Jayanta K. Pal

Role of 5′‐ and 3′‐untranslated regions of mRNAs in human diseases

Protein synthesis is often regulated at the level of initiation of translation, making it a critical step. This regulation occurs by both the cis‐regulatory elements, which are located in the 5′‐ and 3′‐UTRs (untranslated regions), and trans‐acting factors. A breakdown in this regulation machinery can perturb cellular metabolism, leading to various physiological abnormalities. The highly structured UTRs, along with features such as GC‐richness, upstream open reading frames and internal ribosome entry sites, significantly influence the rate of translation of mRNAs. In this review, we discuss how changes in the cis‐regulatory sequences of the UTRs, for example, point mutations and truncations, influence expression of specific genes at the level of translation. Such modifications may tilt the physiological balance from healthy to diseased states, resulting in conditions such as hereditary thrombocythaemia, breast cancer, fragile X syndrome, bipolar affective disorder and Alzheimers disease. This information tends to establish the crucial role of UTRs, perhaps as much as that of coding sequences, in health and disease.

Coordinated regulation of p53 apoptotic targets BAX and PUMA by SMAR1 through an identical MAR element

How tumour suppressor p53 bifurcates cell cycle arrest and apoptosis and executes these distinct pathways is not clearly understood. We show that BAX and PUMA promoters harbour an identical MAR element and are transcriptional targets of SMAR1. On mild DNA damage, SMAR1 selectively represses BAX and PUMA through binding to the MAR independently of inducing p53 deacetylation through HDAC1. This generates an anti‐apoptotic response leading to cell cycle arrest. Importantly, knockdown of SMAR1 induces apoptosis, which is abrogated in the absence of p53. Conversely, apoptotic DNA damage results in increased size and number of promyelocytic leukaemia (PML) nuclear bodies with consequent sequestration of SMAR1. This facilitates p53 acetylation and restricts SMAR1 binding to BAX and PUMA MAR leading to apoptosis. Thus, our study establishes MAR as a damage responsive cis element and SMAR1–PML crosstalk as a switch that modulates the decision between cell cycle arrest and apoptosis in response to DNA damage.

Dose-dependent differential effect of hemin on protein synthesis and cell proliferation inLeishmania donovani promastigotes culturedin vitro

Leishmania donovani requires an exogenous source of heme for growth and transformation. Inin vitro culture of the free-living promastigotes, exogenously added hemin enhances cell proliferation. In this investigation, the question of the function of heme with particular reference to protein synthesis and cell proliferation has been addressed. The results ofin vitro cell culture experiments demonstrated that hemin (10 μM) alone is suitable for supporting optimum level of protein synthesis, and thereby cell proliferation of promastigotes to an extent that it can replace fetal bovine serum. However,in situ labelling experiments along with Western blots revealed that high concentration of hemin (50 μM) reduced the level of protein synthesis in general and of β-tubulin in particular with a concomitant induction of hsp90, and induced consequent morphological changes that are observed duringin situ transformation of promastigotes in mammalian macrophages. These results therefore suggest that sudden exposure to high concentration of heme in mammalian macrophages may be one of the key factors that trigger promastigote to amastigote transformation inL. donovani. Furthermore, hemin with its dual characteristic could be used as a tool to understand molecular mechanism of cell proliferation and transformation in these parasites.

Small molecule modulators of eukaryotic initiation factor 2α kinases, the key regulators of protein synthesis.

Eukaryotic initiation factor 2 alpha kinases (eIF-2α kinases) are key mediators of stress response in cells. In mammalian cells, there are four eIF-2α kinases, namely HRI (Heme-Regulated Inhibitor), PKR (RNA-dependent Protein Kinase), PERK (PKR-like ER Kinase) and GCN2 (General Control Non-derepressible 2). These kinases get activated during diverse cytoplasmic stress conditions and phosphorylate the alpha-subunit of eIF2, leading to global protein synthesis inhibition. Therefore, eIF-2α kinases play a vital role in various cellular processes such as proliferation, differentiation, apoptosis and cell signaling. Deregulation of eIF-2α kinases and protein synthesis has been linked to numerous pathological conditions such as certain cancers, anemia and neurodegenerative disorders. Thus, modulation of these kinases by small molecules holds a great therapeutic promise. In this review we have compiled the available information on inhibitors and activators of these four eIF-2α kinases. The review concludes with a note on the selectivity issue of currently available modulators and future perspectives for the design of specific small molecule probes.

Dioscorea bulbifera mediated synthesis of novel Au core Ag shell nanoparticles with potent antibiofilm and antileishmanial activity

Dioscorea bulbifera is a potent medicinal plant used in both Indian and Chinese traditional medicine owing to its rich phytochemical diversity. Herein, we report the rapid synthesis of novel AucoreAgshell nanoparticles by D. bulbifera tuber extract (DBTE). AucoreAgshell NPs synthesis was completed within 5 h showing a prominent peak at 540 nm. HRTEM analysis revealed 9 nm inner core of elemental gold covered by a silver shell giving a total particle diameter upto 15 nm. AucoreAgshellNPs were comprised of 57.34 ± 1.01% gold and 42.66 ± 0.97% silver of the total mass. AucoreAgshellNPs showed highest biofilm inhibition upto 83.68 ± 0.09% against A. baumannii. Biofilms of P. aeruginosa, E. coli, and S. aureus were inhibited up to 18.93 ± 1.94%, 22.33 ± 0.56%, and 30.70 ± 1.33%, respectively. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) confirmed unregulated cellular efflux through pore formation leading to cell death. Potent antileishmanial activity of AucoreAgshellNPs (MIC = 32 µg/mL) was confirmed by MTT assay. Further SEM micrographs showed pronounced deformity in the spindle shaped cellular morphology changing to spherical. This is the first report of synthesis, characterization, antibiofilm, and antileishmanial activity of AucoreAgshellNPs synthesized by D. bulbifera.

Oxidative stress perturbs cell proliferation in human K562 cells by modulating protein synthesis and cell cycle

Oxidative stress leads to perturbation of a variety of cellular processes resulting in inhibition of cell proliferation. This study has determined the effect of oxidative stress on protein synthesis in human K562 cells using a hydrophilic peroxyl radical initiator, AAPH and H2O2. The results indicated that oxidative stress leads to a significant decrease in the rate of protein synthesis caused due to induced activation as well as expression of the erythroid cell-specific eIF-2α kinase, called the Heme Regulated Inhibitor (HRI). Elevated levels of HRI expression and activity were accompanied by increased lipid peroxidation and decreased cell proliferation. Further, oxidative stress also caused inactivation of p34cdc2 kinase, thereby arresting cell division leading to apoptosis. Thus, the data provides the mechanism of inhibition of protein synthesis and perturbation of a cell cycle regulatory protein leading to inhibition of cell proliferation in K562 cells during oxidative stress.

Effect of sublethal concentrations of flufenoxuron on growth, development and reproductive performance of Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae)

Summary The effect of sublethal concentrations 0.00141%(LC20), 0.00251%(LC30) and 0.00336% (LC40) of a dispersible concentrated formulation of the insect growth regulator flufenoxuron (Cascade®), on larval growth and development, adult reproductive potential and egg hatchability of the red flour beetle, Tribolium castaneum, was investigated. When neonates were subjected to sublethal concentrations of flufenoxuron in artificial diet for 24 h, there were dose-dependent effects on larval weight, percent pupation and percent adult emergence, as well as time taken for adult emergence. A small proportion of larval-pupal as well as pupal-adult intermediates were observed at all concentrations. Adults emerging from the LC20 and LC30 concentrations laid mostly non-viable eggs, and the few larvae which emerged from viable eggs died at the first instar stage. At the LC40 concentration, all the adults that emerged were deformed and subsequently died. Flufenoxuron exhibited transovarial ovicidal activity resulting in the production of non-Viable eggs upon exposure of adults of different ages (2 days old, 3 days old and 4 days old) to treated diet. It was observed that in 2-day-old adults, fecundity decreased with an increased concentration. In the case of 3-day-old adults, there was no difference in fecundity with respect to the concentrations tested, although it was significantly less than the control. In the case of 4-day-old adults there was a drastic reduction in fecundity at LC40 and the eggs laid were abnormal at all concentrations. Topical application of sublethal concentrations of flufenoxuron to adults of either sex reduced the fecundity in a dose-dependent manner. Furthermore, the fecundity was reduced drastically in pairs where both the sexes were treated as compared to the pairs where only one sex was treated. Eggs showed a decrease in hatching percentage with increasing concentrations of flufenoxuron mixed with diet to which the eggs were exposed.

IRESPred: Web Server for Prediction of Cellular and Viral Internal Ribosome Entry Site (IRES).

Cellular mRNAs are predominantly translated in a cap-dependent manner. However, some viral and a subset of cellular mRNAs initiate their translation in a cap-independent manner. This requires presence of a structured RNA element, known as, Internal Ribosome Entry Site (IRES) in their 5′ untranslated regions (UTRs). Experimental demonstration of IRES in UTR remains a challenging task. Computational prediction of IRES merely based on sequence and structure conservation is also difficult, particularly for cellular IRES. A web server, IRESPred is developed for prediction of both viral and cellular IRES using Support Vector Machine (SVM). The predictive model was built using 35 features that are based on sequence and structural properties of UTRs and the probabilities of interactions between UTR and small subunit ribosomal proteins (SSRPs). The model was found to have 75.51% accuracy, 75.75% sensitivity, 75.25% specificity, 75.75% precision and Matthews Correlation Coefficient (MCC) of 0.51 in blind testing. IRESPred was found to perform better than the only available viral IRES prediction server, VIPS. The IRESPred server is freely available at http://bioinfo.net.in/IRESPred/.

Lead exposure and heat shock inhibit cell proliferation in human HeLa and K562 cells by inducing expression and activity of the heme-regulated eIF-2alpha kinase.

We have used human cell lines, namely, K562 and HeLa cells as model systems in understanding the mechanism of lead toxicity and heat shock, that may be mediated by the heme-regulated eIF-2alpha kinase which is also called the heme-regulated inhibitor (HRI). RT-PCR analysis using HRI-specific primers indicated a two- to three-fold increase in HRI expression in K562 and HeLa cells exposed to lead acetate and heat shock, respectively. Further, in vitro eIF-2alpha kinase assay indicated a two- to three-fold increase in HRI kinase activity during lead toxicity in K562 cells. This increase in HRI expression and its activity was accompanied by a significant decrease in cell proliferation and cell viability. This is therefore, the first report indicating that both heavy metal exposure and heat shock cause inhibition of protein synthesis not by activation of HRI alone but by its over-expression as well as activation. Our data indicate further that lead-induced inhibition of cell proliferation may be caused due to inhibition of protein synthesis resulted due to induced expression and activity of HRI.

Stress-induced overexpression of the heme-regulated eIF-2α kinase is regulated by Elk-1 activated through ERK pathway

The heme-regulated eIF-2alpha kinase, also called the heme-regulated inhibitor (HRI), is activated under various cytoplasmic stresses in reticulocytes leading to inhibition of initiation of protein synthesis. Our previous studies indicated that the promoter activity and expression of the human HRI (hHRI) increase in human K562 cells during heat shock and lead exposure. Contrary to this, hemin chloride which inactivates the kinase, downregulates HRI expression. Here, we attempted to understand the mechanism of regulation of hHRI expression in the lead- and hemin-exposed cells. Our results demonstrate the involvement of two transcription factors, Elk-1 and MZF-1 in regulating HRI expression. Chromatin immunoprecipitation assays established further that Elk-1 is involved in upregulating HRI expression during stress along with a co-activator p300, while MZF-1 along with HDAC-1 is instrumental in its downregulation during hemin treatment. We also demonstrate the involvement of ERK pathway in activating Elk-1 during stress resulting in an over expression of hHRI.