Macromolecular Crystallography, Cell Biology, Molecular Genetics, Membrane Biophysics, Genetic Toxicology and Radiation Biology.During the Xth Plan period, the fact that a holistic, high-throughput approach is critical to understand the underlying mechanism of human diseases was envisaged. Keeping that in view, structural genomics, proteomics and gene expression studies in hematological and neurodegenerative disorders, viz., Thalassaemia, Leukemia, Huntington's & Alzheimer's disease, have been given the major emphasis in the project entitled "Structural genomics in human health & disease".  In this rapidly growing area of interdisciplinary research, our scientists have initiated research on identification of target gene/proteins, cloning, expression and purification for subsequent determination of the 3D structure of different target proteins.

Thalassaemia is a genetically determined class of anemia where the defect has been identified in hemoglobin (Hb) synthesis. This results in an inability to manufacture sufficient quantities of globin chains. The alpha and beta globin genes have been identified in chromosome 11 and 16 respectively. A huge number of Indian populations are affected by this inherited blood disorder. In India, specially in the eastern region, Thalassaemia is mainly prevalent among Sindhis, Punjabis, Marwaris, Gujratis and Bengalis. Nearly, 8000-10000 children with thalassaemia alone are born in our country every year. Hematopietic stem cells will be isolated from affected/carrier maternal chord blood using established cell separation techniques such as magnetic beads or fluorescent activated cell sorter (FACS). The major objectives of the project is to study the reconstitution, folding and aggregation of globin chains of pathological Hbs, classify the Hb variants, determine 3D structures of Hb variants, study the protein-protein interactions of intact Hbs and the globin chains with cytoskeletal proteins and the oxidative stress disturbances in the red cells isolated from patients suffering from the disease. The major activities will center on proteomics study and characterization of novel/mutant proteins of the erythrocyte membrane and its cytoskeleton, which will be followed by cloning, and expressing of the protein/protein fragment leading to the determination of its structure by X-ray diffraction and other spectroscopic techniques.

Normal lymphoid cell populations undergo diverse clonal rearrangements of their genes, followed by highly regulated proliferations of the cells that successfully complete these genetic changes. The development process generates B cells and T cells with the specificity needed to support a fully competent immune system. When a lymphoid progenitor cell becomes genetically altered through somatic changes, the result can be deregulated proliferation and clonal expansion, eventually leading to acute lymphoblastic leukemia (ALL) or chronic lymphoblastic leukemia (CLL). Because leukemia blasts represent the clonal expansion of hematopoietic progenitors that are blocked in differentiation at discrete stages of development, they provide large uniform populations for molecular, structural and functional analyses.

Hematopoietic cells are highly specialized cells whose phenotype and functional characteristics are intimately linked to their stage of maturation. However the specific genes whose expression mediates differentiations of pluripotent progenitors to mature lymphoblast are largely undefined. The generation of large number of lymphoid cells has become feasible through in vitro culturing of progenitors using exogenous cytokines to support their growth, differentiation and maturation. We propose to use in vitro culture of CD34+ stem cells isolated from chord blood of normal female in order to analyze systemic gene expression during differentiation and in the process of leukogenesis in comparison with blood collected from leukemic patients.

Few studies have been undertaken that simultaneously analyzed cell population at both RNA and protein levels. Potential source of discordance between RNA and Protein levels that include altered translational control and protein stability, post-translational modification that is not predictable at the RNA level will be addressed in this plan proposal. More specifically we would look into the following groups of proteins: Cell surface proteins (MHC II, HLA-DQ family), secreted proteins (TGF-family) and nuclear proteins (IRF-family).

Expansion of polymorphic glutamine repeats in specific proteins has been implicated in a number of neurodegenerative diseases where loss of neurons is primary consequence of such expansion. The disease includes Huntingtons disease (HD), dentatorubral pallidolysian atrophy, spinal bulbar muscular atrophy and several subtypes of autosomal dominant spinocerebellar ataxias (SCA1, SCA 2, SCA3, SCA6, SCA7, SCA17). All these are progressive, ultimately fatal disorders typically begin at middle age with wide range of variation in the age at onset. Rate of diseases, the age at onset roughly correlated inversely with the number of glutamine repeats in the protein. Various studies indicated that number of glutamines in the repeats is the most important determinant but does not explain all the variations. Given the immense complex biochemical events that occur due to glutamine repeat expansion involving large number of proteins including caspases, chaperons, and poly-glutamine interacting proteins and defects in the degradation of the mis-folded/aggregated proteins, it is likely that small variations (as SNPs) in any one of the these genes would modify the progression and age at onset of these diseases. Apoptosis, a specific type of cell death has also been implicated in neurodegenerative diseases. It is also likely that variation in the genes involved in apoptosis would contribute towards the development and progression of the disease. Thus, the 3D structure determination of the poly-q interacting proteins and detection of the variations in the genes involved in apoptosis together would probably be able to decipher the pathways involved in neurodegeneration and provide possible therapeutic intervention.

In recent years several lines of evidence suggest a common scheme of pathogenicity for amyloidogenic neurodegenerative diseases viz., Alzheimers Disease (AD), Huntingtons Disease (HD), Parkinsons Disease (PD) etc., as they happen to share molecular components along their pathogenic cascades. In case of AD, the attention of clinical research has mainly been focused on its amyloidogenic component, i.e., Abeta peptides, but over the last twenty years or so, it yielded no specific drugs, nor could it mechanistically explain the disease process in totality. Recent focus is therefore being shifted to other peptide fragments (C-terminal Fragments, CTFs) of Amyloid Precursor Protein (APP), which are found to be equally cytotoxic in neurons. Furthermore, this Amyloid Intracellular Domain (AICD) is found to interact with several adaptor proteins, some of which are known to be involved  in other physiological processes, viz., calcium homeostasis, transcriptional transactivation, cholesterol metabolism, long term potentiation etc. It is therefore crucial to organize these pathways involving adaptors as upstream or downstream events in terms of Abeta peptidogenesis and neuropathy, eventually to establish a putative pivotal role for the AICD peptide. The initial goal for the project would be to analyze the expression levels and interactions of the adaptor proteins in samples (blood, CSF, solid tissue) from AD patients as well as AD model systems.