Development of Noninvasive Activity-Based Protein Profiling-Bioluminescence Resonance Energy Transfer Platform Technology Enables Target Engagement Studies with Absolute Specificity in Living Systems.

Noninvasive, real-time, longitudinal imaging of protein functions in living systems with unprecedented specificity is one of the critical challenges of modern biomedical research. Toward that goal, here, we report a platform fusion technology called activity-based protein profiling-bioluminescence resonance energy transfer (ABPP-BRET). This method provides an opportunity to study the post-translational modification of a target protein in real time in living systems in a longitudinal manner. This semisynthetic BRET biosensor method is used for target engagement studies and further for inhibitor profiling in live cells. The simplicity of this method coupled with the critical physical distance-dependent BRET readout turned out to be a powerful method, thus pushing the activity-based protein profiling technology to the next level.

A Universal Chemical Method for Rational Design of Protein-Based Nanoreactors*.

Self-assembly of a monomeric protease to form a multi-subunit protein complex "proteasome" enables targeted protein degradation in living cells. Naturally occurring proteasomes serve as an inspiration and blueprint for the design of artificial protein-based nanoreactors. Here we disclose a general chemical strategy for the design of proteasome-like nanoreactors. Micelle-assisted protein labeling (MAPLab) technology along with the N-terminal bioconjugation strategy is utilized for the synthesis of a well-defined monodisperse self-assembling semi-synthetic protease. The designed protein is programmed to self-assemble into a proteasome-like nanostructure which preserves the functional properties of native protease.

Development of Activity-Based Reporter Gene Technology for Imaging of Protease Activity with an Exquisite Specificity in a Single Live Cell.

Imaging of an active protease with an exquisite specificity in the presence of highly homologous proteins within a living cell is a very challenging task. Herein, we disclose a new method called "Activity-based Reporter Gene Technology" (AbRGT). This method provides an opportunity to study the function of "active protease" with an unprecedented specificity. As a proof-of-concept, we have applied this method to study the function of individual caspase protease in both intrinsic and extrinsic apoptosis signaling pathways. The versatility of this method is demonstrated by studying the function of both the initiator and effector caspases, independently. The modular fashion of this technology provides the opportunity to noninvasively image the function of cathepsin-B in a caspase-dependent cell death pathway. As a potential application, this method is used as a tool to screen compounds that are potent inhibitors of caspases and cathepsin-B proteases. The fact that this method can be readily applied to any protease of interest opens up huge opportunities for this technology in the area of target validation, high-throughput screening, in vivo imaging, diagnostics, and therapeutic intervention.

Isolation and characterization of bacteriophages from India, with lytic activity against Mycobacterium tuberculosis.

Bacteriophages are being considered as a promising natural resource for the development of alternative strategies against mycobacterial diseases, especially in the context of the wide-spread occurrence of drug resistance among the clinical isolates of Mycobacterium tuberculosis. However, there is not much information documented on mycobacteriophages from India. Here, we report the isolation of 17 mycobacteriophages using Mycobacterium smegmatis as the bacterial host, where 9 phages also lyse M. tuberculosis H37Rv. We present detailed analysis of one of these mycobacteriophages - PDRPv. Transmission electron microscopy and polymerase chain reaction analysis (of a conserved region within the TMP gene) show PDRPv to belong to the Siphoviridae family and B1 subcluster, respectively. The genome (69 110 bp) of PDRPv is circularly permuted double-stranded DNA with ∼66% GC content and has 106 open reading frames (ORFs). On the basis of sequence similarity and conserved domains, we have assigned function to 28 ORFs and have broadly categorized them into 6 groups that are related to replication and genome maintenance, DNA packaging, virion release, structural proteins, lysogeny-related genes and endolysins. The present study reports the occurrence of novel antimycobacterial phages in India and highlights their potential to contribute to our understanding of these phages and their gene products as potential antimicrobial agents.