Sushil Chandani

Calcium Binding Properties of γ-Crystallin CALCIUM ION BINDS AT THE GREEK KEY βγ-CRYSTALLIN FOLD

The β- and γ-crystallins are closely related lens proteins that are members of the βγ-crystallin superfamily, which also include many non-lens members. Although β-crystallin is known to be a calcium-binding protein, this property has not been reported in γ-crystallin. We have studied the calcium binding properties of γ-crystallin, and we show that it binds 4 mol eq of calcium with a dissociation constant of 90 μm. It also binds the calcium-mimic spectral probes, terbium and Stains-all. Calcium binding does not significantly influence protein secondary and tertiary structures. We present evidence that the Greek key crystallin fold is the site for calcium ion binding in γ-crystallin. Peptides corresponding to Greek key motif of γ-crystallin (42 residues) and their mutants were synthesized and studied for calcium binding. These peptides adopt β-sheet conformation and form aggregates producing β-sandwich. Our results with peptides show that, in Greek key motif, the amino acid adjacent to the conserved aromatic corner in the “a” strand and three amino acids of the “d” strand participate in calcium binding. We suggest that the βγ superfamily represents a novel class of calcium-binding proteins with the Greek key βγ-crystallin fold as potential calcium-binding sites. These results are of significance in understanding the mechanism of calcium homeostasis in the lens.

β-Carbolines That Accumulate in Human Tissues May Serve a Protective Role against Oxidative Stress

β-Carbolines are tricyclic nitrogen heterocycles formed in plants and animals as Maillard reaction products between amino acids and reducing sugars or aldehydes. They are being detected increasingly in human tissues, and their physiological roles need to be understood. Two β-carboline carboxylates have been reported to accumulate in the human eye lens. We report here on the identification of another β-carboline, namely 1-methyl-1-vinyl -2,3,4-trihydro-β-carboline-3-carboxylic acid, in the lenses of some cataract patients from India. Analysis of these three lenticular β-carbolines using photodynamic and antioxidant assays shows all of them to be inert as sensitizers and effective as antioxidants; they quench singlet oxygen, superoxide and hydroxyl radicals and inhibit the oxidative formation of higher molecular weight aggregates of the test protein, eye lens γ-crystallin. Such antioxidative ability of β-carbolines is of particular relevance to the lens, which faces continual photic and oxidative stress. The β-carboline diacid IV is also seen to display an unexpected ability of inhibiting the thermal coagulation of γ-crystallin and the dithiothreitol-induced precipitation of insulin. These results offer experimental support to earlier suggestions that one of the roles that the β-carbolines have is to offer protection against oxidative stress to the human tissues where they accumulate.

Structural Integrity of the Greek Key Motif in βγ-Crystallins Is Vital for Central Eye Lens Transparency

Background We highlight an unrecognized physiological role for the Greek key motif, an evolutionarily conserved super-secondary structural topology of the βγ-crystallins. These proteins constitute the bulk of the human eye lens, packed at very high concentrations in a compact, globular, short-range order, generating transparency. Congenital cataract (affecting 400,000 newborns yearly worldwide), associated with 54 mutations in βγ-crystallins, occurs in two major phenotypes nuclear cataract, which blocks the central visual axis, hampering the development of the growing eye and demanding earliest intervention, and the milder peripheral progressive cataract where surgery can wait. In order to understand this phenotypic dichotomy at the molecular level, we have studied the structural and aggregation features of representative mutations. Methods Wild type and several representative mutant proteins were cloned, expressed and purified and their secondary and tertiary structural details, as well as structural stability, were compared in solution, using spectroscopy. Their tendencies to aggregate in vitro and in cellulo were also compared. In addition, we analyzed their structural differences by molecular modeling in silico. Results Based on their properties, mutants are seen to fall into two classes. Mutants A36P, L45PL54P, R140X, and G165fs display lowered solubility and structural stability, expose several buried residues to the surface, aggregate in vitro and in cellulo, and disturb/distort the Greek key motif. And they are associated with nuclear cataract. In contrast, mutants P24T and R77S, associated with peripheral cataract, behave quite similar to the wild type molecule, and do not affect the Greek key topology. Conclusion When a mutation distorts even one of the four Greek key motifs, the protein readily self-aggregates and precipitates, consistent with the phenotype of nuclear cataract, while mutations not affecting the motif display ‘native state aggregation’, leading to peripheral cataract, thus offering a protein structural rationale for the cataract phenotypic dichotomy “distort motif, lose central vision”.

The Mutation V42M Distorts the Compact Packing of the Human Gamma-S-Crystallin Molecule, Resulting in Congenital Cataract

Background Human γS-crystallin is an important component of the human eye lens nucleus and cortex. The mutation V42M in the molecule causes severe congenital cataract in children. We compare the structure of the mutant protein with that of the wild type in order to understand how structural changes in the mutant relate to the mechanism of opacification. Methods Both proteins were made using conventional cloning and expression procedures. Secondary and tertiary structural features of the proteins were analyzed using spectral methods. Structural stabilities of the proteins were analyzed using chemical and thermal denaturation methods. Self-aggregation was monitored using extrinsic spectral probes. Molecular modeling was used to compare the structural features of the two proteins. Results While the wild type and mutant have the same secondary structure, molecular modeling and fluorescence analysis suggest the mutant to have a more open tertiary structure, with a larger hydrophobic surface. Experiments using extrinsic probes reveal that the mutant readily self-aggregates, with the suggestion that the aggregates might be similar to amyloidogenic fibrils. Chemical denaturation indicates that while the wild type exhibits the classic two-state transition, V42M goes through an intermediate state, and has a distinctly lower stability than the wild type. The temperature of thermal unfolding of the mutant is also distinctly lower. Further, the mutant readily precipitates and scatters light more easily than the wild type. Conclusion The replacement of valine in position 42 by the longer and bulkier methionine in human γS-crystallin perturbs the compact β-sheet core packing topology in the N-terminal domain of the molecule, exposes nonpolar residues thereby increasing the surface hydrophobicity and weakens the stability of the protein, thus promoting self-aggregation leading to light scattering particles. This set of changes in the properties of the mutant offers a molecular insight into the mechanism of opacification.

Regulation of cell division and malignant transformation.

The problem of regulation of cell division is essentially a problem of understanding regulation of transition from the resting state of a cell to the dividing state and vice versa. In malignancy the ability to revert back to a normal resting state is impaired. A model is presented which attempts to explain the control of the above transitions through control of uptake of essential nutrients by a transport-inhibitory protein. Experimental evidence in favour of the model is given.

Seminalplasmin, a bovine seminal plasma protein, lyses dividing but not resting mammalian cells

Seminal plasmin, an antimicrobial and transcription-inhibitory protein of bovine seminal plasma, is shown to lyse dividing mammalian cells in vitro. It lyses cells in culture such as CHO, Vero, HeLa and L929. It also lyses regenerating rat liver parenchymal cells and cells of two ascitic tumours of rat--the Zajdela ascitic hepatoma and the AK-5. However, it does not lyse resting cells such as adult liver parenchymal cells, erythrocytes, or resting lymphocytes, though it binds to their cell surface. It can be used, therefore, to distinguish cells that are in the division cycle from cells that are in the resting phase. The cell-lytic activity of seminal plasmin is inhibited by Ca2+.

The role of cell membrane in the regulation of cell division and malignant transformation

An earlier model in which uptake of essential nutrients for which the cell is auxotrophic, regulates cell division, is discussed in the light of new experimental findings, specifically the purification of a new type of transport-inhibitory protein from rat liver and the properties of the protein. The possible role of such proteins in malignant transformation is also discussed.