Structural and functional characterization of TRX16, a thioredoxinlike protein and altering substrate specificity of a serine protease inhibitor

Abstract

The causative agents of most diseases like cancer and Alzheimer¿s are proteins. The function of a protein can be fully appreciated only when the complete knowledge of its 3-dimensional structure is known. First part of my thesis deals with the proteins that are involved in gene regulation under stress by interacting with its partners and the second project uses a rational approach for protein design and engineering to change the substrate specificity of a protease inhibitor.

Carcinoscorpius rotundicauda thioredoxin (Trx) like protein (Cr-TRP16) is a Trx like protein that contains a conserved WCPPC motif. We have determined the NMR structure of the reduced form of Cr-TRP16, and elucidated the molecular mechanism by which NF-¿B activation is regulated through Cr-TRP16 (Giri et al., J Biol Chem. M112.379859, 2012). Besides, we have expressed and purified the human thioredoxin like protein-6 (TXNL-6), a homolog of Cr-TRP16, which protects retinal cells from apoptosis under stress and characterized its interaction with NF-kB. These studies help further in understanding the interaction of Trx and Trx-like proteins with other metabolic pathways and will lead to promising therapeutic development.

The second project reports the structural basis for the rational design of altered specificity of a protease inhibitor. Protease inhibitors play a decisive role in maintaining homeostasis and eliciting antimicrobial activities. CrSPI-1 is a protease inhibitor identified from Carcinoscorpius rotundicauda. Here, we illustrate the use its structure to modify CrSPI-1-D1 to a potent thrombin inhibitor with IC50 of 26.3 nM (Giri et al., PloS ONE 5, e15258, 2010). This study will lead to the modification of a multi-domain inhibitor to target several proteases.