Proteomic identification of E6AP as a molecular target of tamoxifen in MCF7 cells

Tamoxifen (Tam) is most widely used selective estrogen receptor modulator (SERM) for treatment of hormone-responsive breast cancer. Despite being regularly used in clinical therapy for breast cancer since 1971, the mechanism of Tam action remains largely unclear. In order to gain insights into Tam-mediated antibreast cancer actions, we applied 2DE and MS based proteomics approach to identify target proteins of Tam. We identified E6-associated protein, i.e. E6AP (UBE3A) among others to be regulated by Tam that otherwise is upregulated in breast tumors. We confirmed our 2DE finding by immunoblotting and further show that Tam leads to inhibition of E6AP expression presumably by promoting its autoubiquitination, which is coupled with nuclear export and subsequent proteasome-mediated degradation. Furthermore, we show that Tam- and siE6AP-mediated inhibition of E6AP leads to enhanced G0-G1 growth arrest and apoptosis, which is also evident from significant upregulation of cytochrome-c, Bax, p21, and PARP cleavage. Taken together, our data suggest that, Tam-targeted E6AP inhibition is in fact required for Tam-mediated antibreast cancer actions. Thus, E6AP may be a therapeutic target in breast cancer.     

Trp-tRNA synthetase bridges DNA-PKcs to PARP-1 to link IFN-γ and p53 signaling

Interferon-γ (IFN-γ) engenders strong antiproliferative responses, in part through activation of p53. However, the long-known IFN-γ-dependent upregulation of human Trp-tRNA synthetase (TrpRS), a cytoplasmic enzyme that activates tryptophan to form Trp-AMP in the first step of protein synthesis, is unexplained. Here we report a nuclear complex of TrpRS with the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) and with poly(ADP-ribose) polymerase 1 (PARP-1), the major PARP in human cells. The IFN-γ-dependent poly(ADP-ribosyl)ation of DNA-PKcs (which activates its kinase function) and concomitant activation of the tumor suppressor p53 were specifically prevented by Trp-SA, an analog of Trp-AMP that disrupted the TrpRS-DNA-PKcs-PARP-1 complex. The connection of TrpRS to p53 signaling in vivo was confirmed in a vertebrate system. These and further results suggest an unexpected evolutionary expansion of the protein synthesis apparatus to a nuclear role that links major signaling pathways.     

Molecular cloning of an actinobacterial-type ClpS gene from Celosia cristata expression library

In proteobacterial cytosol, ClpS protein is known as a molecular adaptor for substrate selectivity and proteolytic activity of the ATP-dependent chaperone-protease complex, ClpAP. ClpA-related ClpS is a small protein usually encoded immediately upstream of ClpA in the genome of proteobacteria. Recent bioinformatics analysis has revealed the presence of cyanobacterial-type ClpS or ClpC-related ClpS in organisms lacking ClpA, including all the plant species sequenced to date. Here we report the identification of an actinobacterial homologue of the ClpS (possibly Clp-related) gene from a plant system. A cDNA, spanning 566 bp with a complete coding region corresponding to 132 amino acids, was isolated from a Celosia cristata expression library constructed on a λ TriplEX2 vector. This cDNA product was considered to be an ATP-dependent Clp protease adaptor and was designated as Celosia actinobacterial-type ClpS, since it contains a highly conserved domain belonging to the ClpS family of proteins from actinobacteria. Celosia ClpS is about 80% identical to actinobacterial ClpS proteins in its overall deduced amino acid sequence. Based on this finding, we may define a novel target of ATP-dependent Clp complex in a plant system or speculate the presence of a second type of molecular chaperone besides ClpC in plants, as predicted for actinobacteria.     

An unconventional form of actin in protozoan hemoflagellate, Leishmania

Leishmania actin was cloned, overexpressed in baculovirus-insect cell system, and purified to homogeneity. The purified protein polymerized optimally in the presence of Mg2+ and ATP, but differed from conventional actins in its following properties: (i) it did not polymerize in the presence of Mg2+ alone, (ii) it polymerized in a restricted range of pH 7.0-8.5, (iii) its critical concentration for polymerization was found to be 3-4-fold lower than of muscle actin, (iv) it predominantly formed bundles rather than single filaments at pH 8.0, (v) it displayed considerably higher ATPase activity during polymerization, (vi) it did not inhibit DNase-I activity, and (vii) it did not bind the F-actin-binding toxin phalloidin or the actin polymerization disrupting agent Latrunculin B. Computational and molecular modeling studies revealed that the observed unconventional behavior of Leishmania actin is related to the diverged amino acid stretches in its sequence, which may lead to changes in the overall charge distribution on its solvent-exposed surface, ATP binding cleft, Mg2+ binding sites, and the hydrophobic loop that is involved in monomer-monomer interactions. Phylogenetically, it is related to ciliate actins, but to the best of our knowledge, no other actin with such unconventional properties has been reported to date. It is therefore suggested that actin in Leishmania may serve as a novel target for design of new antileishmanial drugs.     

Comparison of Metarhizium isolates for biocontrol of Helicoverpa armigera (Lepidoptera: Noctuidae) in chickpea

Metarhizium isolates from soil (53) and insect hosts (10) were evaluated for extracellular production of cuticle degrading enzyme (CDE) activities such as chitinase, chitin deacetylase (CDA), chitosanase, protease and lipase. Regression analysis demonstrated the relation of CDE activities with Helicoverpa armigera mortality. On basis of this relation, ten isolates were selected for further evaluation. Subsequently, based on LT₅₀ of the 10 isolates towards H. armigera, five isolates were selected. Out of these five isolates, three were selected on the basis of higher conidia production (60–75 g/kg rice), faster sedimentation time (ST₅₀) (2.3–2.65 h in 0.1% (w/v) Tween 80) and lower LC₅₀ (1.4–5.7×10³ conidia/mL) against H. armigera. Finally, three Metarhizium isolates were selected for the molecular fingerprinting using ITS sequencing and RAPD patterning. All three isolates, M34412, M34311 and M81123, showed comparable RAPD patterns with a 935G primer. These were further evaluated for their field performance against H. armigera in a chickpea crop. The percent efficacies with the three Metarhizium isolates were from 65 to 72%, which was comparable to the chemical insecticide, endosulfan (74%).     

Arbuscular mycorrhizal (AM) technology for the conservation of <Emphasis Type="Italic">Curculigo orchioides</Emphasis> Gaertn.: an endangered medicinal herb

Curculigo orchioides Gaertn. (family Hypoxidaceae) is an endangered anticarcinogenic and aphrodisiac herb, native of India. This study reports the effect of three arbuscular mycorrhizal (AM) fungal inocula on post-transplanting performance of ‘in vitro’ raised C. orchioides plantlets. The three AM fungal inocula consisted of two monospecific cultures of Glomus geosporum and G. microcarpum and one crude consortium of AM fungal spores isolated from rhizosphere soil of C. orchioides growing in natural habitat. Complete plantlets of C. orchioides were raised by direct organogenesis of leaf explants on half strength Murashige and Skoog’s medium devoid of any growth hormone. C. orchioides plantlets responded significantly different to all three mycorrhizal treatments. Mycorrhization enhanced the survival rate of C. orchioides plantlets to 100%. The inoculated plantlets fared significantly better than the uninoculated ones in terms of biomass production and number of leaves and roots per plant. Mycorrhizal plantlets exhibited higher concentrations of photosynthetic pigments as well as minerals P, Mg, Cu, Zn, Mn and Fe in both shoots and roots. Among the three inocula tested, plantlets inoculated with the mixed consortium of AM fungi consistently performed better in terms of the parameters evaluated. The study suggests use of mixed consortium of AM fungi over monospecific cultures for the sustainable cultivation and conservation of endangered medicinal plant: Curculigo orchioides.     

Conformational behavior of polypeptides derived through simultaneous global conservative site-directed mutagenesis of chymotrypsin inhibitor 2

The natural occurrence of conservative residue substitutions in proteins suggests that side-chain packing schemes in protein interiors can accommodate mutational replacements of residues by others of similar nature. To explore the extent to which such substitutions are tolerated, especially when introduced simultaneously and globally over the entire length of a polypeptide chain, we examined the conformational behavior of a model 65 residues-long protein, wild-type chymotrypsin inhibitor 2 (WTCI2), and two globally-mutated (GM) variants named GMCI2-1 and GMCI2-2, each incorporating 55 conservative residue substitutions. GMCI2-1, was soluble over a wide range of pH, and folded into a compact, spherical, monomer marked by (i) complete absence of surface hydrophobicity, (ii) a WTCI2-like betaII-type CD spectrum, (iii) high WTCI2-like thermal stability, and (d) 1D and 2D NMR spectra characteristic of folded protein structure. GMCI2-2 was insoluble over a wide range of pH, and could be solubilized only at pH 4.0, showing non-WTCI2-like far-UV CD spectra characterized by high helical content. These results tentatively indicate that polypeptides incorporating residues of identical nature at equivalent chain locations can show the potential to fold with similar characteristics. However, further detailed investigations would be required to determine whether indeed the structural fold of GMCI2-1 resembles that of WTCI2, and to evaluate the extent to which it does so.