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Interaction of Proteins with small Molecules and Peptides
No full textUnderstanding the molecular interaction of proteins with small molecules and peptides is essential in drug discovery. This thesis explores the importance of
protein small molecule interactions in biology and its implications in rational drug development. Using combined theoretical and experimental chemistry, author
tried to device new approaches or build upon the existing tools to study the interactions of proteins with small molecules and peptides. New algorithm has been developed based on the existing molecular docking methods to improve the specificity in docking prediction and reduce the false positive results (hits) in virtual high throughput screening. In recent years allosteric drugs have gained much
attention because of the specificity it can achieve due to the evolutionarily diverse binding sites on the target proteins. The author developed a pattern based
approach using molecular docking tools to detect the allosteric binding sites on structured protein targets and analyzed the druggability of these allosteric binding
sites. Detections of binding sites in the intrinsically disordered proteins was also explored using the sequence based analysis tools. Statistical distribution of binding
sites in a library of disordered proteins was characterized. A fluorescent probe molecule has been developed to study the microenvironment of protein binding
sites, which showed increased quantum yield, anisotropy and higher energy emission upon binding to a hydrophobic site of the target protein. Author studied even deeper into the finer details of molecular interactions such as the hydrogen
bonding and its strength on model system (alcohol amine complexes) using isotope labeling, solution state nuclear magnetic resonance spectroscopy and quantum mechanics with implications in protein small molecule interactions
Assessment of virulence potential of uncharacterized Enterococcus faecalis strains using pan genomic approach – Identification of pathogen–specific and habitatspecific genes
Get PDFEnterococcus faecalis, a leading nosocomial pathogen and yet a prominent member of gut microbiome,
lacks clear demarcation between pathogenic and non-pathogenic strains at genome level. Here we
present the comparative genome analysis of 36 E. faecalis strains with different pathogenic features
and from different body-habitats. This study begins by addressing the genome dynamics, which
shows that the pan-genome of E. faecalis is still open, though the core genome is nearly saturated. We
identified eight uncharacterized strains as potential pathogens on the basis of their co-segregation
with reported pathogens in gene presence-absence matrix and Pathogenicity Island (PAI) distribution.
A ~7.4 kb genomic-cassette, which is itself a part of PAI, is found to exist in all reported and potential
pathogens, but not in commensals and other uncharacterized strains. This region encodes four genes
and among them, products of two hypothetical genes are predicted to be intrinsically disordered that
may serve as novel targets for therapeutic measures. Exclusive existence of 215, 129, 4 and 1 genes
in the blood, gastrointestinal tract, urogenital tract, oral cavity and lymph node derived E. faecalis
genomes respectively suggests possible employment of distinct habitat-specific genetic strategies in
the adaptation of E. faecalis in human hos
Tlr and immune response: role of β- (1-4) - galactose terminal glycans in enhancement of immune response and protection against experimental visceral leishmaniasis
No full textKala-azar orvisceral leishmaniasis (VL), caused by the protozoan parasite Leishmania donavani (LD), is gradually making inroads in different continents due to migration of
people and vectors. Production of proinflammatory cytokines by antigen presenting cells and microbicidal molecules (reactive oxygen species or ROS & nitric oxide or NO) by the
host cells are the key regulators for successful clearance of LD parasites. The control of LD infection requires generation of NO and activation of a strong Th1 response, mainly by up regulation IFN-gamma through interleukin-12 or IL-12, while disease progression is promoted by induction of interleukin-10 or IL-10. An increasing number of receptor families are being implicated in the recognition of
pathogen-associated molecular patterns (PAMP) by the cells of immune system. Among these, toll like receptors (TLRs) are key players, linking innate and adaptive arm of
immune system. Following infection, pathogen derived several exogenous components may bind with the toll receptors thereby initiating several signaling pathways and
ultimately leading to the production of pro or anti inflammatory cytokines and generation of inducible Nitric oxide synthase or iNOS. TLR ligands such as LPS, glycolipids, peptidoglycans, lipopeptides are shared by large group of micro organisms and allow recognition of wide range of microbial pathogens. Following receptor ligand association except TLR3, all other TLRs recruits cytoplasmic adaptor molecule myeloid differentiation
primary response protein88 (MyD88) in the Toll/IL-1 receptor domain of TLRs. Although,
unlike other TLRs, TLR4 engagement, cause activation through both the MyD88 dependent and independent pathways, thereby ultimately leading to activation of common signaling cascade involving extracellular signal-regulated kinase (ERK), P38 mitogenactivated protein kinase, stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB).The overall impact of this signaling pathway would be the initiation of a Th-1 type immune response.TLR mediated signalling becomes refractory in LD infection
Amelioration of arsenic toxicity by garlic and its major bioactive components
No full textGround water arsenic contamination has been reported from many countries of the world
including India and Bangladesh. Arsenic exposure in human leads to a wide variety of
adverse health outcomes like skin lesions including melanosis, hyperkeratosis, Bowen’s
disease, basal and squamous cell carcinomas. Previous report from our laboratory
suggested that aqueous garlic extract (AGE), a common Indian household spice
containing a number of organosulphur compounds (OSCs), was found to play an
inhibitory action against arsenic-induced toxicity both in vitro and in vivo. This could
make AGE a better substitute for synthetic chelators which are associated with various
side effects
Studies on the regulation of expression of CDC20 and search for its new molecular interactors
No full textAdvancement in the understanding of the molecular basis of cancer has led to the
emergence of tumor-specific, molecularly targeted agents that specifically kill
malignant cells more efficiently than ever. A number of cell signaling molecules, cellcycle
regulators, metabolic enzymes and cell surface macromolecules are currently
considered as potential targets for the development of novel therapeutic agents for
cancer treatment. Cdc20 a key spindle assembly checkpoint (SAC) protein, activates
APC/C during mitosis. Therefore, the major function of Cdc20 is to coordinate
mitotic progression by facilitating the orderly degradation of mitotic APC/Cb
substrates. Deregulation of Cdc20 causes erroneous mitotic division followed by
chromosomal instability (CIN). CIN is the hallmark of cancer. It is observed that the
overexpression of CDC20 occurs in several oral cell lines and primary head and neck
tumors and provide evidence that such overexpression of CDC20 is associated with
premature anaphase promotion, resulting in chromosome abnormalities in the cell
lines. More over invitro experiments have also shown that mitotic instability may be a
mechanism for developing resistance to cytotoxic drugs. Thus proper Cdc20
expression and function plays a crucial role in the context of the disease like cancer.
In this study we investigated first, the transcriptional regulation of Cdc20, second
Cdc20 mediated regulation of other cell cycle genes and finally we tried to establish
the Cdc20–Mad2 complex as a potential therapeutic target. As a result we proposed
for the first time, an oncogenic regulation of Cdc20. We showed that the protooncogene
Myb transctivates CDC20 in colorectal cancer cells and also in tumour
patient samples. We also reported that Cdc20 may negatively regulate its own
expression when the endogenous Cdc20 level goes up. We hypothesized that this
occurs probably due to maintain its own balance, since the proper level of Cdc20 is
crucial for maintaining the genomic stability of the daughter cells. To validate our
hypothesis we rescued the Myb mediated trans-activation of CDC20 from the Cdc20
auto inhibition, by lowering the endogenous Cdc20 level in the cell. More over we
showed that the Myb trans-activation response site and the Cdc20 auto-inhibition
response site resides very close to each other within 71 bp upstream of CDC20
transcription start site. All these evidences confers that the transcription of CDC20 is
a dual regulated phenomena operated by Myb and Cdc20 itself and the mode of
Summary
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operation depends upon the endogenous level of the Cdc20 protein. We also found the
involvement of p300 in Myb mediated trans-activation. As well as HDAC1 take part
in Cdc20-auto-inhibition. Additionally ubiquitination has a role in the autoregulation
of Cdc20. Unubiquitinated Cdc20 protein was found to be more prone for its own
inhibition. Finally to assess the fuctional significance of this oncogenic regulation of
Cdc20, we showed that over-expression of Myb leads to premature anaphase by over
expressing CDC20, followed by deregulation of SAC.
Cdc20 is reported to participate in the transcriptional regulation of mitotic genes by
CBP/p300 complex. Further, we have dissected the transcriptional role of Cdc20
protein in a global aspect. We performed the whole genome microarray analysis in the
altered Cdc20 expressing condition in HepG2 cells and found 252 differentially
expressed genes. The altered genes mainly belong to cell cycle and apoptotic
pathways. We found Rb, an important tumor suppressor gene is one of the major
downstream target of Cdc20. We have validated our observation in human cancer cell
line.
Finally we established Withaferin A (WA) as an inhibitor of Cdc20-Mad2 complex in
colorectal cancer cells. It causes spindle assembly checkpoint (SAC) arrest in
HCT116 and SW480 colorectal adenocarcinoma cell lines by stabilizing Cyclin B1
and its downstream molecules. Further we dissected the mechanism of action of WA
in these cell lines. We found that WA leads to proteasomal degradation of the Cdc20-
Mad2 complex that actually causes SAC arrest and apoptosis, as consequences. We
also rescued the Cyclin B1 degradation and the cell proliferation by over expression
of Mad2 in presence of WA. Together the experimental results suggests that WA
treated cells are arrested at the metaphase allowing sufficient time to activate the
apoptotic machinery leading to mitotic death of the cells.
Collectively this continuing effort is being made to identify Cdc20 as a new
oncogenic lead based on the knowledge drawn from the molecular analysis of cancer
cell lines and primary tumor that will enrich the repertoire of new molecular targets
for cancer management.
Part of this work has been published in Biochemical Pharmacology, 91 (2014) 31–3
Deamidation and protein repair studies of bio-catalysts from different organisms
No full textProteins undergo certain posttranslational modifications like acetylation, phosphorylation, methylation, deamidation etc as a step in protein biosynthesis. Protein post-translational modifications play a key role in many cellular processes such as cellular differentiation [G. Grotenbreg, H. Ploegh (2007)], protein degradation [R.Geiss-Friedlander, F.Melchior (2007)], signaling and regulatory
processes [Morrison et al (2002)], regulation of gene expression, and protein-protein interactions. Deamidation is a type of posttranslational modification which changes
the chemical nature of amide containing amino acids like asparagines (Asn). The reaction is initiated by the favorable nucleophilic attack on the side chain carbonyl
by the peptide backbone nitrogen of the following amino acid residue. This leads to the formation of an unstable succinimidyl intermediate that non-enzymatically
hydrolyzes into either L-aspartyl (normal) or L-isoaspartyl (abnormal) residue, resulting in a cumulative gain of net negative charge at their specific sites of chemical reaction [Q. Hasan et al (2005)] The metastable succinimide has a typical half life of 4 hours at pH-7.4, 37°C. The rapid hydrolysis of this intermediate mixture generates Asp (~30%) and predominantly isoAsp (~70%) residues. Each
amide has a specific deamidation rate that is genetically determined by the sequence of residues immediately adjacent in the peptide chain and by secondary, tertiary and
quarternary structure. The influence of local sequence and solution environment on the rates of deamidation has been extensively studied by researchers using synthetic
/natual peptides. Isoaspartate forms most readily at sequences in which the side chain of the C flanking amino acid is relatively small and hydrophilic, and is less likely to be formed where bulky or hydrophobic residues are in this position. The most favorable C-flanking amino acids are Gly, Ser and His, leading to so called isoAsp hot spots at Asn-Gly, Asn-Ser, Asn-His and Asp-Gly sequences[A.Cimmino et al (2004)]. Protein deamidation occurs at flexible regions of proteins especially on those sites which are more exposed. Robinson and Robinson have recently designed a computational method which could estimate the probability of deamidation within a folded protein with known crystal structures [N. E.Robinson & A. B. Robinson
(2001)]
A Glimpse on the Shift in Research Focus at IICB (1940-2010)
Get PDFThe study has tried to evaluate the shift in research focus carried out at IICB during the period 1940 to 2010. In this study, the growth has been observed in terms of
publications, patents and Ph.D. theses. During its long journey, IICB published 3570 publications, filed 242 patents and 552 Ph.Ds. supervised. The scholarly outputs have been recorded and categorized based on the diseases focus undertaken for research an
Interaction of Ab peptide with tubulin causes an inhibition of tubulin polymerization and the apoptotic death of cancer cells
No full textWe report in this work that the Ab peptide directly interacts with tubulin close to the vinblastine and GTP/GDP binding site, inhibits the tubulin polymerization rate, induces tubulin aggregation, causes cell shrinking, enhances Mad2, BubR1, p53, and p21 activation in MCF7
cells and induces the apoptotic death of A549, HeLa and MCF7 cells
Direct observation of the growth and shrinkage of microtubules by single molecule Fo¨rster resonance energy transfer
No full textSingle molecule Fo¨rster resonance energy transfer (FRET) has been applied, for the first time, to monitor the growth (polymerization) and the shrinkage (depolymerization) of the dynamic microtubules, employing EGFP (attached to Mal3) as a donor and alexa-568 bound
to tubulin as an accepto
Effects of Transient Hypoxia versus Prolonged Hypoxia on Satellite Cell Proliferation and Differentiation In Vivo
No full textThe microenvironment of the injury site can have profound effects on wound healing. Muscle injury results in ischemia leading to
short-termlocal hypoxia, but there are conflicting reports on the role of hypoxia on themyogenic programin vivo and in vitro. In our
rat model of mitochondrial restoration (MR), temporary upregulation of mitochondrial activity by a cocktail of organelle-encoded
RNAs results in satellite cell proliferation and initiation ofmyogenesis.We now report thatMR leads to a transient hypoxic response
in situ. Inhibition of hypoxia by lowering mitochondrial O2 consumption, either by respiratory electron transport inhibitors, or by
NO-mediated inhibition of O2 binding to cytochrome c oxidase, resulted in exacerbation of inflammation. Lentivirus-mediated
knockdown of hypoxia-inducible factor