The Biochemistry Laboratory is involved in research dealing with biochemical agents that play a crucial role in various eye diseases. One aspect of the work involves identifying substances for use in delaying the progression or onset of age-related eye diseases. Extracts of plants such as tea, Ginkgo biloba and Withania somnifera (Ashwagandha) have been analyzed and found to have a beneficial role. The effect is traced to the antioxidant and cytoprotective molecules present in these extracts
A second project involves the study of the molecular phenotypes in various eye disorders. The mutant gene identified as being associated with the disorder is cloned and expressed in heterologous systems, in order to produce the mutant protein, and the molecular properties of the latter are compared with those of the normal protein. Currently, the mutant crystalline genes in cataractous lenses, and the glaucoma-causing optineurin gene expressed in the trabecular meshwork cells, are being cloned and expressed to understand the molecular phenotypes. In addition, where necessary, the cDNAs of the target proteins are transfected into relevant cells and the differences in the behavior of the normal (wild type) and mutant molecules compared. This gives an idea of the functional alterations that occur in situ due to the mutation. The laboratory is equipped with all the advanced instruments required for biochemical and molecular biological work
Research In Biochemistry
Study of the role of vitreous liquefaction in age-related nuclear cataract
Investigators: D Balasubramanian, Pravin V Krishna
Support: Department of Biotechnology, India (US-India collaborative eye research program)
This is the Indian component of a US-India collaborative study, The US part is being done by Dr David Beebe's group. The goal of this collaborative study is to determine whether nuclear cataracts are associated with increased degeneration of the vitreous body in Indian patients, as they are in patients from the US. At the same time, this study will establish whether or not the brunescent nuclear cataracts, as found in India, have a similar association with the degeneration of the vitreous body. Degeneration of the vitreous body of the eye is a major risk factor for nuclear cataracts. These finding are consistent with the well-known association between the loss of vitreous structure during vitrectomy and nuclear cataracts, and indicates that the gel structure of the vitreous body protects the lens from nuclear cataracts. These studies are being conducted using essentially the same methods as in Harocopos et al. 2004, with some improvements in instrumentation.
Program support on translational research on eye diseases
Support: Department of Biotechnology, India (Program support grant)
India has about 15 million people who are blind. Of these, about 10 million are cataract blind, about 1.2 million are blind due to uncorrected refractive errors, and another 1.0 million due to infection of the eye. All these are easily managed through effective national level programs since, in each of these cases, ready and effective solutions are available, and no new research is needed. But the rest of the blindness burden is due to corneal dystrophies, glaucoma, retinal disorders and inherited problems. In these instances, basic research and its application and translation are badly needed. Fortunately, the status of research in these areas in India is of the same level and quality as elsewhere, and the connection between basic researchers and clinicians is good and mutually productive, particularly in ophthalmology. We need to take advantage of this combination and hence the need for translational research on eye diseases.
The program has the following R&D projects:
Focal Theme 1: Stem Cell Biology and Therapy
Project A: Establishment of a centre for translational research in stem cells
Focal Theme 2: Molecular Genetics of Ocular Diseases
Project B: Translational research in the genetics of retinal diseases
Project C: Molecular genetic study of retinopathy of prematurity
Project D: Identifying candidate genes associated with raised intraocular pressure in primary open angle glaucoma
Project E: Molecular phenotyping of genetic mutations leading to eye disorders - functional studies on the disorderassociated proteins
Functional analysis of the glaucoma-associated protein optineurin
Investigators: D Balasubramanian, Subhabrata Chakrabarti, Ghanshyam Swarup
Support: Department of Biotechnology, India
Optineurin is a multifunctional protein involved in several functions such as vesicular trafficking from the Golgi to the plasma membrane, NF-B regulation, signal transduction and gene expression. Mutations in optineurin are associated with glaucoma, a neurodegenerative eye disease that causes blindness. Genetic evidence suggests that the E50 K (Glu50Lys) is a dominant disease-causing mutation of optineurin. However, functional alterations caused by mutations in optineurin are not known. Earlier, we had shown that the mutant E50K induces retinal ganglion cell death through oxidative and apoptotic mechanisms. In an effort to understand the cell biology of the process, we have analyzed the role of optineurin in endocytic recycling and the effect of E50K mutant on this process.
Structural basis and physiological consequences of congenital nuclear cataracts in humans, associated with mutations in human gamma-D-crystallin
Investigators: D Balasubramanian, Prof N Srinivasan1
Support: Department of Biotechnology, India (Program support grant)
Many human congenital cataracts arise from mutations in the lens proteins, crystallins. We focus on human gamma-D-crystallin, particularly because over 16 mutations are known here, and they neatly group into one group that leads to cortical, peripheral and similar cataracts, which are associated with mutations in the N-terminal domain (N-tD) of the molecule, and the second groups where mutations are in the C-terminal domain (CtD), which are associated with nuclear cataracts. Of the two types of cataracts (nuclear and peripheral), nuclear cataract blocks the central vision of the infant, causing impairment of the development of the eye and the visual system of the growing child, thus requiring the earliest possible intervention.
The questions we ask are: what is the structural rationale behind this interesting dichotomy of N-tD mutants leading to peripheral and C-tD mutants nuclear cataracts, and what are its functional and physiological consequences? Gamma crystallins are known to have two biochemical functions. One is their ability to bind to and sequester calcium ions, and thus inhibit free Ca ions from initiating protein and nucleic acid degradation in situ. The other is to induce the production and release of the growth factor called ciliary neurotrophic factor or CN TF, an important development-relevant factor in the eye. We thus cloned, expressed and isolated pure wild type human gamma-D-crystallin and its N-tD mutant P23T, and C-tD mutants E107A, R140X and W157X, and compared their solution state conformations (secondary and tertiary structures) and their Ca ion binding abilities. We have also done extensive in silico modeling of these proteins, and assessed their aggregational properties in vitro and also in situ in lens epithelial cells.
We find a clear distinction between the properties of the N-tD mutant and the C-tD mutants. While the N-tD maintains the chain conformation of the wild type, and also binds Ca ions just as well as the wild type, each one of the C-tD mutant is seen to lose its chain order (the 'greek key' motif) in the C-terminal domain. This causes the C-tD mutants to form light-scattering aggregate particles, and also to lose the Ca binding ability. We are currently studying their relative abilities to induce CN TF.
1Indian Institute of Science, Bangalore
1Institute of Life Sciences, Bhubaneswar