Title of Thesis

To study the Apoptotic Role of Granzyme H Before and After Chemotherapy of Breast Cancer

Author(s)

Naheed Z. Rizvi

Abstract
Breast cancer is the most common type of cancer—related mortality among women world-wide.Physiological changes of the patients were noted. Comparative study of analytical assay of GzmH was carried out in two different methods using serum samples of normal subjects with breast cancer patients of same age, socio—economic background and environmental conditions.
One method is by using the substrate PARP and isocoumarin inhibitor. Other one is electrophoresis. It is found that the electrophoretic technique as ompared to using substrate can be used for the detection of granzyme H is simple, accurate, and quick and may give better results than enzyme substrate assay. Identification by electrophoresis shows GzmH having a mass of appearance 32 KDa. 3D structure of GzmH was constructed by Modeller 9.0 in order to find out the different sites of granzyme. It showed highest homology with GzmB. The predicted 3D homology models show a conserved two similar domain structure, i.e., an N—terminal domain and a C—terminal domain comprising predominantly of beta—sheet structure with a little alpha—helical content.
The basic mechanism of the role of GzmH like other granzymes especially GzmB, showed that the Gzm having two cationic sites; cs1and cs2.These binding sites participate in the binding of Gzm to cell surface thereby promoting its uptake and release from the cytotoxic lymphocytes to the cell cytoplasm of virus or tumor or cell undergo autolysis. In the cell it causes the cleavage of proteins at its specific site like tyrosine or phenylalanine shows chymotrypsin-like activity. This cleavage stimulate the process of proteolysis
which may cause the mitochondrial disruption (caspase independent pathway), it is predicted that cystiene residues present near the catalytic residues Ser202 and Cys49 may help in triggering the cell death in a caspase dependent manner. Besides this pathway GzmH may stimulate the conversion of procaspase to caspase which acts on the nuclear protein like Poly-amino ribose
polymerase and causes DNA fragmentation that leads to cell death (caspase dependent pathway).
However, significant differences between GzmH and GzmB in the Xray structure and the protein model lie at the important functional sites. In the crystal structure of GzmB the catalytic triad is His57, Ser195 and Asp102, while in GzmH the catalytic triad is His64, Ser202 and Asp108. An ideal peptide present as cs1 site of GzmH. The peptide may promote the conversion of pro-caspase to caspase which successively cause cell death.
A segment of Gly214 to Asn220 is present near the catalytic triad of GzmH. This segment may provide a template for substrate binding bulges out of the active site. On the other hand, a hydrophobic patch of Trp238, Ileu239, Lys240 and Arg241 present in the helical form that provides a site for enzyme substrate interaction.
Enzyme inhibitor study showed that the inhibitor CMK (MAI-Pro-DPN) act as competitive inhibitor for GzmH which totally inhibit the enzyme activity by forming number of H-bonds with catalytic triad. The enzyme inhibitor study may be useful to probe and discuss the disease state with which they are associated.
Present study tried to mutate amino acids of granzyme H but only few showed significant effect of mutation e.g., mutation of Lys222→Ala222 & Pro225Arg225 causes to change their distance with the cs2 which may affect on the stability of cs2. The mutation of Lys222 to Ala markedly decreased the surface accessibility. It is stated that this mutation in turn may affect the uptake of GzmH into target cells; cytoplasmic distribution with reduced accumulation in target cell; and slightly impaired cytotoxicity of GzmH.
Arg55 forms number of H—bonds with other amino acids and thereby showed apoptotic promoting activity, present near the peptide of cationic site cs1. It is observed that the mutation of Arg55Gly55 causes the loss of Hbonds between mutated Gly to Asp57. It is therefore possible that mutation of Arg may affect the cytotoxic activity of GzmH. Mutational effect of Arg116 to Glu also observed. Present study observed that mutation of Arg116 to Glu may lose its H—bonds and salt bridges with Glu115. This shows that the mutation of both Arg55 and Arg116 affects the cytotoxic activity of GzmH.
Asp210 is near to the cs2 binding site of GzmH. This mutation from Asp210—Gly210 may affect the H—bonding pattern of cs2 which may reduce binding to heparin; slightly reduced uptake into target cells; cytoplasmic distribution with reduced accumulation in cell; and in turn may impaired cytotoxicity.
It is therefore concluded that GzmH due to its important functional effects may have diagnostic importance and it may be used as a tumor marker in breast cancer.

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