A nuclear export signal and phosphorylation regulate Dok1 subcellular localization and functions

Dok1 is believed to be a mainly cytoplasmic adaptor protein which down-regulates mitogen-activated protein kinase activation, inhibits cell proliferation and transformation, and promotes cell spreading and cell migration. Here we show that Dok1 shuttles between the nucleus and cytoplasm. Treatment of cells with leptomycin B (LMB), a specific inhibitor of the nuclear export signal (NES)-dependent receptor CRM1, causes nuclear accumulation of Dok1. We have identified a functional NES (348LLKAKLTDPKED359) that plays a major role in the cytoplasmic localization of Dok1. Src-induced tyrosine phosphorylation prevented the LMB-mediated nuclear accumulation of Dok1. Dok1 cytoplasmic localization is also dependent on IKKbeta. Serum starvation or maintaining cells in suspension favor Dok1 nuclear localization, while serum stimulation, exposure to growth factor, or cell adhesion to a substrate induce cytoplasmic localization. Functionally, nuclear NES-mutant Dok1 had impaired ability to inhibit cell proliferation and to promote cell spreading and cell motility. Taken together, our results provide the first evidence that Dok1 transits through the nucleus and is actively exported into the cytoplasm by the CRM1 nuclear export system. Nuclear export modulated by external stimuli and phosphorylation may be a mechanism by which Dok1 is maintained in the cytoplasm and membrane, thus regulating its signaling functions.     

Differential Alterations in Metabolic Pattern of the Spliceosomal UsnRNAs during Pre-Malignant Lung Lesions Induced by Benzo(a)pyrene: Modulation by Tea Polyphenols

The differential alterations of the spliceosomal UsnRNAs (U1, U2, U4, U5, and U6) were reported to be associated with cellular proliferation and development. The attempt was made in this study to analyze the metabolic pattern of the spliceosomal UsnRNAs during the development of pre-malignant lung lesions induced in experimental mice model system by benzo(a)pyrene (BP) and also to see how tea polyphenols, epigallocatechin gallate (EGCG) and epicatechin gallate (ECG), modulate the metabolism of these UsnRNAs during the lung carcinogenesis. No significant changes in the level of the UsnRNAs were seen in the inflammatory lung lesions at 9th week due to treatment of BP. However, there was significant increase in the level of U1 (∼2.5 fold) and U5 (∼47%) in the hyperplastic lung lesions at 17th week. But in the mild dysplastic lung lesions at 26th week, the level of UsnRNAs did not change significantly. Whereas, in the dysplastic lung lesions at 36th week there was significant increase in the level of the U2 (∼2 fold), U4 (∼2.5 fold) and U5 (∼2 fold). Due to the EGCG and ECG treatment the lung lesions at 9th week appeared normal and in the 17th, 26th, and 36th week it appeared as hyperplasia. The level of the UsnRNAs was significantly low in the lung lesions at 9th week (only U2 and U4 by EGCG), at 17th week (only U1 by EGCG/ECG), at 26th week (U1 by ECG; U2, U4 and U5 by EGCG/ECG) and at 36th week (U1 by ECG, U2 and U4 by EGCG/ECG). Whereas, there was significant increase in the level of U5 (by EGCG/ECG) and U6 (by EGCG only) in the lung lesions at 36th and 26th week respectively. This indicates that the metabolism of the spliceosomal UsnRNAs differentially altered during the development of pre-malignant lung lesions by BP as well as during the modulation of the lung lesions by the tea polyphenols.     

Potential of cadmium sulphide nanorods as an optical microscopic probe to the folding state of cytochrome C

The folding behavior of cytochrome C (Cyt-C) conjugated with CdS nanorods (CdSnr) is amenable to monitoring by bright field microscopy, the porosity and percolating behavior of such protein conjugated nanoclusters depending on the folding history prior to the conjugation. The method has been used to predict the thermal melting behavior as well as guanidine hydrochloride induced unfolding of Cyt-C. Dynamic light scattering studies indicate that the size distribution of the nanoforms widens in presence of the protein. Furthermore, there is emergence of clusters with higher conductivity and altered zeta potential. Increase of second virial coefficient of CdS nanoforms in the presence of Cyt-C (obtained from static light scattering experiments) implies presence of protein coat over the hydrophobic nanosurface. The results are supported by morphological changes observed through scanning electron microscopy (SEM). Accordingly, the X-ray diffraction pattern shows a change of crystallographic orientations of CdSnr in presence of Cyt-C.     

Purification and characterization of an enantioselective arylacetonitrilase from Pseudomonas putida

The highly enantioselective arylacetonitrilase of Pseudomonas putida was purified to homogeneity using a combination of (NH₄)₂SO₄ fractionation and different chromatographic techniques. The enzyme has a molecular weight of 412 kDa and consisted of approximately nine to ten identical subunits (43 kDa). The purified enzyme exhibited a pH optimum of 7.0 and temperature optimum of 40°C. The nitrilase was highly susceptible to thiol-specific reagents and metal ions and also required a reducing environment for its activity. These reflected the presence of a catalytically essential thiol group for enzyme activity which is in accordance with the proposed mechanism for nitrilase-catalyzed reaction. The enzyme was highly specific for arylacetonitriles with phenylacetonitrile and its derivatives being the most preferred substrates. Higher specificity constant (k _cat/K _m) values for phenylacetonitrile compared to mandelonitrile also revealed the same. Faster reaction rate achieved with this nitrilase for mandelonitrile hydrolysis was possibly due to the low activation energy required by the protein. Incorporation of low concentration (<5%) of organic solvent increased the enzyme activity by increasing the availability of the substrate. Higher stability of the enzyme at slightly alkaline pH and ambient temperature provides an excellent opportunity to establish a dynamic kinetic resolution process for the production of (R)-(−)-mandelic acid from readily available mandelonitrile.     

Differential induction of Leishmania donovani bi-subunit topoisomerase I-DNA cleavage complex by selected flavones and camptothecin: activity of flavones against camptothecin-resistant topoisomerase I

Emergence of the bi-subunit topoisomerase I in the kinetoplastid family (Trypanosoma and Leishmania) has brought a new twist in topoisomerase research related to evolution, functional conservation and preferential sensitivities to the specific inhibitors of type IB topoisomerase family. In the present study, we describe that naturally occurring flavones baicalein, luteolin and quercetin are potent inhibitors of the recombinant Leishmania donovani topoisomerase I. These compounds bind to the free enzyme and also intercalate into the DNA at a very high concentration (300 µM) without binding to the minor grove. Here, we show that inhibition of topoisomerase I by these flavones is due to stabilization of topoisomerase I–DNA cleavage complexes, which subsequently inhibit the religation step. Their ability to stabilize the covalent topoisomerase I–DNA complex in vitro and in living cells is similar to that of the known topoisomerase I inhibitor camptothecin (CPT). However, in contrast to CPT, baicalein and luteolin failed to inhibit the religation step when the drugs were added to pre-formed enzyme substrate binary complex. This differential mechanism to induce the stabilization of cleavable complex with topoisomerase I and DNA by these selected flavones and CPT led us to investigate the effect of baicalein and luteolin on CPT-resistant mutant enzyme LdTOP1Δ39LS lacking 1–39 amino acids of the large subunit [B. B. Das, N. Sen, S. B. Dasgupta, A. Ganguly and H. K. Majumder (2005) J. Biol. Chem. 280, 16335–16344]. Baicalein and luteolin stabilize duplex oligonucleotide cleavage with LdTOP1Δ39LS. This observation was further supported by the stabilization of in vivo cleavable complex by baicalein and luteolin with highly CPT-resistant L.donovani strain. Taken together, our data suggest that the interacting amino acid residues of topoisomerase I may be partially overlapping or different for flavones and CPT. This study illuminates new properties of the flavones and provide additional insights into the ligand binding properties of L.donovani topoisomerase I.     

sll1981, an acetolactate synthase homologue of Synechocystis sp. PCC6803, functions as l-myo-inositol 1-phosphate synthase

l-myo-inositol 1-phosphate synthase (EC 5.5.1.4; MIPS) catalyzes the first rate limiting conversion of d-glucose 6-phosphate to l-myo-inositol 1-phosphate in the inositol biosynthetic pathway. In an earlier communication we have reported two forms of MIPS in Synechocystis sp. PCC6803 (Chatterjee et al. in Planta 218:989–998, 2004). One of the forms with a ~50 kDa subunit has been found to be coded by an as yet unassigned ORF, sll1722. In the present study we have purified the second isoform of MIPS as a ~65 kDa protein from the crude extract of Synechocystis sp. PCC6803 to apparent homogeneity and biochemically characterized. MALDI-TOF analysis of the 65 kDa protein led to its identification as acetolactate synthase large subunit (EC 2.2.1.6; ALS), the putatively assigned ORF sll1981 of Synechocystis sp. PCC6803. The PCR amplified ~1.6 kb product of sll1981 was found to functionally complement the yeast inositol auxotroph, FY250 and could be expressed as an immunoreactive ~65 kDa MIPS protein in the natural inositol auxotroph, Schizosaccharomyces pombe. In vitro MIPS activity and cross reactivity against MIPS antibody of purified recombinant sll1981 further consolidated its identity as the second probable MIPS gene in Synechocystis sp. PCC6803. Sequence comparison along with available crystal structure analysis of the yeast MIPS reveals conservation of several amino acids in sll1981 essential for substrate and co-factor binding. Comparison with other prokaryotic and eukaryotic MIPS sequences and phylogenetic analysis, however, revealed that like sll1722, sll1981 is quite divergent from others. It is probable that sll1981 may code for a bifunctional enzyme protein having conserved domains for both MIPS and acetolactate synthase (ALS) activities.Anirban Chatterjee and Krishnarup Ghosh Dastidar contributed equally.     

CRTAP is required for prolyl 3- hydroxylation and mutations cause recessive osteogenesis imperfecta

Prolyl hydroxylation is a critical posttranslational modification that affects structure, function, and turnover of target proteins. Prolyl 3-hydroxylation occurs at only one position in the triple-helical domain of fibrillar collagen chains, and its biological significance is unknown. CRTAP shares homology with a family of putative prolyl 3-hydroxylases (P3Hs), but it does not contain their common dioxygenase domain. Loss of Crtap in mice causes an osteochondrodysplasia characterized by severe osteoporosis and decreased osteoid production. CRTAP can form a complex with P3H1 and cyclophilin B (CYPB), and Crtap-/- bone and cartilage collagens show decreased prolyl 3-hydroxylation. Moreover, mutant collagen shows evidence of overmodification, and collagen fibrils in mutant skin have increased diameter consistent with altered fibrillogenesis. In humans, CRTAP mutations are associated with the clinical spectrum of recessive osteogenesis imperfecta, including the type II and VII forms. Hence, dysregulation of prolyl 3-hydroxylation is a mechanism for connective tissue disease.