Title of Thesis
To study the Apoptotic Role of Granzyme H Before and After
Chemotherapy of Breast Cancer
Naheed Z. Rizvi
Institute Of Chemistry / University Of The
|Number of Pages|
|Keywords (Extracted from title, table of contents and
abstract of thesis)|
Cancer, Isocoumarin, Before,
Physiological, Serum, Granzyme, Detection, Breast, Study,
Chemotherapy, After, Role, Methods, Apoptotic
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
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
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
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
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 & Pro225Arg225 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 Arg55Gly55
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.