Deepak Kumar Saini

A tetrafacial water-soluble molecular barrel (1) was synthesized by coordination driven self-assembly of a symmetrical tetrapyridyl donor (L) with a cis-blocked 90° acceptor [cis-(en)Pd(NO₃)₂] (en = ethane-1,2-diamine). The open barrel structure of (1) was confirmed by single crystal X-ray diffraction. The presence of a hydrophobic cavity with large windows makes it an ideal candidate for encapsulation and carrying hydrophobic drug like curcumin in an aqueous medium. The barrel (1) encapsulates curcumin inside its molecular cavity and protects highly photosensitive curcumin from photodegradation. The photostability of encapsulated curcumin is due to the absorption of a high proportion of the incident photons by the aromatic walls of 1 with a high absorption cross-sectional area, which helps the walls to shield the guest even against sunlight/UV radiations. As compared to free curcumin in water, we noticed a significant increase in solubility as well as cellular uptake of curcumin upon encapsulation inside the water-soluble molecular barrel (1) in aqueous medium. Fluorescence imaging confirmed that curcumin was delivered into HeLa cancer cells by the aqueous barrel (1) with the retention of its potential anticancer activity. While free curcumin is inactive toward cancer cells in aqueous medium at room temperature due to negligible solubility, the determined IC₅₀ value of ∼14 μM for curcumin in aqueous medium in the presence of the barrel (1) reflects the efficiency of the barrel as a potential curcumin carrier in aqueous medium without any other additives. Thus, two major challenges of increasing the bioavailability and stability of curcumin in aqueous medium even in the presence of UV light have been addressed by using a new supramolecular water-soluble barrel (1) as a drug carrier.

The MMP (matrix metalloproteinases) family of endopeptidases are involved in cleavage induced remodelling of the extracellular matrix including collagen, fibrinogen, elastin, and gelatin. Owing to their proteolytic activity which can cleave and degrade multiple intracellular substrates, the overexpression and purification of these proteins tends to be toxic. Here we describe a novel “matrix assisted refolding” protocol to overcome the technical challenges associated with overexpression and purification of full-length MMPs. The toxicity issue associated with MMP expression, is circumvented by expressing the recombinant protein in Escherichia coli in an inactive insoluble form. The methodology used for obtaining full-length MMP2 protein from these inclusion bodies, by its subsequent purification and refolding using affinity chromatography, through a single-step matrix based refolding protocol is presented here. The protocol described yields high concentrations of pure full-length and active MMP2 protein useful for downstream applications.

Two-component signaling systems (TCSs) are composed of two proteins, sensor kinases and response regulators, which can cross-talk and integrate information between them by virtue of high-sequence conservation and modular nature, to generate concerted and diversified responses. However, TCSs have been shown to be insulated, to facilitate linear signal transmission and response generation. Here, we discuss various mechanisms that confer specificity or cross-talk among TCSs. The presented models are supported with evidence that indicate the physiological significance of the observed TCS signaling architecture. Overall, we propose that the signaling topology of any TCSs cannot be predicted using obvious sequence or structural rules, as TCS signaling is regulated by multiple factors, including spatial and temporal distribution of the participating proteins.

Two-component systems (TCSs), which contain paired sensor kinase and response regulator proteins, form the primary apparatus for sensing and responding to environmental cues in bacteria. TCSs are thought to be highly specific, displaying minimal cross-talk, primarily due to the co-evolution of the participating proteins. To assess the level of cross-talk between the TCSs of Mycobacterium tuberculosis, we mapped the complete interactome of the M. tuberculosis TCSs using phosphotransfer profiling. Surprisingly, we found extensive cross-talk among the M. tuberculosis TCSs, significantly more than that in the TCSs in Escherichia coli or Caulobacter crescentus, thereby offering an alternate to specificity paradigm in TCS signalling. Nearly half of the interactions we detected were significant novel cross-interactions, unravelling a potentially complex signalling landscape. We classified the TCSs into specific ‘one-to-one’ and promiscuous ‘one-to-many’ and ‘many-to-one’ circuits. Using mathematical modelling, we deduced that the promiscuous signalling observed can explain several currently confounding observations about M. tuberculosis TCSs. Our findings suggest an alternative paradigm of bacterial signalling with significant cross-talk between TCSs yielding potentially complex signalling landscapes.

Cells exposed to genotoxic stress induce cellular senescence through a DNA damage response (DDR) pathway regulated by ATM kinase and reactive oxygen species (ROS). Here, we show that the regulatory roles for ATM kinase and ROS differ during induction and maintenance of cellular senescence. Cells treated with different genotoxic agents were analyzed using specific pathway markers and inhibitors to determine that ATM kinase activation is directly proportional to the dose of the genotoxic stress and that senescence initiation is not dependent on ROS or the p53 status of cells. Cells in which ROS was quenched still activated ATM and initiated the DDR when insulted, and progressed normally to senescence. By contrast, maintenance of a viable senescent state required the presence of ROS as well as activated ATM. Inhibition or removal of either of the components caused cell death in senescent cells, through a deregulated ATM–ROS axis. Overall, our work demonstrates existence of an intricate temporal hierarchy between genotoxic stress, DDR and ROS in cellular senescence. Our model reports the existence of different stages of cellular senescence with distinct regulatory networks.

Matrix metalloproteinases expression is used as biomarker for various cancers and associated malignancies. Since these proteinases can cleave many intracellular proteins, overexpression tends to be toxic; hence, a challenge to purify them. To overcome these limitations, we designed a protocol where full length pro-MMP2 enzyme was overexpressed in E. coli as inclusion bodies and purified using 6xHis affinity chromatography under denaturing conditions. In one step, the enzyme was purified and refolded directly on the affinity matrix under redox conditions to obtain a bioactive protein. The pro-MMP2 protein was characterized by mass spectrometry, CD spectroscopy, zymography and activity analysis using a simple in-house developed ‘form invariant’ assay, which reports the total MMP2 activity independent of its various forms. The methodology yielded higher yields of bioactive protein compared to other strategies reported till date, and we anticipate that using the protocol, other toxic proteins can also be overexpressed and purified from E. coli and subsequently refolded into active form using a one step renaturation protocol.