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Non-structural protein interaction reporter (NSPIR) system
Non-structural protein interaction reporter (NSPIR) system
to combat DENGUE
to combat DENGUE
One of the important mechanisms of dengue viral pathogenesis manifestation is through myriad protein-protein interactions of viral Non-structural (NS) and host proteins. We propose to design biomolecules (peptide inhibitors) which targets NS proteins specifically and disrupts all their pathogenesis pathways. We would try to achieve this via a reporter system based on principles of CID and FRET.
CHEMICALLY INDUCED DIMERIZATION
CHEMICALLY INDUCED DIMERIZATION
Our aim is to study the Protein-protein interaction between the NS3 and NS2B viral proteins thoroughly. In an actual cell, this interaction may be difficult to implement or observe. So in order to ensure that this is not a problem, we will use our first reporter system, which employs the process of Chemically Induced Dimerization, wherein two particular proteins which can be induced to dimerize with one another, are expressed in a complex with our selected NS proteins. In this way, our chosen proteins can be made to interact by bringing them in close proximity.
In our system we aim to use FKBP12 and FRB protein domains and Rapamycin, that will induce their dimerization (Chemical Inducer of Dimerization). With the CID Reporter, we can ensure that our chosen PPI takes place in the cell and we can localize and observe it more closely.
CID SYSTEM USING FKBP AND FRB DOMAINS
CID SYSTEM USING FKBP AND FRB DOMAINS
Forster’s Resonance Energy Transfer
Forster’s Resonance Energy Transfer
However, to quantify the extent of the interactions, we need a second reporter system, for which we plan on using a FRET Reporter. FRET is a phenomenon that essentially functions as a spectroscopic ruler, by employing two fluorophores (donor and acceptor) wherein the excitation energy of the donor is transmitted to the acceptor in its vicinity, resulting in the distinctive fluorescence emission spectra of the acceptor, which is extremely sensitive to distance of separation between molecules.
Since PPI is also dependent on molecular proximity, in our project, we are going to use a FRET-CID coupled system where, after the CID reporter ensures the PPI, the FRET reporter will measure the extent of interaction and act as an assay for the peptide inhibitor by showing a change in the acceptor fluorophore’s emission spectra. The measurable parameters generated from the FRET system, namely the efficiency and dynamic range will be our tools in gauging the interaction and its subsequent inhibition.
FRET PRINCIPLE AND SPECTRAL OVERLAPS
FRET PRINCIPLE AND SPECTRAL OVERLAPS
SCHEMATIC OF OUR STRATEGY (REPORTER SYSTEM)
SCHEMATIC OF OUR STRATEGY (REPORTER SYSTEM)
Project outcomes
Project outcomes
The project will eventually result in the construction of a mammalian reporter system that can mimic viral protein interaction with host or other viral proteins, in an exogenous system. Although, we propose this for studying DENV interacting proteome, the reporter can be easily adapted to any viral proteins including SARS-COV-2. Additionally, the project will also result in validation of peptide inhibitors as a potential intervention strategy for dengue infections. This methodology will thus pave new vistas for anti-viral research.
RESources that will be generated:
RESources that will be generated:
CID system for DENV NS PPI, individual components cloned in bacterial system
FRET reporter system, cloned in bacterial system
FRET reporter coupled CID system for DENV NS PPI, transfected and expressed in mammalian cells.
Peptide inhibitor for DENV NS PPI (NMR validation of the same)
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