ethyl-1,3di-thiopropionate (sulfo-SBED), and then complexed with ADH at 48 degrees C for 1 h in the dark. The complex was photolyzed and digested with protease, and the biotinylated peptide fragments were isolated using an avidin column and then analyzed. The amino acid sequencing and mass spectral analysis revealed the sequence YSGVCHTDLHAWHGDWPLPVK (yeast ADH(40-60)) as the alpha-crystallin binding site in ADH. The interaction was further confirmed by demonstrating complex formation between alpha-crystallin and a synthetic peptide representing the binding site of ADH.

Hydroimidazolone modification of the conserved Arg12 in small heat shock proteins: studies on the structure and chaperone function using mutant mimics.

Methylglyoxal (MGO) is an α-dicarbonyl compound present ubiquitously in the human body. MGO reacts with arginine residues in proteins and forms adducts such as hydroimidazolone and argpyrimidine in vivo. Previously, we showed that MGO-mediated modification of αA-crystallin increased its chaperone function. We identified MGO-modified arginine residues in αA-crystallin and found that replacing such arginine residues with alanine residues mimicked the effects of MGO on the chaperone function. Arginine 12 (R12) is a conserved amino acid residue in Hsp27 as well as αA- and αB-crystallin. When treated with MGO at or near physiological concentrations (2–10 µM), R12 was modified to hydroimidazolone in all three small heat shock proteins. In this study, we determined the effect of arginine substitution with alanine at position 12 (R12A to mimic MGO modification) on the structure and chaperone function of these proteins. Among the three proteins, the R12A mutation improved the chaperone function of only αA-crystallin. This enhancement in the chaperone function was accompanied by subtle changes in the tertiary structure, which increased the thermodynamic stability of αA-crystallin. This mutation induced the exposure of additional client protein binding sites on αA-crystallin. Altogether, our data suggest that MGO-modification of the conserved R12 in αA-crystallin to hydroimidazolone may play an important role in reducing protein aggregation in the lens during aging and cataract formation.

A peptide sequence-YSGVCHTDLHAWHGDWPLPVK [40-60]-in yeast alcohol dehydrogenase prevents the aggregation of denatured substrate proteins.

The structural and functional characteristics of a yeast alcohol dehydrogenase (ADH) peptide (YSGVCHTDLHAWHGDWPLPVK, residues 40-60) have been studied in detail. The peptide is hydrophobic in nature, binds the hydrophobic probe bis-ANS, and is mostly present in a random coil conformation. It shows chaperone-like activity by preventing dithiothreitol (DTT)-induced aggregation of insulin at 27 degrees C, oxidation-induced aggregation of gamma-crystallin at 37 degrees C, and aggregation of thermally denatured ADH and beta(L)-crystallins at 52 degrees C. However, the ADH peptide does not solubilize protein aggregates as do surfactants. Substitution of Pro for His in the ADH peptide leads to diminished anti-aggregation activity. Further, analysis of ADH incubated at 47 degrees C suggests that a significant portion of the enzyme remains as soluble inactive protein with negligible conformational change. Therefore, we propose that the residues 40-60 in native protein may be an intramolecular chaperone site of yeast ADH.

The Role of the Cysteine Residue in the Chaperone and Anti-Apoptotic Functions of Human Hsp27

The small heat shock protein Hsp27 is a molecular chaperone and an anti‐apoptotic protein. Human Hsp27 has one cysteine residue at position 137. We investigated the role of this cysteine residue in the chaperone and anti‐apoptotic functions of Hsp27 by mutating the cysteine residue to an alanine (Hsp27C137A) and comparing it to wild‐type protein (Hsp27WT). Both proteins were multi‐subunit oligomers, but subunits of Hsp27WT were disulfide‐linked unlike those of Hsp27C137A, which were monomeric. Hsp27C137A was indistinguishable from Hsp27WT with regard to its secondary structure, surface hydrophobicity, oligomeric size and chaperone function. S‐thiolation and reductive methylation of the cysteine residue had no apparent effect on the chaperone function of Hsp27WT. The anti‐apoptotic function of Hsp27C137A and Hsp27WT was studied by overexpressing them in CHO cells. No difference in the caspase‐3 or ‐9 activity was observed in staurosporine‐treated cells. The rate of apoptosis between Hsp27C137A and Hsp27WT overexpressing cells was similar whether the cells were treated with staurosporine or etoposide. However, the mutant protein was less protective relative to the wild‐type protein in preventing caspase‐3 and caspase‐9 activation and apoptosis induced by 1 mM H2O2 in CHO and HeLa cells. These data demonstrate that in human Hsp27, disulfide formation by the lone cysteine does not affect its chaperone function and anti‐apoptotic function against chemical toxicants. However, oxidation of the lone cysteine in Hsp27 might at least partially affect the anti‐apoptotic function against oxidative stress. J. Cell. Biochem. 110: 408–419, 2010.

Histone deacetylase inhibitors trichostatin A and vorinostat inhibit TGFβ2-induced lens epithelial-to-mesenchymal cell transition.

PURPOSE Posterior capsule opacification (PCO) after cataract surgery is due in part to proliferation of the adhering lens epithelial cells and transdifferentiation into mesenchymal cells. The histone deacetylase (HDAC) inhibitors, trichostatin A (TSA) and vorinostat (suberoylanilidehydroxamic acid [SAHA]) are known to modulate cell proliferation and epithelial-mesenchymal transition (EMT). Studies have shown that TGFβ2 can induce EMT similar to that seen during PCO. This study evaluated the effects of TSA and SAHA on TGFβ2-induced EMT in lens epithelial explants. METHODS Epithelial cells adherent to lens capsules were isolated from fresh pig lenses and human donor lenses and cultured for 12 hours. Explants were pretreated with TSA or SAHA for 1 hour and then treated with TGFβ2 for up to 3 days. Scratch wound healing assay was used to determine epithelial cell proliferation and migration in the samples. The effects of TSA and SAHA on histone acetylation and HDAC 1 to 6 levels were analyzed by Western blotting. RESULTS Western blotting and immunocytochemistry demonstrated high expression of α-SMA in lens epithelial cells treated with TGFβ2. The HDAC inhibitors exerted dose-dependent inhibition of α-SMA expression, with complete inhibition occurring with 0.5 μM of TSA and 2.5 μM of SAHA. Transforming growth factor β2-induced EMT was suppressed by TSA and SAHA. Histone deacetylase inhibition in pig lens epithelia led to increased acetylation of histone 3 and 4 at multiple sites. CONCLUSIONS Histone deacetylase inhibitors, TSA, and SAHA prevent EMT in lens epithelial explants. The results also suggest that the epigenetic modifiers are the potential targets to control PCO after cataract surgery.