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.     

Bax translocation mediated mitochondrial apoptosis and caspase dependent photosensitizing effect of Ficus religiosa on cancer cells

The main aim of the present work was to investigate the potential effect of acetone extract of Ficus religosa leaf (FAE) in multiple apoptosis signalling in human breast cancer cells. FAE treatment significantly induced dose and time dependent, irreversible inhibition of breast cancer cell growth with moderate toxicity to normal breast epithelial cells. This observation was validated using Sulforhodamine B assay. Cell cycle analysis by Flow cytometry showed cell cycle arrest in G1 phase and induction of sub-G0 peak. FAE induced chromatin condensation and displayed an increase in apoptotic population in Annexin V-FITC/PI (Fluorescein isothiocyanate/Propidium iodide) double staining. FAE stimulated the loss of mitochondrial membrane potential in multiple breast cancer cell lines when compared to normal diploid cells. To understand the role of Bax in FAE induced apoptosis, we employed a sensitive cell based platform of MCF-7 cells expressing Bax-EGFP. Bax translocation to mitochondria was accompanied by the disruption of mitochondrial membrane potential and marked elevation in LEHDase activity (Caspase 9). Consistent with this data, FAE induced Caspase activation as evidenced by ratio change in FRET Caspase sensor expressing MCF-7 cell line and cleavage of prominent Caspases and PARP. Interestingly, FAE accelerated cell death in a mitochondrial dependent manner in continuous live cell imaging mode indicating its possible photosensitizing effect. Intracellular generation of reactive oxygen species (ROS) by FAE played a critical role in mediating apoptotic cell death and photosensitizing activity. FAE induced dose and time dependent inhibition of cancer cell growth which was associated with Bax translocation and mitochondria mediated apoptosis with the activation of Caspase 9 dependent Caspase cascade. FAE also possessed strong photosensitizing effect on cancer cell line that was mediated through rapid mitochondrial transmembrane potential loss and partial Caspase activation involving generation of intracellular ROS.     

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.     

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 (Hsp27_C137A) and comparing it to wild‐type protein (Hsp27_WT). Both proteins were multi‐subunit oligomers, but subunits of Hsp27_WT were disulfide‐linked unlike those of Hsp27_C137A, which were monomeric. Hsp27_C137A was indistinguishable from Hsp27_WT 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 Hsp27_WT. The anti‐apoptotic function of Hsp27_C137A and Hsp27_WT 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 Hsp27_C137A and Hsp27_WT 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. © 2010 Wiley‐Liss, Inc.     

Endoplasmic reticulum targeted Bcl2 confers long term cell survival through phosphorylation of heat shock protein 27

Overexpression of anti-apoptotic Bcl2 family proteins is often seen in cancers rendering them insensitive to apoptosis inducing anticancer strategies. Anti-apoptotic Bcl2 family proteins are associated with different organelles like mitochondria and endoplasmic reticulum (ER) and exert their anti-apoptotic activity by inhibiting the release of Cyt.C from mitochondria irrespective of its localization. Here, we have identified a long term survival function for Bcl2 targeted at ER in mammalian system compared to wild type Bcl2 that is mediated by enhanced phosphorylation of heat shock protein 27 at ser 15, 78 and 82 sites with inhibition of caspase9 activity. Phosphorylation of hsp27 was prevented and the survival of ER-Bcl2 cells was reversed by inhibiting p38 and MEK suggesting that these kinases can act as the upstream targets for hsp27 phosphorylation. The results suggest that Bcl2 possess additional survival function in the regulation of apoptosis which is primarily regulated by its association with the ER in an hsp27 dependent manner. The interplay of both hsp27 and ER-Bcl2 in providing long term survival to cancer cells is interesting since both of these proteins are overexpressed in tumors with aggressive phenotype. The results suggest that spatial localization of Bcl2 family proteins also play a key role in long term survival of cancers indicating another level of functional regulation of Bcl2 in cancer cell survival.     

Evaluation of stem aqueous extract and synthesized silver nanoparticles using Cissus quadrangularis against Hippobosca maculata and Rhipicephalus (Boophilus) microplus

The present study was to determine the efficacies of anti-parasitic activities of synthesized silver nanoparticles (Ag NPs) using stem aqueous extract of Cissus quadrangularis against the adult of hematophagous fly, Hippobosca maculata (Diptera: Hippoboscidae), and the larvae of cattle tick, Rhipicephalus (Boophilus) microplus (Acari: Ixodidae). Contact toxicity method was followed to determine the potential of parasitic activity. Twelve milliliters of stem aqueous extract of C. quadrangularis was treated with 88ml of 1mM silver nitrate (AgNO(3)) solution at room temperature for 30min and the resulting solution was yellow-brown color indicating the formation extracellular synthesis of Ag NPs. The synthesized Ag NPs were characterized with UV-visible spectroscopy, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), Field emission scanning electron microscope (FESEM) and energy dispersive X-ray (EDX) spectroscopy. The synthesized Ag NPs were recorded by UV-visible spectrum at 420nm and XRD patterns showed the nanoparticles crystalline in nature. FTIR analysis confirmed that the bioreduction of Ag((+)) ions to Ag NPs were due to the reduction by capping material of plant extract. FESEM image of Ag NPs showed spherical and oval in shape. By using the Bragg's Law and Scherrer's constant, the average mean size of synthesized Ag NPs was 42.46nm. The spot EDX analysis showed the complete chemical composition of the synthesized Ag NPs. The mortality obtained by the synthesized Ag NPs from the C. quadrangularis was more effective than the aqueous extract of C. quadrangularis and AgNO(3) solution (1mM). The adulticidal activity was observed in the aqueous extract, AgNO(3) solution and synthesized Ag NPs against the adult of H. maculata with LC(50) values of 37.08, 40.35 and 6.30mg/L; LC(90) values of 175.46, 192.17 and 18.14mg/L and r(2) values of 0.970, 0.992 and 0.969, respectively. The maximum efficacy showed in the aqueous extract, AgNO(3) solution and synthesized Ag NPs against the larvae of R. (B.) microplus with LC(50) values of 50.00, 21.72 and 7.61mg/L; LC(90) values of 205.12, 82.99 and 22.68mg/L and r(2) values of 0.968, 0.945and 0.994, respectively. The present study is the first report on antiparasitic activity of the experimental plant extract and synthesized Ag NPs. This is an ideal eco-friendly and inexpensive approach for the control of H. maculata and R. (B.) microplus.     

Effect of Trifluoroethanol on the Structural and Functional Properties of α-Crystallin

Alpha crystallin is an eye lens protein with a molecular weight of approximately 800 kDa. It belongs to the class of small heat shock proteins. Besides its structural role, it is known to prevent the aggregation of β- and γ-crystallins and several other proteins under denaturing conditions and is thus believed to play an important role in maintaining lens transparency. In this communication, we have investigated the effect of 2,2,2-trifluoroethanol (TFE) on the structural and functional features of the native α-crystallin and its two constituent subunits. A conformational change occurs from the characteristic β-sheet to the α-helix structure in both native α-crystallin and its subunits with the increase in TFE levels. Among the two subunits, αA-crystallin is relatively stable and upon preincubation prevents the characteristic aggregation of αB-crystallin at 20% and 30% (v/v) TFE. The hydrophobicity and chaperone-like activity of the crystallin subunits decrease on TFE treatment. The ability of αA-crystallin to bind and prevent the aggregation of αB-crystallin, despite a conformational change, could be important in protecting the lens from external stress. The loss in chaperone activity of αA-crystallin exposed to TFE and the inability of peptide chaperone—the functional site of αA-crystallin—to stabilize αB-crystallin at 20–30% TFE suggest that the site(s) involved in subunit interaction and chaperone-like function are quite distinct.     

Phe71 is essential for chaperone-like function in alpha A-crystallin

Experiments with mini-alphaA-crystallin (KFVIFLDVKHFSPEDLTVK) showed that Phe(71) in alphaA-crystallin could be essential for the chaperone-like action of the protein (Sharma, K. K., Kumar, R. S., Kumar, G. S., and Quinn, P. T. (2000) J. Biol. Chem. 275, 3767-3771). In the present study we replaced Phe(71) in rat alphaA-crystallin with Gly by site-directed mutagenesis and then compared the structural and functional properties of the mutant protein with the wild-type protein. There were no differences in molecular size or intrinsic tryptophan fluorescence between the proteins. However, 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid interaction indicated a higher hydrophobicity for the mutant protein. Both wild-type and mutant proteins displayed similar secondary structure during far UV CD experiments. Near UV CD signal showed a slight difference in the tertiary structure around the 285-295 region for the two proteins. The mutant protein was totally inactive in suppressing the aggregation of reduced insulin, heat-denatured citrate synthase, and alcohol dehydrogenase. However, a marginal suppression of beta(L)-crystallin aggregation was observed when mutant alphaA-crystallin was included. These results suggest that Phe(71) contributes to the chaperone-like action of alphaA-crystallin. Therefore we conclude that the 70-88-region in alphaA-crystallin, identified by us earlier, is the functional chaperone site in alphaA-crystallin.     

Identification of a region in alcohol dehydrogenase that binds to alpha-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.     

Nutripriming of Seeds with Manganese Sulphate for Better Germination and Seedling Vigour in the East Indian Sandalwood (Santalum album L.)

Journal of Plant Growth Regulation - Poor and staggered seeds germination is the major hurdle in plantation establishment of sandalwood (Santalum album L) which is one among the esteemed timber...