The biochemical mechanism of caspase-2 activation

A unified model for initiator caspase activation has previously been proposed based on the biochemical analysis of caspase-8 and -9. Caspase-2 is structurally related to caspase-9, but its mechanism of activation is not known. Using an uncleavable mutant of caspase-2, we show that dimerization (and not processing) is the key event that drives initial procaspase-2 activation. Following dimerization, caspase-2 undergoes autocatalytic cleavage that promotes its stable dimerization and further enhances the catalytic activity of caspase-2. Although the caspase-2 zymogen does not require cleavage for the initial acquisition of activity, intersubunit cleavage is required to generate levels of activity required to induce cell death by overexpression. We also provide evidence that the reported disulfide bond linkage between two caspase-2 monomers is dispensable for caspase-2 dimerization. As caspase-2 does not require cleavage for its initial activation, our findings confirm caspase-2 to be a bona fide initiator caspase.     

Competitive binding of EF1 and EF2 by mammalian ribosomes: role of GTP hydrolysis in overcoming inhibition by EF2 of aminoacyl-tRNA binding

Preincubation of mammalian ribosomes with EF2 retards subsequent binding of EF1-leucyl tRNA when GTP hydrolysis does not occur. This indicates that both the elongation factors have a common binding site on the ribosome. Since the conditions permitting hydrolysis of GTP overcome this inhibition, the possible regulatory role of GTP at the common site on the ribosome is discussed.     

Estimation of protein sulfhydryl groups with 5,5'-(35S) dithio-bis(2-nitrobenzoate)

³⁵S-Labeled dithio-bis(2-nitrobenzoate) has been prepared and its usefulness for the measurement of protein sulfhydryl groups has been investigated. The technique is at least 20 times more sensitive than when dithio-bis(2-nitrobenzoate) is used colorimetrically. The adduct formed between the reagent and sulfhydryl groups is stable between pH 3–9 which should enable use of the reagent for the tagging of sulfhydryl-containing proteins in a wide variety of biochemical procedures. Removal of the adduct from a protein is achieved simply by treatment with a thiol reagent, with concomitant restoration of enzymic activity of the protein. The procedure has been successfully tested on alcohol dehydrogenase and mammalian ribosomes.     

Anti-neoplastic pharmacophore benzophenone-1 coumarin (BP-1C) targets JAK2 to induce apoptosis in lung cancer

Reigning of the abnormal gene activation associated with survival signalling in lung cancer leads to the anomalous growth and therapeutic failure. Targeting specific cell survival signalling like JAK2/STAT3 nexus has become a major focus of investigation to establish a target specific treatment. The 2-bromobenzoyl-4-methylphenoxy-acetyl hydra acetyl Coumarin (BP-1C), is new anti-neoplastic agent with apoptosis inducing capacity. The current study was aimed to develop antitumor phramacophore, BP-1C as JAK2 specific inhibitor against lung neoplastic progression. The study validates and identifies the molecular targets of BP-1C induced cell death. Cell based screening against multiple cancer cell lines identified, lung adenocarcinoma as its specific target through promotion of apoptosis. The BP-1C is able to induce, specific hall marks of apoptosis and there by conferring anti-neoplastic activity. Validation of its molecular mechanism, identified, BP-1C specifically targets JAK2^Tyr1007/1008 phosphorylation, and inhibits its downstream STAT3^Tyr705 signalling pathway to induce cell death. As a consequence, modulation in Akt/Src survival signal and altered expression of interwoven apoptotic genes were evident. The results were reproducible in an in-vivo LLC tumor model and in-ovo xenograft studies. The computational approaches viz, drug finger printing confers, BP-1C as novel class JAK2 inhibitor and molecular simulations studies assures its efficiency in binding with JAK2. Overall, BP-1C is a novel JAK2 inhibitor with experimental evidence and could be effectively developed into a promising drug for lung cancer treatment.

Graphical abstract

     

The role of a 70 kDa nuclear protein in fetal haemoglobin synthesis in K562 cells

Abstract. Previously, we reported that a 70 kDa nuclear protein may regulate fetal haemoglobin gene expression in haemin treated K562 cells. To obtain further evidence of the specific role of this 70 kDa nuclear protein, we compared the nuclear fractions isolated from phenylacetate, hydroxyurea and haemin treated K562 cells. Both phenylacetate and hydroxyurea have been used to induce fetal haemoglobin synthesis in K562 cells. Cell growth was measured by biochemical events including DNA, RNA and protein synthesis. Differentiation of K562 cells was determined by both [³H]-leucine incorporation into fetal haemoglobin and scoring benzidine-stained positive cells. Unlike the haemin treated cells, phenylacetate and hydroxyurea induced growth arrest and increased fetal haemoglobin gene expression in K562 cells. After four days of treatment with phenylacetate and hydroxyurea more than 50% of the cells stained positive with benzidine. The SDS-Polyacrylamide gel electrophoretic analysis of nuclear proteins isolated from phenylacetate and hydroxyurea treated K562 cells showed that the 70 kDa protein was reduced in nuclear protein extract in both groups similar to haemin treated cells. These results suggest that the loss of the 70 kDa protein from a nuclear protein extract is not restricted to only haemin treated cells but also occurs in hydroxyurea and phenylacetate treated cells. Our results provide further evidence that the 70 kDa nuclear protein may be involved in regulating fetal haemoglobin expression through a negative control mechanism.     

Inhibition of T-lymphocyte activation by amiloride

The T-lymphocyte activation process involves a series of coordinately coupled biochemical events occurring in response to antigen or mitogen. These events have not been completely characterized. The present studies investigate the mechanism of protein synthesis during the initial phase of T-cell activation. Among the early biochemical changes, induction of protein synthesis was observed as early as 10 minutes after mitogen stimulation of T-lymphocytes. This early protein synthesis was inhibited by cycloheximide but was insensitive to actinomycin-D, indicating the presence of preformed mRNA in resting lymphocytes. Since early protein synthesis parallels the increase in protein kinase C activity in activated T-lymphocytes, these two biochemical events may be related. In the present report, amiloride, an inhibitor of Na+/H+ antiport and protein kinase C, significantly inhibited [3H]leucine and [3H]thymidine incorporation in a dose-dependent manner into phytohemagglutinin (PHA)-stimulated T-lymphocytes. Furthermore, when T-lymphocytes were stimulated by phorbol myristate acetate, a known activator of protein kinase C, a similar inhibition of protein and DNA synthesis by amiloride was observed. The partially purified cytosol fraction isolated from PHA-activated T-lymphocytes showed a 75% decrease in protein kinase C-mediated [32P] incorporation from ATP in the presence of 100 microM amiloride. These results suggest that the T-cell activation process following exposure to mitogens involves early protein synthesis, which may be mediated by protein kinase C.     

A simple method for hemoglobin measurement in cell culture system.

A simple method of hemoglobin analysis in a cell culture system is described. Hemoglobins synthesized in cell cultures are labeled with radioactive amino acids. The cell extract containing radiolabeled hemoglobin is mixed with A, F, S, C, hemoglobin markers and separated by cellulose acetate electrophoresis. Individual bands of hemoglobin are cut from the gel and analyzed for radioactivity. This method is especially useful for determination of newly synthesized minute amount of hemoglobin in cell extracts that are difficult to visualize by staining procedure.     

On-line monitoring of ethanol: in relation to the rate of carbon dioxide evolved

A simple method for rapid on-line measurement of ethanol, produced in a continuous bioreactor, in a steady state, is described. Soap film meter measures the rate of CO² evolved. This value is equated to the equimolar production of ethanol. A mean 3.3% error was observed, as compared to the gas chromatography method.     

Status of tRNA charging, trinucleotide acceptor sequence and tRNA nucleotidyltransferase activity in the human placenta.

Samples of tRNA isolated from the cell sap of full-term human placenta were found to have a low capacity for accepting amino acids in the presence of partially purified synthetase preparations made from placental or rat liver cell sap. Gel electrophoresis of placental tRNA showed that part of this could be accounted for by gross degradation. The proportion of chargeable tRNA carrying amino acids was estimated by periodate oxidation followed by stripping and then charging with labeled amino acids. Only 50% of chargeable placental tRNA was in the charged state when isolated, whereas 87% of freshly isolated rat liver tRNA was found to be charged with amino acids. A fraction from placental cell sap was shown to have tRNA nucleotidyltransferase activity. When placental tRNA was incubated with this fraction and [3H]ATP or [3H]CTP, ATP was incorporated into about 12% of the tRNA molecules and CTP into 5-7%. When rat liver tRNA was used in place of placental tRNA, [3H]ATP was incorporated into less than 5% of the tRNA molecules. By using snake-venom diesterase over short periods of incubation, it was confirmed that the ATP had been incorporated terminally as AMP into the placental tRNA. These observations show that, in contrast to rat liver tRNA, tRNA prepared from human placenta is poorly charged with amino acids, many of the molecules lack the acceptor trinucleotide and there is extensive degradation beyond this stage.